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Influence of some physiologic and dietary factors on plasma sterol concentration in bile acid excretion… Lindsay, Owen Burnett 1968

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THE  INFLUENCE OF SOME PHYSIOLOGIC AND DIETARY FACTORS ON PLASMA STEROL CONCENTRATION AND BILE ACID EXCRETION IN THE DOMESTIC CHICKEN  by  OWEN BURNETT LINDSAY B.S.A., U n i v e r s i t y o f B r i t i s h Columbia, 1960 M.S.A., U n i v e r s i t y o f B r i t i s h Columbia, 1963  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  i n the Department of Poultry  We accept t h i s required  Science  t h e s i s as conforming t o the  standard  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1968  In p r e s e n t i n g  for  that  this  an a d v a n c e d  the  Study.  thesis  thesis  degree  Library shall  I f u r t h e r agree  for  Department  at  in p a r t i a l  f u l f i l m e n t of  the U n i v e r s i t y of  make  the  requirements  British  Columbia,  I  it f r e e l y available for  reference  and  that permission  for  extensive  copying  of  agree  this  s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my  o r b y h.i)s  or p u b l i c a t i o n of  w i t h o u t my w r i t t e n  representatives.  this  thesis  for  permission.  Department The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  Columbia  It  is  understood  f i n a n c i a l gain  shall  that  copying  n o t be a l l o w e d  ABSTRACT  ^  v i v o technique  n  was  developed f o r the purpose of  the r a t e o f a b s o r p t i o n of b i l e s a l t s by the m e s e n t e r i c the chickeno f the  Using  t h i s technique,  i t was  i n t e s t i n e f o r sodium t a u r o c h o l a t e  small  determining  i n t e s t i n e of  found t h a t the a b s o r p t i v e  and  sodium g l y c o c h o l a t e  capacity  increases  distally. The reabsorbed  p o s s i b i l i t y was  from the  i n the c h i c k e n .  i n v e s t i g a t e d t h a t the amount o f b i l e  i n t e s t i n e determines the l e v e l of c i r c u l a t i n g  I n t e s t i n a l a b s o r p t i o n of sodium t a u r o c h o l a t e was  determined i n b i r d s s e l e c t e d to show a wide range i n l e v e l s o f sterols.  Absorption  taurocholate  of the b i l e s a l t was  e q u i v a l e n t to 0.15  of i n t e s t i n e .  Data o b t a i n e d  a t i n g s t e r o l s and  and  sterols accordingly  circulating  t e s t e d with c o n c e n t r a t i o n s  of  0.757* c h o l i c a c i d , u s i n g open segments  from 20 b i r d s showed t h a t the l e v e l of  the r a t e of b i l e s a l t  correlated.  The  probably  i n p a r t to v a r i a t i o n  due  salt  l a c k of a s i g n i f i c a n t  a b s o r p t i o n were not c o r r e l a t i o n was  circul-  significantly  considered  i n the e f f e c t of d i e t on the  to  be  availability  of b i l e s a l t s f o r r e a b s o r p t i o n . The  r a t e o f b i l e a c i d e x c r e t i o n and  the c o n c e n t r a t i o n o f  s t e r o l s were t h e r e f o r e determined i n b i r d s f e d d i e t s supplemented t r i g l y c e r i d e s , of v a r y i n g degree of u n s a t u r a t i o n , sitosterol. induced  I t was  was  with beta  found t h a t a d i e t supplemented w i t h 15% coconut o i l  an e l e v a t i o n i n the l e v e l o f plasma s t e r o l s when s u b s t i t u t e d f o r a  l o w - f a t d i e t but induced  s o y a l e c i t h i n and  circulating  little  t h a t d i e t s supplemented w i t h c o r n o i l and h e r r i n g o i l ,  or no change i n t h i s l e v e l .  The  change to a f a t - e n r i c h e d d i e t  a s s o c i a t e d w i t h a decrease i n the f e c a l output  of b i l e a c i d s .  The  decrease was g r e a t e r when coconut o i l r a t h e r than c o r n o i l o r h e r r i n g o i l was f e d , i n d i c a t i n g t h a t the plasma s t e r o l l o w e r i n g fats  i s due a t l e a s t  property  of unsaturated  i n p a r t t o t h e i r enhancing e f f e c t on b i l e a c i d e x c r e t i o n .  D i e t s t o which 2.57o s o y a l e c i t h i n and 0.57o b e t a added, e f f e c t e d a s l i g h t r e d u c t i o n  s i t o s t e r o l were  i n t h e l e v e l o f plasma s t e r o l s .  a d d i t i o n of s o y a l e c i t h i n to the d i e t r e s u l t e d i n a s i g n i f i c a n t (P < 0.01) i n the growth r a t e of t h e b i r d s s t u d i e d . excreted  was i n c r e a s e d  l e c i t h i n to the d i e t . a l s o i n an i n c r e a s e  significantly  The  increase  The amount o f b i l e  acids  (P < 0.01) by the a d d i t i o n o f soya-  The a d d i t i o n o f b e t a  s i t o s t e r o l to the d i e t r e s u l t e d  i nb i l e acid excretion.  The i n c r e a s e  approached  s i g n i f i c a n c e a t the 57« l e v e l o f s i g n i f i c a n c e (F r a t i o c a l c u l a t e d = 7.53, s i g n i f i c a n t F r a t i o = 7.71).  The i n c r e a s e  i nb i l e acid excretion resulting  from the a d m i n i s t r a t i o n o f the s o y a l e c i t h i n supplemented d i e t was c o n s i d e r e d to be the n e t r e s u l t o f two opposing e f f e c t s . i n the r e a b s o r p t i o n o f b i l e a c i d s .  One e f f e c t was an i n c r e a s e  I t was evidenced by a f u r t h e r  increase  i n t h e plasma s t e r o l l e v e l o f t h e l i t h o c h o l i c a c i d - f e d c h i c k i n response t o dietary soyalecithin.  The o t h e r  e f f e c t was reasoned t o be an i n c r e a s e i n  the b i l i a r y e x c r e t i o n o f b i l e a c i d s and/or b a c t e r i a l acids i n the d i g e s t i v e t r a c t .  alteration of b i l e  iii TABLE OF CONTENTS Page INTRODUCTION  PART I .  1  RELATIONSHIP BETWEEN THE ABSORPTIVE CAPACITY OF THE INTESTINE FOR BILE SALT AND THE LEVEL OF CIRCULATING STEROLS REVIEW OF LITERATURE  4  EXPERIMENT 1. DEVELOPMENT OF IN VIVO TECHNIQUE FOR MEASURING BILE SALT ABSORPTION AND ASSESSMENT OF THE ABILITY OF VARIOUS PARTS OF THE SMALL INTESTINE TO ABSORB BILE SALTS 12 M a t e r i a l s and Methods 12 R e s u l t s and D i s c u s s i o n 17 EXPERIMENT 2. MEASUREMENT OF THE CAPACITY OF THE SMALL INTESTINE OF MATURE COCKERELS TO ABSORB BILE SALT AND THE LEVEL OF CIRCULATING STEROLS M a t e r i a l s and Methods 22 Experimental 27 R e s u l t s and D i s c u s s i o n 29  22  SUMMARY - PART ONE  39  PART I I . THE EFFECT OF DIETARY FATS ON THE FECAL OUTPUT OF BILE ACIDS AND THE LEVEL OF CIRCULATING STEROLS  40  INTRODUCTION  40  DETERMINATION OF BILE ACIDS IN FECES A Note on A n a l y t i c a l Problems 41 Gas L i q u i d Chromatography 44 T h i n L a y e r Chromatography 49 A n a l y s i s o f Sample 55 S c a l i n g o f the Procedure 69 Suggestions f o r F u t u r e A n a l y s i s 7 1  41  EXPERIMENT 3. THE EFFECT OF DIETARY TRIGLYCERIDES OF VARYING DEGREE OF UNSATURATION ON THE FECAL OUTPUT OF BILE ACIDS AND THE LEVEL OF CIRCULATING STEROLS IN MATURE COCKERELS 73 Review o f L i t e r a t u r e 73 Experimental 75 R e s u l t s and D i s c u s s i o n 78  iv EXPERIMENT 4. THE EFFECT OF DIETARY LECITHIN AND BETA SITOSTEROL ON THE FECAL OUTPUT OF BILE ACIDS AND THE LEVEL OF CIRCULATING STEROLS IN GROWING COCKERELS 95 Review of Literature 95 Experimental 97 Results and Discussion 98 SUMMARY - PART TWO  117  PART III. PARTIAL ELUCIDATION OF MECHANISMS EXPERIMENT 5. THE EFFECT OF DIETARY BETA SITOSTEROL AND SOYALECITHIN ON THE LEVEL OF CIRCULATING STEROLS IN THE LITHOCHOLIC ACID-FED COCKEREL Experimental 119 Results and Discussion 121 EXPERIMENT 6. PLASMA STEROL LEVELS OF GROWING COCKERELS ADMINISTERED POTASSIUM LITHOCHOLATE WHILE RECEIVING A SOYALECITHIN OR BETA SITOSTEROL SUPPLEMENTED DIET HJT FED EQUAL AMOUNTS OF EACH DIET AT 12 HOUR INTERVALS Experimental 128 Results and Discussion 129 SUMMARY - PART THREE  119  119  128  135  GENERAL SUMMARY  137  SUGGESTIONS FOR FUTURE WORK  141  BIBLIOGRAPHY  143  V  LIST OF  TABLES  T a b l e No. 1.1.  Page Rate of uptake of sodium t a u r o c h o l a t e (as mg. c h o l i c a c i d ) by the lower m e s e n t e r i c s m a l l i n t e s t i n e o f the c h i c k e n d u r i n g a 90 minute p e r i o d (experiment l a )  18  Uptake of sodium t a u r o c h o l a t e and sodium g l y c o c h o l a t e (as mg. c h o l i c a c i d / h r . / l O O gm. wet t i s s u e ) by proximal and d i s t a l segments of upper and lower m e s e n t e r i c s m a l l i n t e s t i n e of the c h i c k e n  20  2.1.  Composition o f the d i e t  28  2.II.  Plasma c h o l e s t e r o l l e v e l s (mg.7») of c o c k e r e l s used f o r the f i r s t s e l e c t i o n  30  C o n c e n t r a t i o n (mg.7o) of c h o l e s t e r o l and DPS i n plasma of c o c k e r e l s r e t a i n e d i n the f i r s t s e l e c t i o n  31  C o n c e n t r a t i o n of DPS (mg.%) i n plasma of b i r d s of groups A and B at i n t e r v a l s throughout experiment 2  33  Weekly body weights of b i r d s of groups A and B  34  Plasma DPS l e v e l s and r a t e o f t a u r o c h o l a t e a b s o r p t i o n by segments of s m a l l i n t e s t i n e of t e s t c o c k e r e l s i n experiment 2  35  l.II.  2.III.  2.IV.  2.V.  2.VI.  2.VII.  f e d i n experiment 2  Degree o f c o r r e l a t i o n between v a r i a b l e s measured i n experiment 2  37  3.1.  Composition of b a s a l d i e t  3.II.  C o n c e n t r a t i o n (mg.7o) o f DPS i n plasma of c o c k e r e l s f e d v a r i o u s experimental d i e t s  79  Body weights (grams) of c o c k e r e l s f e d v a r i o u s experimental d i e t s  81  Body weights (grams) and f e c a l output (grams l y o p h i l i z e d f e c e s ) of subgroups of c o c k e r e l s fed v a r i o u s experimental d i e t s  84  3.III.  3.IV.  f e d i n experiment 3  76  VI  T a b l e No. 3.V.  Page F e c a l output o f b i l e a c i d s on the experimental d i e t s f e d  85  Changes i n c o n c e n t r a t i o n o f plasma DPS, f e c a l b i l e a c i d s and output o f b i l e a c i d s r e s u l t i n g from the v a r i o u s d i e t a r y substitutions  86  F e c a l output and change i n f e c a l output r e s u l t i n g from d i e t a r y exchanges  88  4.1.  Composition o f d i e t s f e d i n experiment 4  99  4.II.  C o n c e n t r a t i o n (mg.%) o f DPS i n plasma of c o c k e r e l s f e d d i e t s o f experiment 4 f o r 15 days  100  Body weights (grams) and g a i n i n body weights o f c o c k e r e l s f e d d i e t s o f experiment 4 f o r 17 days  102  4.III.ii.  Summary o f body weight data  104  4.IV.  A n a l y s i s o f v a r i a n c e o f g a i n i n body weight  105  Feed consumption and f e c a l output o f c o c k e r e l s f e d the e x p e r i m e n t a l d i e t s  107  B i l e a c i d e x c r e t i o n on d i e t s f e d i n experiment 4  108  Percentage o f i n d i v i d u a l and grouped b i l e a c i d s and percentage change i n t h e i r r a t e of e l i m i n a t i o n i n feces o f experimental birds  113  5.1.  Composition o f d i e t s f e d i n experiment 5  120  5.II.  Plasma DPS l e v e l s fed various d i e t s  (mg.%) o f c o c k e r e l s i n experiment 5  125  Plasma DPS l e v e l s (mg.%) o f c o c k e r e l s administered potassium l i t h o c h o l a t e on d i f f e r e n t d i e t s  130  Twelve (12) hour feed consumption o f experimental cockerels  133  3.VI.  3. V I I .  4.III.i.  4. V.  4.VI.  4.VII.  6.1.  6.II.  VX1  LIST OF  FIGURES  F i g u r e No. 1.1.  3.1.  3.2.  3.3.  3.4.  3.5.  3.6.  3.7.  3.8.  Page Diagrammatic r e p r e s e n t a t i o n of a prepared open segment o f m e s e n t e r i c s m a l l i n t e s t i n e  14  Connections to gas chromatograph column and source of vacuum d u r i n g p a c k i n g of column  47  Chamber u t i l i z e d f o r development of l a y e r chromatogram  52  thin  T h i n l a y e r chromatogram assembled and f o r development  ready  Apparatus f o r e v a p o r a t i o n of s o l v e n t s reduced p r e s s u r e under n i t r o g e n  at  53  57  M a n i f o l d and c o n n e c t i o n s u t i l i z e d i n the e v a p o r a t i o n of s o l v e n t s under n i t r o g e n at atmospheric p r e s s u r e  58  Gas chromatographic p a t t e r n of the TMS ethers o f the methyl e s t e r s of the f e c a l b i l e a c i d s of a d u l t c o c k e r e l s . A n a l y t i c a l c o n d i t i o n s : 1.0% SE-30 on 100-120 mesh Gas Chromosorb Q; 6'6" x 1/4" s t a i n l e s s s t e e l column; column temperature, 218° f o r 5 mins., 218° - 240° at l°/min.; i n j e c t i o n p o r t , 270°; d e t e c t o r , 260°; n i t r o g e n , 80 c.c./min.  67  Gas chromatographic p a t t e r n of the t r i f l u o r o a c e t a t e s of the methyl e s t e r s of F i g . 3.6. The r e a c t i o n m i x t u r e was i n j e c t e d i n t o the gas chromatograph. Column and oven c o n d i t i o n s as g i v e n i n F i g . 3.6. I n j e c t i o n p o r t and d e t e c t o r temp., 245°.  68  Gas chromatographic p a t t e r n of the TMS ethers of methyl e s t e r s of f e c a l b i l e a c i d s of c o c k e r e l s f e d the b a s a l d i e t of experiment 3. Analytical conditions: 1.0% SE-30 on 100-120 mesh Gas Chromosorb Q; 6'6" s t a i n l e s s s t e e l column; column temperature, 230° f o r 8 mins., 230° - 250° at l°/min.; i n j e c t i o n p o r t , 270°; d e t e c t o r , 260°; n i t r o g e n , 80 c.c./min.  89  viii F i g u r e No. 3.9.  3.10.  3.11.  4.1.  4.2.  4.3.  Page Gas chromatographic p a t t e r n of the TMS e t h e r s of the methyl e s t e r s of the f e c a l b i l e a c i d s o f c o c k e r e l s f e d a c o r n o i l supplemented d i e t . A n a l y t i c a l c o n d i t i o n s as g i v e n i n F i g . 3.8.  90  Gas chromatographic p a t t e r n o f the TMS ethers of the methyl e s t e r s o f the f e c a l b i l e a c i d s o f c o c k e r e l s f e d a h e r r i n g o i l supplemented diet. A n a l y t i c a l c o n d i t i o n s as g i v e n i n F i g . 3.8.  91  Gas chromatographic p a t t e r n of the TMS e t h e r s of the methyl e s t e r s o f the f e c a l b i l e a c i d s of c o c k e r e l s f e d a coconut o i l supplemented diet. A n a l y t i c a l c o n d i t i o n s as g i v e n i n F i g . 3.8.  92  Gas chromatographic p a t t e r n o f the TMS e t h e r s o f the methyl e s t e r s o f the f e c a l b i l e a c i d s o f c o c k e r e l s f e d the b a s a l d i e t o f experiment 4. Analytical conditions: 1.0% SE-30 on 100-120 mesh Gas Chromosorb Q; 6'6" s t a i n l e s s s t e e l column; column temperature, 245°; i n j e c t i o n p o r t , 275°; d e t e c t o r , 260°; n i t r o g e n , 80 c . c . / min. Gas chromatographic p a t t e r n o f the TMS e t h e r s of the methyl e s t e r s o f the f e c a l b i l e a c i d s of c o c k e r e l s f e d a b e t a s i t o s t e r o l supplemented diet. A n a l y t i c a l c o n d i t i o n s as g i v e n i n F i g . 4.1. Gas chromatographic p a t t e r n of the TMS e t h e r s of the methyl e s t e r s of the f e c a l b i l e a c i d s of c o c k e r e l s f e d a s o y a l e c i t h i n supplemented diet. A n a l y t i c a l c o n d i t i o n s as g i v e n i n F i g . 4.1.  110  I l l  112  ix  ACKNOWLEDGEMENTS  I wish t o extend  a f u l l measure o f a p p r e c i a t i o n t o the members  of my T h e s i s Committee: Professor J . Biely  (Chairman)  Chairman, Department o f P o u l t r y Science  P r o f e s s o r B. E. March  Department o f P o u l t r y S c i e n c e  Dr. W. D. K i t t s  Head, Department o f Animal Science Dean E m e r i t u s , F a c u l t y o f Agriculture  Dr. B. A. Eagles  Dr. S. H. Zbarsky  P r o f e s s o r , Department o f Biochemistry  Dr. M. Darrach  Head, Department o f B i o c h e m i s t r y  Dr. J . J . R. Campbell  Head, Department o f M i c r o b i o l o g y  To P r o f e s s o r s Jacob B i e l y and B e r y l March I would l i k e t o express my g r a t i t u d e f o r t h e i r c o n s t a n t encouragement i n my s t u d i e s and r e s e a r c h over the years o f my a s s o c i a t i o n w i t h them. thanks  I would l i k e t o a c c o r d  special  t o them a l s o f o r i n t r o d u c i n g me t o the i n t e r e s t i n g s u b j e c t o f  c h o l e s t e r o l metabolism as i n f l u e n c e d by d i e t . of r e s e a r c h has been most  My e x p e r i e n c e  i n this  area  satisfying.  I would a l s o l i k e t o thank the l a b o r a t o r y t e c h n i c i a n s , r e s e a r c h a s s o c i a t e s and farm o p e r a t o r s f o r t h e i r c o o p e r a t i o n which was a t no time perfunctory. I am sure t h a t my many y e a r s o f a s s o c i a t i o n w i t h the s t a f f o f t h i s department w i l l c o n t i n u e t o  p r o v i d e me w i t h a source o f g r e a t d e l i g h t .  INTRODUCTION  The  manner i n which the plasma s t e r o l l e v e l may be r e g u l a t e d has  been f o r many y e a r s , a s u b j e c t  o f widespread i n t e r e s t .  been due i n l a r g e measure t o a long cholesterolemia  The i n t e r e s t has  r e c o g n i z e d a s s o c i a t i o n between hyper-  and a t h e r o s c l e r o s i s .  This  a s s o c i a t i o n has prompted numerous  i n v e s t i g a t o r s t o undertake i n t e n s i v e r e s e a r c h  i n t o the means whereby plasma  c h o l e s t e r o l l e v e l s may be lowered. Emerging from t h e r e s u l t s o f r e s e a r c h plasma c h o l e s t e r o l l e v e l first the  consists  are two b a s i c  approaches to t h i s problem.  i n i n h i b i t i n g the b i o s y n t h e s i s  enzymes concerned i n c h o l e s t e r o l e x c r e t i o n  When i n h i b i t i o n o f s y n t h e s i s i n a manner which n e i t h e r  i n t o t h e l o w e r i n g o f the The  o f c h o l e s t e r o l and/or  stimulating  and d e g r a d a t i o n t o b i l e  i s the e f f e c t d e s i r e d ,  acids.  i t should be accomplished  i n t e r f e r e s with the synthesis  o f substances having  p r e c u r s o r s i n common w i t h c h o l e s t e r o l n o r causes t h e accumulation o f steroidal  intermediates with atherogenic p r o p e r t i e s .  In t h i s approach, i t  i s e s s e n t i a l t h a t t h e agents employed induce no p a t h o l o g i c a l hormonal i n b a l a n c e . rapid  These r i g i d requirements have not been conducive to  progress. The  second approach i n v o l v e s  s u i t a b l e composition. tested  Diets  containing  the a d m i n i s t r a t i o n  coincident  the d i e t a r y  intake  where c h o l e s t e r o l i n t a k e i s  inadequate where the i n t a k e  with hypercholesterolemia.  p r a c t i c e to r e s t r i c t  of d i e t s of  l i t t l e o r no c h o l e s t e r o l have been  and found t o be b e n e f i c i a l i n i n s t a n c e s  h i g h , but have been found t o be g r o s s l y and  changes o r  I t has t h e r e f o r e  i s low  been a common  o f c h o l e s t e r o l and e i t h e r manipulate  the normal components o f t h e d i e t o r a d m i n i s t e r substances which i n t e r f e r e  2 w i t h the r e a b s o r p t i o n recent  o f c h o l e s t e r o l and b i l e a c i d s .  There have been  d i s c o v e r i e s o f a number o f substances which are n o n t o x i c  and non-  absorbable and which d i s p l a y a remarkable a b i l i t y t o reduce the r e a b s o r p t i o n of b i l e a c i d s and c o n s e q u e n t l y c h o l e s t e r o l , by s e q u e s t e r i n g Perhaps the most s i g n i f i c a n t d i s c o v e r y  sterol  When t h i s r e s i n i s a d m i n i s t e r e d  o f e x p e r i m e n t a l animals, a f a l l  l e v e l ensues, w i t h an i m p r e s s i v e n e s s which c l e a r l y  e f f e c t i v e c o n t r o l o f the plasma s t e r o l  acids.  o f the type mentioned i s t h a t o f  c h o l e s t y r a m i n e , an i o n exchange r e s i n . to humans and some s p e c i e s  the b i l e  i n the plasma indicates that  l e v e l w i l l c o n t i n u e t o depend on  the use o f a d i e t a r y approach t o t h i s problem. The  study o f the plasma s t e r o l l e v e l i n r e l a t i o n t o the f e c a l  e x c r e t i o n o f endogenous s t e r o l s and b i l e a c i d s thus appears t o be a subject  of f a r reaching  importance.  T h i s study i s o f s p e c i a l v a l u e i n  those s p e c i e s where the r e s u l t s o b t a i n e d nutrition.  These s p e c i e s  a decisive f a l l  are o f r e l e v a n c e  i n c l u d e the c h i c k e n  and dog.  t o human  I n these  i n the plasma s t e r o l l e v e l o c c u r s upon d i v e r s i o n o f the  b i l e a c i d s t o the f e c e s .  In o t h e r s ,  and  level  p i g the plasma s t e r o l  on the o t h e r hand, such as the r a t .  i s r e f r a c t o r y t o such treatment.  u n d e r s t a n d i n g o f the f a c t o r s which govern the amount o f b i l e reabsorbed from the gut o f the c h i c k e n only  species,  from t h e p o i n t o f view t h a t  An  acids  would t h e r e f o r e be v a l u a b l e , n o t  i t would expand our knowledge o f s t e r o l  metabolism i n t h i s s p e c i e s , but a l s o because i t may suggest means o f c o n t r o l l i n g the plasma s t e r o l l e v e l o f humans through the e n t e r o h e p a t i c circulation. In the p u r s u i t o f t h i s u n d e r s t a n d i n g , r e s e a r c h  i n t o some  p h y s i o l o g i c a l and d i e t a r y f a c t o r s l i k e l y t o i n f l u e n c e the plasma  sterol  3 level,  secondary t o a f f e c t i n g b i l e  w i t h the c h i c k e n .  a c i d e x c r e t i o n have been undertaken  4 PART I RELATIONSHIP BETWEEN THE FOR  BILE SALT AND  THE  ABSORPTIVE CAPACITY OF THE  LEVEL OF CIRCULATING STEROLS  REVIEW OF  Many r e p o r t s  i n the  INTESTINE  LITERATURE  l i t e r a t u r e suggest t h a t v a r i a t i o n i n the  amount of b i l e s a l t s reabsorbed, c o n t r i b u t e i m p o r t a n t l y  to i n d i v i d u a l  d i f f e r e n c e s i n the l e v e l of c i r c u l a t i n g s t e r o l s i n some s p e c i e s . v a l i d i t y of t h i s suggestion b i l e salts homeostatically of c h o l e s t e r o l to b i l e  duct  fistulas,  d e r i v e s from the knowledge t h a t reabsorbed r e g u l a t e the h e p a t i c s y n t h e s i s  a c i d s through a n e g a t i v e  Bergstrom and  Daniellson  (1958) showed t h a t  oxidation  effect.  i n r a t s with  the r a t e of b i l e a c i d e x c r e t i o n which was  a s o l u t i o n of sodium t a u r o c h o l a t e .  and  feedback  r e s u l t of f i s t u l a t i o n , c o u l d be reduced to normal, by duodenum w i t h  The  bile  i n c r e a s e d as a  i n f u s i o n of  the  These workers were  a b l e to reduce the e l e v a t e d r a t e s of b i l e a c i d e x c r e t i o n i n p r o p o r t i o n to the amount of b i l e s a l t o f reabsorbed b i l e  infused.  Consistent  with  a homeostatic f u n c t i o n  s a l t s are a l s o the e a r l i e r o b s e r v a t i o n s  and Vars  (1954) and  Eriksson  in rats,  i n c r e a s e d by  of Thompson  (1957) t h a t the r a t e of b i l e s a l t  a f a c t o r of 20  - 30 when b i l e was  Measurement o f the c o n c e n t r a t i o n  excretion  diverted.  of c i r c u l a t i n g s t e r o l s and  the r a t e of h e p a t i c s y n t h e s i s of c h o l e s t e r o l , r e v e a l s t h a t t h e r e  are  important s p e c i e s d i f f e r e n c e s i n the response to i n t e r r u p t i o n of  the  enterohepatic  circulation.  undergoes no net and Eder, 1961).  In the r a t , the plasma c h o l e s t e r o l l e v e l  change f o l l o w i n g f i s t u l a t i o n o f the b i l e duct A n o r m o c h o l e s t e r o l e m i c response to the  (Myant  administration  5 of c h o l e s t y r a m i n e , a b i l e a c i d s e q u e s t r a n t of b i l e a c i d s and 1963).  The  which p r e v e n t s the  c h o l e s t e r o l i s a l s o shown by  a b i l i t y of the r a t to m a i n t a i n a constant  cholesterol, despite  excessive  of c h o l e s t e r o l (Huff e_t a l . , 1963). administered,  rates  was  b i l e a c i d s has  i n the r a t e o f h e p a t i c  increased  12  fold.  Under c o n d i t i o n s where c h o l e s t y r a m i n e  In o l d e r r a t s a s i m i l a r i n c r e a s e noted by  i n c h o l e s t e r o l synthesis  was  been  synthesis  excretion  3 f o l d whereas c h o l e s t e r o l b i o s y n t h e s i s  of e x c r e t i o n of b i l e a c i d s was increase  circulating  H u f f and h i s a s s o c i a t e s observed t h a t b i l e a c i d  i n young r a t s i n c r e a s e d  acetate  (Huff et a l . ,  l e v e l of  l o s s e s of c h o l e s t e r o l and  a t t r i b u t e d to a l a r g e compensatory i n c r e a s e  is  t h i s species  reabsorption  these authors but  the  from  i n the  rate  corresponding  5 fold.  I n i t s a b i l i t y to cope w i t h l a r g e endogenous l o s s e s of c h o l e s t e r o l and b i l e a c i d s by p i g has  a c c e l e r a t i n g the r a t e o f s y n t h e s i s  been found to resemble the r a t .  t h a t when c h o l e s t y r a m i n e was r a t e of h e p a t i c  synthesis  e x c r e t i o n of b i l e acids l e v e l s were not  incorporated  a l t e r e d by  S c h n e i d e r et al^. (1966) r e p o r t e d i n t o the d i e t of young p i g s ,  of c h o l e s t e r o l i n c r e a s e d  increased  o f c h o l e s t e r o l , the  the  19  f o l d as the r a t e of  to 10 times normal.  Plasma c h o l e s t e r o l  a d m i n i s t r a t i o n o f the r e s i n .  In some s p e c i e s , on the o t h e r hand, the plasma c h o l e s t e r o l l e v e l i s s e n s i t i v e to i n t e r r u p t i o n of the e n t e r o h e p a t i c administration  of c h o l e s t y r a m i n e to man,  chickens  (Bergen et a l . , 1959;  resulted  in a f a l l  circulation.  (Bergen e_t a_l., 1959;  Tennant, 1960)  and  The Datta,  dogs (Tennant,  i n the plasma c h o l e s t e r o l c o n c e n t r a t i o n .  The  1960) addition  of f e r r i c c h l o r i d e , a b i l e s a l t p r e c i p i t a n t , to d i e t s supplemented w i t h c h o l e s t e r o l or c h o l e s t e r o l and  b i l e concentrate  1963)  l a r g e l y suppressed  the  6 h y p e r c h o l e s t e r o l e m i a developed by b i r d s et a l . , 1953).  receiving  (Siperstein  Based upon the r e s u l t s o f a comparative study by G a l l o  et a l . (1966) i t would appear t h a t  these e f f e c t s a r e due t o an i n s u f f i c i e n c y  i n compensatory s y n t h e s i s o f c h o l e s t e r o l . p r e s e n t e d data showing t h a t synthesis of c h o l e s t e r o l 3 fold  these d i e t s  Gallo  i n rats receiving  and h i s  collaborators  cholestyramine, hepatic  from a c e t a t e was i n c r e a s e d 12 - 15 f o l d b u t o n l y  i n c h i c k e n s s u b j e c t e d t o the same treatment. Depending on the s p e c i e s ,  the a d d i t i o n  of s p e c i f i c b i l e  acids  to t h e d i e t leads t o noteworthy changes i n t h e l e v e l o f c i r c u l a t i n g sterols.  According to the report  o f Howe e t a l . (1960) mice fed  supplemented w i t h c h o l i c a c i d become h y p e r c h o l e s t e r o l e m i c . (1959) r e p o r t e d a s i m i l a r e f f e c t o f c h o l i c a c i d  i n man.  diets  Yamasaki  Beher e t a l .  (1963) rendered guinea p i g s h y p e r c h o l e s t e r o l e m i c by t h e a d m i n i s t r a t i o n a c h o l i c a c i d supplemented d i e t . shows that  dietary  o f Yamasaki et al.  c h o l i c and chenodeoxycholic a c i d s  esterolemia i n r a t s . regard.  The r e p o r t  Beher ej: al.  of  (1959)  induced h y p e r c h o l -  The l a t t e r b i l e a c i d was the more e f f e c t i v e i n t h i s (1963) were unable t o demonstrate a h y p e r c h o l e s t e r o l e m i c  e f f e c t o f c h o l i c a c i d on r a t s  as r e p o r t e d by Yamasaki e t a l . (1959).  investigators  f e d c h o l i c a c i d supplemented d i e t s  noted t h a t  rats  These  remained  n o r m o c h o l e s t e r o l e m i c b u t became h y p e r c h o l e s t e r o l e m i c when c h e n o d e o x y c h o l i c a c i d was s u b s t i t u t e d The and  for cholic  incorporation  acid.  of cholic acid  (Portman and Bruno, 1961; Howe  Hutchinson, 1962) o r c h o l i c a c i d t o g e t h e r w i t h c h e n o d e o x y c h o l i c  (Howe and Hutchinson, 1962) i n t o a c h o l e s t e r o l a marked e l e v a t i o n  o f the plasma c h o l e s t e r o l  acid  e n r i c h e d d i e t promoted  l e v e l o f the r a t .  The con-  c l u s i o n reached by K r i t c h e v s k y and Tepper (1966) o f a h y p e r c h o l e s t e r o l e m i c  7 e f f e c t o f c h o l i c a c i d on r a t s f e d d i e t s c o n t a i n i n g w i t h t h a t o f Portman and Bruno (1961). Howe and H u t c h i n s o n (1962) K r i t c h e v s k y administration  was w i t h o u t ' e f f e c t  cholesterol-fed The  But c o n t r a r y  agrees  t o the f i n d i n g s o f  and Tepper found t h a t  chenodeoxycholate  on t h e plasma c h o l e s t e r o l l e v e l o f the  rat.  e f f e c t o f d i e t a r y d e o x y c h o l i c a c i d on t h e plasma c h o l e s t e r o l  l e v e l o f r a t s and humans was s t u d i e d hypocholesterolemic.  Contradictory  by Yamasaki (1959) and found to be r e s u l t s were o b t a i n e d by Portman and  Bruno (1961) who noted a c h o l e s t e r o l e l e v a t i n g in rats.  added c h o l e s t e r o l  The l a t t e r workers a l s o showed t h a t  hypocholesterolemic  i n this  e f f e c t of t h i s b i l e  acid  l i t h o c h o l i c acid i s  species.  Beher e_t al_. (1963) showed t h a t  l i t h o c h o l i c acid  displays  c h a r a c t e r i s t i c s which a r e n o r m o c h o l e s t e r o l e m i c i n mice and h y p e r c h o l e s t e r o l e m i c i n r a t s and hamsters. l e v e l of rabbits  This b i l e  a c i d a l s o r a i s e s the plasma c h o l e s t e r o l  (Hunt e_t al., 1964).  A pronounced r i s e  i n t h e plasma  c h o l e s t e r o l l e v e l o f c h i c k e n s f e d a l i t h o c h o l i c a c i d supplemented d i e t has been r e p o r t e d species by  by Edwards (1961) whose r e s u l t s i n d i c a t e d t h a t  h y o d e o x y c h o l i c a c i d antagonizes the h y p e r c h o l e s t e r o l e m i a  l i t h o c h o l i c acid  induced  administration.  A c c o r d i n g t o Beher e_t al.  (1963) h y o d e o x y c h o l i c a c i d depresses  the plasma c h o l e s t e r o l l e v e l o f mice and r a i s e s pigs.  i n this  i t i n hamsters and guinea  In experiments o f Howe e_t al_. (1960) l i t h o c h o l i c and h y o d e o x y c h o l i c  acids n e u t r a l i z e d  the h y p e r c h o l e s t e r o l e m i c e f f e c t o f c h o l i c a c i d i n mice.  These r e s u l t s suggest t h a t depending on the b i l e a c i d  composition  of b i l e , changes i n t h e plasma s t e r o l l e v e l may a r i s e as a r e s u l t o f an increase  i n the rate of reabsorption  of b i l e  salts.  8 The  concept of a plasma c h o l e s t e r o l r e g u l a t i n g f u n c t i o n o f  reabsorbed b i l e  s a l t s r e c e i v e s added support from the r e s u l t o f  concerned w i t h the s y n t h e s i s under c o n d i t i o n s  where b i l e  and  studies  o x i d a t i o n of c h o l e s t e r o l to b i l e  i s d i v e r t e d or b i l e a c i d s  acids  administered  in  the d i e t . Whitehouse and  Staple  (1959) showed t h a t the  increased  r a t e of  b i l e a c i d e x c r e t i o n observed when the b i l e duct o f r a t s i s d r a i n e d , due  to an  increase  i n o x i d a t i o n of c h o l e s t e r o l to b i l e a c i d s .  Eder (1961) demonstrated t h a t t h i s i s accompanied by  an  increased  increased  is  Myant  and  r a t e of c h o l e s t e r o l o x i d a t i o n  r a t e of c h o l e s t e r o l b i o s y n t h e s i s  i n the  liver. These changes are not due  to an e f f e c t of mass a c t i o n alone  a l s o to a r e l e a s e of i n h i b i t i o n exerted metabolic s i t e s . bile salts  inhibit  Myant and  i n c h o l e s t e r o l synthesis  but  t h a t such treatment induced no  p h y s i o l o g i c a l concentrations inhibition  markedly i n h i b i t e d by  100.  The  the  s p e c i f i c i t y of  addition the  i n these experiments by  (2 x 10 ^M)  which induced almost complete and  by  the  by  synthesis  illustrated  (98.0%) of mevalonate s y n t h e s i s  the d e t e r g e n t T r i t o n X -  differences  (1965) which show t h a t mevalonate  conjugated b i l e s a l t s to the medium. s a l t s was  synthesis  T h e i r r e s u l t s are supported  from a c e t a t e by r a t l i v e r homogenates was  a c t i o n of the b i l e  drainage  above c o n t r o l s , i n the r a t e of  from mevalonate.  Rodwell  the  of c h o l e s t e r o l at some s t e p p r i o r to  increase,  of c h o l e s t e r o l from a c e t a t e  of f r e e and  specific  Working w i t h r a t s these workers showed t h a t  of the b i l e duct caused an  those of F i m o g n a r i and  the b i l e s a l t s at  Eder (1961) advanced evidence showing t h a t  the s y n t h e s i s  mevalonate f o r m a t i o n .  by  but  the  l a c k of e f f e c t of  9 That b i l e  s a l t s exert  o x i d a t i o n of c h o l e s t e r o l was  a specific  i n h i b i t o r y e f f e c t on  shown by Whitehouse and  Staple  the  (1959)  who  found t h a t the a d d i t i o n of c h o l i c a c i d conjugates to f o r t i f i e d r a t mitochondrial the  terminal  preparations  acid formation. Daniellson  bile fistula  These o b s e r v a t i o n s  are  of carbon d i o x i d e  from 3<K , 7oc ,  which i s a l a t e r i n t e r m e d i a t e  on the pathway to  al.,  of the plasma c h o l e s t e r o l l e v e l .  Gould and T a y l o r  r a t s , d i e t a r y c h o l e s t e r o l exerted  a profound e f f e c t on  obtained  Morris  reduction cell  S i p e r s t e i n and  that i n  suppressing  hepatic  inhibition  of  brought about by d i e t a r y c h o l e s t e r o l occurs at  the  Working w i t h a  from r a t l i v e r these workers found t h a t c h o l e s t e r o l  without e f f e c t on the  b e t a h y d r o x y m e t h y l g l u t a r a t e but mevalonate to  regulator  Fagan (1966) more r e c e n t l y  of b e t a h y d r o x y m e t h y l g l u t a r a t e to mevalonate.  f e e d i n g was  the  (1950) r e p o r t e d  r e s u l t s which were taken as evidence t h a t the  free preparation  1958).  T h e i r r e s u l t s have been confirmed by F r a n z et_ a l .  et a l . , 1957).  c h o l e s t e r o l synthesis  With  been shown to decrease the r a t e of  been made f o r c h o l e s t e r o l i t s e l f b e i n g  cholesterol synthesis.  bile  of taurochenodeoxycholate to  a c i d s from c h o l e s t e r o l (Mosbach et  A case has  (1952) and  Hoc—  r a t s lowered the r a t e of e x c r e t i o n of t a u r o c h o l a t e .  of b i l e  from  i n l i n e w i t h those of Bergstrom  (1958) t h a t a d m i n i s t r a t i o n  dogs c h o l i c a c i d a d m i n i s t r a t i o n has formation  formation  carbon atoms of c h o l e s t e r o l but not  trihydroxycoprostane  and  depressed the  liver  i n c o r p o r a t i o n of l a b e l l e d a c e t a t e  t h a t i t reduced the  incorporation  into  into  3.0%.  Attempts to induce d i r e c t i n h i b i t i o n of c h o l e s t e r o l b i o s y n t h e s i s i n v i t r o by  the a d d i t i o n of c h o l e s t e r o l or c h o l e s t e r o l e s t e r s to  s l i c e s or homogenates have however been u n s u c c e s s f u l  (Siperstein,  liver 1960;  10 F i m o g n a r i and Rodwell, 1965). F u r t h e r evidence a g a i n s t of c h o l e s t e r o l b i o s y n t h e s i s  cholesterol  p e r se b e i n g the r e g u l a t o r  has been adduced by Beher e_t al. (1959).  These  workers showed u s i n g r a t s m a i n t a i n e d on a c h o l i c a c i d supplemented d i e t t h a t maximum i n h i b i t i o n o f h e p a t i c s y n t h e s i s 3 days when the l e v e l o f serum b i l e cholesterol After  acids  o f c h o l e s t e r o l o c c u r r e d at  a l s o reached a maximum.  Hepatic  c o n c e n t r a t i o n was found t o reach a maximum on the f i r s t day.  3 days, t h e r e were no f u r t h e r  i t i o n o r i n the l e v e l o f serum b i l e The  changes e i t h e r  i n t h e degree o f i n h i b -  acids.  b u l k o f e x i s t i n g evidence i s t h e r e f o r e  of t h e b i l e a c i d s b e i n g the r e g u l a t o r s  weighted i n f a v o u r  of hepatic synthesis  of c h o l e s t e r o l .  There i s l i t t l e doubt t h a t v a r i a t i o n i n the r a t e o f c h o l e s t e r o l reabsorption could cholesterol  give r i s e to i n d i v i d u a l differences  level.  i n t h e plasma  But because the r a t e o f a b s o r p t i o n o f c h o l e s t e r o l  would be expected t o c o r r e l a t e h i g h l y w i t h t h a t o f b i l e s a l t , i n t h e plasma c h o l e s t e r o l should coincide  l e v e l , due t o d i f f e r e n c e s  w i t h that due t o d i f f e r e n c e s  T h i s v i e w i s based on the knowledge that  i n cholesterol  i n bile salt  cholesterol  variation absorption  absorption.  is first  absorbed  from the gut i n t o the mucosa i n the f r e e form (Vahounny and T r e a d w e l l , 1964;  Hyun e t al_., 1964) and t h e r e e s t e r i f i e d by enzymes dependent on  bile salts foractivity 1962;  (Hernandez and C h a i k o f f ,  Swell e t a l . , 1953).  This  1957; Murthy and Ganguly,  e s t e r i f i c a t i o n step i s mandatory to the  a b s o r p t i o n o f b o t h f r e e and e s t e r i f i e d c h o l e s t e r o l .  Drainage o f the common  b i l e duct o f r a t s has been shown to r e s u l t i n the d i s a p p e a r a n c e o f c h o l e s t e r o l e s t e r i f y i n g a c t i v i t y of i n t e s t i n a l homogenates and a b o l i s h a b s o r p t i o n o f labelled cholesterol  i n t o the t h o r a c i c  duct lymph.  The a d m i n i s t r a t i o n  of  11 pancreatic  j u i c e together with b i l e restored  cholesterol  a b s o r p t i o n to normal ( B o r j a  cholesterol  exists  and  ejt al_., 1960)  e s t e r i f i e d and limiting factor  the  could  be  endogenous c h o l e s t e r o l  i t was  should be  r a t e of a b s o r p t i o n of b i l e a c i d s a b s o r p t i o n on  absorptive capacity  a major determinant o f the  portal vein  e_t al_., 1964).  Since  and  biliary  e_t <al.,  1966)  are u n f a v o u r a b l e to c h o l e s t e r o l e s t e r i f i c a t i o n  in cholesterol  S i n c e the  esterifying activity  i n the n o n e s t e r i f i e d form ( N e i d e r h i s e r  i n t e s t i n a l conditions  (Swell  the  c o n s i d e r e d of  non-  i n t o the mucosa  a d i e t d e v o i d of  of the  the  cholesterol.  intestine for b i l e  salt  amount of b i l e s a l t s r e t u r n e d v i a  i n t e r e s t to determine i f t h e r e i s a  r e l a t i o n s h i p between plasma s t e r o l l e v e l s and m e s e n t e r i c s m a l l i n t e s t i n e of the  principally  i n t r i n s i c a b i l i t y of  c h i c k e n to absorb b i l e s a l t s .  the  the  12 EXPERIMENT 1 DEVELOPMENT OF IN VIVO TECHNIQUE FOR MEASURING BILE SALT ABSORPTION AND ASSESSMENT OF THE ABILITY OF VARIOUS PARTS OF THE SMALL INTESTINE TO ABSORB BILE SALTS Up t o the time when t h i s study was i n i t i a t e d t h e r e were no r e p o r t s on b i l e s a l t a b s o r p t i o n i n the c h i c k e n . to  develop  an i n v i v o technique  I t was t h e r e f o r e  a p p r o p r i a t e t o the c h i c k e n t h a t would  p r o v i d e q u a n t i t a t i v e data on b i l e s a l t a b s o r p t i o n . permitted  necessary  The technique  developed  the measurement o f the r a t e o f b i l e s a l t a b s o r p t i o n from open  segments and c l o s e d sacs o f the m e s e n t e r i c  small  intestine.  M a t e r i a l s and Methods Experiment l a Chemicals Sodium t a u r o c h o l a t e was o b t a i n e d C o r p o r a t i o n and was used without  from N u t r i t i o n a l  Biochemical  further purification.  Reagents and S o l v e n t s F u r f u r a l was used without r e f r i g e r a t i o n immediately  I t was p l a c e d under  f o l l o w i n g purchase and brought t o room temperature  j u s t p r i o r t o u s e . The reagent (darkening)  being d i s t i l l e d .  showed no v i s i b l e s i g n s o f d e t e r i o r a t i o n  over the p e r i o d used.  A c e t i c a c i d was g l a s s Determination  distilled.  of Taurocholate  A n a l y s i s o f f l u i d recovered  from i n t e s t i n a l segments  prepared  as l a t e r d e s c r i b e d was c a r r i e d out u s i n g the procedure o f P e t t e n k o f e r as m o d i f i e d by I r v i n ejt a l . (1944).  P r i o r t o c o l o r development the s m a l l  amount o f mucosal p r o t e i n removed by the f l u i d was p r e c i p i t a t e d as recommended by L a c k and Weiner (1961).  The r a t i o o f f l u i d  (33% t r i c h l o r a c e t i c a c i d ) was 20:1. c h o l a t e was as f o l l o w s .  t o the p r e c i p i t a t i n g  solution  In b r i e f the d e t e r m i n a t i o n o f t a u r o -  One m l . o f f l u i d was p l a c e d i n a t e s t tube t o which  13 6.0 ml. o f 16.0  N s u l f u r i c a c i d were then added.  The tube c o n t e n t s were  mixed w i t h a V o r t e x mixer f o l l o w i n g which 2.0 ml. o f a s o l u t i o n o f f u r f u r a l were added. minutes  A f t e r f u r t h e r m i x i n g the tubes were heated f o r 13  i n a water bath m a i n t a i n e d at 65°C.  Upon c o o l i n g , 5.0 ml. o f  a c e t i c a c i d were added f o l l o w e d by m i x i n g . measured at 660 mu. is specific  0.5%  The c o l o r i n t e n s i t y was  w i t h a Beckman Model B spectrophotometer.  f o r c h o l i c a c i d and y i e l d s the same c o l o r  equimolar amounts o f f r e e and conjugated c h o l i c  The  then reaction  intensity for  acid.  A b s o r p t i o n o f T a u r o c h o l a t e from Open Segments The r a t e o f i n t e s t i n a l a b s o r p t i o n o f sodium s t u d i e d i n t h r e e mature White Leghorn c o c k e r e l s s u i t a b l e l e n g t h o f time over which  t a u r o c h o l a t e was  i n o r d e r to a r r i v e at a  to measure b i l e s a l t a b s o r p t i o n .  b i r d s were s t a r v e d f o r 36 hours p r i o r to b e i n g s t u d i e d .  This  The  procedure  ensured the removal o f P e t t e n k o f e r p o s i t i v e m a t e r i a l from the mucosa (Lack and Weiner, 1961). sodium p e n t o b a r b i t a l  The b i r d s were i n i t i a l l y  (1 ml./5  l b . body wt.)  anaesthetized with  and h e l d under a n a e s t h e s i a  w i t h d i e t h y l e t h e r throughout the experiment  a f t e r which they were k i l l e d .  The procedure f o r measurement o f a b s o r p t i o n was Each b i r d was  p l a c e d on i t s l e f t  r i g h t s i d e o f the abdomen.  side.  An  L i g a t u r e s were t i e d around the i n t e s t i n e at  y o l k s t a l k and j u s t above the i l e o - c e c a l j u n c t i o n .  the  l i g a t u r e s was 1.1.  The segment between  c a n n u l a t e d at both ends as shown d i a g r a m m a t i c a l l y i n  The p r o x i m a l end was  connected through a two-way stopcock t o a  r e s e r v o i r of p h y s i o l o g i c a l s a l i n e warmed to 40°C. was  follows:  i n c i s i o n was made along the  the  Fig.  as  The  i n t e s t i n a l segment  f l u s h e d out w i t h the s a l i n e under the f o r c e of g r a v i t y u n t i l  washings appeared  clear.  Entrapped s o l u t i o n was  the  blown out by g e n t l y  15 a p p l y i n g p r e s s u r e by mouth through the t u b i n g connected to the With the stopcock taurocholate cannula  at the p r o x i m a l  end open to the atmosphere, sodium  s o l u t i o n , warmed to 4 0 ° C , was  introduced  s o l u t i o n was  equivalent  amount o f f l u i d  approximately  2 cm.  i n t r o d u c e d brought the l e v e l s above the l i g a t u r e s .  throughout the a b s o r p t i o n p e r i o d by the b u r e t t e as r e q u i r e d .  was  periods.  The  intestinal  as b e f o r e .  s o l u t i o n i n t o the  to volume w i t h 5 ml.  glucose.  f l a s k with segment was  i n the  intestinal  checked by  con-  segment.  i n t e s t i n a l segment were the a i d of 70 ml.  of  saline  r e f i l l e d w i t h the s o l u t i o n  f o l l o w e d f o r s i x 15-minute  i n t r o d u c i n g 25 ml.  of the of  sixth  bile  i n t e s t i n a l segment and withdrawing i t immediately of s a l i n e as d e s c r i b e d . of 33%  cholic acid concentration  The  s o l u t i o n s were made  t r i c h l o r o a c e t i c a c i d and  s a l i n e and  their  determined. Experiment l b  Chemicals, Reagents and  Solvents  These were the same as i n experiment l a .  Determination  of  of  maintained  A f t e r removal of the b i l e s a l t s o l u t i o n at the end  w i t h the a i d of 70 ml. up  of the  T h i s procedure was  a b s o r p t i o n p e r i o d r e c o v e r y was salt  of  percent  i n the cannulae to  T h i s l e v e l was  thus maintained  i n t o a 100 ml. v o l u m e t r i c  of b i l e s a l t  sodium  i n t e r m i t t e n t a d d i t i o n of s o l u t i o n from  A f t e r 15 minutes, the contents  from the r e s e r v o i r .  percent  distal  Throughout the procedure, an approximately  stant h y d r o s t a t i c pressure  collected  The  i n c o n c e n t r a t i o n to 0.15  c h o l i c a c i d i n p h y s i o l o g i c a l s a l i n e c o n t a i n i n g 0.3 The  i n t o the  from a b u r e t t e which emptied i n t o i t as shown.  taurocholate  stopcock.  Taurocholate  A l i q u o t s of f l u i d  recovered  from the upper and  lower s m a l l  16  intestine  (1 and 2 ml.) r e s p e c t i v e l y were used.  To the 2.0 m l . a l i q u o t s  5 m l . o f 19.2 N IL^SO^ were added and the a n a l y s i s completed as d e s c r i b e d f o r 1 m l . a l i q u o t s i n experiment l a .  Absorption  of Taurocholate The  from C l o s e d  Sacs  r e l a t i v e a b s o r p t i o n o f sodium t a u r o c h o l a t e was measured i n  d i f f e r e n t segments o f the m e s e n t e r i c White Leghorn c o c k e r e l s .  s m a l l i n t e s t i n e o f twelve 12-week-old  The b i r d s were s t a r v e d and a n a e s t h e t i z e d  Each b i r d was p l a c e d on i t s l e f t  side.  An i n c i s i o n o f l e n g t h  as b e f o r e .  sufficient  to l o c a t e t h e c e c a l j u n c t i o n and duodenum was made along the r i g h t of the abdomen.  L i g a t u r e s were t i e d j u s t below the duodenum and above  the c e c a l j u n c t i o n . stalk. and  The i s o l a t e d  i n t e s t i n e was c u t i n two a t the y o l k  Both t h e upper and lower p o r t i o n s o f the i n t e s t i n e were  cannulated  f l u s h e d w i t h warm p h y s i o l o g i c a l s a l i n e s o l u t i o n as d e s c r i b e d i n  experiment l a .  The cannulae were then removed.  Each p o r t i o n o f the  i n t e s t i n e was then c u t t o g i v e two segments o f approximately One  end o f each segment was l i g a t u r e d .  was  introduced  As  side  equal  The s o l u t i o n o f sodium  length.  taurocholate  a t the open end w i t h a s y r i n g e f i t t e d w i t h a b l u n t  needle.  the needle was withdrawn a second l i g a t u r e , making a s a c from the  segment, was t i e d . experiment l a .  The b i l e s a l t  The amount p l a c e d  per kgm. body weight.  s o l u t i o n was s i m i l a r t o t h a t used i n i n each s a c was approximately  3 ml.  A f t e r 45 minutes the sacs were removed from the body,  extraneous t i s s u e c a r e f u l l y removed, and the o u t s i d e cleaned w i t h t i s s u e paper moistened w i t h s a l i n e .  The f i n a l contents  o f each s a c were d r a i n e d  i n t o a 15 m l . graduated tube.  The i n t e s t i n a l mucosa was r i n s e d w i t h 1.0 m l .  of warm s a l i n e i n t o the tube.  Each segment was l i g h t l y b l o t t e d w i t h  filter  17 paper, and  the t i s s u e o u t s i d e the l i g a t u r e s making the sac, cut away.  remaining  t i s s u e which had  weighed.  C h o l i c a c i d was  c o n s t i t u t e d the a c t u a l a b s o r b i n g determined on the f l u i d  recovered  The  t i s s u e was from the  then sacs.  Experiment l c Chemicals Sodium g l y c o c h o l a t e was C o r p o r a t i o n and  Reagents and  was  used without  obtained  solvents  of The  i n experiment l a .  Glycocholate r e l a t i v e a b s o r p t i o n of sodium g l y c o c h o l a t e was  c l o s e d sacs of the m e s e n t e r i c Leghorn c o c k e r e l s . The  Biochemical  further purification.  These were the same as  Absorption  from N u t r i t i o n a l  The  measured i n  s m a l l i n t e s t i n e i n e i g h t 10-week-old White  technique  used was  t h a t d e s c r i b e d i n experiment l b .  s o l u t i o n of sodium g l y c o c h o l a t e c o n t a i n e d  the e q u i v a l e n t of 0.15  c h o l i c a c i d i n p h y s i o l o g i c a l s a l i n e with glucose  added at a l e v e l o f  percent 0.3  percent. R e s u l t s and  Discussion  Experiment l a The  r e s u l t s of experiment l a i n which the amounts of sodium  t a u r o c h o l a t e absorbed by the lower h a l f of the m e s e n t e r i c  small  intestine  d u r i n g s i x s u c c e s s i v e 15-minute p e r i o d s are shown i n T a b l e 1.1. of the t h r e e b i r d s s t u d i e d , the r a t e of a b s o r p t i o n o f the b i l e decreased  w i t h time.  15 minute p e r i o d was first.  The  The  In each salt  amount of b i l e s a l t absorbed d u r i n g the  approximately  55 p e r c e n t  last  o f t h a t absorbed i n the  r e s u l t s of the t e s t show t h a t , u s i n g the technique  described  18  TABLE 1.1.  Rate of uptake of sodium t a u r o c h o l a t e (as mg. c h o l i c a c i d ) by the lower m e s e n t e r i c s m a l l i n t e s t i n e o f the c h i c k e n d u r i n g a 90 minute p e r i o d (experiment l a )  Absorption period (each o f 15 minutes duration)  Bird 1  Bird 2  Bird 3  Ayg.  1  4.15  7.47  6.26  5.96  2  3.90  7.22  6.00  5.71  3  3.29  4.27  3.92  3.83  4  2.67  5.38  4.83  4.29  5  2.30  4.12  4.70  3.70  6  2.18  3.79  3.92  3.30  19 a p e r i o d of one  hour i s p r o b a b l y  a s u i t a b l e l e n g t h of time over which to  study b i l e s a l t a b s o r p t i o n from i s o l a t e d the  i n t e s t i n a l segments i n v i v o i n  chicken. The  r e s u l t s of the r e c o v e r y  t e s t s c a r r i e d out  a f t e r the  a b s o r p t i o n p e r i o d showed t h a t e s s e n t i a l l y a l l of the t a u r o c h o l a t e duced i n t o the  i n t e s t i n a l segment was  o b t a i n e d were 101,  100  and  99%  immediately r e c o v e r a b l e .  final intro-  Recoveries  (average 100%).  Experiment l b The first  technique  experiment i n t h a t the  as c l o s e d sacs taurocholate mesenteric The  used i n experiment l b d i f f e r e d from t h a t i n the i s o l a t e d segments of i n t e s t i n e were  i n s t e a d of as open systems.  absorbed d u r i n g one  The  prepared  amounts of sodium  hour by each o f the f o u r r e g i o n s o f  the  s m a l l i n t e s t i n e i n i n d i v i d u a l b i r d s are shown i n T a b l e l . I I .  term "mesenteric  s m a l l i n t e s t i n e " has  been used as suggested by McLeod  et a l . (1964), to d e s c r i b e the p o r t i o n of the s m a l l i n t e s t i n e between the duodenum and  the l a r g e i n t e s t i n e .  w i t h the terms jejunum and c o n s i s t s of two  ileum,  parts d i f f e r i n g  I t s use  avoids  the i m p l i c a t i o n a s s o c i a t e d  t h a t t h i s p o r t i o n of the  i n microscopic  structure.  comparisons among the d i f f e r e n t  l e v e l s of the  i n t e s t i n e and  birds,  i s expressed  . b i l e s a l t absorption  t i s s u e present  intestine To  enable  among d i f f e r e n t  on the b a s i s of the weight of  i n each p r e p a r a t i o n .  In g e n e r a l , the amount of b i l e s a l t absorbed from the lumen of the i n t e s t i n e was  greatest  i n the d i s t a l  successive quarters proximally. showed any  q u a r t e r and  decreased  in  Of the 12 b i r d s s t u d i e d o n l y 1, 2 and  d e v i a t i o n from the g e n e r a l Subsequent to the completion  12  observation. of t h i s study r e p o r t s have appeared  TABLE l . I I .  Uptake o f sodium t a u r o c h o l a t e and sodium g l y c o c h o l a t e (as mg. c h o l i c a c i d / h r . / l O O gm. \<ret t i s s u e ) by p r o x i m a l and d i s t a l segments of upper and lower m e s e n t e r i c s m a l l i n t e s t i n e o f the c h i c k e n  T a u r o c h o l a t e a b s o r p t i o n (experiment l b ) Portion of i n t e s t i n e  B i r d no.  Upper Proximal Distal  Lower Proximal Distal  1 2 3 4 5 6 7 8 9 10 11 12  142 127 093 125 120 077 094 080 099 077 078 104  118 084 125 133 124 115 096 099 114 108 103 098  115 133 185 208 242 166 176 179 181 164 166 137  241 266 285 353 264 262 202 264 241 246 219 259  Average  101  110  171  259  G l y c o c h o l a t e a b s o r p t i o n (experiment l c ) 8 Birds Average Range  88 73-113  109 95-120  167  266  149-182  221-310  21 i n d i c a t i n g t h a t the a b i l i t y of the i n t e s t i n e of the c h i c k e n to absorb bile salts  increases d i s t a l l y .  Webling  taurocholate  to p o l y e t h y l e n e  p a r t s of the  i n t e s t i n e f o l l o w i n g a d m i n i s t r a t i o n of a m i x t u r e of  substances and  glycol  (1966) measured the r a t i o  i n i n t e s t i n a l contents  found a s i m i l a r g r a d i e n t w i t h r e s p e c t to b i l e  His r e s u l t s are c o n s i s t e n t w i t h those comparative study  employing e v e r t e d  t r a n s p o r t of t a u r o c h o l a t e occurs intestine.  The  reported  of G l a s s n e r  different these  salt  absorption.  e_t aJL. (1965) who,  in a  sacs of i n t e s t i n e showed t h a t a c t i v e  o n l y i n the d i s t a l h a l f of the  r e s u l t s o f the p r e s e n t  w i t h r e p o r t e d d a t a on b i l e to those  at  of  small  i n v e s t i g a t i o n are thus i n agreement  s a l t absorption  i n the c h i c k e n ,  and  are  similar  f o r mammalian s p e c i e s .  Experiment l c T h i s was not  Sjovall  average v a l u e s  are a l s o g i v e n was  to determine whether g l y c o c h o l a t e , which i s  a normal c o n s t i t u e n t of the b i l e of a v i a n s p e c i e s a c c o r d i n g  Haslewood and The  designed  to  (1954) and Wiggins (1955), i s absorbed to any  obtained  i n Table  f o r each of the f o u r r e g i o n s o f the  l . I I . The  intestine  r a t e of a b s o r p t i o n of g l y c o c h o l a t e  s i m i l a r to t h a t of t a u r o c h o l a t e .  a l s o e x h i b i t e d by the m e s e n t e r i c  extent.  small  A s i m i l a r absorption gradient intestine.  was  22 EXPERIMENT 2 MEASUREMENT OF THE CAPACITY OF THE TO ABSORB BILE SALT AND  SMALL INTESTINE OF MATURE COCKERELS  THE LEVEL OF CIRCULATING STEROLS  The t e c h n i q u e d e s c r i b e d i n experiment  1 made i t p o s s i b l e t o  determine i f t h e r e i s a s i g n i f i c a n t r e l a t i o n s h i p between plasma  sterol  l e v e l s and a b s o r p t i v e c a p a c i t y o f the m e s e n t e r i c s m a l l i n t e s t i n e o f the chicken f o r b i l e s a l t .  The r e s u l t s o f the p r e v i o u s experiments showed  t h a t a p e r i o d o f 1 hour was  a s u i t a b l e p e r i o d of time over which t o measure  b i l e s a l t a b s o r p t i o n from open segments o f i n t e s t i n e and t h a t b o t h upper and lower m e s e n t e r i c s m a l l i n t e s t i n e s h o u l d be u t i l i z e d  i n such a s t u d y .  M a t e r i a l s and Methods Blood  Sampling Blood was  o b t a i n e d from the wing v e i n a f t e r i t was  a number 11 Bard P a r k e r s c a l p e l .  Three ml. samples  punctured w i t h  were c o l l e c t e d  into  5.0 m l . t e s t tubes c a l i b r a t e d to 3.0 ml. and c o n t a i n i n g 0.3 ml. o f a s o l u t i o n of 0.1% h e p a r i n i n 0.9% and i n v e r t e d 6 t i m e s .  sodium c h l o r i d e .  The tubes were then s t o p p e r e d  The samples were c e n t r i f u g e d f o r 8 minutes a t  1200 r.p.m. and the plasma  t r a n s f e r r e d by p i p e t t e t o tubes o f a s i m i l a r  size.  Plasma D i g i t o n i n P r e c i p i t a b l e S t e r o l s  (DPS)  These were determined i n d i r e c t l y by the c o l o r i m e t r i c method of Vahouny e t a l .  (1960a).  T h i s procedure u t i l i z e s  anthrone reagent to  measure the amount o f d i g i t o n i n i n the d i g i t o n i n - c h o l e s t e r o l  complex  o b t a i n e d f o l l o w i n g p r e c i p i t a t i o n o f the e x t r a c t e d s t e r o l s w i t h d i g i t o n i n and subsequent  r e c r y s t a l l i z a t i o n o f the complex, t w i c e from a c i d i f i e d  to which aluminum c h l o r i d e i s added  in solution.  methanol  Using t h i s procedure the  23 problem o f i n t r o d u c i n g  e r r o r i n t o the measurement o f the s t e r o l s because  of v a r i a t i o n i n t h e i r chromogenic b e h a v i o u r towards a g i v e n not  arise. a.  Extraction of Sterols The  by  reagent does  s t e r o l s were e x t r a c t e d  Sperry and Webb (1950).  volumetric  f l a s k using  from plasma e s s e n t i a l l y as d e s c r i b e d  One m l . o f plasma was e x t r a c t e d  i n a 25 m l .  15 i n s t e a d o f 10 m l . o f a l c o h o l acetone 1:1. The  e x t r a c t i o n was c a r r i e d out i n b o i l i n g water i n s t e a d o f a steam b a t h . Bumping was prevented by t h e a d d i t i o n o f 2 s m a l l p i e c e s filter  of solvent  paper (1.0 x 0.5 cm.) j u s t p r i o r t o t h e a p p l i c a t i o n o f h e a t .  f l a s k was h e l d w i t h a t e s t tube h o l d e r b a t h , was t i l t e d g e n t l y  and upon b e i n g p l a c e d  extracted The  i n the water  from s i d e t o s i d e through a p p r o x i m a t e l y 90°  so t h a t t h e p r e c i p i t a t e d p r o t e i n would n o t adhere t o the bottom o f t h e f l a s k and cause bumping.  At the f i r s t  i n d i c a t i o n o f b o i l i n g t h e motion  was  discontinued.  45°  and t h e c o n t e n t s brought t o a f u l l b o i l .  and  returned  cooled  The f l a s k was then h e l d a t an angle o f a p p r o x i m a t e l y I t was o c c a s i o n a l l y removed  t o t h e b a t h i n o r d e r t o p r e v e n t too v i g o r o u s b o i l i n g .  When  i t was made up t o volume w i t h a l c o h o l acetone m i x t u r e and r e p e a t e d l y  inverted. b.  P r e c i p i t a t i o n of Digitonides Better  overnight  r e p l i c a t i o n was o b t a i n e d when t h e s t e r o l s were p r e c i p i t a t e d  under c o n d i t i o n s  by Vahouny e t a l . (1960a). aliquots of extracts. to s e t t l e and 10 ml.  recommended by S p e r r y and Webb (1950) than those The s t e r o l s were p r e c i p i t a t e d from 2.0 m l .  P r i o r t o t h e i r p r e c i p i t a t i o n the e x t r a c t was allowed o f the c l e a r e r p o r t i o n t r a n s f e r r e d by p i p e t t e  into a  24 150 x 15 mm. for  t e s t tube.  6 minutes  at 1500  centrifugation. filtering to  The tube was  r.p.m.  T h i s step was  the e x t r a c t .  No  capped w i t h t i n f o i l and  centrifuged  l o s s o f s o l v e n t o c c u r r e d as a r e s u l t o f  used to r e p l a c e the more t e d i o u s one of  The tube was  then a l l o w e d a few minutes  to come  room temperature f o l l o w i n g which 2.0 ml. o f the c l e a r e x t r a c t were  removed by p i p e t t e to 15 ml. c e n t r i f u g e tubes.  The e x t r a c t was  further  t r e a t e d by the method o f S p e r r y and Webb (1950).  I t was  saponified,  and brought to 4.0 ml. w i t h a l c o h o l acetone 1:1.  Upon a c i d i f i c a t i o n w i t h  107. a c e t i c a c i d , 2.0 ml. o f a s o l u t i o n o f 0.57. d i g i t o n i n was discharged  were l e f t  forcefully  i n t o the c o n t e n t s o f the tube by blowing i n t o a 2.0  volumetric pipette.  The c o n t e n t s o f the tube were not mixed  at room temperature o v e r n i g h t .  cooled  ml.  further  and  M i x i n g o f the c o n t e n t s w i t h the  V o r t e x mixer used i n the a n a l y s i s would have p r o b a b l y l e f t  enough d i g i t o n i n  on the w a l l s o f the tube t o render d i g i t o n i n removal the f o l l o w i n g  day  difficult. c.  P u r i f i c a t i o n of D i g i t o n i d e s The time f o r each o f the two r e c r y s t a l l i z a t i o n s was  from 20 - 35 minutes. more aggregated. the of  At 35 minutes  extended  the d i g i t o n i d e s appeared to have become  T h i s would p r o b a b l y have r e s u l t e d  i n t i g h t e r packing of  p r e c i p i t a t e when c e n t r i f u g e d and c o n s e q u e n t l y a r e d u c t i o n i n the l o s s d i g i t o n i d e s when the solvent., used f o r r e c r y s t a l l i z a t i o n was b e i n g  decanted f o l l o w i n g d.  centrifugation.  C o l o r Development w i t h Anthrone  ( a c c o r d i n g to Vahouny et  al.  1960a)  Anthrone reagent c o n s i s t e d o f 0.057. anthrone and 3.07. t h i o u r e a i n 667. s u l f u r i c a c i d .  I t was  prepared at 33° i n s t e a d o f at room temperature  25 i n o r d e r t o e f f e c t d i s s o l u t i o n o f the anthrone and t h i o u r e a over a reasona b l e p e r i o d o f time  (1 h r . ) , u s i n g an e l e c t r i c a l l y d r i v e n g l a s s r o d .  reagent may be prepared at temperatures has been shown t o reduce i t s s t a b i l i t y  The  up t o 80° but treatment w i t h heat (Vahouny £t al_. 1960b).  When the  reagent was allowed t o s t a n d o v e r n i g h t , some a p p a r e n t l y i n s o l u b l e m a t e r i a l s e t t l e d t o the bottom.  The c l e a r s o l u t i o n was t h e r e f o r e siphoned o f f .  B e f o r e the reagent was added t o the d i g i t o n i d e s , the l a t t e r were dispersed  i n g l a c i a l a c e t i c a c i d a t 50° i n s t e a d o f 4 5 ° as i n s t r u c t e d .  temperature  was i n c r e a s e d i n o r d e r t o d i s l o d g e a l l o f the d i g i t o n i d e s  the t i p o f the cone o f the c e n t r i f u g e tube. room temperature.  T h i s treatment was a l s o a p p l i e d t o a b l a n k and a s t a n d a r d  s t a n d a r d was made up i n the f o l l o w i n g manner. i n a vacuum oven.  f l a s k made up t o volume w i t h g l a c i a l t o 100 ml. u s i n g g l a c i a l  s o l u t i o n was used as a s t a n d a r d . pipette directly  One hundred  The  and f i f t y n i n e m i l l i g r a m s I t was d i s s o l v e d and the  acetic acid. acetic acid.  F o l l o w i n g m i x i n g , 20 m l . One ml. o f t h i s  final  The c o l o r reagent was added from a 10 m l .  i n t o the a c e t i c a c i d s u s p e n s i o n and the d i g i t o n i d e s  i n t o s o l u t i o n by i n v e r t i n g the tube 15 t i m e s . pressing  acetic acid.  D i g i t o n i n was d r i e d a t  were t r a n s f e r r e d t o a 100 ml. v o l u m e t r i c f l a s k .  were d i l u t e d  from  The tubes were then c o o l e d to  c o n t a i n i n g 0.318 mg. o f d i g i t o n i n i n 1.0 m l . o f g l a c i a l  80° f o r 1% hours  The  brought  The tube was stoppered by  i t s mouth a g a i n s t a s m a l l rubber b a l l which was wrapped w i t h  polyethylene sheeting.  T h i s p r a c t i c e was adopted  over the recommendation  of blowing the reagent out o f a p i p e t t e i n t o the a c e t i c a c i d  suspension,  d e v e l o p i n g the c o l o u r and r e a d i n g the o p t i c a l d e n s i t y without p r i o r m i x i n g . I t was found t h a t when the recommendation o f Vahouny e t al_. (1960a) was  26 f o l l o w e d , a d d i t i o n a l mixing of the tube c o n t e n t s f o l l o w i n g development o f the c o l o r became n e c e s s a r y replicates.  M i x i n g j u s t p r i o r t o r e a d i n g the o p t i c a l d e n s i t y , i n t r o d u c e d  a d e l a y i n t o the procedure bubbles.  i n order t o o b t a i n best agreement between  as a r e s u l t of the f o r m a t i o n of p e r s i s t e n t a i r  T h i s i n n o v a t i o n was  t h e r e f o r e performed  a d d i t i o n of the c o l o r r e a g e n t . condenser i t was  Instead of f i t t i n g  the tube w i t h an a i r  capped w i t h a h o l l o w serum stopper through which a s m a l l  h o l e was  made w i t h a number 0 c o r k b o r e r .  inserted  i n t o the outer p a r t o f the s t o p p e r .  the r i m o f the tube was was  immediately f o l l o w i n g  A l o o s e p l u g of c o t t o n Any reagent  f i r s t removed u s i n g moistened  was  a d h e r i n g to  t i s s u e paper.  There  no change i n the volume of the tube f o l l o w i n g development of the c o l o r  at 100°C u s i n g a b o i l i n g water bath f o r 12 minutes. procedure was  not  The remainder  of the  altered.  Plasma C h o l e s t e r o l A n a l y s i s of 0.1 m l . samples o f plasma f o r c h o l e s t e r o l was out u s i n g the c o l o r i m e t r i c procedure by R o s e n t h a l e_t al. of a s o l u t i o n of 8.0 acid diluted  (1957).  o f Z l a t k i s e_t a l . (1953) as m o d i f i e d  The reagent used  ml. of 2.5%  f o r c o l o r development c o n s i s t e d  f e r r i c c h l o r i d e hexahydrate  to 100 m l . w i t h c o n c e n t r a t e d s u l f u r i c a c i d .  reagent were added to a s o l u t i o n of the plasma i n 2.5 acid and  i n a 150 X 20 mm.  t e s t tube.  The  r e a d from a s t a n d a r d  The  Two  i n phosphoric ml. o f  ml. of g l a c i a l  tube c o n t e n t s were immediately  the c o l o r formed, read a f t e r 30 minutes at 560 mu.  spectrophotometer.  carried  c o n c e n t r a t i o n of c h o l e s t e r o l  this acetic mixed  i n a Beckman Model B  i n each sample  was  curve.  The r e a c t i o n ( s ) l e a d i n g t o c o l o r f o r m a t i o n are not r e s t r i c t e d c h o l e s t e r o l and hence t h i s method g i v e s h i g h v a l u e s when used w i t h whole  to  27 plasma.  I t i s a u s e f u l method however when r e l a t i v e d i f f e r e n c e s a r e b e i n g  estimated.  Cholic Acid T h i s was measured as i n experiment 1 u s i n g the method o f L e v i n et  a l . (1944).  Absorption  of Taurocholate  from Open Segments  The _in v i v o technique the r a t e o f t a u r o c h o l a t e  described  absorption.  i n experiment 1 was used t o measure  The r a t e o f a b s o r p t i o n was measured over  a p e r i o d o f 1 hour u s i n g segments prepared of the m e s e n t e r i c  small i n t e s t i n e .  of c h o l i c a c i d prepared  The segments were f i l l e d  as the sodium t a u r o c h o l a t e  c o n t a i n i n g 300 mg.7. g l u c o s e . 150  from the upper and lower  equivalent  with a s o l u t i o n i n saline  The c o n c e n t r a t i o n o f c h o l i c a c i d used was  and 750 mg.7. w i t h b i r d s from group A and B r e s p e c t i v e l y .  are d e s c r i b e d  halves  These groups  i n a later section.  Experimental Plasma c h o l e s t e r o l l e v e l s were determined on 80 S i n g l e Comb White Leghorn c o c k e r e l s which were b e i n g r e a r e d on l i t t e r a l l - v e g e t a b l e d i e t o f Table  and had been f e d the  2.1 f o r 1 week ( p r e - e x p e r i m e n t a l  period).  On the b a s i s o f the l e v e l s found 40 b i r d s were s e l e c t e d and t r a n s f e r r e d to m e t a l b a t t e r i e s w i t h wire  screen f l o o r s .  Twenty b i r d s were s e l e c t e d at  each extreme o f the range o f c h o l e s t e r o l found ( i . e . 20 e x h i b i t i n g the highest concentrations  and 20 e x h i b i t i n g the l o w e s t ) .  They were then  randomized i n t o 8 l o t s o f 5 and vermifuged w i t h a s i n g l e P r a t t s A c t i o n " capsule.  "Split  At the end o f 1 week the b i r d s were weighed and b l o o d  samples a g a i n taken.  The c o n c e n t r a t i o n o f d i g i t o n i n - p r e c i p i t a b l e  sterols  TABLE 2.1.  Composition o f the d i e t  Ingredients  f e d i n experiment 2  % of diet  Corn o i l  3.00  Ground wheat  86.01  Soyabean meal (45.07. p r o t e i n )  5.49  Dried d i s t i l l e r s '  2.00  solubles  Bonemeal  2.00  Limestone  1.00  Iodized s a l t  0.50  mg./kgm. o f d i e t Manganese s u l f a t e Menadione  110.00 0.50  units/kgm. o f d i e t Vitamin  A (I.U.)  Vitamin  D,  (I.C.U.)  4400.00 199.76  i n the plasma was  then determined  e t a l . (1960a) d e s c r i b e d above.  a c c o r d i n g t o the method of Vahouny On the b a s i s of the v a l u e s o b t a i n e d  groups of 10 b i r d s were s e l e c t e d .  These were d e s i g n a t e d A and B.  s e l e c t i o n was  such t h a t each group e x h i b i t e d the widest  plasma s t e r o l  l e v e l s and  to t h i s range. and  The  at the same time resembled  The  p o s s i b l e range of  the other w i t h r e s p e c t  s e l e c t e d b i r d s were then p l a c e d i n i n d i v i d u a l cages  c o n t i n u e d on the same d i e t f o r 3 weeks.  weights  two  were determined  l e v e l s and  body  a t weekly i n t e r v a l s f o r 3 weeks i n group A and  the end of the t h i r d week i n group B. b i l e s a l t a b s o r p t i o n was  Plasma s t e r o l  At the end  at  of 3 weeks the r a t e of  measured i n each b i r d as d e s c r i b e d .  R e s u l t s and D i s c u s s i o n The experiment  plasma c h o l e s t e r o l l e v e l s of b i r d s used  are g i v e n i n T a b l e 2 . I I .  to i n i t i a t e  S i m i l a r data are a l s o presented as a  magnitude a r r a y f o r the b i r d s r e t a i n e d i n the f i r s t  selection  (Table 2 . I I I ) .  A g a i n s t the c h o l e s t e r o l l e v e l s of the l a t t e r are t a b u l a t e d the l e v e l s of d i g i t o n i n p r e c i p i t a b l e s t e r o l s ment o f the DPS  was  undertaken  (DPS)  found  r e p r e s e n t a l a r g e r p o r t i o n of 3 beta-hydroxy  corresponding  i n the plasma.  i n s t e a d of c h o l e s t e r o l as they  t o b i l e a c i d s (Blohm e t a l . , 1960;  the  Measure  collectively  s t e r o l s which are c o n v e r t i b l e  Goodman e_t al_., 1962;  S w e l l and  Treadwell  1961). I t may  be seen from T a b l e 2 . I l l t h a t the c h o l e s t e r o l l e v e l s  were h i g h e r than the c o r r e s p o n d i n g r e a d i l y e x p l a i n e d by the presence  l e v e l s o f DPS.  T h i s anomaly may  ( R o s e n t h a l , 1957).  be  of pigmented m a t e r i a l i n plasma which  c o n t r i b u t e t o the c o l o r formed by the r e a c t i o n of c h o l e s t e r o l w i t h chloride  found  Although  the i r o n reagent was  ferric  of l i t t l e  i n d e t e r m i n i n g the a b s o l u t e c o n c e n t r a t i o n of c h o l e s t e r o l i t was  value  nonetheless  TABLE 2 . I I .  Plasma c h o l e s t e r o l used f o r the f i r s t  B i r d no.  Plasma cholesterol level  2953 8593 8566 2955 9131 9200 8642 9218 8542 8660 9147 2972 8658 9119 2960 9206 8594 8570 9226 9229 8640 2951 2974 9134 8644 9224 9210 2980 8591 2990 9227 8562 8641 9324 9266 9275 9304 9320 9263 9321  134.2 127.5 161.0 162.3 191.3 134.7 178.6 104.8 174.2 134.1 134.6 181.5 162.4 165.3 142.1 159.3 166.2 134.5 149.2 129.1 178.0 102.4 162.4 166.4 172.6 176.5 170.2 142.3 169.1 144.3 162.4 168.9 134.5 143.5 147.3 149.8 142.0 158.0 154.1 159.4  l e v e l s (mg.7.) of c o c k e r e l s selection  B i r d no.  Plasma cholesterol level  2961 9308 9315 9241 9317 9128 2991 8544 8650 9170 9130 8588 9221 9217 9214 8549 8585 8575 9138 8555 8550 9164 9264 9118 9240 9318 9145 8548 8551 9220 9168 8576 9142 8564 9165 9204 8598 9270 8584  144.2 148.4 157.4 160.1 157.5 144.3 142.6 144.3 180.1 134.2 180.4 134.4 129.4 120.1 150.1 166.5 172.6 164.7 184.6 170.0 134.1 161.4 104.6 136.8 171.3 131.5 138.1 172.5 172.4 148.7 161.3 134.3 138.9 129.1 138.4 159.4 129.1 140.1 164.2  '  31  TABLE 2. I I I .  C o n c e n t r a t i o n (mg.7.) o f c h o l e s t e r o l and DPS i n plasma o f c o c k e r e l s r e t a i n e d i n the f i r s t s e l e c t i o n  Bird number  Cholesterol  2951 9264 9218 9217 8593 9229 9221 9318 9147 9170 9200 9118 9145 9142 9165 9270 9304 9324 9128 2961  102.4 104.6 104.8 120.1 127.5 129.1 129.4 131.5 134.6 134.2 134.7 136.8 138.1 138.9 138.4 140.1 142.0 143.5 144.3 144.2  DPS 62.3 58.4 54.6 54.2 54.7 58.7 64.6 60.8 64.1 74.2 58.5 71.0 71.2 74.8 61.1 64.8 80.4 89.4 80.1 82.7  Bird number  Cholesterol  9227 8584 8575 8549 8562 8591 9210 8555 9240 8548 8644 8542 9224 8640 8642 8650 9130 2972 9138 9131  162.4 164.2 164.7 166.5 168.9 169.1 170.2 170.0 171.3 172.5 172.6 174.2 176.5 178.0 178.6 180.1 180.9 181.5 184.6 191.3  DPS 80.5 81.7 83.5 84.4 99.2 94.1 96.7 94.0 97.1 84.8 86.2 99.8 110.2 97.5 100.2 90.4 104.2 87.5 100.5 100.3  32 s a t i s f a c t o r y f o r measuring the r e l a t i v e It f u l f i l l e d  l e v e l s as shown i n t h e r a n k i n g .  one o f the o b j e c t i v e s o f the e x p e r i m e n t - - t h a t o f o b t a i n i n g  a r a p i d s e l e c t i o n o f two groups o f b i r d s e x h i b i t i n g a wide range i n l e v e l s of DPS. The c o n c e n t r a t i o n o f DPS and body weights measured p e r i o d i c a l l y throughout the experiment f o r the two groups  (A and B) r e s u l t i n g from t h e  second s e l e c t i o n a r e shown i n T a b l e s 2 . I V and 2.V r e s p e c t i v e l y . of plasma DPS l e v e l s was wide i n b o t h i n s t a n c e s . DPS  l e v e l was a p p r o x i m a t e l y twice the lower.  on a l l o c c a s i o n s when DPS were measured. observed b u t these d i d not r e s u l t ranking order. stabilized  The range  F o r b o t h ranges the upper  This r e l a t i o n s h i p held  S l i g h t weekly f l u c t u a t i o n s were  i n any change worthy o f note i n t h e  The l e v e l s o f DPS a t 3 weeks t h e r e f o r e r e p r e s e n t e d the  l e v e l on t h e d i e t  fed.  They were t h e r e f o r e used t o make the  n e c e s s a r y c a l c u l a t i o n s r e l a t i n g plasma s t e r o l l e v e l s t o b i l e s a l t rates.  true  absorption  The plasma DPS range a t 3 weeks was 58.1 - 101.9 mg.% f o r group A  and 52.0 - 101.6 mg.% f o r group B. As  indicated  i n T a b l e 2.V, body weights f l u c t u a t e d  slightly  throughout the e x p e r i m e n t a l p e r i o d but n o t t o the e x t e n t o f r e n d e r i n g the c a l c u l a t i o n s u n r e p r o d u c i b l e when the f i n a l weights e n t e r e d i n t o them. The amount o f t a u r o c h o l a t e absorbed from t h e sacs p r e p a r e d from b i r d s o f groups A and B a r e shown i n T a b l e 2.VI.  The b i r d s o f group A  which were s t u d i e d u s i n g a t a u r o c h o l a t e s o l u t i o n e q u i v a l e n t to 150 mg.% of c h o l i c a c i d showed an a b s o r p t i o n r a t e of 43.4 - 83.4 mg./kgm. body wt./hr.  Those o f group B absorbed t h e b i l e s a l t  at a r a t e o f 54.0 - 186.6 mg./kgm. body wt./hr.  from a 750 mg.7. s o l u t i o n There was thus a decrease  33  TABLE 2.IV.  C o n c e n t r a t i o n o f DPS (mg.%) i n plasma of b i r d s o f groups A and B a t i n t e r v a l s throughout experiment 2  GROUP A D u r a t i o n o f experiment Bird number 2951 8593 9318 9145 2961 9131 9240 8642 8542 9130  2  1 62.3 54.7 60.8 71.2 82.7 100.3 97.1 100.2 99.8 104.2  60.4 58.1 68.3 70.4 88.1 86.2 98.4 104.1 110.3 108.2  (weeks)  3 58.1 60.8 71.9 73.4 84.9 91.4 91.8 94.8 101.7 101.9  GROUP B 9218 9134 9229 9170 9270 9304 9128 9210 9224 9138  54.6 64.1 58.7 74.2 64.8 80.4 80.1 96.7 110.2 100.5  52.0 57.8 63.4 66.7 70.8 72.4 86.6 89.4 97.6 101.6  34  TABLE 2.V.  Weekly body weights o f b i r d s o f groups A and B  GROUP A  GROUP B  B i r d no.  Week 1  Week 2  Week 3  B i r d no.  Week 1  Week 2  Week 3  2951  1460  1461  1469  9218  1751  1762  1772  8593  1494  1515  1505  9134  1954  1934  1925  9318  1380  1388  1395  9229  1953  1957  1962  9145  1729  1751  1743  9170  1834  1840  1815  2961  1658  1661  1672  9270  1671  1660  1658  9131  1664  1471  1480  9304  1945  1958  1965  9240  1590  1589  1603  9128  1701  1700  1704  8642  1381  1372  1394  9210  1783  1774  1778  8542  1448  1439  1446  9224  1635  1638  1635  9130  1492  1490  1498  9138  1731  1744  1724  TABLE 2.VI.  Plasma DPS l e v e l s and r a t e o f t a u r o c h o l a t e a b s o r p t i o n by segments o f s m a l l i n t e s t i n e o f t e s t c o c k e r e l s i n experiment 2  GROUP A ( T a u r o c h o l a t e  e q u i v a l e n t o f 150 mg.% c h o l i c  Rate o f t a u r o c h o l a t e B i r d no.  DPS  Upper i n t e s t i n e mg./kgm. body wt./hr.  2951 8593 9318 9145 2961 9131 9240 8642 8542 9130  58.1 60.8 71.9 73.4 84.9 91.4 91.8 94.8 101.7 101.9  28.3 25.7 25.1 15.2 15.7 16.9 37.8 36.8 18.2 26.1  mg./hr. 41.6 38.7 35.0 26.5 26.3 25.0 60.6 51.3 26.3 39.1  GROUP B ( T a u r o c h o l a t e 9218 9134 9229 9170 9270 9304 9128 9210 9224 9138  52.0 57.8 63.4 66.7 70.8 72.4 86.6 89.4 97.6 101.6  32.6 84.1 65.0 86.5 25.1 52.7 25.0 75.0 48.5 26.5  57.8 161.9 127.5 157.0 41.6 103.6 42.6 133.4 79.3 45.7  acid)  absorption Lower  mg./kgm. body wt./hr. 29.8 34.6 44.3 28.2 34.5 31.1 45.6 33.4 35.2 26.7  Upper and lower intestine  intestine  mg./hr.  mg./kgm. body wt./hr.  mg./hr.  58.1 60.3 69.4 43.4 50.2 48.0 83.4 70.2 53.4 52.8  85.4 90.8 96.8 75.7 84.0 71.0 133.7 97.9 77.2 79.1  90.2 143.4 186.6 161.9 54.0 125.0 71.9 152.9 133.0 119.3  159.9 276.1 366.1 293.9 89.5 245.7 122.5 271.9 217.5 205.7  43.8 52.1 61.8 49.2 57.7 46.0 73.1 46.6 50.9 40.0  e q u i v a l e n t o f 750 mg.% c h o l i c a c i d ) 57.6 59.3 121.6 75.4 28.9 72.3 46.9 77.9 84.5 92.8  102.1 114.2 238.6 136.9 47.9 142.1 79.9 138.5 138.2 160.0  36 i n the percentage uptake from the segments when the b i l e s a l t was  increased  five-fold.  The  those found i n the upper and given  i n Table  above r a t e s were a r r i v e d at by lower i n t e s t i n e s e p a r a t e l y .  I t may  found between the l e v e l of plasma DPS i n t e s t i n e on the one i s low  ence of a b s o r p t i o n Absorption The  was  These are  be  seen t h a t the degree of c o r r e l a t i o n  and  absorption rates  hand and  and n o n s i g n i f i c a n t .  different  w i t h the t o t a l r a t e of The  data  r a t e s between the upper and  however g r e a t e r  i n the  i n d i c a t e a l s o an  lower p a r t s o f the  independ-  intestine.  i n the lower p a r t as found i n experiment  1.  inmg./hr. Assuming t h a t b i l e s a l t s r e g u l a t e the l e v e l o f plasma DPS  data may  be  i n t e r p r e t e d as i n d i c a t i n g t h a t t h e r e may  s a l t s which p e r f o r m t h i s f u n c t i o n .  The  the determinant of plasma l e v e l s of DPS from the gut It not be  s p e c i f i c pools  of  into this pool(s)  p r o v i d i n g t h e i r rate of  being  absorption  i s adequate. i s a l s o p o s s i b l e t h a t the r a t e of a b s o r p t i o n  from the  gut  a good index o f the r a t e at which these s a l t s e n t e r the p o r t a l  circulation. w a l l and  be  these  o p e r a t i o n of such a mechanism  would a l l o w f o r the r a t e of e n t r y of b i l e s a l t s  may  absorption  above r e l a t i o n s h i p s remained u n a f f e c t e d when the a b s o r p t i o n r a t e s were  expressed  bile  also  2.VII l i s t s a number of c o r r e l a t i o n c o e f f i c i e n t s c a l c u l a t e d  from the v a r i a b l e s measured.  on the o t h e r ,  summation of  2.VI.  Table  p a r t s of the  concentration  I f f o r example t h e r e were a r a p i d uptake by the  a slower r e l e a s e i n t o the c i r c u l a t i o n and  c l o s e l y c o r r e l a t e d i t would be d i f f i c u l t the r e l a t i o n s h i p i n q u e s t i o n .  i f not  intestinal  these r a t e s were not  impossible  to demonstrate  37  TABLE 2.VII.  Degree o f c o r r e l a t i o n between v a r i a b l e s measured i n experiment 2  Variables  Correlation GROUP A  coefficient GROUP B  Plasma DPS and r a t e o f t a u r o c h o l a t e a b s o r p t i o n by upper i n t e s t i n e i n mg./kgm. body wt./hr.  0.047 n s . *  0.312 n s .  Plasma DPS and r a t e o f t a u r o c h o l a t e by upper i n t e s t i n e i n mg./hr.  0.038 n s .  0.370 ns.  Plasma DPS and r a t e o f t a u r o c h o l a t e a b s o r p t i o n by lower i n t e s t i n e i n mg./kgm. body wt./hr.  0.013 n s .  0.193 ns.  Plasma DPS and r a t e o f t a u r o c h o l a t e by lower i n t e s t i n e i n mg./hr.  0.003 n s .  0.056 ns,  Rate o f t a u r o c h o l a t e a b s o r p t i o n i n upper and lower i n t e s t i n e i n mg./kgm. body wt./hr.  0.409 n s .  0.358 ns.  Rate o f t a u r o c h o l a t e a b s o r p t i o n lower i n t e s t i n e i n mg./hr.  0.372 n s .  0.349 ns.  * nonsignificant  absorption  absorption  i n upper and  38 It  i s l i k e l y also that v a r i a t i o n  i n i n t e s t i n a l m o t i l i t y and  consequently, rate of t r a n s i t of i n t e s t i n a l contents, l e d to d i f f e r e n c e s i n the amount o f b i l e s a l t s made a v a i l a b l e d i f f e r e n c e s i n the plasma s t e r o l l e v e l s .  f o r r e a b s o r p t i o n , and hence t o When t h i s type o f v a r i a t i o n i s  g r e a t the a b s o r p t i v e c a p a c i t y o f the i n t e s t i n e  toward b i l e s a l t s would n o t  n e c e s s a r i l y be a major determinant o f the plasma s t e r o l t h e r e i s an i n v e r s e r e l a t i o n s h i p  between m o t i l i t y  l e v e l except when  and a b s o r p t i v e c a p a c i t y .  Another l i k e l y p o s s i b i l i t y i s t h a t v a r i a t i o n  i n hormone metabolism  c o n t r i b u t e d markedly t o the d i f f e r e n c e s observed i n the plasma l e v e l s , without h a v i n g n e c e s s a r i l y the  sterol  i n f l u e n c e d the a b s o r p t i v e c a p a c i t y o f  intestine. On  the b a s i s o f the d a t a p r e s e n t e d the v i e w may a l s o be advanced  t h a t a l i m e n t a r y substances govern the amount o f b i l e s a l t s made  available  f o r a b s o r p t i o n and were i n d i r e c t l y r e s p o n s i b l e f o r e s t a b l i s h i n g  the l e v e l s o f  circulating  sterols  noted on the d i e t f e d .  39 SUMMARY - PART A method f o r d e t e r m i n i n g  ONE  the r a t e of a b s o r p t i o n of b i l e s a l t  from  open segments or c l o s e d sacs of i n t e s t i n e of the c h i c k e n i s d e s c r i b e d .  The  r a t e of a b s o r p t i o n o f sodium t a u r o c h o l a t e from open segments was over s i x c o n s e c u t i v e  15 minute p e r i o d s and was  found  time to have d e c l i n e d to about 507. of t h a t observed  at the end of  i n t e s t i n e f o r t a u r o c h o l a t e and g l y c o c h o l a t e .  the a b s o r p t i v e c a p a c i t y of the i n t e s t i n e was and decreased  this  i n the f i r s t .  sacs were used to measure the r e l a t i v e a b s o r p t i v e c a p a c i t y o f p a r t s of the  followed  Closed  different  F o r both  g r e a t e s t i n the d i s t a l p o r t i o n  proximally.  Experiments were a l s o conducted to determine i f the l e v e l circulating sterols  i n mature c o c k e r e l s bore a s i g n i f i c a n t  found  two  c o n c e n t r a t i o n s of t a u r o c h o l a t e , 0.15  and  Using  findings is discussed.  open  0.757. i t was  t h a t the two v a r i a b l e s were independent of each o t h e r .  of these  of  relationship  to the a b s o r p t i v e c a p a c i t y of the i n t e s t i n e f o r b i l e s a l t . segments and  substances  The  significance  40 PART I I THE  EFFECT OF DIETARY FATS ON AND  THE  LEVEL OF  THE  FECAL OUTPUT OF  BILE ACIDS  CIRCULATING STEROLS  INTRODUCTION  The variation  results  of experiment 2 d i d not  i n the a b s o r p t i v e c a p a c i t y of the  be r e f l e c t e d hypothesis  intestine  the h y p o t h e s i s  that  for bile salt  should  as d i f f e r e n c e s i n the plasma s t e r o l l e v e l s .  appears to be w e l l founded, one  advanced f o r the r e s u l t s to  support  obtained,  or more of the  c o u l d be v a l i d and  Since  explanations  t h e r e f o r e any  i d e n t i f y the v a l i d e x p l a n a t i o n ( s ) would be of importance.  nutritional  standpoint,  d i e t a r y f a c t o r s played d i e t which was  fed.  the e x p l a n a t i o n a role  was  exerts probably  I t would have been most f i t t i n g  s i n g l e d out  for  t h a t the l a t t e r substances i n f l u e n c e the  variation  T h i s judgement i s triglycerides  l e v e l of c i r c u l a t i n g  sterols  However, because o f the r i s k of i n t r o d u c i n g  i n the p h y s i c a l s t a t e of the t e s t  preparation with t r i g l y c e r i d e s  investigation.  to f i r s t determine i f d i e t a r y  based on the knowledge t h a t b i l e s a l t s promote the d i g e s t i o n of  dietarily.  the  the most v a r i a b l e e f f e c t  t r i g l y c e r i d e s have an e f f e c t on b i l e s a l t a b s o r p t i o n .  when a d m i n i s t e r e d  that  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 on  Since d i e t  attempts  From a  found most i n t e r e s t i n g  on the plasma s t e r o l l e v e l , t h i s e x p l a n a t i o n was  and  this  emulsions d u r i n g  their  of v a r y i n g degree of u n s a t u r a t i o n ,  from measuring b i l e s a l t a b s o r p t i o n to b i l e s a l t e x c r e t i o n was p h y s i c a l s t a t e of the emulsions prepared, amount of b i l e s a l t absorbed from them and  could conceivably  made.  The  i n f l u e n c e the  thus obscure the exact  to which a b s o r p t i o n of b i l e s a l t s take p l a c e when v a r i o u s  a change  extent  triglycerides  41 are f e d and  when d i g e s t i o n i s p r o c e e d i n g n o r m a l l y .  I t was  hoped t h a t  i n v i v o t e c h n i q u e developed, would be used to f u r t h e r study any  the  e f f e c t on  b i l e a c i d e x c r e t i o n induced by d i e t a r y t r i g l y c e r i d e s .  DETERMINATION OF  BILE ACIDS IN FECES  A Note on A n a l y t i c a l Problems In s t e r o l e x c r e t i o n s t u d i e s employing n o n i s o t o p i c a major m e t h o d o l o g i c a l problem has b i l e acids  i n feces.  been the  techniques,  quantitative determination  With the advent o f t h i n l a y e r and  gas  of  liquid  chromatography, a s o l u t i o n to t h i s problem appears c l o s e r than b e f o r e continues  to elude the a n a l y s t  f o r many r e a s o n s .  In the c o l o n and  but  cecum  the p r i m a r y b i l e a c i d s are c o n v e r t e d by the a c t i o n of microorganisms to a m u l t i p l i c i t y of compounds (Norman and in physical properties  Sjovall,  (Bergstrom, 1962).  1958)  w i t h marked d i f f e r e n c e s  There are b i l e a c i d s  i n feces  w i t h a p o l a r i t y s i m i l a r to t h a t of f a t t y a c i d s which have thus f a r d e f i e d complete s e p a r a t i o n  from the  l a t t e r by  the common techniques of  such as column chromatography, t h i n l a y e r chromatography and distribution  (Grundy et a l . , 1965;  have p o i n t e d  out  grossly unsuitable be  lost  as up  extraction  f o r the removal of f r e e f a t t y a c i d s i s  to 307. o f the t o t a l b i l e a c i d s  i n the f a t t y a c i d e x t r a c t .  f u r t h e r complicated  countercurrent  A l i et a l . (1966)  t h a t the c l a s s i c a l method of p e t r o l e u m ether  of crude b i l e a c i d p r e p a r a t i o n s  may  A l i et a l . , 1966).  analysis  The  i n human f e c e s  problem of d e t e r m i n a t i o n  by the presence o f a c i d i c substances d e r i v e d  from  haemoglobin w i t h v i r t u a l l y the same p o l a r i t y c h a r a c t e r i s t i c s as the a c i d s and  therefore  accompany the  latter  account of these substances t h e r e has  i n most a n a l y t i c a l s t e p s .  been a g e n e r a l  is  bile On  tendency to o v e r e s t i m a t e  42 the f e c a l output  of b i l e a c i d s .  from r a t f e c e s has 1963)  but  i t has  S u c c e s s f u l removal of these  been achieved  been r e p o r t e d  that t h e i r  f e c e s of v a r i o u s s p e c i e s renders (1965). searching  using charcoal  (Roscoe and  substances Fahrenbach,  variable concentration  i n the  t h i s method u n r e l i a b l e (Grundy,et a l .  T h i s c r i t i c i s m appears to o b t a i n a l s o w i t h c h i c k e n f e c e s . f o r a method of d e t e r m i n i n g  In  b i l e a c i d s i n c h i c k e n f e c e s , i t was  found t h a t c h a r c o a l treatment l a c k e d e f f e c t i v e n e s s i n removing a l l of f e c a l pigments.  That the  i n e f f e c t i v e n e s s o f c h a r c o a l was  probably  the  due  to  l a r g e amounts o f pigments i n the f e c e s , i s suggested i n the h i g h r a t e of b i l e a c i d e x c r e t i o n found f o r the c h i c k e n when t i t r i m e t r i c methods of a n a l y s i s are used to measure the f e c a l b i l e a c i d s . who  appear to have p u b l i s h e d  (1961)  the o n l y r e p o r t on the r a t e of f e c a l e x c r e t i o n  of b i l e a c i d s i n the c h i c k e n , gms./kgm. body wt./24 h r .  L e v e i l l e ejt al.  found an e x c r e t i o n r a t e o f 3.06  The  -  4.05  r e s u l t s of the experiments undertaken here  p l a c e t h i s r a t e i n the range of 15.7  - 79.7  mg./kgm. body wt./24 h r . which  compares f a v o u r a b l y w i t h t h a t r e p o r t e d f o r s m a l l e r l a b o r a t o r y animals such as the r a t (7.7  - 52.5)  and  r a b b i t (10.9  - 27.0)  (Grundy et a l . , 1965).  Lewis (1957) d e s c r i b e d a method f o r removal of these pigments by hydroxide  p r e c i p i t a t i o n but  a number of workers have expressed  w i t h t h i s method because of the low r e c o v e r i e s o b t a i n e d w i t h et a l . , 1965; pigments u s i n g but  S j o v a l l , 1964).  they have f a i l e d  fecal  g l a s s f i b e r chromatography have been r e p o r t e d  i n the hands of many workers.  d i s c u s s e d by Grundy e t a l . (1965).  Apart  These methods are  from c o n f e r r i n g v a r y i n g  on the b i l e a c i d s , the a c t i o n of i n t e s t i n a l microorganisms a l s o s u s c e p t i b i l i t y to d e g r a d a t i o n  dissatisfaction  i t (Grundy  Other methods f o r the removal of  i o n exchange and  zinc  by hot  polarity imparts  a l k a l i which i n t r o d u c e s a f u r t h e r  43 problem-  The  f o r m a t i o n of a 3-keto group on the b i l e a c i d molecule as a  r e s u l t of b a c t e r i a l dehydrogenation leads to e x t e n s i v e d e s t r u c t i o n of b i l e a c i d s under c o n d i t i o n s employed f o r s a p o n i f i c a t i o n of t h e i r (1.25  - 2.0  normal a l k a l i f o r 2 - 4  et aJL., 1954;  L e v i n et a l . , 1961).  r a t s and r a b b i t s , t h i s l o s s  i s not  hours at 120° and  In the a n a l y s i s of f e c e s from humans, a s e r i o u s one  (Grundy e t a l . , 1965).  i n s t e r o l balance  as 3-keto b i l e  in this species.  of q u a l i t a t i v e  F o r the same reason,  a c i d s as done i n the p r e s e n t of the e s t i m a t e d mainly  output.  The  analyses  acids  of b i l e  acids  I f these l o s s e s are p e r m i s s i b l e  s t u d i e s employing the domestic c h i c k e n ,  because of the sparseness  conjugates  15 p . s . i . ) (Mosbach  c o n s t i t u t e o n l y a n e g l i g i b l e f r a c t i o n of the d a i l y output by these s p e c i e s  i s not known,  i n f o r m a t i o n on b i l e a c i d e x c r e t i o n  n e g l e c t of any h i g h l y nonpolar a l s o opens to q u e s t i o n the  c o r r e c t n e s s of t h i s p r e s e n t  estimate  (3 hours) and would p r o b a b l y not  f o r e x t e n s i v e a l t e r a t i o n s of the primary In the p r e s e n t  relies  t h i n l a y e r and gas (hydroxy  T h i s method employs the use of column, substances  f a t t y a c i d s and pigments) which i n the past have l e d to  e s t i m a t i o n o f the f e c a l b i l e a c i d s .  g r e a t e r than  over-  I t s s p e c i f i c i t y has been checked u s i n g  I t however f a i l s  2.07. of the t o t a l b i l e a c i d s which migrate l a y e r chromatography and  allow  the method of Grundy et a l .  l i q u i d chromatography f o r the removal o f  radioisotope techniques.  gut  b i l e acids.  s e r i e s of a n a l y s e s  (1965) i s used as a main g u i d e .  bile  validity  on the knowledge t h a t the time o f passage of f e e d through the  of the c h i c k e n i s r e l a t i v e l y s h o r t  the  to measure i n human f e c e s , w i t h the f a t t y a c i d s d u r i n g  thin  a n e g l i g i b l e amount of b i l e a c i d s w i t h a p o l a r i t y  t h a t of c h o l i c a c i d .  F o r reasons  a l r e a d y c i t e d , a comparison  w i t h the c h i c k e n o f t h a t f r a c t i o n o f b i l e a c i d s which escapes being measured  44 cannot be made a t the p r e s e n t time.  Gas L i q u i d Chromatography (GLC)  An F and M model 5750 d u a l column gas chromatograph, w i t h d u a l hydrogen  equipped  flame i o n i z a t i o n d e t e c t o r s , a d i f f e r e n t i a l e l e c t r o m e t e r ,  a l i n e a r temperature programmer, and a Hewlett Packard, m u l t i s p e e d , 1 m.v. s t r i p c h a r t r e c o r d e r model 7127A w i t h p l u g i n module 17503A was used. column p a c k i n g c o n s i s t e d o f a commercial p r e p a r a t i o n L a b o r a t o r i e s ) o f 17. SE-30 on the s i l a n i z e d mesh s i z e 100 - 120.  was  (Applied Science  support gas chromosorb  Columns were made from s t a i n l e s s s t e e l  x 1/4 i n . ) purchased from W i l k i n s o n ' s S t e e l  The  Q, o f  (6 f t . 6 i n s .  (Vancouver, B. C ) . N i t r o g e n  used as c a r r i e r gas, a t a f l o w r a t e o f 80 cc./min.  a i r flows were 70 - 80 and 550 cc./min. r e s p e c t i v e l y .  The hydrogen and Column temperature  was m a i n t a i n e d at e i t h e r 245° o r was programmed as f o l l o w s :  230°  isothermally  f o r 8 minutes, 230° t o 250° a t a r a t e o f 1° p e r minute, 250° f o r 30 mins. The d e t e c t o r temperature was 260° and the i n j e c t i o n p o r t 270°.  Injections  s i m u l a t e d the on-column technique i n t h a t the sample was d i s c h a r g e d l e s s than 0.5 cm. away from p a c k i n g which was i n t r o d u c e d of 1/4 i n . I.D. column.  i n t o the i n s e r t  liner  There was no dead volume between the i n s e r t l i n e r and  P r e p a r a t i o n o f the gas chromatograph  e n t a i l e d the f o l l o w i n g  f o r a n a l y s i s o f samples  steps.  P r e p a r a t i o n o f Column Newly purchased s t a i n l e s s s t e e l tubes were t h o r o u g h l y washed u s i n g s u c t i o n and about 6 l i t e r s o f a hot s o l u t i o n o f haemosol d e t e r g e n t . The tubes were r i n s e d f o r % hour by c o n n e c t i n g them to a hot water tap  45 w i t h a p i e c e o f c l e a n rubber of methanol. and  tubing.  C l e a n i n g was completed w i t h 250 ml.  A l l t r a c e s o f s o l v e n t were then removed, f i r s t by d r a i n i n g  then by f l o w o f a i r s u p p l i e d from a c y l i n d e r w h i l e the tubes were  p l a c e d on two hot p l a t e s separated  by a few f e e t .  a f t e r the e x p i r a t i o n o f approximately repeated.  The ends were r e v e r s e d  15 minutes and the d r y i n g  process  F o l l o w i n g c o o l i n g , the columns were s l o w l y s i l a n i z e d by s u c k i n g  through 150 m l . o f a 3.07. s o l u t i o n o f d i m e t h y l d i c h l o r o s i l a n e i n c h l o r o f o r m . Excess d i m e t h y l d i c h l o r o s i l a n e was s l o w l y removed w i t h 200 m l . o f methanol. The  column was then d r i e d by p l a c i n g i t a c r o s s the top o f a b a s i n o f b o i l i n g  water and blowing  a i r through as b e f o r e .  the column o v e r n i g h t  Packing  in a vertical  D r y i n g was completed by s t a n d i n g  position.  o f Column The  column was packed i n the u p r i g h t p o s i t i o n u s i n g vacuum, a  v i b r a t o r and g e n t l e t a p p i n g . A Y connector  Resort was made t o the f o l l o w i n g  was j o i n e d t o a t r a p w i t h h i g h vacuum rubber  technique.  tubing.  This  t r a p was p l a c e d i n c o n t i n u i t y w i t h a vacuum gauge which was f u r t h e r connected t o a water t r a p .  The l a t t e r made a f i n a l  connection  t o a water  aspirator.  were made w i t h h i g h vacuum t u b i n g .  Two s h o r t  A l l connections  p i e c e s o f t u b i n g about 6 inches Y.  A number 13 gauge needle  to i t s l i m i t At t h i s The  limit  i n l e n g t h were f i t t e d  t o the arms o f the  c u t t o a l e n g t h o f about 1 i n c h was f o r c e d  ( p o i n t f i r s t ) down the t u b i n g and i n t o the arm o f the Y. the l a r g e r end o f the needle  r e s t e d on the r i m o f the g l a s s .  t u b i n g around the arm was then t i g h t l y wrapped w i t h wide e l a s t i c bands.  A s m a l l amount o f s t e e l wool which r e s i s t e d displacement greater  by the vacuum to a  extent than d i d g l a s s wool was used t o s e a l the entrance  t o the  46 needle.  A s m a l l amount of s i l a n i z e d g l a s s wool was  the p l u g formed, p r e s s e d p a c k i n g was  added.  p o s i t i o n was  The  Finally,  empty column was  introduced  and  a s m a l l amount of column  then f o r c e d i n t o t h i s p l u g .  are shown d i a g r a m m a t i c a l l y  apparatus t h e r e was  a c h o i c e of p a c k i n g  In the l a t t e r case,  p l u g o r clamp.  i n F i g . 3.1.  the two  With  this  columns s i m u l t a n e o u s l y  the tube from 1 arm was  2.5  cm.  or  c l o s e d by means of a  With the column i n p l a c e , s m a l l amounts of packing m a t e r i a l  were i n t r o d u c e d r e p e a t e d l y w i t h the a i d of a s p a t u l a through a s m a l l of  Its  made secure by wrapping i t to the arm w i t h e l a s t i c bands."  These c o n n e c t i o n s  singly.  into place.  then  i n diameter.  A f t e r each i n t r o d u c t i o n , the m a t e r i a l was  funnel  packed  w i t h the movement of a v i b r a t o r along the a p p r o p r i a t e l e n g t h o f column. T h i s was and  f o l l o w e d by g e n t l e t a p p i n g .  the ends i d e n t i f i e d by d e s i g n a t i n g t h a t f i r s t  other  inlet.  The  e x i t was  connector  1/4  i n . to 1/4  welding.  The  u n i o n was  then f i t t e d  both  sources  There was  thus no  (Swagelok s e a l e d o f f by  f u r t h e r added as r e q u i r e d .  The  loss  i n q u a l i t y of end.  By means  F i n a l packing  fitting  of the column  was  Packing m a t e r i a l  was  from the i n l e t end  of the column  r e p l a c e d w i t h a p l u g of s i l a n i z e d g l a s s wool ( A p p l i e d  S c i e n c e L a b o r a t o r i e s ) which was  the  connected through a s h o r t p i e c e o f  under 20 l b . / s q . i n c h of p r e s s u r e .  removed and  the  the column m a t e r i a l from  a p p l i e d at the i n l e t  end was  copper t u b i n g to a tank of n i t r o g e n .  was  as e x i t and  of which was  of column to u n i o n  subsequently  Swagelok f i t t i n g s the i n l e t  accomplished  filled  removed  t i g h t l y packed w i t h column m a t e r i a l i n such a manner  became c o n t i n u o u s .  column when p r e s s u r e was  the column was  to a s t r a i g h t u n i o n  i n . ) the t e r m i n a l nut  t h a t upon making the c o n n e c t i o n  of  When f i l l e d ,  i n s e r t e d 0.5  cm.  i n t o the column.  The  1. COLUMN 2. RUBBER  TUBING  3.ELASTIC  W D  /•.SHORTENED 5 .WATER 6. Y  WRAPPING SYRINGE  NEEDLE  ASPIRATOR  CONNECTOR  F i g . 3.1.  Connections t o gas chromatograph column and source o f vacuum d u r i n g p a c k i n g o f column  •p-  48 f o l l o w i n g procedure was  used i n p l u g g i n g  removed to a d i s t a n c e of 0.5 by r e p a c k i n g A pad The  w i t h the f l a t  cm.  and  the column.  the r e s u l t i n g d i s t u r b a n c e  made and p l a c e d over the end  c l e a n s u r f a c e of a f l a t p i e c e of m e t a l was applied with clockwise  and  diameter.  of the column.  p l a c e d on t h i s pad  a n t i c l o c k w i s e motion.  was  rectified  s u r f a c e of a g l a s s rod of a p p r o p r i a t e  of s i l a n i z e d g l a s s wool was  pressure  Column m a t e r i a l  and  T h i s caused  a dense p l u g of g l a s s wool of diameter matching t h a t o f the column to be cut and  automatically inserted.  wool which c o u l d not be  Upon removal o f the f l a t  accommodated and which t h e r e f o r e p r o t r u d e d  the end o f the column, was  removed a f t e r repeated  it  not of s u f f i c i e n t  The  failed.  I f the pad was  e x i t end  object, glass above  attempts to accommodate  t h i c k n e s s t h i s step was  repeated.  of the column-was then plugged i n a s i m i l a r manner.  Shaping of Column One  end  of the column was  i n s e r t e d i n t o a l a t c h screwed to  s i d e s u r f a c e of the top o f a bench or t a b l e of s u i t a b l e h e i g h t . was  then shaped by wrapping i t around a gas  the  The  column  cylinder.  C o n d i t i o n i n g o f Column The  column was  c o n d i t i o n e d f o r 16 hours w i t h the oven and  p o r t temperatures maintained o f 100  cc./min.  oven compartment. capping obtained  During  injection  at 280° and w i t h n i t r o g e n f l o w i n g at a r a t e  t h i s time, column contaminants were vented to  Contamination of the d e t e c t o r b l o c k was  the d e t e c t o r entrances  w i t h aluminum f o i l .  at the working s e n s i t i v i t y  of t h i s conditioning period.  (range 10,  prevented  the by  A s t a b l e base l i n e  a t t e n u a t i o n 32)  at the  was end  49 Injection  Technique All  i n j e c t i o n s were made w i t h a 10 u l . Hamilton s y r i n g e #701N  f i t t e d w i t h a 26 gauge 2 i n c h n e e d l e o f 0.010 i n . i n t e r n a l  diameter.  Sampling w i t h the s m a l l e r diameter needle o f the s e r i e s 701N was made i m p o s s i b l e by the presence o f ammonium c h l o r i d e c r y s t a l s reagent  i n the s i l y l a t i o n  (the c r y s t a l s do not i n t e r f e r e w i t h d e r i v a t i v e f o r m a t i o n o r gas  chromatographic a n a l y s i s ) .  A l l samples  n e e d l e p r i o r to i n j e c t i o n .  The needle was u s u a l l y h e l d  port u n t i l  were withdrawn from the s y r i n g e  emergence o f the s o l v e n t f r o n t .  t e c h n i q u e was used. plunger depressed.  i n the i n j e c t i o n  I n some i n s t a n c e s the f o l l o w i n g  The n e e d l e was f i l l e d w i t h carbon d i s u l f i d e and the The s o l v e n t remaining i n the needle was then withdrawn  f o r a s h o r t d i s t a n c e and sampling c a r r i e d out i n the u s u a l manner.  Because  o f the v e r y low s e n s i t i v i t y o f the hydrogen' flame i o n i z a t i o n d e t e c t o r to carbon d i s u l f i d e t h e r e was a s u b s t a n t i a l r e d u c t i o n i n t a i l i n g o f the s o l v e n t f r o n t caused by slow e v a p o r a t i o n o f r e s i d u a l sample from the n e e d l e . T a i l i n g was however n o t c o m p l e t e l y e l i m i n a t e d on account o f the h i g h s e n s i t i v i t y used d u r i n g a n a l y s i s , and because reactant  o f the h i g h reagent t o  ratio.  T h i n L a y e r Chromatography  Preparation of Plates S i l i c a g e l H ( b i n d e r f r e e s i l i c a g e l ) was mixed i n a mortar w i t h d i s t i l l e d water was  i n the p r o p o r t i o n 40 grams g e l t o 93 m l . water.  The s l u r r y  t r a n s f e r r e d t o a Desaga a p p l i c a t o r and spread as a l a y e r 0.700 mm.  t h i c k on f i v e 20 x 20 cm. g l a s s p l a t e s .  The p l a t e s were f i r s t  l a i d on a  50 plastic  template w i t h r e t a i n i n g edges i n descending  t h e i r s u r f a c e s moistened dried overnight.  p r i o r to s p r e a d i n g .  The  On the f o l l o w i n g day 2 x 2.0  cm.  o r d e r of t h i c k n e s s and  coated p l a t e s were a i r o u t e r and 4.x  i n n e r l a n e s were d e s c r i b e d on each p l a t e w i t h vacuum u s i n g the tip of  o f P a s t e u r p i p e t t e which was rubber t u b i n g .  connected  4.0  cm.  curved  t o a water a s p i r a t o r v i a a p i e c e  The p l a t e s were then developed  with a mixture  of  methanol and a c e t i c a c i d as i n s t r u c t e d by Grundy et a_l. (1965) i n o r d e r t h a t contaminants coated was  used  be moved to the top.  as the o r i g i n .  solvent.  The  end o f the p l a t e t h a t was  last  F i f t e e n minutes were allowed f o r e v a p o r a t i o n  of  excess  The p l a t e s were then p l a c e d i n a d r y i n g r a c k and  in  a w e l l v e n t i l a t e d oven at 120° f o r 1 h r .  The door of the oven  activated  was  i n t e r m i t t e n t l y opened u n t i l a c e t i c a c i d vapours c o u l d no l o n g e r be  detected.  Development of P l a t e s The  chromatograms were developed  u s i n g the h o r i z o n t a l  A Gelman development chamber which i s designed sheets of adsorbents  and g l a s s f i b r e paper,  t h i s t e c h n i q u e and was s t e e l used  t h e r e f o r e used.  to h o l d the p r e - p r e p a r e d  f o r use w i t h  fulfilled  The  technique.  pre-prepared  the requirements  t h r e e narrow sheets of  for  stainless  chromatograms i n p l a c e by means of a  magnet were removed t o f a c i l i t a t e proper c l o s u r e of the tank when the t h i c k e r g l a s s p l a t e s were used. the tanks were r e p l a c e d .  A wick p e r m i t t i n g t r a n s f e r of s o l v e n t from  s o l v e n t trough t o the adsorbent Whatman No.  The p i n s which f a s t e n e d these sheets to  was  made as f o l l o w s :  1 f i l t e r paper of dimension  22.0  x 3.5  cm.  Two  p i e c e s of  were cut and sewn  t o g e t h e r a c r o s s t h e i r e n t i r e l e n g t h w i t h c o t t o n t h r e a d at 0.5, from one o f the l o n g edges.  The paper was  the  1.5,  and 2.0  then f o l d e d at r i g h t angles at  cm.  0.9  cm.  x 0.3  from the o t h e r  cm.  was  long edge.  A p i e c e of g l a s s measuring 22.0  then s e c u r e l y a t t a c h e d  with  of the s e c t i o n of paper measuring 22.0 by going  through and  thread  x 0.9  to the o u t s i d e  cm.  The  done without g i v i n g r i s e t o w r i n k l e s  when t h i s a t t a c h e d  on a f l a t  t h e i r r e l a t i o n to one o f the wick was  surface.  The  o b j e c t which r a i s e d i t 5.0  T h i s was  cm.  dimension 1.0  x 0.3  2.0  The cm.  the wick and  cm.  were p l a c e d  then covered w i t h  the g l a s s s t r i p . silica  g e l and  8.0  cm.)  placed  was  f i l l e d with  in position.  disturbance  and  3.3.  and  efficiency  made at p o i n t s  centered  on the  of  a second g l a s s p l a t e 22.5 and g l a s s s t r i p  of  x 19.75  from the c e n t r a l one o f s o l v e n t and  outer  by 18.0  cm.  0.4  between as  trough o f (other  the  by  the assembled chromatogram  of the p l a t e s or adsorbent m a i n l y on account of the  d i f f e r e n c e i n width of the c a r r i e r and  x  protruded  r e s u l t e d i n even t r a n s f e r of s o l v e n t  90 ml.  the  T h i s arrangement maximized c o n t a c t  The  lower  of  T h i s assembly c o u l d be handled f r e e l y without  the same width a s h i f t  was  the  The  i n the upper c o r n e r s  i n d i c a t e d by the u n i f o r m i t y of the s o l v e n t f r o n t . chamber which i s separated  s e c t i o n of  spacers made from g l a s s and  cover p l a t e r e s t i n g on the spacers past  This  a r e s u l t o f poor c o n t a c t  or more, the wick was  At t h i s p o i n t two  cm.  a  On p l a c i n g the c a r r i e r p l a t e on a s u i t a b l e  edge o f the p l a t e . x 1.0  made  b a s i c p a r t s of t h i s apparatus  d r a s t i c a l l y reduced when attachment was  the adsorbent.  p l a t e which was  from i t s s h o r t edges.  another are shown i n F i g s . 3.2  making c o n t a c t w i t h the adsorbent. the wick w i t h  surface  around the paper at the edges of the g l a s s w i t h cm.  placed  1.2  attachment was  threaded needle at a d i s t a n c e of 0.5  wick was  x  cover p l a t e s .  causing  slight  When both were of  i n p o s i t i o n of the cover p l a t e u s u a l l y  occurred  F i g . 3.2.  Chamber u t i l i z e d f o r development of t h i n l a y e r chromatogram  F i g . 3.3.  T h i n l a y e r chromatogram f o r development  assembled and ready  54 upon l o w e r i n g the chromatoplate i n t o the tank. r e l e a s i n g the f i n g e r s and tank.  T h i s o c c u r r e d when  i n the narrow space between the edges o f the p l a t e  The use of t h i c k e r cover p l a t e s rendered p r o p e r c l o s i n g o f the  tank i m p o s s i b l e on account o f c o n t a c t between the tank's cover and cover plate.  The tank was  then covered a f t e r  chromatogram allowed s u f f i c i e n t t e r m i n a t i o n o f the development  i t was  lowered i n p o s i t i o n and  time to develop at room temperature. the assembly was  the Upon  removed and the cover  p l a t e removed w h i l e u s i n g the index f i n g e r s to p r e s s downwards s l i g h t l y the  ends o f the g l a s s s t r i p s .  T h i s m a n i p u l a t i o n w i t h the f i n g e r s  o b l i g a t o r y when f u r t h e r development to  solvents  Disregard f o r i t led  o f the cover p l a t e to the g l a s s s t r i p was  Adhesion o f the wick t o the adsorbent was  s a t u r a t i o n o f the s i l i c a g e l w i t h s o l v e n t .  wick the excess s o l v e n t was  left  intact.  due  also  P r i o r t o the removal o f the  t h e r e f o r e a l l o w e d to evaporate  3 minutes) from the wick and o r i g i n . was  due to evaporated  (due to heat o f a d s o r p t i o n ) condensing on the g l a s s and c r e e p i n g  between t h e i r s u r f a c e s .  ent  to f o l l o w .  was  the wick and some adsorbent accompanying the c o v e r p l a t e on i t s removal.  The adherence  to  was  on  (time - approx.  In so doing the s u r f a c e of the adsorb-  When these p r e c a u t i o n s a g a i n s t damage were taken  s e v e r a l r e p e a t e d developments  were made p o s s i b l e .  Development w i t h Iodine A l a r g e aluminum cooker 10% inches deep and 11% inches wide utilized.  was  In i t were arranged two columns o f b r i c k s on which was p l a c e d  a w i r e basket measuring 4% on i t s f l o o r .  i n s . x 5% i n s .  The cooker was  and heated, f o r a few minutes.  A s m a l l m e t a l can was  also placed  then p l a c e d on a hot p l a t e i n a fume hood A few c r y s t a l s o f i o d i n e were then added  55 to the c a n and t h e p l a t e p l a c e d on t h e wire b a s k e t . covered w i t h  a sheet  The cooker was then  o f g l a s s 15 i n s . x 15 i n s . through which c o l o r  development was viewed.  A n a l y s i s o f Sample  Solvents All used:  s o l v e n t s used were g l a s s d i s t i l l e d .  chloroform,  benzene, i s o o c t a n e ,  methanol, e t h a n o l ,  The f o l l o w i n g were  isopropyl alcohol, ethyl  acetate,  p e t r o l e u m e t h e r , a c e t i c a c i d , acetone, p y r i d i n e and  n-heptane.  Reagents i.  57o HC1 i n Superdry Methanol t o the procedure o f S t o f f e l et al_. (1959).  T h i s was made a c c o r d i n g The  reagent was d i s c a r d e d  a f t e r 1 month o f storage  at l e a s t 2 months has been r e p o r t e d ii.  (Kishimoto  although a s h e l f l i f e of  and Radin,  1965).  T r i m e t h y l s i l y l a t i o n M i x t u r e (TMS) T h i s was a commercial p r e p a r a t i o n  Laboratories  i n boxes o f 10 s e a l e d  1 m l . ampoules.  anhydrous p y r i d i n e , h e x a m e t h y l d i s i l a z a n e p r o p o r t i o n 9:3:1.  s u p p l i e d by A p p l i e d Each ampoule  contained  and t r i m e t h y l c h l o r o s i l a n e i n the  B i l e acids react q u a n t i t a t i v e l y with  form t h e t r i m e t h y l s i l y l e t h e r s .  Science  t h i s reagent t o  The r e a c t i o n i s q u a n t i t a t i v e w i t h  respect  to both keto and h y d r o x y l  groups.  p l a c e v i a the e n o l form.  The r e a c t i o n proceeds r e a d i l y a t room temperature  and  With the former i t i s b e l i e v e d t o take  i s r e p o r t e d l y complete i n 10 minutes  (Makita  and W e l l s ,  recommended r e a c t i o n p e r i o d o f 1 h r . was used i n the p r e s e n t  1963).  The  series of  56 experiments.  The  d e r i v a t i v e s , once formed may  d e t e r i o r a t e r a p i d l y upon  s t a n d i n g owing to a b s o r p t i o n of m o i s t u r e from the atmosphere. t h e r e f o r e used w i t h i n a few hours of t h e i r  Evaporation  of  They were  formation.  Solvents  For evaporation  o f s o l v e n t s under reduced p r e s s u r e , the vacuum  d i s t i l l a t i o n apparatus of F i g . 3.4  was  designed.  I t was  c o n s t r u c t e d by  Vancouver S c i e n t i f i c Glass Blowing Company.  For evaporation  pressure  When a s i n g l e sample was  the m a n i f o l d  o f F i g . 3.5  was  used.  t r e a t e d the f l o w of n i t r o g e n through the remaining use  of p i n c h  tubes was  the  at atmospheric being  a r r e s t e d by  clamps.  A n a l y t i c a l Procedure The method of Grundy et_ al_. (1965) r e q u i r e s as a f i r s t direct  p r i o r e x t r a c t i o n of the b i l e a c i d s .  that with chicken subjected  T h i s was  due  f e c e s had  to severe  s a p o n i f i c a t i o n under p r e s s u r e . i n a Soxhlet  I t soon became e v i d e n t  f e c e s an e x t r a c t i o n step i s to be p r e f e r r e d as glassware  to use w i t h u n e x t r a c t e d  determinations.  a usable  life  of o n l y  e t c h i n g o c c u r r i n g as a r e s u l t  Samples of 800 mg.  round bottom f l a s k .  bacteria (Sjovall,  1963).  The  under a stream of n i t r o g e n and described.  The  e x t r a c t was  s o l v e n t was  using  T h i s treatment  then evaporated at  reduced p r e s s u r e  then s u b j e c t e d  of  were t h e r e f o r e e x t r a c t e d  apparatus f o r 48 hours w i t h c h l o r o f o r m methanol 1:1  of s o l v e n t i n a 250 ml.  1-2  has  been found to g i v e complete r e c o v e r y of b i l e a c i d s i n c l u s i v e of those to  the  s a p o n i f i c a t i o n of m i l l i g r a m amounts of human f e c e s i n c e n t r i f u g e  b o t t l e s without  160 ml.  step,  u s i n g the  bound  50°  apparatus  to m i l d s a p o n i f i c a t i o n , a  new  a n a l y t i c a l f e a t u r e i n b i l e a c i d a n a l y s i s i n t r o d u c e d by Grundy et a l . (1965).  F i g . 3.4.  Apparatus f o r e v a p o r a t i o n o f s o l v e n t s a t reduced p r e s s u r e under n i t r o g e n  U1  F i g . 3.5.  M a n i f o l d and c o n n e c t i o n s u t i l i z e d i n the e v a p o r a t i o n of s o l v e n t s under n i t r o g e n at atmospheric p r e s s u r e .  00  59 Twenty ml.  o f N-NaOH i n 907. e t h a n o l were added and the m i x t u r e was r e f l u x e d  on a sandbath f o r 1 hour.  Upon c o o l i n g 10 m l . o f water were added and the  n e u t r a l s t e r o l s e x t r a c t e d w i t h 4 x 50 ml. E x t r a c t i o n was performed by v i g o r o u s i n s t e a d o f shaking,  f o r 1 minute.  portions o f petroleum  ether.  s w i r l i n g o f the f l a s k h e l d a t an angle  Although only 3 e x t r a c t i o n s are s p e c i f i e d  i n t h e procedure a f o u r t h was i n c l u d e d s i n c e some d i e t s c o n t a i n e d beta s i t o s t e r o l . c o n t a i n i n g 10.  Each e x t r a c t was siphoned  i n t o 500 ml.  m l . o f N NaOH i n 507. e t h a n o l .  added  separatory  funnels  E n t r a i n e d b i l e a c i d s were  backwashed i n t o the a l k a l i phase and added t o t h e roundbottom f l a s k .  The  m i l d s a p o n i f i c a t i o n step made p o s s i b l e the complete e x t r a c t i o n o f n e u t r a l s t e r o l s from a 60% e t h a n o l i c s o l u t i o n .  An i n c r e a s e i n a l k a l i n i t y o r change  i n e t h a n o l c o n c e n t r a t i o n has been shown by Grundy e_t al_. (1965) t o s e v e r e l y impair the e x t r a c t i o n o f n e u t r a l s t e r o l s w i t h p e t r o l e u m e t h e r . combined a l k a l i n e phases 2.0 ml. were added.  o f a 10 N s o l u t i o n o f sodium  The f l a s k s were then covered  To the hydroxide  w i t h 50 ml. beakers and t h e  m i x t u r e s a p o n i f i e d at 15 p . s . i . and 120° f o r 3 hours as d i r e c t e d . S a p o n i f i c a t i o n was c a r r i e d out i n a 16 l i t e r p r e s s u r e o f which was maintained  through t h e use o f an a d j u s t a b l e screw clamp p l a c e d  on t h e rubber t u b i n g o f a bunsen b u r n e r .  Following  m i x t u r e was c o o l e d and a c i d i f i e d w i t h c o n c e n t r a t e d of 2.0, as r e q u i r e d i n the procedure.  s a p o n i f i c a t i o n , the HC1 t o pH 1.0, i n s t e a d  A c i d i f i c a t i o n t o pH 1.0 was found  to improve t h e r e p r o d u c i b i l i t y o f the a n a l y s i s .  The a c i d i f i e d m i x t u r e was  then e x t r a c t e d as b e f o r e w i t h 75 m l . o f e t h y l a c e t a t e . siphoned water.  i n t o a 500 ml.  cooker, the temperature  separatory  E x t r a c t i o n was repeated  The e x t r a c t was  f u n n e l c o n t a i n i n g 10 ml.  w i t h 2 x 60 ml.  of d i s t i l l e d  portions of solvent.  The  60 combined e x t r a c t s were washed by i n v e r t i n g the s e p a r a t o r y f u n n e l and swirling of  it.  When the e x t r a c t was  the o r g a n i c phase was  ventionally.  The  c o n s i d e r a b l y l e s s than when i t was  aqueous phase was  r e p e a t e d twice more.  washed i n t h i s manner, e m u l s i f i c a t i o n  d i s c a r d e d and the washing  A white p r e c i p i t a t e was  aqueous wash i n amounts which decreased p r e c i p i t a t e d i d not appear  washed conprocedure  observed t o form i n the  i n each s u c c e s s i v e wash.  The  i n the f o u r t h wash and f o r t h i s reason t h r e e  washings were c o n s i d e r e d to be adequate.  T h i s method of e x t r a c t i o n  gave r e s u l t s s i m i l a r to those o b t a i n e d w i t h the method of Grundy £t a l . (1965).  In the l a t t e r method, the b i l e a c i d s are e x t r a c t e d from the  a c i d i f i e d mixture contained i n a c e n t r i f u g e b o t t l e , f i r s t with  a'mixture  of  The  are  c h l o r o f o r m and methanol  (2:1), then twice w i t h c h l o r o f o r m .  extracts  combined i n t o a round bottom f l a s k and then evaporated to d r y n e s s .  The r e s i d u e i s then d i s s o l v e d water.  The  i n e t h y l a c e t a t e and washed w i t h  distilled  aqueous phase i s next t r a n s f e r r e d by p i p e t t e t o a 40 ml.  c e n t r i f u g e tube where i t i s backwashed w i t h e t h y l a c e t a t e .  The  scheme o f  e x t r a c t i o n f o l l o w e d here e l i m i n a t e d long p e r i o d s o f w a i t i n g (sometimes o v e r n i g h t ) r e q u i r e d f o r phase s e p a r a t i o n to o c c u r when the above s o l v e n t s were used.  A l t h o u g h t h i s problem c o u l d have been r e a d i l y s o l v e d by  centrifugation, f a c i l i t i e s  f o r a c c o m p l i s h i n g t h i s u s i n g b o t t l e s o f 250  c a p a c i t y were not then a v a i l a b l e .  E x t r a c t i o n of the b i l e a c i d s from the  a c i d i f i e d aqueous phase d i r e c t l y w i t h e t h y l a c e t a t e proved s u i t a b l e , as the l a y e r s s e p a r a t e d out almost The washed e x t r a c t was evaporated to d r y n e s s .  ml.  immediately  extremely  following mixing.  then added to a 250 ml. round bottom f l a s k R e s i d u a l water was  and  removed by adding 10 ml. o f  acetone 5% HC1  and e v a p o r a t i n g to d r y n e s s .  To the r e s i d u e was  i n s u p e r d r y methanol and the f l a s k f i t t e d w i t h a r e d u c i n g adapter  ( Q u i c k f i t B24/29 t o B12/26) and a m i c r o e s t e r i f i c a t i o n E s t e r i f i c a t i o n was  performed  (1965) i . e . 100° f o r 2 hours. equipped  condenser.  under c o n d i t i o n s p r e s c r i b e d by Grundy e_t a l . An o i l bath was  used.  Each condenser  was  w i t h a m o i s t u r e t r a p and a l l c o n n e c t i o n s were of ground g l a s s .  F o l l o w i n g e s t e r i f i c a t i o n the methanol was until  added 5 ml. o f  the s m e l l o f HC1  evaporated  and e v a p o r a t i o n c o n t i n u e d  c o u l d no l o n g e r be d e t e c t e d i n the f l a s k .  e s t e r s were then q u a n t i t a t i v e l y t r a n s f e r r e d  to 40 ml. c e n t r i f u g e  w i t h 4 x 10 ml. p o r t i o n s o f c h l o r o f o r m u s i n g a 10 ml. p i p e t t e . p o r t i o n o f c h l o r o f o r m was  evaporated  The tubes Each 10 ml.  at 60° under a stream o f n i t r o g e n .  F o r m u l t i p l e samples the m a n i f o l d d e s c r i b e d was  used.  A f t e r the  last  t r a n s f e r and e v a p o r a t i o n , the w a l l s o f the tube were t h o r o u g h l y washed down w i t h 4.0  ml. o f c h l o r o f o r m methanol 1:1.  The  s o l v e n t s were evaporated  under a slow stream of n i t r o g e n .  TLC of Mixed Methyl E s t e r s and GLC Methyl E s t e r s The methyl methanol 4:1  e s t e r s were d i s s o l v e d  and a p p l i e d dropwise  edge o f the p l a t e .  of D e r i v a t i v e s o f the B i l e  T r a n s f e r was  cm.  from i t s t i p .  by  cm.  from the  lower  e f f e c t e d w i t h the use o f a 1.0 ml. s y r i n g e at an angle o f a p p r o x i m a t e l y  Complete t r a n s f e r was  c e n t r i f u g e tube w i t h s e p a r a t e 0.4, of s o l v e n t .  ml. o f c h l o r o f o r m  to form 3 bands 1.5  f i t t e d with a long 26 gauge n e e d l e , bent 0.5  i n 0.5  Acid  0.3  60°  ensured by r i s i n g o f the  and 0.3 ml. p o r t i o n s r e s p e c t i v e l y  During the t r a n s f e r p r o c e s s the s y r i n g e i t s e l f was  also  rinsed  i n v e r s i o n and movement of the p l u n g e r upwards and downwards a number of  62 times.  The  bands were d i s c o n t i n u e d  of each l a n e .  To  one  and methyl c h e l a t e  at a p p r o x i m a t e l y 0.5  o f the o u t e r lanes  i n c h l o r o f o r m was  120  ug.  cm.  from the  each o f methyl l i t h o c h o l a t e  a p p l i e d a l s o as a band 1 cm. i n o r d e r t h a t the  evaporate, a f t e r which the p l a t e was  developed i n benzene a c c o r d i n g  Following  development, the  to evaporate at room temperature and vapor as d e s c r i b e d .  The  the p l a t e s  solvent  wide.  A few minutes were allowed to elapse  Grundy e t al.. (1965).  exposed b r i e f l y to  as r e p o r t e d  i n p y r i d i n e and conditions  a 15 ml.  subjected  and  to a s i n g l e peak on the gas  longer  chromatography.  as a l r e a d y  chromatograph.  i t was  than 5 oc - c h o l e s t a n e and  0.50.  The  described.  o r i g i n a t e d from the  l a y e r chromatography i n benzene i t was It i s u n l i k e l y that  with  dissolved  gas  chromatographic  The  sample gave  rise of  a r e t e n t i o n time  zone between the f a t t y  (R^ <^ 0.1)  as not  a neutral  e x t r a c t i o n of the n e u t r a l s t e r o l s w i t h 50 ml.  during  being a b i l e  s t e r o l since  An  attempt was  determine whether t h i s compound possessed a h y d r o x y l group..  thin  acid  further  of p e t r o l e u m ether was  i n t e n s i t y of s t a i n i n g of the band.  e l u t i o n from the p l a t e 2 x 12 ml.  eluted  Nonetheless, because of i t s  dismissed  i t was  large  Upon  I t s a t i s f i e d the c r i t e r i a  much g r e a t e r m o b i l i t y compared to m e t h y l l i t h o c h o l a t e  e f f e c t on the  I t was  a b i l e a c i d i . e . i t had  a c i d methyl e s t e r band and methyl c h o l a t e .  methyl .ester.  iodine  a f a i n t c o l o r with  a l i q u o t evaporated to d r y n e s s ,  to gas  were i s o t h e r m a l  Grundy et_ al_. (1965) t h a t  allowed  by Grundy et a l . (1965).  seen behind t h i s l a r g e band at R^  o f methanol and  to  excess benzene was  p r o l o n g e d exposure however, a band which produced o n l y  25 ml.  would  f a t t y a c i d methyl e s t e r s appeared as a s i n g l e  band at a p p r o x i m a t e l y R j 0.65  i o d i n e c o u l d be  edges  without  made to  Following  a l i q u o t s were prepared f o r GLC  as  follows:  63 F i v e ml. ug.  o f a s o l u t i o n o f 5 °<- - c h o l e s t a n e  per ml.  tube.  The  were added to each a l i q u o t c o n t a i n e d a l i q u o t s were evaporated to dryness.  200  u l . of p y r i d i n e and  The  l a t t e r tube was  hour.  At the end  i n p y r i d i n e was  t i g h t l y stoppered  o f t h i s time a 2.0  subjected  sample exposed to the TMS  graph.  to gas  left  which may  be of no v a l u e  compound.  tube was  added  was  A f u r t h e r 2.0  then taken up  i n t o the s y r i n g e  obtained.  hydroxylated  the  and  chromato-  Thus evidence  reagent  again  u l . of  i n j e c t e d i n t o the gas  f u r n i s h e d w i t h the TMS  Firstly,  reagent.  Another sample was  1 u l . of a i r .  Whether t h i s compound was reasons.  centrifuge  at room temperature f o r 1  chromatography.  reagent  group c o u l d not be  however f o r two  To one  40  u l . sample of the compound d i s s o l v e d  i t s withdrawal from the t i p , was  l i q u i d phase.  may  and  In both i n s t a n c e s a s i n g l e peak was  hydroxyl  i n a 40 ml.  to the o t h e r a s i m i l a r q u a n t i t y o f TMS  drawn i n t o the s y r i n g e f o l l o w e d by  following  i n e t h y l acetate containing  for a  on an SE-30  is s t i l l  not known  SE-30 i s a n o n s e l e c t i v e l i q u i d  i n s e p a r a t i n g the d e r i v a t i v e from the  phase  unsubstituted  Secondly, the c o n d i t i o n s used f o r s i l y l a t i o n of t h i s compound  not have been i d e a l .  unesterified  That t h i s band c o u l d not have been due  f a t t y a c i d s was  to  borne out by the o b s e r v a t i o n t h a t i n c r e a s i n g  the amount o f e s t e r i f i c a t i o n reagent  by 507. d i d not  reduce or e l i m i n a t e i t .  In o r d e r to e f f e c t a sharp s e p a r a t i o n of the b i l e a c i d e s t e r s from the contaminating  pigments, a second development i n benzene was  carried  out.  P r i o r to the second development the p l a t e was  placed  and most of the benzene and  i o d i n e removed by  a p p l i c a t i o n of vacuum to  desiccator.  s u p p l i e d through use  R  £  The  v a l u e of 0.68  vacuum was was  obtained  i n a vacuum d e s i c c a t o r the  of a water a s p i r a t o r .  f o r the u n i d e n t i f i e d band and  0.84  f o r the  An  64 l a r g e f a t t y a c i d methyl e s t e r band.  the f a t t y a c i d e s t e r s as done by Grundy £t al.  i n s t e a d o f behind The  A l i n e was t h e r e f o r e drawn a t R^ 0.60  p l a t e was then developed t o t h i s l i n e w i t h the m i x t u r e ,  i s o p r o p y l a l c o h o l : a c e t i c a c i d , 120:40:1 i n o r d e r t o separate e s t e r s from the pigment e s t e r s . An  isooctane: the b i l e a c i d  I t was a g a i n exposed t o i o d i n e v a p o u r s .  improvement i n s e p a r a t i o n o f these  substances was  upon r e p e a t i n g the development i n t h e second s o l v e n t system. proceeding  (1965).  achieved Before  w i t h t h i s step most o f t h e s o l v e n t s were removed by a p p l i c a t i o n  o f vacuum f o r 15 minutes.  At the end o f the f i n a l development, the area  between the l i n e and methyl c h o l a t e was scraped a razor blade.  onto a sheet  o f paper u s i n g  The s i l i c a g e l was then t r a n s f e r r e d t o a g l a s s column  55 cm. x 1.0 cm. i n t o which a s o l v e n t e x t r a c t e d p i e c e o f c o t t o n was i n t r o d u c e d down t o the t i p . advantage o f r e d u c i n g  The use o f a column o f t h i s l e n g t h had the  l o s s o f the g e l as a t h i c k dust d u r i n g t r a n s f e r - -  a problem i n v a r i a b l y encountered d u r i n g t r a n s f e r , except when a zone e x t r a c t o r i s used. gel  w i t h the a i d o f a long-stemmed f u n n e l w h i l e h o l d i n g the column a t an  angle  such t h a t the adsorbent r a n down the s i d e s o f the column i n s t e a d o f  falling. The  The l o s s e s c o u l d be f u r t h e r reduced by i n t r o d u c i n g the  Gentle  tapping during  t r a n s f e r expedited  e s t e r s vrere then e l u t e d w i t h approximately  25 ml. v o l u m e t r i c  flask.  3 l b . / s q . i n . of pressure tank o f n i t r o g e n . made up t o volume.  the process  appreciably.  25 ml. o f methanol i n t o a  The e l u t i o n was c a r r i e d out under  approximately  s u p p l i e d through a rubber t u b i n g connected t o a  The t i p o f the tube was then r i n s e d and the f l a s k s To a 40 ml. c e n t r i f u g e tube was added 15 m l . o f t h i s  e l u a n t and 200 ug. of 5<*- - c h o l e s t a n e  (5 ml. o f a 47. s o l u t i o n i n e t h y l  65 acetate).  The s o l v e n t s were evaporated under a stream o f n i t r o g e n .  the r e s i d u e was added 10 ml. o f n-heptane which was a l s o evaporated. a d d i t i o n and e v a p o r a t i o n o f n-heptane f a c i l i t a t e d  To The  the removal o f a c e t i c  a c i d which was e l u t e d from the adsorbent and which proved d i f f i c u l t t o evaporate w i t h t h e low b o i l i n g s o l v e n t s .  I t s accumulation was the r e s u l t  of an i n c r e a s e i n the impregnation o f the adsorbent upon r e p e a t i n g development w i t h the s o l v e n t m i x t u r e c o n t a i n i n g a c e t i c a c i d .  The b i l e a c i d  methyl  e s t e r s were then c o n c e n t r a t e d t o the bottom o f the tube as d e s c r i b e d previously. reagent.  To the almost pure b i l e a c i d e s t e r s was added 200 u l . o f TMS  To guarantee  complete m i x i n g o f the sample the reagent was added  i n the f o l l o w i n g manner. The  A 1.0 m l . p i p e t t e was f i l l e d  c e n t r i f u g e tube was h e l d at a sharp a n g l e .  approximating 1/3 the s i z e o f a normal of the p i p e t t e .  t o the 0.8 ml. mark.  An amount o f reagent  drop was allowed t o r e a c h the t i p  By making c o n t a c t o f t h i s d r o p l e t w i t h the w a l l s o f the  tube j u s t above the l e v e l o f the e s t e r s , a s m a l l amount o f reagent was made t o f l o w down t o the bottom o f the tube.  T h i s was r e p e a t e d w i t h  r o t a t i o n o f the tube i n such a manner t h a t t h e r e was s u c c e s s i v e o v e r l a p w i t h the l a s t  area washed.  The c o n t e n t s o f the tube were then mixed by  s w i r l i n g and the tube t i g h t l y stoppered and l e f t desiccator f o r 1 hr.  at room temperature  ina  A t the end o f t h i s time a 1.0 u l . sample was i n j e c t e d  i n t o the gas chromatograph u s i n g a Hamilton s y r i n g e o f 10 u l . c a p a c i t y . A f t e r each i n j e c t i o n , the s y r i n g e was r i n s e d w i t h p y r i d i n e .  Gas chromato-  g r a p h i c r e c o r d s made up to 2 hours f o l l o w i n g a d d i t i o n o f the reagent showed t h a t the r e a c t i o n was complete TMS reagent f a i l e d  a f t e r 1 hour.  I n c r e a s i n g the amount o f  t o a l t e r the r e l a t i v e amount o f each peak o r the peak  66 areas r e l a t i v e to 5o<. - c h o l e s t a n e demonstrating used.  Subsequent c a l c u l a t i o n s  the adequacy of the amount  i n d i c a t e d t h a t % t h i s amount o f TMS  would have been s u f f i c i e n t t o q u a n t i t a t i v e l y r e a c t w i t h the b i l e methyl  esters,  r e t a i n the use o f 200 u l . f o r GLC  acid  (Grundy et al_. s p e c i f i e d 100 ul./mg. o f e s t e r s ) but  o f the l a r g e s i z e o f the r e a c t i o n tube used The  i t was  reagent  i n view  c o n s i d e r e d j u d i c i o u s to  compounds p r e s e n t i n the sample p r e p a r e d  were t e s t e d f o r h y d r o x y l groups by t r i f l u o r o a c e t y l a t i o n f o l l o w e d  by gas chromatography.  T r i f l u o r o a c e t y l a t i o n was  by S j o v a l l  t r i f l u o r o a c e t y l a t i o n mixture was  (1964).  The  c a r r i e d out as d e s c r i b e d injected  directly  i n t o the gas chromatograph under c o n d i t i o n s s i m i l a r to those used f o r a n a l y s i s o f the TMS  e t h e r s w i t h the e x c e p t i o n t h a t the i n j e c t i o n p o r t and  d e t e c t o r were m a i n t a i n e d methylchenodeoxycholate  at 245° i n o r d e r to prevent decomposition derivatives.  The  thermal l i m i t o f s t a b i l i t y o f  these compounds i s r e p o r t e d to be 250° (Kuksis e t a l . , chromatographic  of  1964).  r e c o r d s o b t a i n e d are shown i n F i g s . 3.6  The  and 3.7.  gas They  show that, the major components at l e a s t , o f the m i x t u r e , r e a c t e d w i t h the reagent as i n d i c a t e d by the marked r e d u c t i o n i n r e t e n t i o n times o f the more volatile trifluoroacetates.  Poor r e s o l u t i o n prevented  i d e n t i f i c a t i o n o f a l l components but  i t may  a  comparative  be seen t h a t t h e r e was  a general  s h i f t o f the components i n c l u d i n g a l l major ones towards s h o r t e r r e t e n t i o n times. There  seems to be l i t t l e  doubt t h e r e f o r e , t h a t most, i f not a l l  peaks appearing on the gas chromatographic than 5 <X.-cholestane  represent b i l e  acids.  r e c o r d w i t h r e t e n t i o n times l o n g e r  c o a—> CO  a>  if)  Z  o Q.  U2  QJ Q  cr: o o QJ  T  15  25 RETENTION  F i g . 3.6.  35  30 TIME  70  (MINSJ  Gas chromatographic p a t t e r n o f the TMS e t h e r s o f t h e methyl e s t e r s o f the f e c a l b i l e acids of adult cockerels. Analytical conditions: 1.07o SE-30 on 100-120 mesh Gas Chromosorb Q; 6'6" x 1/4" s t a i n l e s s s t e e l column; column temperature, 218° f o r 5 mins. 218° - 240° a t l°/min.; i n j e c t i o n p o r t , 270°; d e t e c t o r , 260; n i t r o g e n , 80 c.c./min.  OJ  c o,  in'Q) '  O  RETENTION F i g . 3.7.  TIME  (MIN5.)  Gas chromatographic p a t t e r n o f the t r i f l u o r o a c e t a t e s of the methyl e s t e r s o f F i g . 3.6. The r e a c t i o n m i x t u r e was i n j e c t e d i n t o the gas chromatograph. Column and oven c o n d i t i o n s as g i v e n in, F i g . 3.6. I n j e c t i o n p o r t and d e t e c t o r temp., 245°.  oo  69 Calculations Peak areas were approximated as the product o f the peak h e i g h t and  the width  at h a l f h e i g h t .  on the c h a r t paper were counted (sq.  mm.)  u s i n g the r e l a t i o n :  F o r u n r e s o l v e d peaks the i n d i v i d u a l and converted  was  t o the u n i t o f measurement  100 u n i t s = 645 s q . mm.  t o t a l area o f peaks r e p r e s e n t i n g b i l e  squares  The r a t i o o f the  a c i d s t o t h a t o f 5 oL - c h o l e s t a n e  used to q u a n t i t a t e the b i l e a c i d s as a group.  The a c c u r a c y o f the data  o b t a i n e d i n t h i s manner i s based on the o b s e r v a t i o n t h a t the response hydrogen flame  o f the  i o n i z a t i o n d e t e c t o r t o t h e TMS d e r i v a t i v e i s i n d i r e c t  p r o p o r t i o n t o the a b s o l u t e weight o f the u n s u b s t i t u t e d compound (Makita and Wells,  1963).  S c a l i n g o f the Procedure  Tediousness chromatoplate  which c h a r a c t e r i z e d the t r a n s f e r o f e s t e r s t o the  made i t d e s i r a b l e t o make t h i s step i n the procedure  t e c h n i c a l l y simpler.  The procedure  amount o f f e c e s i n i t i a l l y of  used w i t h a v i e w t o sampling  t r a n s f e r r i n g them q u a n t i t a t i v e l y .  success.  was t h e r e f o r e a p p l i e d t o twice t h e  The f i r s t  the e s t e r s i n s t e a d  attempts met w i t h  limited  T h i s was due t o a v a r i a b l e l o s s o f the b i l e a c i d showing the  l o n g e s t r e t e n t i o n time on the gas chromatographic  record.  The cause o f  t h i s poor r e p r o d u c i b i l i t y was t r a c e d t o the e s t e r i f i c a t i o n s t e p and was reasoned  t o be due t o e i t h e r f a i l u r e to e s t e r i f y t h i s compound o r l o s s o f i t  following e s t e r i f i c a t i o n .  The r e s u l t s remained unchanged when new  e s t e r i f i c a t i o n reagent made w i t h the utmost care was used.  Evidence was  o b t a i n e d t h a t the l o s s was due t o p r e f e r e n t i a l e v a p o r a t i o n o f methanol  70 resulting  i n an i n c r e a s e i n the c o n c e n t r a t i o n o f HC1 d u r i n g  o f the HC1 methanol m i x t u r e . to  evaporation  these  evaporation  When the b u l k o f t h e HC1 was removed p r i o r  l o s s e s were no l o n g e r s u f f e r e d .  of t h e b a t h had f a l l e n t o 70°, 0.8 ml. m i x t u r e through t h e condenser.  When the temperature  o f p y r i d i n e was s l o w l y added t o t h e  The b a t h was then unplugged and e v a p o r a t i o n  begun under a slow stream o f n i t r o g e n .  P y r i d i n e h y d r o c h l o r i d e must have  been formed under these new c o n d i t i o n s because a t h i c k paste became e v i d e n t when e v a p o r a t i o n  was about 507. complete.  Evaporation  reached  completion  when the temperature o f t h e o i l bath had f a l l e n t o approximately t h i s time 5 ml. o f e t h y l a c e t a t e were added and evaporated. then removed from t h e b a t h and c o o l e d . the contents  60°.  At  The f l a s k was  U n l i k e when p y r i d i n e was not added  o f t h e f l a s k appeared as a s o l i d .  s o l i d m a t e r i a l was i n s o l u b l e i n e t h y l a c e t a t e .  A considerable part of this The methyl e s t e r s o f the  b i l e a c i d s were t h e r e f o r e t r a n s f e r r e d t o 40 ml. c e n t r i f u g e tubes u s i n g 4 x 10 m l . p o r t i o n s o f e t h y l a c e t a t e w i t h f i l t e r i n g c o n t a i n i n g about 0.5 grams o f s i l i c i c and  the " i n s o l u b l e m a t e r i a l avoided.  acid.  through a s h o r t column  A 10 ml. p i p e t t e was used  The use o f t h i s method o f f i l t e r i n g  c o u l d not have e n t a i l e d any l o s s o f e s t e r s as i t has a l r e a d y been shown t h a t e t h y l a c e t a t e r e a d i l y e l u t e s b i l e a c i d methyl e s t e r s from a c i d columns (Eneroth for  silicic  e t a l . , 1966).  a c i d was b r i e f l y  silicic  The use o f f l o r i s i l as a s u b s t i t u t e  investigated.  No a n a l y s i s was c a r r i e d out on  the f i l t r a t e but j u d g i n g from i t s c o l o r i t appeared t h a t f l o r i s i l would be o f g r e a t e r v a l u e than s i l i c i c  a c i d i n removing some o f t h e pigmented  contaminants p r i o r t o t h i n l a y e r chromatography. was  evaporated and t h e r e s i d u e c o n c e n t r a t e d  described e a r l i e r .  The e t h y l a c e t a t e  extract  t o the bottom o f the tube as  The tube was then c o o l e d and t h e sample d i s s o l v e d i n  71 e x a c t l y 1.5 ml. o f a m i x t u r e o f c h l o r o f o r m methanol 4:1.  An a l i q u o t o f  0.75 m l . was taken up by s y r i n g e and chromatographed on t h i n l a y e r as before.  Omission o f Column Chromatography Because o f the d i f f i c u l t y experienced  i n removing a c e t i c a c i d  from the b i l e a c i d e l u a t e w i t h the d i s t i l l a t i o n apparatus  constructed,  the column chromatography step which was i n c o r p o r a t e d by Grundy e_t a l . (1965) i n o r d e r t o remove the b u l k o f the f a t t y a c i d s and some o f t h e contaminating  pigments p r i o r t o TLC was o m i t t e d .  I t d i d n o t appear t h a t  p a r t i c u l a r b e n e f i t s were t o be d e r i v e d from adherence t o t h i s  step as  i n c r e a s i n g the adsorbent t h i c k n e s s o f the t h i n l a y e r p l a t e t o 0.700 mm. permitted  s a t i s f a c t o r y s e p a r a t i o n o f the b i l e a c i d methyl e s t e r s from the  o t h e r substances.  These p l a t e s showed no s i g n s o f o v e r l o a d .  Suggestions f o r F u t u r e  Analysis  F o r each s e t o f a n a l y s i s performed, i t was a n t i c i p a t e d t h a t the p e t r o l e u m e t h e r e x t r a c t s might be subsequently  examined.  In instances  where the n e u t r a l s t e r o l s a r e n o t t o be s t u d i e d i t would p r o b a b l y  be o f  g r e a t e r advantage t o remove them t o g e t h e r w i t h the f a t t y a c i d methyl e s t e r s f o l l o w i n g e s t e r i f i c a t i o n by use o f column chromatography on s i l i c i c florisil.  T h i s would u t i l i z e  the methyl e s t e r s .  t o g r e a t e r advantage the step used f o r f i l t e r i n g  There was some s u g g e s t i o n  t h a t t h i s step might a l s o  serve t o remove a l a r g e r f r a c t i o n o f t h e pigments than t h a t o b t a i n e d the f r e e a c i d s a r e chromatographed a c c o r d i n g et a l . (1965).  a c i d or  This suggestion  when  t o the procedure o f Grundy  a r i s e s out o f the o b s e r v a t i o n t h a t the  72 ethyl acetate  i n s o l u b l e m a t e r i a l obtained  f o l l o w i n g e s t e r i f i c a t i o n ranged  i n c o l o r through many shades o f brown i n d i c a t i n g t h a t at l e a s t some subs t a n c e s were d e r i v e d  from the pigments.  form are f r e e l y s o l u b l e i n e t h y l a c e t a t e  Since  the pigments i n t h e i r a c i d  i t appeared unusual t h a t the l e s s  p o l a r e s t e r s d i d not e x h i b i t the same o r a g r e a t e r solvent.  These c o n s i d e r a t i o n s  therefore  c h l o r i d e s were p r o b a b l y formed d u r i n g s u p e r d r y methanol.  solubility  in this  suggest t h a t some pigment hydro-  t h e i r e s t e r i f i c a t i o n using  I f , i n f a c t , these h y d r o c h l o r i d e s  t h e r e would have been a s e l e c t i v e i n c r e a s e  HC1 i n  were formed, then  i n the p o l a r i t y o f some o f the  pigments many o f which are a l r e a d y more p o l a r than the b i l e a c i d s . i n c r e a s e c o u l d have been s u f f i c i e n t  This  to r e s u l t i n the removal o f l a r g e r  amounts of pigments than r e a l i z e d when the f r e e a c i d s are chromatographed. The exact  amount which c o u l d be removed by column chromatography  which d e s e r v e s f u r t h e r s t u d y . e t h y l acetate acetate  is a  question  The low s o l u b i l i t y o f these compounds i n  i s a good i n d i c a t i o n t h a t s o l v e n t m i x t u r e s c o n t a i n i n g  would serve w e l l , the f u n c t i o n o f s e p a r a t i n g  ethyl  the b i l e a c i d e s t e r s  from the contaminants, n e u t r a l s t e r o l s and normal f a t t y a c i d e s t e r s .  73  EXPERIMENT 3  THE  EFFECT OF DIETARY TRIGLYCERIDES OF VARYING DEGREE OF ON  THE  FECAL OUTPUT OF BILE ACIDS AND  THE  UNSATURATION  LEVEL  OF  CIRCULATING STEROLS IN MATURE COCKERELS  Review of L i t e r a t u r e  The  study o f the e f f e c t i v e n e s s of d i e t a r y f a t i n e l i c i t i n g  cholesterolemic the  response i n man  i n t e r e s t of many workers.  o f f a t s i t i s now Ahrens et al.,  and Due  e x p e r i m e n t a l animals has to a c t i v e r e s e a r c h  g e n e r a l l y agreed t h a t i n man,  1954;  Kinsell,  1954;  Beveridge et a l . , 1956)  Gopalan et al_., 1960;  (Rowsell  r a b b i t s , (Lambert et a l . , 1958;  Gresham, 1962) 1967)  dogs (Grande, 1960,  the a d d i t i o n o f s a t u r a t e d  results  1962)  and  gerbil  property 1952;  swine,  Wigand,  1959;  (Hegsted and  added f a t i s u n s a t u r a t e d .  The  as opposed to a  r e s u l t s of experiments  i l l u s t r a t e , Weiss and F i s h e r  (1957) and  (1958) showed t h a t a d i e t supplemented w i t h s a t u r a t e d plasma c h o l e s t e r o l l e v e l of the L e v e i l l e and  Sauberlich  of u n s a t u r a t i o n  l a y i n g hen  using  in this  the e f f e c t of d i e t a r y f a t i s analagous to t h a t observed i n those To  Gallagher,  f a t s to a l o w - s t e r o l or s t e r o l - f r e e d i e t  the domestic fowl do not however i n d i c a t e w i t h c e r t a i n t y t h a t  mentioned.  engaged  monkeys,  Jaganathan, 1962)  i n a r i s e i n plasma c h o l e s t e r o l c o n c e n t r a t i o n  f a l l when the  into this  (Groen et a l . ,  (Portman et a l . , 1956; et a l . , 1965)  long  a  species  species  L e v e i l l e arid F i s h e r f a t elevates  the  but Adamson et_ a l . (1961) and  (1963) p r e s e n t e d r e s u l t s i n d i c a t i n g t h a t the  degree  of the d i e t a r y f a t i s without i n f l u e n c e on the plasma  c h o l e s t e r o l l e v e l of the growing c o c k e r e l .  The  r e s u l t s of W h i t e s i d e et a l . ,  74 (1965) showed t h a t the plasma c h o l e s t e r o l l e v e l of c o c k e r e l s supplemented w i t h coconut o i l from 1 - 6 greater  than those f e d d i e t s c o n t a i n i n g  weeks of age corn o i l .  was  fed d i e t s  significantly  A search  of  the  l i t e r a t u r e r e v e a l s t h a t s i m i l a r experiments have not been undertaken u s i n g mature c o c k e r e l s .  That such experiments would be o f i n t e r e s t , d e r i v e s  o n l y from t h e i r absence from the l i t e r a t u r e but the e a r l y p e r i o d o f l i f e  of the c o c k e r e l  not  a l s o from the knowledge t h a t  ( d u r i n g which many experiments  c h o l e s t e r o l metabolism have been conducted) i s p r o b a b l y an  on  inappropriate  time to study the e f f e c t of d i e t a r y f a c t o r s on c h o l e s t e r o l metabolism because of the c a r r y over of c h o l e s t e r o l from the (1940) have shown t h a t the c o n c e n t r a t i o n hatched c h i c k i s of the o r d e r t h i s l e v e l to the l a y i n g hen  adult  of 300  Entenman ejt a l .  of plasma c h o l e s t e r o l of the newly  mg.7.  and  l e v e l of 110 mg.7.  a l s o does not  egg.  undergoes a change from  over a p e r i o d of 36 days.  appear s u i t e d to the  b o l i s m when hormonal f a c t o r s are not b e i n g  The  study of c h o l e s t e r o l meta-  i n v e s t i g a t e d because the  estrogens  have added p r o b a b l y too l a r g e a hormonal component to the normal l e v e l  of  circulating sterols.  I t would thus appear t h a t f u r t h e r e x p e r i m e n t a t i o n  using  i s the a p p r o p r i a t e  adult cockerels  step toward an u n d e r s t a n d i n g  the plasma c h o l e s t e r o l r e g u l a t i n g p r o p e r t y In view of the numerous r e p o r t s lowering  of d i e t a r y f a t i n t h i s  and Friedman, 1958;  acids  (Gordon ejt a l . , 1957a, 1957b; Lewis, 1957;  Haust and  and  the s u g g e s t i o n  may  a f f e c t the a b s o r p t i o n  i n v e s t i g a t e the  made by  species.  a t t r i b u t i n g the c h o l e s t e r o l  e f f e c t of u n s a t u r a t e d f a t s to t h e i r a b i l i t y to a c c e l e r a t e  e x c r e t i o n of b i l e  of  Beveridge, 1958;  Goldsmith et a l . ,  the Byers 1960)  the r e s u l t s of experiment 2 t h a t d i e t a r y f a c t o r s of b i l e s a l t s  i t was  considered  of i n t e r e s t to  i n t e r r e l a t i o n s h i p between the degree of u n s a t u r a t i o n  of  75 d i e t a r y f a t , t h e plasma s t e r o l i n the adult  l e v e l and the r a t e of b i l e a c i d  excretion  cockerel.  Experimental  T h i r t y - s i x a d u l t S i n g l e Comb White Leghorn c o c k e r e l s reared  on l i t t e r were randomly s e l e c t e d and a s s i g n e d t o t h r e e  c o n s i s t i n g o f 12 i n d i v i d u a l cages w i t h w i r e s c r e e n f l o o r s . then vermifuged w i t h a s i n g l e P r a t t s v e g e t a b l e r a t i o n o f T a b l e 3.1. p r o t e i n and was s u p p l i e d  A l l b i r d s were  " S p l i t A c t i o n " capsule,  ad l i b i t u m w i t h water.  were f e d l a t e r i n the experiment. i n t o 4 subgroups o f 3.  b a t t e r i e s each  and f e d the a l l  T h i s r a t i o n was formulated t o c o n t a i n 13.07=,  a s s i g n e d a treatment which c o n s i s t e d  divided  which had been  of three  Each b a t t e r y was then randomly  d i f f e r e n t h i g h - f a t d i e t s which  The b i r d s from each treatment were then  The r e l a t i v e p o s i t i o n o f b i r d s b e l o n g i n g t o  the same subgroup was s i m i l a r on a l l 3 l e v e l s o f a g i v e n b a t t e r y arrangement).  T h i s arrangement e l i m i n a t e d  have proven u s e f u l i n p r e v e n t i n g collection. period  A t the end o f the f i r s t  their  and second week o f the e x p e r i m e n t a l  the b i r d s were weighed and samples of blood taken f o r d e t e r m i n a t i o n Blood was drawn from t h e wing  o f i n d i v i d u a l b i r d s i n t o 5.0 m l . t e s t tubes c o n t a i n i n g  a s o l u t i o n o f 0.17c, h e p a r i n i n experiment 2.  0.3 m l . o f  i n 0.97o sodium c h l o r i d e and t r e a t e d  as d e s c r i b e d  The plasma from b i r d s w i t h i n each subgroup was then  pooled t o g i v e 4 samples per t r e a t m e n t . determined a l s o as i n experiment 2. the  the need f o r b l a n k t r a y s which  cross-contamination o f feces during  o f d i g i t o n i n p r e c i p i t a b l e s t e r o l s (DPS). vein,  (vertical  The s t e r o l s were e x t r a c t e d and  The c o n c e n t r a t i o n  plasma a t t h e end o f the two c o n s e c u t i v e  periods  as the c o n t r o l l e v e l o f t h e e x p e r i m e n t a l b i r d s .  o f s t e r o l s found i n  was averaged and used  F o r 48 hours  following  TABLE 3.1.  Composition o f b a s a l  d i e t fed  i n experiment 3  7. o f d i e t  Ingredients Dextrose  15.00  Ground wheat  71.65  Soyabean meal (457. p r o t e i n )  8.85  Dried d i s t i l l e r s '  2.00  solubles  Bone meal  2.00  Iodized  0.50  salt  mg./kgm. o f d i e t Manganese  sulfate  Menadione  110.00 0.50  units/kgm. o f d i e t V i t a m i n A (I.U.) V i t a m i n Do (I.C.U.)  4400.00 199.76  the second  s e t of d e t e r m i n a t i o n s , f e c e s were c o l l e c t e d on sheets o f  p o l y e t h y l e n e and p o o l e d on the same b a s i s as the plasma.  The  f e c e s were  t r a n s f e r r e d d a i l y from these sheets t o l a r g e aluminum d i s h e s and f r o z e n . A t the end o f the c o l l e c t i o n p e r i o d the d i e t o f T a b l e 3.1 oil,  h e r r i n g o i l and coconut  o i l were s e p a r a t e l y i n c o r p o r a t e d at a l e v e l  of 15.07. at the expense o f d e x t r o s e was p r e v i o u s l y mentioned.  The  coconut  f e d as the t h r e e  o i l was  p l a t e p r i o r to i t s a d d i t i o n to the d i e t . were removed by r u b b i n g the feed through d i e t was  to which c o r n  f i n a l l y o b t a i n e d by mixing  d r i v e n mixer o f the h o r i z o n t a l type.  treatments  l i q u e f i e d by warming on a hot The  lumps formed upon i t s a d d i t i o n  a sieve.  A homogenous mix  f o r 15 minutes i n an  of the  electrically  Corn o i l and h e r r i n g o i l which present  no problems when b e i n g added to b a s a l d i e t s were n e v e r t h e l e s s warmed to the same extent as the coconut  oil.  A l l b i r d s were c o n t i n u e d on  r e s p e c t i v e d i e t s f o r 2 weeks at the end of which time r e p e a t of (a) plasma s t e r o l c o n c e n t r a t i o n s (b) body weights  and  their  determinations  (c) f e c a l  output  were c a r r i e d out as b e f o r e . The  e n t i r e c o l l e c t i o n of f e c e s was  l y o p h i l i z e d to c o n s t a n t weight  and homogenized i n a Waring b l e n d o r under an atmosphere of n i t r o g e n . were then taken and s t o r e d under n i t r o g e n i n g l a s s j a r s .  These were used  f o r b i l e a c i d a n a l y s i s a f t e r f u r t h e r p o o l i n g to g i v e 1 sample per F u r t h e r p o o l i n g became n e c e s s a r y when i t was  r e a l i z e d t h a t the  f o r b i l e a c i d a n a l y s i s does not r e a d i l y l e n d i t s e l f to s e r i a l All  a n a l y s e s were c a r r i e d out  in duplicate.  Samples  treatment.  procedure analyses.  78 R e s u l t s and D i s c u s s i o n  The  c o n c e n t r a t i o n o f DPS found i n the plasma o f c o c k e r e l s when  the v a r i o u s d i e t s were f e d a r e summarized i n T a b l e t h a t f o r each i n t e r v a l o f the experimental  3.II.  These data show  p e r i o d the t e r m i n a l l e v e l s o f  DPS were dependent on the type o f d i e t which had been f e d .  Plasma DPS  remained unchanged i n c o n c e n t r a t i o n o r i n c r e a s e d when the b a s a l d i e t was exchanged f o r one e n r i c h e d  in fat.  T h i s exchange was  low-fat without  e f f e c t when a d d i t i o n t o the d i e t was made u s i n g c o r n o i l but promoted an i n c r e a s e o f d i f f e r i n g magnitude when made w i t h coconut o i l and h e r r i n g o i l . The substantial  i n c o r p o r a t i o n o f coconut o i l i n t o the b a s a l d i e t  i n c r e a s e o f 45.37. i n plasma DPS c o n c e n t r a t i o n .  l e d to a  An i n c r e a s e o f  9.27. was noted when the h e r r i n g o i l supplemented d i e t was f e d .  The  a d m i n i s t r a t i o n o f a f a t supplemented d i e t d u r i n g the second d i e t a r y p e r i o d was expected t o produce some i n c r e a s e i n the l e v e l o f plasma s t e r o l s .  This  e x p e c t a t i o n had as i t s b a s i s the o b s e r v a t i o n t h a t c h o l e s t e r o l l e v e l s are lower i n the plasma o f the r a t ( A l f i n S l a t e r et a_l., 1954a) and c h i c k e n (Dam fed.  e t al_., 1955) when a l o w - f a t compared t o a f a t supplemented d i e t i s The f a i l u r e o f c o r n o i l t o induce  s t e r o l s was t h e r e f o r e p r o b a b l y l e v e l i n the b a s a l  a change i n the l e v e l o f c i r c u l a t i n g  due t o the presence o f f a t above a c r i t i c a l  diet.  A s a t i s f a c t o r y assessment o f the s m a l l d i f f e r e n c e i n s t e r o l e m i c e f f e c t observed between c o r n and h e r r i n g o i l was h i n d e r e d samples r e s u l t i n g  i n only 4 observations  per treatment.  by p o o l i n g o f the The l o s s i n  v a r i a b i l i t y c o u l d have been compensated by the d e t e r m i n a t i o n  o f an  e q u i l i b r i u m range o f the plasma s t e r o l s over a p e r i o d o f time as was done  79  TABLE 3 . I I .  C o n c e n t r a t i o n (mg.7.) o f DPS i n plasma o f cockerels fed various experimental d i e t s  Diets Group number  Sub-group number  Fat Basal Week 1  1  Week 2  supplemented Corn o i l  Average  Week 4  1 2 3 4  86.1 98.2 78.4 91.8  84.2 103.2 75.1 89.6  85.2 100.7 76.8 90.7  85.9 99.6 74.3 96.1  Avg.  88.6  88.0  88.3  89.0  Herring o i l 2  1 2 3 4  92.3 80.5 87.6 80.7  86.6 83.2 94.9 80.8  89.5 81.9 91.3 80.8  99.6 95.8 102.7 77.7  Avg.  85.3  86.4  85.9  94.0 Coconut o i l  1  3  1 2 3 4  80.2 92.9 88.3 76.7  87.5 91.3 93.0 69.5  83.9 92.1 90.7 73.1  124.8 119.2 133.7 113.6  Avg.  84.5  85.3  84.9  122.8  80 on the b a s a l d i e t .  However, because o f the u n e q u i v o c a l d i f f e r e n c e found  between s a t u r a t e d and u n s a t u r a t e d f a t s i n the f i r s t  set of determinations  f o r the second d i e t a r y p e r i o d , such a range was not determined.  Should  t h e r e be i n f a c t , a s m a l l d i f f e r e n c e between the s t e r o l e m i c e f f e c t s o f c o r n and h e r r i n g o i l t h i s d i f f e r e n c e may be r a t i o n a l i z e d w i t h c o n s i d e r a t i o n of the s t e r o l c o n t e n t o f the f a t supplemented d i e t s .  The h e r r i n g o i l  supplemented d i e t would no doubt c o n t a i n some c h o l e s t e r o l whereas the c o r n oil  supplemented d i e t would be expected t o c o n t a i n some p h y t o s t e r o l s .  The  l a t t e r s t e r o l s which are themselves p o o r l y absorbed, would have reduced the endogenous a b s o r p t i o n o f c h o l e s t e r o l  ( P e t e r s o n , 1951) w i t h the r e s u l t a n t  e f f e c t o f l o w e r i n g the plasma s t e r o l l e v e l .  C h o l e s t e r o l , which i s r e a d i l y  absorbed i n the presence o f u n s a t u r a t e d f a t (Huang produces the o p p o s i t e e f f e c t on the plasma' s t e r o l  and K u k s i s , 1965) level.  These r e s u l t s show c l e a r l y t h a t i n the mature l o w - f a t d i e t , the a d d i t i o n o f s a t u r a t e d f a t t o the d i e t o f the plasma s t e r o l  cockerel fed a l e a d s t o an e l e v a t i o n  l e v e l whereas the a d d i t i o n o f u n s a t u r a t e d f a t i s  e s s e n t i a l l y without e f f e c t . Body weight d a t a which were o b t a i n e d a t times c o i n c i d i n g w i t h those when s t e r o l a n a l y s i s were made are p r e s e n t e d i n T a b l e 3 . I I I . fluctuations  Weekly  i n weight were observed on t h e b a s a l d i e t but these had minimal  e f f e c t s on the mean weight o f each group.  The s u b s t i t u t i o n o f f a t f o r  c a r b o h y d r a t e a f f e c t e d body weight f a v o u r a b l y when c o r n and coconut o i l were used.  The magnitude o f the r e s u l t a n t changes were 97 and 71 grams  respectively. supplemented  Body weight was u n a f f e c t e d by consumption o f the h e r r i n g o i l diet.  81  TABLE 3 . I I I .  Body weights (grams o f c o c k e r e l s f e d various experimental d i e t s  Diets Group number  Sub-group number  Bird number  F a t supplemented Corn o i l  Basal Week 1  Week 2  Week 4  9248 7306 9250  2330 2335 2284 2316  2328 2325 2267 2307  2455 2345 2425 2408  7307 9295 7308  2038 2163 1964 2055  2045 2177 1950 2057  2075 2285 2052 2137  7309 7310 9210  2804 2154 2130 2363  2817 2164 2143 2375  3042 2134 2338 2505  7311 7324 7312  2817 2745 2210 2591  2810 2735 2245 2597  2800 2852 2370 2674  Sub-avg.  Sub-avg.  Sub-avg.  Sub-avg.  2331  Avg.  2431  2334  Herring o i l 9298 8525 8409  2381 2548 2315 2415  2400 2553 2306 2420  1712 2456 2157 2108  9208 8544 7313  2059 2271 2090 2140  2051 2283 2093 2142  2564 2280 2156 2333  Sub-avg.  Sub-avg.  82  TABLE 3 . I l l (Cont'd) Body weights (grams) o f c o c k e r e l s f e d v a r i o u s d i e t s i n experiment 3  Diets Group number  Sub-group number  Bird number  F a t supplemented H e r r i n g o i l (cont'd)  Basal Week 1  Week 2  Week 4  7314 7315 9308  2484 2251 2208 2314  2493 2262 2231 2328  2564 2280 2156 2333  8402 7316 9222  2320 2495 2349 2388  2325 2488 2345 2386  2339 2488 2288 2372  Sub-avg.  Sub-avg.  2314  Avg.  2317  2319  Coconut o i l 7317 9191 7318  2599 1958 2412 2323  2603 1946 2430 2326  2265 2045 2647 2319  7319 7320 7321  2121 1774 2080 1992  2130 1769 2062 1987  2350 1983 2270 2201  7322 8453 9155  2152 2401 2139 2231  2160 2408 2127 2232  1872 2453 2242 2189  9276 7323 9241  2364 2117 1931 2137  2365 2104 1917 2129  2555 2162 2037 2251  Sub-avg.  Sub-avg.  Sub-avg.  Sub-avg. Avg.  2171  2169  2240  83 Shown i n T a b l e s 3.IV and 3.V a r e the r a t e s o f f e c a l output and b i l e a c i d e x c r e t i o n r e s p e c t i v e l y on t h e d i e t s f e d . these d a t a t h a t these r a t e s were s u b j e c t Greater  I t i s apparent from  t o the influence o f d i e t .  e x c r e t i o n r a t e s were observed on t h e b a s a l d i e t than on the f a t  supplemented d i e t s .  These changes a r e i n accord  humans ( A l i e t aJL., 1966).  w i t h those r e p o r t e d  On t h e b a s a l d i e t the output o f f e c e s  with  ranged  between 13.83 and 15.58 grams/kgm. body wt./24 h r . b u t changed t o 10.1 10.83  grams/kgm. body wt./24 h r . when f a t was an added i n g r e d i e n t .  The  c o r r e s p o n d i n g r a t e s o f e x c r e t i o n o f b i l e a c i d s were 15.45 - 19.12 mg./kgm. body wt./24 h r . and 11.83 - 14.33 mg./kgm. body wt./24 h r . r e s p e c t i v e l y . Both components o f the r a t e o f b i l e a c i d e x c r e t i o n , namely, the concentration  of b i l e acids  were r e s p o n s i b l e  f o r i n t e r g r o u p v a r i a t i o n observed i n t o t a l b i l e  e x c r e t i o n on the b a s a l d i e t . b i l e acids  i n the f e c e s and t h e output o f f e c e s  Groups 1 and 2 e x c r e t e d  (Table 3.V) acid  s i m i l a r amounts o f  inasmuch as they showed a d i f f e r e n c e i n the magnitude o f these  components.  T h i s was t h e r e s u l t o f the opposing d i r e c t i o n o f t h e d i f f e r e n c e s  between l i k e components.  As expected, group 3 which e x h i b i t e d the h i g h e s t  r a t e o f output o f f e c e s as w e l l as t h e h i g h e s t i n the f e c e s  excreted  concentration  the l a r g e s t q u a n t i t i e s of b i l e  of b i l e  acids.  T a b l e 3.VI summarizes t h e changes i n f e c a l output o f b i l e brought about by t h e d i e t a r y m a n i p u l a t i o n s .  acids  acids  These d a t a show t h a t t h e  percentage decrease i n t o t a l output o f b i l e a c i d s p e r kgm./body wt./24 h r . which r e s u l t e d from f e e d i n g (-7.3  t h e c o r n and h e r r i n g o i l supplemented d i e t s  and -19.5 r e s p e c t i v e l y ) was due t o a f a l l  because i n b o t h i n s t a n c e s  the c o n c e n t r a t i o n  i n the output o f f e c e s ,  of b i l e acids  i n the feces  TABLE 3.IV.  Body weights (grams) and f e c a l output (grams l y o p h i l i z e d f e c e s ) o f subgroups o f c o c k e r e l s fed v a r i o u s experimental d i e t s  F a t supplemented  Basal Group number  Sub-group number  Fecal T o t a l body wt.  Fecal  output  24 h r . output  24 h r . output /kgm. body wt.  T o t a l body wt.  output  24 h r . output  24 h r . output /kgm. body wt.  Corn o i l 1 2 3 4 Avg.  6920 6172 7124 7790 7001  100.75 88.15 96.20 101.10 96.55  14.56 14.28 13.50 12.98 13.83  7225 6412 7514 8022 7293  83.45 68.70 71.85 92.95 79.24  11.55 10.71 9.56 11.59 10.83  Herring o i l 1 2 3 4 Avg.  7259 6427 6986 7158 6958  110.95 81.65 103.35 125.55 105.38  15.29 12.70 14.79 17.54 15.08  7367 6325 7000 7115 6952  85.95 59.90 63.55 72.45 70.46  11.67 9.47 9.08 10.18 10.10  Coconut o i l 1 2 3 4 Avg.  6979 5961 6695 6386 6505  117.40 108.70 88.25 90.10 101.11  16.82 18.24 13.18 14.10 15.58  6957 6603 6567 6755 6721  91.45 67.25 61.60 71.60 72.98  13.15 10.19 9.38 10.60 10.83  00 •p-  TABLE 3.V. F e c a l output o f b i l e a c i d s on the experimental d i e t s f e d  Diets Group  Basal mg./gm. feces  mg./bird /24 h r .  F a t supplemented mg./kgm. body wt./24 h r .  mg./gm. feces  mg./bird /24 h r .  mg./kgm. body wt./24 h r .  Corn o i l 1  1.12  36.05  15.45 (13.83)  1.32  34.83  14.33 (10.83)  Herring o i l 2  1.04  36.53  15.75 (15.08)  1.25  29.36  12.68 (10.10)  Coconut o i l 3  1.23  (  )  41.46  The v a l u e s  19.12 (15.58)  i n parentheses r e p r e s e n t  1.09  the output  26.51  11.83 (10.83)  o f f e c e s i n gms./kgm. body wt./24 h r .  oo  86  TABLE 3.VI.  Changes i n c o n c e n t r a t i o n o f plasma DPS, f e c a l b i l e a c i d s and output o f b i l e a c i d s r e s u l t i n g from the v a r i o u s d i e t a r y substitutions  i.  Digitonin-precipitable sterols  D i e t s exchanged f o r b a s a l Sub-group number  Corn o i l mg.% %  Herring o i l mg.% %  Coconut o i l mg.% %  1  +0.7  0.8  +10.1  +11.3  +40.9  +48.8  2  -1.1  -1.1  +13.9  +17.0  +27.1  +29.4  3  -2.5  -^3.3  +11.4  +12.5  +43.0  +47.4  4  +5.4  6.0  - 3.1  - 3.8  +40.5  +55.9  +0.7  +0.6  +- 8.1  + 9.3  +37.9  +45.4  T r t . avg.  ii.  Fecal b i l e  acids  D i e t a r y change  Per gram of d r i e d feces mg. %  Per b i r d per,24 h r s . mg. %  Per kgm./body wt./24 h r s . mg. %  Basal  to corn o i l  +0.20  +17.86  - 1.22  - 3.38  -1.12  - 7.25  Basal  to herring o i l  +0.21  +20.19  - 7.17  -19.63  -3.07  =19.49  B a s a l t o coconut o i l  -0.14  -11.38  -14.95  -36.06  -7.29  -38.13  87 increased.  F o r the c o r n and h e r r i n g o i l supplemented d i e t s the change i n  output of f e c e s was  -21.7 and -33.07. r e s p e c t i v e l y (Table 3 . V I I ) .  ponding changes i n the c o n c e n t r a t i o n and +20.2%. in  o f b i l e a c i d s i n f e c e s were  Thus r e l a t i v e d i f f e r e n c e s i n c o n c e n t r a t i o n  of b i l e  Corres+17.9 acids  the f e c e s were m a i n t a i n e d from one d i e t a r y p e r i o d t o another when d i e t s  containing unsaturated  f a t s were f e d .  The d i f f e r e n c e i n the amount o f  change i n the r a t e o f e l i m i n a t i o n o f b i l e  a c i d s between these two treatments  was t h e r e f o r e due t o a d i f f e r e n c e i n the amount of change i n output o f feces. The s t e e p e s t  fall  i n the output of b i l e a c i d s o c c u r r e d  coconut o i l r e p l a c e d dextrose -38.17. (Table 3.VI). concentration  i n the d i e t .  Accompanying t h i s f a l l  when  The amount o f change was was a decrease a l s o i n the  o f b i l e a c i d s i n the f e c e s which c o n t r a s t s w i t h the change  when u n s a t u r a t e d  f a t was f e d .  The amount o f decrease was  The d e c l i n e i n f e c a l output o f b i l e a c i d s a s s o c i a t e d w i t h coconut o i l f o r dextrose  3.VI).  s u b s t i t u t i o n of  was t h e r e f o r e due t o a decrease both i n the output  of f e c e s and i n the c o n c e n t r a t i o n Table  11.4% (Table  o f b i l e a c i d s i n the f e c e s .  3.VII i n d i c a t e s t h a t d i f f e r e n c e s i n the output o f f e c e s  when the b i r d s were consuming the f a t - e n r i c h e d d i e t s were s m a l l  (10.10 -  10.83 gms./kgm. body wt./24 h r . ) compared to when they were consuming the basal diet.  The data  in  (1.09 - 1.32 mg./gm. f e c e s ) was m a i n l y r e s p o n s i b l e f o r  the f e c e s  intertreatment  t h e r e f o r e show t h a t the c o n c e n t r a t i o n  of b i l e  v a r i a t i o n i n the amount o f b i l e a c i d s e x c r e t e d  consuming the h i g h - f a t  acids  by b i r d s  diets.  The p a t t e r n o f b i l e a c i d s i n f e c e s o f b i r d s s u b j e c t e d v a r i o u s d i e t s i s shown i f F i g s . 3.8 to 3.11.  to the  TABLE 3.VII.  Basal d i e t gms./24 h r .  gms./kgm. body wt./24 h r .  F e c a l output and change i n f e c a l output r e s u l t i n g from d i e t a r y exchanges  Change  F a t supplemented d i e t gms./24 h r .  gms./kgm. body wt./24 h r .  gms./24 h r .  7, change  gms./kgm. body wt./24 h r .  gms./24 h r .  gms./kgm. body wt./24 h r .  Corn o i l 96.55  13.83  79.24  10.83  -17.31  -3.00  -17.92  -21.69  -34.92  -4.98  -33.14  -33.02  -28.12  -4.75  -27.81  -30.49  Herring o i l 105.38  15.08  70.46  10.10  Coconut o i l 101.10  15.58  72.98  10.83  oo oo  F i g . 3.8.  Gas chromatographic p a t t e r n o f the TMS e t h e r s o f methyl e s t e r s o f f e c a l b i l e a c i d s of c o c k e r e l s f e d the b a s a l d i e t o f experiment 3. A n a l y t i c a l c o n d i t i o n s : 1.0% SE-30 on 100-120 mesh Gas Chromosorb Q; 6'6" s t a i n l e s s s t e e l column; column temperature, 230° f o r 8 mins., 230° - 250° at l°/min.; i n j e c t i o n p o r t , 270°; d e t e c t o r , 260°; n i t r o g e n , 80 c.c./min. OO vo  Fig. 3 . 9 .  Gas chromatographic p a t t e r n of the TMS ethers o f the methyl e s t e r s o f the f e c a l b i l e a c i d s of c o c k e r e l s fed a c o r n o i l supplemented d i e t . Analytical conditions as g i v e n i n F i g . 3 . 8 .  o  C  o OJ  QJ CD Z  o  CL  CO Lu  cr cr  QJ Q  cn  o o  QJ  cr  1 0  1 5  2 0  RETENTION F i g . 3.10.  3 0  2 5  TIME  (MINS.)  Gas chromatographic p a t t e r n o f the TMS ethers o f the methyl e s t e r s o f the f e c a l b i l e a c i d s of c o c k e r e l s f e d a h e r r i n g o i l supplemented d i e t . Analytical conditions as g i v e n i n F i g . 3.8.  5 5  Q) C  o  -*—»  CJ  2 0  RETENTION F i g . 3.11.  (MINS.)  Gas chromatographic p a t t e r n o f the TMS ethers of the methyl e s t e r s o f the f e c a l b i l e a c i d s of c o c k e r e l s f e d a coconut o i l supplemented d i e t . Analytical conditions as g i v e n i n F i g . 3.8. vo  N>  93 On  the b a s i s of these c a l c u l a t i o n s the  f o l l o w i n g order  of  e f f e c t i v e n e s s of the d i e t a r y f a t s i n promoting the e x c r e t i o n of b i l e has  been a r r i v e d a t : The  e x c r e t i o n has  corn o i l )  herring o i l y  been undertaken on numerous o c c a s i o n s  f e c a l e l i m i n a t i o n of b i l e  acids.  the  acid  conclusions  i n the  literature  of the d i e t a r y f a t favours  In these experiments r e p o r t e d ,  c o l o r i m e t r i c techniques were u t i l i z e d .  The  i n a d e q u a c i e s i n the p u r i f i c a t i o n of the b i l e a t i o n has  but  E a r l y experiments ( c i t e d  review) have i n d i c a t e d t h a t u n s a t u r a t i b n  and  coconut o i l .  e v a l u a t i o n of d i e t a r y f a t s f o r t h e i r e f f e c t s on b i l e  reached have been d i v e r g e n t .  acids  the  titrimetric  subsequent r e c o g n i t i o n of  a c i d s p r i o r to t h e i r  quantit-  however c a s t s e r i o u s doubts on the v a l i d i t y of the r e s u l t s  reported.  In l a t e r experiments, i n which more s p e c i f i c methods of a n a l y s i s have been employed a g e n e r a l  agreement has  Roels and  Hashim (1964) u s i n g  chromatography r e p o r t e d the  an a p p r e c i a b l e  amount of both d e o x y c h o l i c  subjects  s t i l l not been reached.  and  i o n exchange and  r e d u c t i o n , which was  paper  reversible, in  chenodeoxycholic a c i d s e x c r e t e d  by human  t r a n s f e r r e d from a d i e t supplemented w i t h c o r n o i l to one  mented w i t h coconut o i l .  acids excreted  when c o r n o i l r e p l a c e d b u t t e r  o t h e r hand, A v i g a n and c o u l d not  supple-  In b a l a n c e s t u d i e s employing r a d i o a c t i v e c h o l e s t e r o l  Moore et ajL. (1962) found t h a t t h e r e were s i g n i f i c a n t  Steinberg,  increases  in bile  i n the d i e t of humans.  (1965) u s i n g  t r a c e r techniques  On  the  i n humans  demonstrate a change i n f e c a l output of b i l e a c i d s when d i e t a r y  changes d e s c r i b e d  above were made.  between s a t u r a t e d  and  et a l . (1965) who  prepared h i g h l y p u r i f i e d b i l e  the use  glass  of column and  F a i l u r e to demonstrate a d i f f e r e n c e  u n s a t u r a t e d f a t s has  a l s o been r e p o r t e d  by S p r i t z  a c i d s from f e c e s  t h i n l a y e r chromatography and  then t i t r a t e d  through them.  94 These workers r e p o r t e d non-sterol  a mean output i n 5 s u b j e c t s  diet containing  o f 101 mg./day on a  coconut o i l and 134 mg./day when c o r n o i l was  s u b s t i t u t e d f o r coconut o i l .  The d i f f e r e n c e found was however n o n s i g n i f i c a n t ,  because o f l a r g e i n d i v i d u a l v a r i a t i o n . The  r e s u l t s o f S p r i t z e_t al.  et a l . (1965) who d e v i s e d  (1965) were confirmed by Grundy  a combination o f t h i n l a y e r and gas l i q u i d  chromatography f o r measuring the f e c a l b i l e a c i d s . Ali  Using s i m i l a r  techniques,  e_t al_. (1966b) i n v e s t i g a t e d the problem i n q u e s t i o n but were unable t o  draw any c o n c l u s i o n s  on account o f l a r g e i n d i v i d u a l v a r i a t i o n i n the  e x c r e t i o n r a t e s o f the few s u b j e c t s The  r e s u l t s o f the p r e s e n t  o r i g i n a l l y reported  studied. i n v e s t i g a t i o n a r e comparable t o those  by Lewis e_t a_l. (1957) f o r humans.  i n the mature c o c k e r e l , s a t u r a t e d  They i n d i c a t e t h a t  and u n s a t u r a t e d f a t s a f f e c t the plasma  s t e r o l l e v e l d i v e r g e n t l y and that the depressant e f f e c t o f u n s a t u r a t e d f a t s on the plasma s t e r o l l e v e l  i s c r e d i t a b l e , at l e a s t p a r t l y , to  enhancement o f b i l e a c i d e x c r e t i o n .  95  EXPERIMENT 4  THE EFFECT OF DIETARY LECITHIN AND BETA SITOSTEROL ON THE FECAL OUTPUT OF BILE ACIDS AND THE LEVEL OF CIRCULATING STEROLS IN GROWING COCKERELS  T h i s experiment  was conducted  have an e f f e c t on b i l e a c i d e x c r e t i o n .  to a s c e r t a i n i f d i e t a r y phospholipids Although  t h e r e i s some  evidence  i m p l i c a t i n g p h o s p h o l i p i d s i n b i l e a c i d metabolism a s e a r c h o f the l i t e r a t u r e f a i l e d t o show any r e p o r t s d e a l i n g s p e c i f i c a l l y response  to d i e t a r y phospholipids.  with b i l e acid e x c r e t i o n i n  Because these substances  a r e more  h y d r o p h i l i c than the t r i g l y c e r i d e s and r e a d i l y form mixed m i c e l l e s w i t h bile  s a l t s any e f f e c t s t h a t they may have on b i l e a c i d e x c r e t i o n c o u l d be  f u r t h e r s t u d i e d w i t h g r e a t e r ease than w i t h the t r i g l y c e r i d e s u s i n g the i n v i v o technique  developed.  Review o f L i t e r a t u r e  Isalcsson (1954) d e s c r i b e d a complex o f l e c i t h i n and b i l e o b t a i n e d by e x t r a c t i o n o f l y o p h i l i z e d b i l e w i t h c h l o r o f o r m . (1956) and V e r s c h u r e cholesterol b i l i r u b i n  and M i j n l i e f f  Verschure  (1956) showed t h a t l e c i t h i n , b i l e  salts,  and a p o l y p e p t i d e o f human g a l l b l a d d e r b i l e moved  w i t h the same e l e c t r o p h o r e t i c m o b i l i t y on paper. (1965) s u b j e c t e d human g a l l b l a d d e r b i l e G-200 and found these substances weight f r a c t i o n .  salts  Nakayama and Miyake  t o g e l f i l t r a t i o n on sephadex  a s s o c i a t e d i n the i n t e r m e d i a t e m o l e c u l a r  N e i d e r h i s e r e t al_. (1966) f r a c t i o n a t e d human and hog  g a l l b l a d d e r b i l e on sephadex G-75 and found t h a t l e c i t h i n  accompanied  96 c h o l e s t e r o l a t the s o l v e n t  f r o n t which was e s s e n t i a l l y f r e e from b i l e  s a l t s , p r o t e i n and b i l i r u b i n .  The l e c i t h i n and c h o l e s t e r o l were not  e l e c t r o p h o r e t i c a l l y mobile u n t i l concentration mobility  o f 0.04 M.  taurocholate  was added t o a f i n a l  The importance o f b i l e  salts for electrophoretic  o f l e c i t h i n and c h o l e s t e r o l has a l s o been demonstrated by Norman  (1964). I t has been p o s t u l a t e d owe t h e i r a b i l i t y t o move d u r i n g associated  t h a t g a l l b l a d d e r l e c i t h i n and c h o l e s t e r o l electrophoresis  w i t h them (Verschure, 1956).  Immunoelectrophoresis  t o the presence o f p r o t e i n  Attempts u s i n g  the techniques o f  (Rawson, 1962) and a n t i g e n i n d u c t i o n  ( R u s s e l l and  B u r n e t t , 1963; R u s s e l l e t a l . , 1964) t o demonstrate the presence o f p r o t e i n at the s i t e t o which the l i p i d s m i g r a t e d u r i n g been u n s u c c e s s f u l . explanation  have however  Consequently some i n v e s t i g a t o r s now s u b s c r i b e  o f mixed m i c e l l e  The  electrophoresis,  f o r m a t i o n (Hoffman, 1965; J u n i p e r ,  t o the  1965).  f a c i l i t y w i t h which l e c i t h i n , b i l e s a l t s and c h o l e s t e r o l  i n t o m o l e c u l a r a s s o c i a t i o n suggests t h a t a c t i n g e f f e c t s on t h e i r a b s o r p t i o n .  This  these substances may exert  enter  inter-  suggestion i s p a r t i c u l a r l y  m e a n i n g f u l i n the l i g h t o f c o m p e l l i n g evidence put forward by Hoffman (1962, 1964) t h a t  the a b s o r p t i o n  Hoffman has d e s c r i b e d  o f f a t s takes p l a c e  evidence c o n s i s t e n t  phase o f i n t e s t i n a l c o n t e n t s d u r i n g I t was t h e r e f o r e  from m i c e l l a r  solution.  w i t h the presence o f a m i c e l l a r  f a t digestion.  considered of i n t e r e s t to i n v e s t i g a t e  of d i e t a r y l e c i t h i n on the r a t e o f output o f b i l e a c i d s l e v e l o f plasma s t e r o l s i n the domestic c h i c k e n .  the e f f e c t  i n the f e c e s  The e f f e c t o f l e c i t h i n  when b e t a s i t o s t e r o l i s added to the d i e t has a l s o been c o n s i d e r e d . s t e r o l s a r e known t o i n h i b i t e i t h e r the a b s o r p t i o n  and the  Plant  o f c h o l e s t e r o l from the  97 gut  lumen ( P o l l a k , 1953)  or the t r a n s p o r t of t h i s s t e r o l out of the  mucosa ( S w e l l et a l . , 1959).  The  s t r u c t u r a l s i m i l a r i t y of c h o l e s t e r o l  the p l a n t s t e r o l s suggests t h a t an p o s s i b l y a r i s e as a r e s u l t of an formation.  i n h i b i t i o n of the former type  could  through t h i s  influence,  changes i n the amount o f b i l e a c i d s e x c r e t e d .  A  f u r t h e r r e a s o n f o r i n v e s t i g a t i n g the e f f e c t of b e t a s i t o s t e r o l on b i l e e x c r e t i o n i s t h a t there may  are a number of r e p o r t s  have an e f f e c t on b i l e a c i d e x c r e t i o n .  suggesting  d i e t a r y f a t contained when the  f a t was  contrasted  unsaturated.  A l i and  p r e s e n t e d d a t a i n d i c a t i n g an o p p o s i t e with i n d i v i d u a l subjects,  the t o t a l output of f e c a l b i l e  mg./day when the  These d i f f e r e n c e s were reproduced co-workers (1966b) on the o t h e r effect.  In t h r e e  these workers found t h a t the  p l a n t s t e r o l s i n t o the d i e t was  sterol  f a t , s t e r o l - f r e e regimen  w i t h an average of 247  plant s t e r o l s .  that t h i s  acid  S p r i t z et a l . (1965) r e p o r t e d  t h a t b i l e a c i d e x c r e t i o n by humans on a s a t u r a t e d averaged 101 mg./day and  and  i n f l u e n c e of the p l a n t s t e r o l s on m i c e l l e  Beta s i t o s t e r o l c o u l d p r o b a b l y t h e r e f o r e ,  induce c o n s e q u e n t i a l  intestinal  separate  hand,  studies  incorporation  of  a s s o c i a t e d w i t h an apparent decrease i n acids.  Experimental  S i n g l e Comb White Leghorn c o c k e r e l s d i e t from h a t c h i n g  to 3 weeks of age.  heated b a t t e r i e s w i t h wire s c r e e n access to feed and  water.  At  were m a i n t a i n e d on a commercial  They were r e a r e d  f l o o r s and  3 weeks of age  in electrically  at a l l times allowed they were weighed and  free assigned  to e i g h t l o t s of 8 b i r d s i n such a manner t h a t the mean weights of the were a p p r o x i m a t e l y the same.  They were then f e d the a l l v e g e t a b l e  lots  basal  98 r a t i o n and i t s m o d i f i c a t i o n s shown i n T a b l e 4.1. assigned  a t random to d u p l i c a t e l o t s .  were made a t the expense o f c o r n o i l .  A l l treatments were  The a d d i t i o n s t o the b a s a l  diet  Beta s i t o s t e r o l was added by f i r s t  dissolving  i t i n e t h e r and a minimum amount o f c h l o r o f o r m  separatory  f u n n e l , then a l l o w i n g the s o l u t i o n to d r i p onto the b a s a l  w i t h m i x i n g i n a l a r g e trough. to  evaporate (approximately  in a 3 liter  Ample time was then allowed  f o r the s o l v e n t s  20 hours) w i t h o c c a s i o n a l m i x i n g .  Homogenization  of the d i e t m i x t u r e was completed with an a u t o m a t i c a l l y operated Soyabean l e c i t h i n corn o i l .  diet  mixer.  (Vancouver Pure Foods L t d . ) was added as a m i x t u r e  with  The supplements were added a t a l e v e l o f 0.5 and 2.5% r e s p e c t i v e l y .  A f t e r the e x p e r i m e n t a l  d i e t s had been f e d f o r 15 days b l o o d samples were  taken from the wing v e i n and a composite sample o f plasma made f o r each lot. The  T h i s was a n a l y s e d feces voided  f o r DPS as d e s c r i b e d  i n the p r e v i o u s  d u r i n g the 48 h r . p e r i o d p r e c e d i n g  were c o l l e c t e d and f r o z e n .  experiments.  withdrawal o f b l o o d  Feed consumption was a l s o measured c o n c u r r e n t l y  w i t h the c o l l e c t i o n o f f e c e s .  Immediately f o l l o w i n g t h i s 48 hour p e r i o d  the b i r d s were a g a i n weighed.  The f r o z e n f e c e s were t r e a t e d as d e s c r i b e d  i n experiment 3 p r i o r t o a n a l y s i s f o r b i l e a c i d s .  Results  The  and D i s c u s s i o n  l e v e l s o f plasma DPS which r e s u l t e d from f e e d i n g the e x p e r i -  mental d i e t s are shown i n T a b l e 4 . I I . The d i f f e r e n c e s found were not s t a t i s t i c a l l y s i g n i f i c a n t , but as may be seen the plasma DPS l e v e l s o f b i r d s f e d the b a s a l d i e t tended to be somewhat h i g h e r birds.  than those o f t r e a t e d  The l a t t e r were c l e a r l y i n d i s t i n g u i s h a b l e on a d i e t a r y b a s i s .  TABLE 4.1.  Composition of d i e t s f e d i n experiment  4  Basal diet Ingredients Corn o i l Ground y e l l o w c o r n Soyabean meal Dehydrated c e r e a l g r a s s Dried d i s t i l l e r s ! solubles Limestone Bonemeal Iodized s a l t C h o l i n e c h l o r i d e (377.) D-L methionine  7. o f d i e t 8.0 51.54 31.46 2.00 2.00 1.00 3.00 0.50 0.22 0.15  mg./kgm. of d i e t Manganese s u l f a t e Folacin Riboflavin C a l c i u m pantothenate Niacin  220.00 1.10 6.60 18.48 44.00  units/kgm. o f d i e t V i t a m i n A (I.U.) Vitamin D (I.C.U.) 3  Diet  number 1 2 3 4  1997.60 199.76  Supplements none 2.57. s o y a l e c i t h i n 0.57. b e t a s i t o s t e r o l 2.57. s o y a l e c i t h i n + 0.57. beta s i t o s t e r o l  TABLE 4 . I I .  C o n c e n t r a t i o n (mg.7.) o f DPS i n plasma of c o c k e r e l s f e d d i e t s of experiment 4 f o r 15 days  Concentration Dietary  supplement  none  2.57. soyabean  0.57. b e t a  lecithin  sitosterol  2.57. soyabean l e c i t h i n + 0.5% b e t a s i t o s t e r o l  Replicate  Treatment  116.7 128.0  122.4  110.7 114.6  112.7  120.8 106.0  113.4  111.3 113.1  112.2  101 D i f f e r e n c e s were a n t i c i p a t e d on the b a s i s o f p r e v i o u s r e p o r t s . a l r e a d y been demonstrated  by P e t e r s o n et a l .  o r a t i o n of beta s i t o s t e r o l  (1951,  I t has  1952a) t h a t the  into a cholesterol enriched diet  inhibits  a b s o r p t i o n of c h o l e s t e r o l and the h y p e r c h o l e s t e r o l e m i a developed c h i c k e n s f e d t h i s d i e t without supplementary the consumption al.,  1958)  1952;  of b e t a s i t o s t e r o l  and l e c i t h i n  M o r r i s o n , 1958)  cholesterol level.  beta s i t o s t e r o l .  (Farquhar et a_l., 1956;  ( S t e i n e r and Domanski, 1944;  incorpthe  by  In humans  Beveridge et  P o t t e n g e r and Krohn,  has been shown to cause a d e c l i n e i n the plasma  The r e s u l t s o f experiment  3 revealed a s i m i l a r i t y  s t e r o l e m i c response o f humans and c h i c k e n s to d i e t a r y f a t . a n t i c i p a t e d d i f f e r e n c e s were not f u l l y r e a l i z e d a t t r i b u t a b l e to the low l e v e l s o f supplements l e v e l s were used i n o r d e r to minimize  That  i n the  these  i s therefore probably  which were f e d .  the r e s t r i c t i o n s  These  frequently  low  imposed  on the i n t e r p r e t a t i o n of d a t a o b t a i n e d by use of i n o r d i n a t e amounts o f dietary  constituents. Body weight measurements made at the s t a r t and  p r e c e d i n g c o m p l e t i o n o f the experiment and 4 . I I I . i i .  are c o n t a i n e d i n T a b l e s 4 . I I I . i  The r e s u l t s o f s t a t i s t i c a l  a n a l y s i s performed  g a i n s are g i v e n i n the a n a l y s i s o f v a r i a n c e T a b l e 4.IV. r e p l i c a t e s was  immediately  When treatment  c o n s i d e r e d as a source o f v a r i a t i o n i n the a n a l y s i s i t  a t t a i n e d s i g n i f i c a n c e at the 5.07» l e v e l of s i g n i f i c a n c e and treatment e f f e c t s n o n s i g n i f i c a n t . was  on the weight  rendered  Because the s i z e o f the r e p l i c a t e s  o b v i o u s l y inadequate f o r s t u d y i n g a h i g h l y v a r i a b l e parameter  body weight,  r e p l i c a t e v a r i a t i o n was  sampling e r r o r .  The new  such as  d i s p e n s e d w i t h by p o o l i n g i t w i t h the  t e s t i n g term uncovered  i n the growth r a t e s o f the b i r d s .  used  significant  differences  L e c i t h i n supplementation was  found t o  TABLE 4 . I I I . i .  Body weights (grams) and g a i n i n body weights o f c o c k e r e l s f e d d i e t s o f experiment 4 f o r 17 days  Diets Basal Lot number  I  1  Basal + l e c i t h i n  Bird number  Initial weight  Final weight  We i g h t  1601 1712 1711 1742 1716 1729 1743 1706  197 181 186 185 181 188 182 187  414 342 435 415 390 416 391 416  217 161 249 230 209 228 209 229  Avg.  186  402  217  1798 1764 1780 1795 1788 1761 1778 1769  184 192 184 177 172 197 196 182  451 455 383 393 407 442 393 400  267 263 199 216 235 245 197 218  Avg.  186  416  230  avg.  186  409  224  Rain  Lot number  Bird number  Initial weight  Final weight  Weight gain  2  1720 1722 1749 1738 1755 1739 1612 1707  185 188 170 206 197 198 197 175  425 441 360 444 431 440 432 411  240 253 190 238 234 242 235 236  Avg.  190  423  234  1603 1615 1606 1762 1701 1757 1607 1790  175 202 180 194 206 176 177 180  377 477 400 448 472 402 390 427  202 275 220 254 266 226 213 247  Avg.  186  424  238  188  424  236  2  1  T r t . avg.  o  K3  TABLE 4 . I I I . i (cont'd) Body weights (grams) and g a i n i n body weights o f c o c k e r e l s f e d d i e t s o f experiment 4 f o r 17 days  Diets Basal Lot number  Basal + l e c i t h i n + beta s i t o s t e r o l  + beta s i t o s t e r o l  Bird number  Initial weight  Final weight  Weight gain  1792 1602 1796 1784 1608 1613 1618 1777  216 208 205 176 172 195 177 186  457 427 450 410 382 405 417 420  241 219 245 234 210 210 240 234  Avg.  192  421  229  1794 1735 1730 1772 1775 1757 1759 1610  189 207 184 192 203 182 175 175  397 482 394 395 421 432 380 385  208 275 210 203 218 250 205 210  196  Avg.  188  411  222  204  T r t . avg.  190  416  226  Bird number  Initial we i g h t  Final weight  Weight Gain  1725 1710 1779 1713 1754 1719 1614 1751  186 197 197 175 178 178 190 196  435 447 402 324 386 356 395 436  249 250 205 149 208 178 205 240  Avg.  187  398  211  1793 1746 1786 1724 1708 1753 1703 1732  201 191 210 150 202 165 186 178  418 390 423 322 416 332 364 388  217 199 213 172 214 167 178 210  Avg.  .185  382  avg.  186  390  Lot number 4  4  o  104  TABLE 4 . I I I . i l .  Dietary  supplement  Summary o f body weight  Avg. i n i t i a l weight (gms.)  Avg. f i n a l weight (gms.)  data  Avg. g a i n i n weight (gms.)  none  186  409  224  2.57. s o y a l e c i t h i n  188  424  236  0.57. b e t a  sitosterol  186  390  204  2.57. s o y a l e c i t h i n + 0.57. b e t a s i t o s t e r o l  190  416  226  TABLE 4.IV.  S.S.  Source o f v a r i a t i o n Treatment  Analysis  8,797.81  of variance  of g a i n  i n body weight  P  df.  M.S.  F  3  2932.60  4.773  <C o . o i  Lecithin  4847.60  1  4847.60  7.890  < 0.01  Sitosterol  3555.10  1  3555.10  5.786  < 0.05  395.11  1  395.11  0.643  ns.  Lec.  x sit.  Experimental Sampling Pooled  error  error  error  Model:  10,597.80  4  2649.45  26,267.39  56  469.06  36,865.19  60  614.42  Y..,  = u + 1 • + s . + (Is).'. + e. .,  106 significantly  increase  the r a t e o f growth(P <^ O.Ol) and  s i g n i f i c a n t l y depress i t (P < 0.05).  beta s i t o s t e r o l to  This m i l i t a t i n g e f f e c t o f beta  s i t o s t e r o l on the r a t e o f growth has p r e v i o u s l y been r e p o r t e d Slater,  1954) f o r r a t s but no r e p o r t s  a favourable  c o u l d be found i n the l i t e r a t u r e o f  e f f e c t o f soya l e c i t h i n on growth performance.  In T a b l e 4.V. a r e p r e s e n t e d the feed those f o r t h e f e c a l o u t p u t . the  consumption d a t a along w i t h  The d i f f e r e n c e s noted i n f e e d  by b i r d s a d m i n i s t e r e d between feed  l e c i t h i n as the o n l y  supplement.  r a t e o f consumption Despite the p a r a l l e l  consumption and f i n a l body weight the l e c i t h i n t r e a t e d  m a i n t a i n e d the narrow feed for differences those i n feed  consumption margin when consumption was c o r r e c t e d  i n body weight.  The d i f f e r e n c e s  i n f e c a l output  r a t e o f b i l e a c i d e x c r e t i o n on the v a r i o u s  i n T a b l e 4.VI.  resembled  d i e t s are given  I t may be seen t h a t t h i s r a t e v a r i e d markedly w i t h d i e t .  a d d i t i o n o f b e t a s i t o s t e r o l t o the d i e t was a s s o c i a t e d  increase  birds  consumption.  The  with a d e f i n i t e  i n the f e c a l output o f b i l e a c i d s , by a l l groups r e c e i v i n g t h i s  supplement.  This  i n c r e a s e , (307.), bordered on s i g n i f i c a n c e a t the 57. l e v e l  o f s i g n i f i c a n c e (F r a t i o c a l c u l a t e d = 7.53, Compared t o c o n t r o l s which e x c r e t e d wt.  consumption over  t e r m i n a l 48 hours o f t h e t e s t were not s t a t i s t i c a l l y s i g n i f i c a n t b u t  as may be seen t h e r e was some i n d i c a t i o n o f an i n c r e a s e d  The  (Alfin-  s i g n i f i c a n t F r a t i o = 7.71).  a d a i l y average o f 34.15 mg./kgm. body  the s i t o s t e r o l - f e d b i r d s e x c r e t e d  an average o f 44.88 mg./kgm. body wt.  These r e s u l t s agree w i t h those o f S p r i t z et_ al_. (1965) b u t stand i n c o n t r a s t to those o f A l i e t a l . (1966). were e x c r e t e d  S t r i k i n g l y large quantities of b i l e  by b i r d s consuming l e c i t h i n .  In t h e b i r d s consuming  acids lecithin  as a s i n g l e supplement t h e average d a i l y e x c r e t i o n was 70.11 mg./kgm. body  TABLE 4.V.  Feed consumption and f e c a l output o f c o c k e r e l s f e d the e x p e r i m e n t a l d i e t s  Feed consumption  D i e t a r y supplement  Avg. (gms.)/24 h r . Replicate Treatment  106 116  50.8 48.9  Avg. (gms.)724 h r . Replicate Treatment  111  8.9 11.1  49.9  120 115  43.9 47.0  45.5  46.3 45.4  45.9  42.5 48.3  2.57. s o y a l e c i t h i n  2.57. s o y a l e c i t h i n + 0.57. b e t a s i t o s t e r o l  gms./kgm. body wt./24 h r . R e p l i c a t e Treatment  45.4  none  0.57. b e t a s i t o s t e r o l  F e c a l output gms./kgm. body wt./24 h r . Replicate Treatment  10.0  22.1 26.7  24.3  118  11.7 11.7  11.7  27.7 27.5  27.6  110 108  109  10.6 9.7  10.2  26.7 25.4  26.1  110 110  110  11.2 11.2  11.2  26.7 27.2  27.0  TABLE 4.VI.  B i l e a c i d e x c r e t i o n on d i e t s f e d i n experiment 4  B i l e acids excreted  Dietary  supplement  none  2.57. s o y a l e c i t h i n  0.5% b e t a  sitosterol  2.57. s o y a l e c i t h i n + 0.57. b e t a s i t o s t e r o l  (mg.)  /gm. Replicate  feces Treatment  1.34 1.45  1.40  11.91 16.08  14.00  29.60 38.70  34.15  2.39 2.69  2.54  28.02 31.37  29.70  66.25 73.98  70.11  1.72 1.69  1.70  18.28 16.37  17.32  45.96 42.99  44.88  3.06 2.85  2.96  34.39 31.89  33.14  81.64 77.58  79.66  Avg./24 hours Replicate Treatment  /kgm. body wt./24 h r . Replicate Treatment  109 wt. r e p r e s e n t i n g The  a 1007. i n c r e a s e o f s t a t i s t i c a l  s i g n i f i c a n c e (P <_ 0.01).  e x c r e t i o n r a t e o f b i r d s f e d l e c i t h i n and b e t a  reflected  an a d d i t i v e e f f e c t o f these s u b s t a n c e s .  sitosterol  together  The i n c r e a s e s i n b i l e  a c i d output o f 10.73 and 35.96 mg./kgm. body wt./24 h r . ( t o t a l occasioned  by the a d d i t i o n o f beta  sitosterol  46.69)  and l e c i t h i n s e p a r a t e l y t o  the d i e t were accounted f o r i n the i n c r e a s e o f 45.51 mg./kgm. body wt./ 24 h r . brought about by simultaneous supplementation w i t h The  gas chromatographic p a t t e r n s  the two s u b s t a n c e s .  of f e c a l b i l e acids obtained f o r  the v a r i o u s d i e t s a r e shown i n F i g s . 4.1 to 4.3. f i g u r e s t h a t q u a l i t a t i v e changes a l s o were induced  I t i s apparent from these by d i e t .  Table  4.VII  summarizes the e f f e c t o f d i e t on the amount o f change i n t h e e x c r e t i o n o f i n d i v i d u a l and grouped b i l e a c i d s and on the r e l a t i v e p r o p o r t i o n s i n the feces.  o f these  The b i l e a c i d s a r e i d e n t i f i e d on the b a s i s o f t h e i r r e t e n t i o n  times r e l a t i v e t o the i n t e r n a l standard scheme drawn up i n T a b l e  5 ^ -cholestane.  4.VII and shown p i c t o r i a l l y  The c l a s s i f i c a t i o n  i n F i g . 4.1 i s a  p r o v i s i o n a l one o n l y , and i s based on t h e degree o f r e s o l u t i o n o b t a i n e d i n the a n a l y s e s separated together  by gas chromatography.  B i l e a c i d s which were n o t s a t i s f a c t o r i l y  and which appeared as a continuum o f u n r e s o l v e d as a s i n g l e e n t r y .  i n sequence and r e p r e s e n t  peaks a r e c o n s i d e r e d  Where these s e r i e s o f u n r e s o l v e d  peaks appear  o n l y a s m a l l p r o p o r t i o n o f the t o t a l b i l e  they a r e i n c l u d e d as a s i n g l e e n t r y , as i n the case o f e n t r y 2. acids are f u r t h e r placed  acids  The b i l e  i n t o two c a t e g o r i e s ; one c o n s i s t i n g o f a s i n g l e minor  b i l e a c i d and two groups o f minor b i l e a c i d s a l l o f which comprise 257. o f the t o t a l output, t h e o t h e r o f the .3 major b i l e a c i d s r e p r e s e n t i n g t h i s output.  This s u b c l a s s i f i c a t i o n i s also tentative.  757. o f  The data o f  T a b l e 4.VII c l e a r l y i n d i c a t e t h a t t h e d i e t a r y treatments caused a m o d i f i c a t i o n  RETENTION  TIME (MINS.)  Gas chromatographic p a t t e r n o f the TMS e t h e r s o f the methyl e s t e r s o f the f e c a l b i l e a c i d s o f c o c k e r e l s f e d t h e b a s a l d i e t of experiment 4. A n a l y t i c a l c o n d i t i o n s 1.0% SE-30 on 100-120 mesh Gas Chromosorb Q; 6'6" s t a i n l e s s s t e e l column; column temperature, 245°; i n j e c t i o n p o r t , 275°; d e t e c t o r , 260°; n i t r o g e n , 80 c.c./min.  F i g . 4.2.  Gas chromatographic p a t t e r n of the TMS ethers o f the methyl e s t e r s of the f e c a l b i l e a c i d s of c o c k e r e l s f e d a b e t a s i t o s t e r o l supplemented d i e t . Analytical c o n d i t i o n s as g i v e n i n F i g . 4.1.  RETENTION  F i g . 4.3.  TIME (MINSJ  Gas chromatographic p a t t e r n o f the TMS e t h e r s o f the methyl e s t e r s o f the f e c a l b i l e a c i d s o f c o c k e r e l s f e d a s o y a l e c i t h i n supplemented d i e t . Analytical c o n d i t i o n s as g i v e n i n F i g . 4.1.  TABLE 4.VII.  Percentage o f i n d i v i d u a l and grouped b i l e a c i d s and percentage change i n t h e i r r a t e o f e l i m i n ation i n feces of experimental b i r d s  Dietary Relative retention times (Rj.) 1.15-1.26 1.64-2.39 2.98  °k 13.74 9.76 1.52 25.02  2.67 3.43 9.16  35.38 18.87 20.76  None 7. change _ —  —  --  ---  75.01  R a t i o o f 7. change i n minor:major  Soyalecithin 7. change  k  9.31 6.36 9.22 24.89  Avg.  34.46 31.47 9.19 75.12  Avg.  supplements Beta s i t o s t e r o l 7. change  k  38.94 33.93 1142.31  12.99 8.91 2.60  405.06  24.50  100.00 242.55 -9.17  36.47 18.31 20.79  111.12  75.57  3.65  Avg.  Avg.  Beta s i t o s t e r o l + s o y a L 7. change  °k  24.04 20.12 125.00  8.93 6.54 9.56  56.38  25.03  35.51 27.48 31.59  32.86 34.22 7.90  31.53  74.98  1.79  51.28 56.46 1362.46 Avg.  490.07  116.72 323.29 -11.28 Avg.  142.91  3.43  114 of the r a t e o f e x c r e t i o n o f a l l the b i l e a c i d s when these were i n d i v i d u a l l y w i t h the two b a s i c groups.  considered  F o r any g i v e n treatment t h e average  r a t e o f e x c r e t i o n o f t h e minor b i l e a c i d s was g r e a t e r than t h a t o f the major. T h i s e f f e c t v a r i e d markedly w i t h d i e t . supplement a d m i n i s t e r e d  the r a t i o o f the average percentage i n c r e a s e i n the  amount o f minor b i l e a c i d s e x c r e t e d r a t i o was 1.79 when beta  l e c i t h i n was a d m i n i s t e r e d o f the b i l e a c i d s .  resulted  i n t h e major was 3.65.  the r a t i o was 3.43.  This  When both  Only when soya-  was t h e r e a decrease i n the r a t e o f e x c r e t i o n o f  The consumption o f the l e c i t h i n supplemented d i e t  i n a decrease o f 9.17 - 11.28% i n the r a t e o f e x c r e t i o n o f t h e  major b i l e a c i d with. by  to t h a t  s i t o s t e r o l alone was a supplement.  supplements were f e d s i m u l t a n e o u s l y  any  When s o y a l e c i t h i n was the o n l y  R  t  9.16.  Beta s i t o s t e r o l a d m i n i s t r a t i o n  increased i t  31.59%. A g a i n as i n experiment 3, d i f f e r e n c e s i n the c o n c e n t r a t i o n  acids  of b i l e  i n the f e c e s and i n the r a t e o f output o f f e c e s accounted f o r the  v a r i a t i o n observed i n the r a t e o f e x c r e t i o n o f b i l e differences  i n the concentration  acids.  Treatment  o f b i l e a c i d s exceeded by f a r s i m i l a r  d i f f e r e n c e s i n the f e c a l output, and were t h e r e f o r e m a i n l y r e s p o n s i b l e f o r the d i s s i m i l a r i t y observed i n the t o t a l output o f b i l e Because d i e t a r y l e c i t h i n r e s u l t s suggest t h a t a l i m e n t a r y  acids.  i s known t o be h y p o c h o l e s t e r o l e m i c ,  phospholipids  may p l a y a r o l e i n e s t a b l i s h i n g  the l e v e l of plasma DPS through e f f e c t s on b i l e a c i d metabolism. suggest t h a t t h e p h o s p h o l i p i d s  species.  They a l s o  o f endogenous o r i g i n may p l a y some p a r t i n  e s t a b l i s h i n g the spectrum o f f e c a l b i l e a c i d s which t y p i f i e s or  these  an animal,  115 On present  the b a s i s of the evidence p r o v i d i n g  the  changes were a sequel e f f e c t s on t r a n s p o r t not been e x c l u d e d .  process.  to m o d i f i c a t i o n o f the  The  increase  An  e f f e c t on t r a n s p o r t  and/or enzyme systems appears expected to r e s u l t from  of the end p r o d u c t s of d i g e s t i o n .  the a d d i t i o n o f 2.57.  s m a l l , because the d i e t was adequate amounts of  l e c i t h i n to the d i e t would be  formulated  to c o n t a i n  Any  increase  c h o l i n e brought relatively  8.07 of u n s a t u r a t e d f a t o  choline.  That b e t a s i t o s t e r o l may  have a f f e c t e d the r e a b s o r p t i o n  acids i s i m p l i c i t  a l s o i n the c o n s i d e r a t i o n s  the  sterol.  i s t i c s of beta s i t o s t e r o l Pollak  to  and/or enzyme systems i n the animal body have however  i n absorption  study of t h i s  these  i n t e s t i n a l m i c r o f l o r a or  i n the e f f e c t s of l i b e r a t e d g l y c e r i d e s , f r e e f a t t y a c i d s and about by  lecithin  p o s s i b i l i t i e s that  u n l i k e l y however as such a marked e f f e c t would not be  and  the  i n v e s t i g a t i o n i t i s tempting to b e l i e v e t h a t the e f f e c t s of  were mediated through the a b s o r p t i v e  an  incentives for  Further  i m p l i c a t i o n may  evidence f o r the  complex between c h o l e s t e r o l and  b i l e a c i d s share some s t r u c t u r a l f e a t u r e s  be  Since  for  charactervalid.  i n v i t r o formation  beta s i t o s t e r o l .  bile  the b a s i s  c o n s i d e r a t i o n of the p h y s i c a l  suggests t h a t t h i s  (1953) advanced s t r o n g  which p r o v i d e d  of  of a  c h o l e s t e r o l and  the  i n common, i t i s p o s s i b l e that  the  p r o p e r t i e s which endow b e t a s i t o s t e r o l w i t h the a b i l i t y to complex w i t h c h o l e s t e r o l allowed t h i s s t e r o l to form s i m i l a r complexes w i t h the acids rendering An  stimulus  them l e s s a b s o r b a b l e .  explanation  beta s i t o s t e r o l  bile  a l s o of the observed e f f e c t s of s o y a l e c i t h i n and  i s t h a t these substances evoked an i n t e n s i f i c a t i o n of  f o r the f l o w o f b i l e .  Since  the  i t i s known t h a t the presence of f a t  I  116 i n the duodenum induces  the f l o w of b i l e through  a r e f l e x mechanism, t h i s  e x p l a n a t i o n i s more p l a u s i b l e i n the case o f the p h o s p h o l i p i d s , because the d e t e r g e n t p r o p e r t i e s of these l i p i d s c o u l d have caused  e m u l s i f i c a t i o n of  i n g e s t e d f a t at an e a r l i e r than normal stage d u r i n g d i g e s t i o n , w i t h  the  r e s u l t of i n c r e a s i n g the s u r f a c e area o f the f a t and p r o v i d i n g f o r g r e a t e r and more e f f e c t i v e c o n t a c t o f i n g e s t e d f a t w i t h the duodenal mucosa. An  important  suggestion contained  v a r i a t i o n i n the b e t a s i t o s t e r o l and administered  i n experiment 3, may  i n these r e s u l t s ,  i s , that  l e c i t h i n content of the d i e t a r y f a t s  have been p a r t l y r e s p o n s i b l e f o r the v a r y i n g  e f f e c t s these f a t s e x e r t e d on b i l e a c i d e x c r e t i o n .  Support  s u g g e s t i o n t h a t b e t a s i t o s t e r o l may  c o n t r i b u t o r to the  be an important  f o r the  r a t e of b i l e a c i d e x c r e t i o n a s s o c i a t e d w i t h the a d m i n i s t r a t i o n of l a r g e amounts of d i e t a r y f a t , may (1957).  These workers found  the h y p o c h o l e s t e r o l e m i c  be found  i n the work of Beveridge  that hydrogenation  p r o p e r t y of c o r n  oil.  et a l .  d i d not " e n t i r e l y  destroy  117  SUMMARY - PART TWO  Experiments were conducted t o e v a l u a t e the a b i l i t y o f d i e t a r y triglycerides,  s o y a l e c i t h i n and b e t a s i t o s t e r o l t o i n f l u e n c e the l e v e l  o f d i g i t o n i n p r e c i p i t a b l e s t e r o l s i n the plasma and the r a t e o f f e c a l excretion of b i l e acids utilizing and  i n cockerels.  The f e c a l b i l e a c i d s were measured  the techniques o f t h i n l a y e r chromatography o f the methyl  gas l i q u i d  chromatography o f the t r i m e t h y l s i l y l e t h e r s  I t was found t h a t when a b a s a l  low-fat  then exchanged f o r one e n r i c h e d  unaffected but  w i t h t r i g l y c e r i d e at a l e v e l o f 157. t h a t  oil  fat.  The l e v e l s were d i e t with corn o i l ,  by 9.3 and 45.4% when replacement was made w i t h h e r r i n g and  coconut o i l r e s p e c t i v e l y . by  o f the s u b s t i t u t e d  t o an extent dependent  by replacement o f c a r b o h y d r a t e i n the b a s a l  increased  esters.  d i e t was f e d t o mature c o c k e r e l s and  plasma s t e r o l l e v e l s remained unchanged o r i n c r e a s e d on the degree o f u n s a t u r a t i o n  o f these  esters  The change t o a f a t - r i c h d i e t was accompanied  a decrease i n the r a t e o f output o f b i l e a c i d s .  With b i r d s f e d a c o r n  supplemented d i e t t h i s decrease was o f the o r d e r o f 7.37..  Corres-  ponding d e c r e a s e s when b i r d s were f e d h e r r i n g and coconut o i l supplemented d i e t s were 19.5 and 38.17. r e s p e c t i v e l y . t r i g l y c e r i d e s studied,  The o r d e r o f e f f e c t i v e n e s s o f the  i n promoting the f e c a l e l i m i n a t i o n o f b i l e  acids--  c o r n o i l y h e r r i n g o i l ^ > coconut o i l was found t o r e l a t e i n v e r s e l y t o t h a t f o r i n c r e a s i n g the l e v e l o f c i r c u l a t i n g s t e r o l s - - c o c o n u t  o i l ^>  h e r r i n g oil")>  corn o i l . Beta s i t o s t e r o l and s o y a l e c i t h i n were s t u d i e d  i n a separate  experiment at d i e t a r y l e v e l s o f 0.5 and 2.57. r e s p e c t i v e l y u s i n g  growing  118 cockerels.  The two supplements caused a s l i g h t n o n s i g n i f i c a n t  i n the l e v e l o f c i r c u l a t i n g s t e r o l s and promoted an i n c r e a s e fecal excretion of b i l e acids.  i n the r a t e o f  I t was found t h a t the a d m i n i s t r a t i o n o f  b e t a s i t o s t e r o l l e d t o an i n c r e a s e o f 307. i n the r a t e o f b i l e excretion. ificance  This  reduction  acid  i n c r e a s e bordered on s i g n i f i c a n c e a t the 57. l e v e l o f s i g n -  (F r a t i o c a l c u l a t e d =7.53, s i g n i f i c a n t F r a t i o = 7.71).  significant  i n c r e a s e o f 100% i n the r a t e o f e l i m i n a t i o n o f b i l e a c i d s was  found t o r e s u l t from the a d m i n i s t r a t i o n  of s o y a l e c i t h i n .  soyalecithin also resulted i n a highly s i g n i f i c a n t growth o f the b i r d s s t u d i e d . increased,  A highly-  I n both i n s t a n c e s  Treatment w i t h  increase  i n the r a t e o f  when b i l e a c i d e x c r e t i o n was  the i n c r e a s e was found t o be due p r i n c i p a l l y t o an i n c r e a s e  concentration  of b i l e acids  i n the f e c e s .  a c i d s were t e n t a t i v e l y c l a s s i f i e d b o t h supplements i n c r e a s e d extent t h a t the major ones.  i n the  I t was found t h a t when the b i l e  i n t o two groups o f major- and minor, t h a t  the e x c r e t i o n o f t h e minor b i l e a c i d s t o a g r e a t e r T h i s d i f f e r e n t i a l e f f e c t was more pronounced  when i t o r i g i n a t e d w i t h supplementation o f the d i e t w i t h s o y a l e c i t h i n .  119 PART I I I PARTIAL ELUCIDATION OF MECHANISMS EXPERIMENT 5 THE  EFFECT OF DIETARY BETA SITOSTEROL AND OF CIRCULATING STEROLS IN THE  SOYALECITHIN ON THE  LEVEL  LITHOCHOLIC ACID-FED COCKEREL  The mechanism(s) r e s p o n s i b l e f o r the  increases  in bile acid  e x c r e t i o n which were brought about by a d d i t i o n of s o y a l e c i t h i n and  beta  s i t o s t e r o l to the d i e t of c o c k e r e l s i n experiment 4 were not d e f i n e d by r e s u l t s obtained.  The  evidence  on which the study of these  based suggested t h a t an e f f e c t on b i l e a c i d r e a b s o r p t i o n was  the  changes.  t h a t these  f u r t h e r i n v e s t i g a t e d f o r e f f e c t s on b i l e a c i d  absorption.  Because h y p e r c h o l e s t e r o l e m i a  therefore considered  may  acid-fed chick provides  important  be r e a d i l y induced  c h i c k e n by the a d m i n i s t r a t i o n o f d i e t a r y l i t h o c h o l i c a c i d , the  i n the  lithocholic  a good model f o r i n v e s t i g a t i n g the e f f e c t s  d i e t a r y substances on b i l e a c i d a b s o r p t i o n .  Information  regarding  of this  would r e s i d e i n the plasma s t e r o l l e v e l s when the agents under study added t o the l i t h o c h o l i c a c i d supplemented d i e t . the l i t h o c h o l i c a c i d - f e d c o c k e r e l f o r s t u d y i n g and  was  causal  f a c t o r i n v o l v e d i n these substances be  I t was  substances  the  T h i s experiment  the e f f e c t of b e t a  effect  are  utilizes sitosterol  s o y a l e c i t h i n on b i l e a c i d a b s o r p t i o n .  Experimental S i n g l e Comb White Leghorn c o c k e r e l s 37 days o l d were at random to 8 l o t s of 15 b i r d s and Beta s i t o s t e r o l was first  crushed  incorporated  assigned  f e d the v a r i o u s d i e t s shown i n Table  i n t o the d i e t  at a l e v e l o f 1.0%.  It  i n t o a f i n e powder u s i n g a mortar and p e s t l e and passed 3  5.1. was  TABLE 5.1.  Composition  of d i e t s f e d i n experiment  Ingredients  5  7. of d i e t 8.00 51.54 31.46 2.00 2.00 1.00 3.00 0.50 0.22 0.15  Corn o i l Ground y e l l o w c o r n Soyabean meal Dehydrated c e r e a l g r a s s Dried d i s t i l l e r s ' solubles Limestone Bonemeal Iodized s a l t C h o l i n e c h l o r i d e (377.) D-L methionine  mg./kgm. o f d i e t 220.00 1.10 6.60 18.48 44.00  Manganese s u l f a t e Folacin Riboflavin C a l c i u m pantothenate Niacin  units/kgm. of d i e t 1997.60 199.76  V i t a m i n A (I.U.) V i t a m i n D- (I.C.U.)  D i e t number  Supplements  1  none  2  2.07.  3  1.0%  4  2.07. s o y a l e c i t h i n + 1.07. b e t a s i t o s t e r o l  5  0.2%  l i t h o c h o l i c acid  6  0.2% 2.0%  l i t h o c h o l i c acid soyalecithin  +  7  0.2% 1.0%  l i t h o c h o l i c acid beta s i t o s t e r o l  +  8  0.27. l i t h o c h o l i c a c i d 2.07. s o y a l e c i t h i n + 1.0% b e t a s i t o s t e r o l  +  soyalecithin beta s i t o s t e r o l  121 times through a fine-meshed s i e v e . 2.07c.  S o y a l e c i t h i n was added at a l e v e l of  I t was premixed w i t h c o r n o i l to f a c i l i t a t e m i x i n g .  randomly a s s i g n e d t o s i n g l e  The d i e t s  l o t s o f b i r d s and f e d f o r 12 days.  were  At the end  o f t h i s time blood samples were taken from the wing v e i n o f the b i r d s and pooled on a treatment b a s i s . as  The plasma o b t a i n e d was analysed f o r DPS  i n the p r e v i o u s experiments.  R e s u l t s and D i s c u s s i o n Part a  In t h i s experiment, the change i n the l e v e l of c i r c u l a t i n g of the l i t h o c h o l i c a c i d - f e d c h i c k i n response to d i e t a r y w i t h b e t a s i t o s t e r o l and s o y a l e c i t h i n effect  sterols  supplementation  has been used as a c r i t e r i o n o f an  o f these supplements on b i l e a c i d  absorption.  For this  criterion  to be v a l i d the f o l l o w i n g two c o n d i t i o n s should be s a t i s f i e d . 1.  That a b s o r p t i o n of s t e r o l s  to the l e v e l o f c i r c u l a t i n g  makes o n l y a minor  contribution  sterols.  Both l e c i t h i n and b e t a s i t o s t e r o l a r e known t o a f f e c t of c h o l e s t e r o l . the  the a b s o r p t i o n  The former has been shown t o enhance i t ( P e t e r s o n , 1952b)  l a t t e r to interfere  w i t h i t (see r e v i e w experiment 4 ) . A change induced  i n the plasma s t e r o l l e v e l s when b e t a s i t o s t e r o l and l e c i t h i n a r e f e d and when t h i s c o n d i t i o n i s not f u l f i l l e d would not s p e c i f y or b i l e a c i d  a b s o r p t i o n were a f f e c t e d .  employed i n the present s t u d y .  whether  A low c h o l e s t e r o l  cholesterol  d i e t was t h e r e f o r e  In a d d i t i o n , a h i g h d i e t a r y l e v e l o f 0.27c.  l i t h o c h o l i c a c i d was u t i l i z e d although a l e v e l as low as 0.17» would have sufficed  t o induce a l e s s  severe h y p e r c h o l e s t e r o l e m i a .  L e v e i l l e and  122 Sauberlich induced  (1966) have advanced evidence  hypercholesterolemia  i s due  showing t h a t l i t h o c h o l i c  t o a derangement i n l i p o p r o t e i n metabolism  and hence not p r i n c i p a l l y t o s t e r o l r e a b s o r p t i o n . induced  acid-  The  high c h o l e s t e r o l levels  by d i e t a r y l i t h o c h o l i c a c i d would t h e r e f o r e be expected  t o mask the  r e l a t i v e l y s m a l l c o n t r i b u t i o n made by endogenous s t e r o l s to the plasma sterol  levels. 2.  That the substances  under study be unabsorable  or of  low  absorbability. It  i s e v i d e n t t h a t the a b s o r p t i o n of a substance  s i t o s t e r o l would i n v a l i d a t e the index of b i l e plasma s t e r o l l e v e l s .  such as  a c i d a b s o r p t i o n , namely,  With r e s p e c t t o t h i s s t e r o l however, i t has been shown  t o be v i r t u a l l y u n a b s o r b a b l e .  In a balance  study conducted w i t h r a t s and  r a b b i t s f e d b e t a s i t o s t e r o l , the s t e r o l c o u l d not be d e t e c t e d and was  recovered  q u a n t i t a t i v e l y i n the f e c e s  i n the t i s s u e s  (Schoenheimer, 1931).  r e s u l t s o f a r e c e n t study have i n d i c a t e d t h a t b e t a s i t o s t e r o l i s but o n l y to a n e g l i g i b l e e x t e n t .  The  absorbable  S w e l l e t a l . (1959) showed t h a t o n l y  o f a t r a c e r does of b e t a s i t o s t e r o l a d m i n i s t e r e d r e c o v e r a b l e i n the lymph 6 hours a f t e r . c a r r i e d out  beta  i n t h i s l a b o r a t o r y and  o r a l l y to r a t s  0.3%  was  Experiments w i t h mature c o c k e r e l s  i n v o l v i n g the d e t e r m i n a t i o n  o f plasma  s t e r o l l e v e l s on a s i t o s t e r o l - e n r i c h e d  d i e t have a l s o l e d t o the c o n c l u s i o n  that beta s i t o s t e r o l i s unabsorbable.  Although  b e t a s i t o s t e r o l has i n r a t s (Gerson  intraperitoneally  been shown to a c c e l e r a t e the f o r m a t i o n of b i l e  e t a l . , 1965), i t does not appear l i k e l y t h a t t h i s  would be o f importance when the s t e r o l i s a d m i n i s t e r e d The  injected  i n the  acids effect  diet.  l i k e l i h o o d o f an enhancement o f s i t o s t e r o l a b s o r p t i o n o c c u r r i n g  i n the presence of a d i e t a r y excess  of l i t h o c h o l i c a c i d may  be  discounted  123 when the experiment of Vahouny e_t al_. (1959) i s taken i n t o These workers found t h a t whereas, c h o l i c , t a u r o c h o l i c and increased  r  deoxycholic  and  cholanic acids did  augmentation of the amount of c h o l e s t e r o l absorbed by  cholantes  the  g l y c o c h o l i c acids  the c h o l e s t e r o l content of t h o r a c i c duct lymph of r a t s when f e d  with c h o l e s t e r o l , l i t h o c h o l i c , The  consideration.  appears to be  the  not.  trihydroxy-  c l o s e l y r e l a t e d to t h e i r a b i l i t y to a c t i v a t e  enzymes concerned i n c h o l e s t e r o l a b s o r p t i o n .  Cholesterol esterase  c h o l e s t e r o l e s t e r i f y i n g enzyme have been shown to e x h i b i t a s p e c i f i c ment f o r t h i s group of b i l e a c i d s Gauguly, 1962;  Swell  (Hernandez and  et a l . , 1953).  ations  i t s esters  i s not  due  require-  Murthy  ( S w e l l e_t al_.,  1953) and  to d i f f e r e n c e s i n a b s o r p t i o n mechanisms but  interference with absorption.  i t would appear t h e r e f o r e  t h a t the  l i t h o c h o l i c a c i d on c h o l e s t e r o l a b s o r p t i o n  On  and  also,  t h a t the d i f f e r e n c e i n a b s o r b a b i l i t y between c h o l e s t e r o l  beta s i t o s t e r o l structural  1957;  These enzymes show a c t i v i t y  (though l e s s ) towards b e t a s i t o s t e r o l and suggesting  Chaikoff,  and  to  the s t r e n g t h o f these observ-  f i n d i n g o f a l a c k of e f f e c t of would apply  a l s o to b e t a  sito-  sterol . The proviso  use  of l e c i t h i n as a d i e t a r y supplement d i d not  of n o n a b s o r b a b i l i t y  the d i e t a l r e a d y i t would not be absorption  contained  of the d i e t a r y supplements. a high  of f a t .  c h o l i n e would serve  However, because  increase  i n the  f a t t y a c i d s r e s u l t i n g from s u p p l e m e n t a t i o n of  the d i e t w i t h l e c i t h i n would add absorption  the  l e v e l o f u n s a t u r a t e d f a t (8% c o r n o i l )  expected t h a t the r e l a t i v e l y s m a l l  of g l y c e r i d e s and  satisfy  f u r t h e r to a s t e r o l e m i c  response due  to  By the same argument, f o r t i f i c a t i o n of the d i e t with to minimize any  sterolemic  e f f e c t of the  choline  124 l i b e r a t e d d u r i n g the d i g e s t i o n .of  lecithin.  Part b  The  concentration  of DPS  found i n the plasma of c o c k e r e l s  days f o l l o w i n g f e e d i n g o f the v a r i o u s d i e t s are shown i n T a b l e data  of DPS  i n plasma o f c o c k e r e l s  s i t o s t e r o l but  no  experiment 4.  hypocholesterolemic  s i t o s t e r o l and  characteristics.  i n c r e a s e of 2287«.  i n plasma DPS  When b e t a  s i t o s t e r o l was  by  sitosterol  a c i d may natively,  added to  the  the  i n moderating  the  and  interest.  of plasma s t e r o l s  intestinal  excretion  f u r n i s h s t r o n g evidence t h a t  have suppressed the endogenous e x c r e t i o n of c h o l e s t e r o l . i t may  a result  inhibits  i s p a r t i c u l a r l y so, because an e l e v a t e d c o n c e n t r a t i o n  These o b s e r v a t i o n s  occurred  mg.7.  l i t h o c h o l i c a c i d i s of  should have givenx'ise to an i n c r e a s e i n h e p a t i c  the  f u r t h e r changes i n t h i s  of c h o l e s t e r o l i t s complete l a c k of e f f e c t induced  As  reached a l e v e l o f 377  With the knowledge t h a t beta  degree of h y p e r c h o l e s t e r o l e m i a  i n l i n e with  lithocholic acid.  d i e t supplemented w i t h l i t h o c h o l i c a c i d alone no  of c h o l e s t e r o l .  lower than  soyalecithin display  A marked r i s e  of t h i s d i e t a r y m o d i f i c a t i o n plasma DPS  absorption  beta  tended to be  c o n s i s t e n t d i f f e r e n c e s are  upon s u p p l e m e n t a t i o n of the b a s a l d i e t w i t h  l e v e l were n o t e d .  concentration  These r e s u l t s bear a c l o s e resemblance to those of  These s m a l l but  an  The  These  clearly  f e d d i e t s c o n t a i n i n g l e c i t h i n and  reports p r e v i o u s l y c i t e d that beta  representing  induced.  l i t h o c h o l i c a c i d were s i m i l a r and  t h a t of c o n t r o l s .  This  5.II.  show t h a t when l i t h o c h o l i c a c i d x^as excluded from the d i e t  d e f i n e d d i f f e r e n c e s of d i e t a r y o r i g i n were not  12  lithocholic Alter-  have suppressed the e x c r e t i o n of some substance r e q u i r e d  TABLE 5.II.  Plasma DPS l e v e l s (mg.%) o f c o c k e r e l s fed v a r i o u s d i e t s i n experiment 5  D i e t a r y supplement  DPS  None  115  2.0% s o y a l e c i t h i n  101  1.0% b e t a s i t o s t e r o l  102  2.0% s o y a l e c i t h i n + 1% b e t a s i t o s t e r o l  99  0.2% l i t h o c h o l i c a c i d  377  0.2% l i t h o c h o l i c a c i d + s o y a l e c i t h i n  565  0.2% l i t h o c h o l i c a c i d + b e t a s i t o s t e r o l  377  0.27c. l i t h o c h o l i c a c i d + b e t a s i t o s t e r o l + soyalecithin  484  126 for  i t to e x e r t  i t s hypocholesterolemic  action e f f e c t i v e l y  The  f a i l u r e of b e t a s i t o s t e r o l to a l l e v i a t e the h y p e r c h o l e s t e r o l e m i a  i n t e r f e r i n g with l i t h o c h o l i c a c i d absorption i s not  e f f e c t i v e against  presence of a s p e c i f i c  gut. by  suggests t h a t e i t h e r the  l i t h o c h o l i c acid absorption  agent i n the gut  i n the  or again  that  sterol  the  is obligatory for i t s effective  action. The  i n c o r p o r a t i o n of l e c i t h i n  i n t o the l i t h o c h o l i c a c i d  mented d i e t f o s t e r e d a pronounced i n c r e a s e of 188 concentration about by  of c i r c u l a t i n g DPS.  the a b s o r p t i o n  f o r reasons a l r e a d y of c h o l e s t e r o l was  o f the end  cited.  That t h i s  endogenous c h o l e s t e r o l was  and  (49.9%) i n the c o u l d have been brought  p r o d u c t s of l e c i t h i n d i g e s t i o n i s d o u b t f u l t h a t augmented  p r o b a b l y lowered as evidenced by  e f f e c t of s o y a l e c i t h i n on  r e s u l t s therefore point  l i t h o c h o l i c acid absorption.  a d m i n i s t r a t i o n o f l e c i t h i n promoted an of b i l e a c i d s  i n the p r e v i o u s  These f u n c t i o n s  to an  Because  i n the f e c a l  One  o f the b i l e a c i d s the o t h e r  a c i d s and/or r e d u c i n g  b a c t e r i a l a c t i o n i n the d i g e s t i v e  high  enhancing the  elimination the  f u n c t i o n i n b i l e a c i d metabolism i n  are opposed to each o t h e r .  i n c r e a s i n g the r e a b s o r p t i o n b i l i a r y output of b i l e  increase  an a l r e a d y  experiment, these r e s u l t s a l s o convey  meaning t h a t l e c i t h i n performs a d u a l the gut.  inability  appear l i k e l y t h a t endogenous  s t e r o l s c o u l d have superimposed such a l a r g e increment on The  the  l i t h o c h o l i c a c i d induced hyper-  s e c o n d l y because i t does not  l e v e l of c i r c u l a t i n g s t e r o l s .  reabsorption  f i r s t l y because the r a t e of e x c r e t i o n of  o f b e t a s i t o s t e r o l alone to a l l e v i a t e the cholesterolemia,  increase  It is equally doubtful  responsible,  mg.%  supple-  tract.  consists in  in increasing  their reabsorption  by  the  promoting  127 On the l i t h o c h o l i c  a c i d supplemented d i e t  the DPS c o n c e n t r a t i o n  i n plasma o f b i r d s r e c e i v i n g l e c i t h i n and b e t a s i t o s t e r o l  simultaneously  (484 mg.%) f e l l between those o f b i r d s r e c e i v i n g l e c i t h i n a l o n e (565 mg.7o) and those r e c e i v i n g n e i t h e r supplement l e v e l accounts  (377 mg.7.).  This intermediate  f o r an i n c r e a s e o f 107 mg.7. (28.47.) above the l e v e l  plasma o f c o c k e r e l s f e d the d i e t  supplemented w i t h l i t h o c h o l i c  acid  i n the alone.  I t may be reasoned t h a t beta s i t o s t e r o l made the l e c i t h i n l e s s  available  f o r p o t e n t i a t i n g the h y p e r c h o l e s t e r o l e m i a .  lecithin  On the o t h e r hand,  c o u l d have made p o s s i b l e the i n t e r f e r e n c e w i t h l i t h o c h o l i c by beta s i t o s t e r o l .  I t i s not p o s s i b l e t o d i f f e r e n t i a t e  mechanisms u s i n g the data o b t a i n e d .  acid absorption  between the two  128 EXPERIMENT 6 PLASMA STEROL LEVELS OF  GROWING COCKERELS ADMINISTERED POTASSIUM  LITHOCHOLATE WHILE RECEIVING A SOYALECITHIN OR SUPPLEMENTED DIET BUT AT The  and  that  r e s u l t s were not  EACH DIET  12 HOUR INTERVALS  b e t a s i t o s t e r o l on  gave s t r o n g i n d i c a t i o n s the  EQUAL AMOUNTS OF  r e s u l t s of experiment 5 d i d not  of s o y a l e c i t h i n  but  FED  BETA SITOSTEROL  a f f o r d p r o o f of an  l i t h o c h o l i c acid absorption.  l e c i t h i n enhanced the  definitive.  undertaken, to throw more l i g h t on  effect  a b s o r p t i o n of t h i s  A second p i l o t  the mechanism  They  experiment was  acid  therefore  involved.  Experimental Forty-eight and  within  On  12 b i r d s the  l e c i t h i n at the  c o n t a i n i n g 93.3  To  o f 0.28  ml.  of  and  fed  to two  The  lots.  The  ml.  potassium s a l t was  grams of  f l a s k which was  then p l a c e d  I f s o l u t i o n was  of a  centrifuged  equivalent follows:  36.11  ml.  then added to j u s t below  the  i n a beaker of b o i l i n g water u n t i l  complete, s m a l l amounts o f f r e e  The  to 5 x 40 ml.  at 1500  lots  solution  made as  f l a s k u n t i l no more went i n t o s o l u t i o n .  removal of excess a l k a l i .  3.0%  remaining  l i t h o c h o l i c a c i d and  D i s t i l l e d water was  i t s contents t r a n s f e r r e d  s o l u t i o n was  t h i s d i e t supplemented w i t h  S i x hours l a t e r 5.0  to a l l b i r d s .  were added back to the ensured the  at 10 a.m.  v o l u m e t r i c f l a s k 3.75  acid dissolved.  grams were a s s i g n e d at random  l i t h o c h o l i c a c i d as the p o t a s s i u m s a l t  N-K0H were added.  neck o f the the  day  the b a s a l d i e t .  mg.  - 350  days o l d  fed the b a s a l d i e t of experiment 5 f o r  expense o f c o r n o i l was  g i v e n by p i p e t t e a 200  and  fourth  were c o n t i n u e d on  was  Comb White Leghorn c o c k e r e l s 42  the body weight range of 300  to 4 l o t s of 3 days.  Single  f l a s k was  This  then made up  centrifuge  r.p.m. f o r 5 minutes.  tubes. The  The clear  acid step  to volume hot salt  129 s o l u t i o n was then removed by p i p e t t e from the s p a r i n g l y s o l u b l e a c i d at the bottom o f the tube.  The d i e t s were a l t e r n a t e d a t 12 hour i n t e r v a l s but  no changes i n the time o f p i p e t t i n g were made.  Under these c o n d i t i o n s the  b i r d s r e c e i v e d s i m i l a r amounts o f each d i e t but one group r e c e i v e d the b i l e s a l t s o l u t i o n d u r i n g i n g e s t i o n o f t h e b a s a l d i e t whereas the o t h e r r e c e i v e d i t d u r i n g i n g e s t i o n o f the l e c i t h i n supplemented d i e t . c o n t i n u e d f o r 7 days,  Treatment was  a t the end o f which time, pooled samples o f plasma  were o b t a i n e d from each l o t o f b i r d s . Feed consumption d a t a were a l s o  These were a n a l y s e d f o r DPS as b e f o r e .  recorded.  R e s u l t s and D i s c u s s i o n  The  c o n c e n t r a t i o n o f DPS found  c o c k e r e l s a f t e r 7 days o f potassium i n T a b l e 6.1.  i n the plasma o f the experimental  l i t h o c h o l a t e a d m i n i s t r a t i o n are given  From these d a t a i t may be seen t h a t plasma s t e r o l l e v e l s were  u n a f f e c t e d by t h e d i e t consumed w i t h the b i l e s a l t . results  i t s h o u l d be concluded  On the b a s i s o f these  t h a t l e c i t h i n d i d not enhance the a b s o r p t i o n  o f l i t h o c h o l i c a c i d i n experiment  4.  T h i s c o n c l u s i o n has n o t been reached  however, because i n t e r p r e t a t i o n o f these d a t a has been made, b e a r i n g i n mind t h a t the amount o f b i l e s a l t a d m i n i s t e r e d was p r o b a b l y o f a q u a n t i t y l a r g e enough t o p r e v e n t  the i n g e s t e d f a t from e x e r t i n g a d i s c e r n i b l e e f f e c t on the  a b s o r p t i o n o f the b i l e s a l t .  I t i s r e a s o n a b l e t o b e l i e v e t h a t the amount  of i n g e s t e d f a t which was p r e s e n t  i n the crop at the moment when the b i l e  s a l t was i n t r o d u c e d was g r o s s l y d i s p r o p o r t i o n a t e t o the dosage.  The b i r d s  were b e i n g f e d ad 1 i b i t u m and hence were consuming the d i e t s i n s m a l l quantities.  More m e a n i n g f u l  d a t a would have been o b t a i n e d i f the b i l e  salt  TABLE 6.1.  Plasma DPS l e v e l s (mg.7.) o f c o c k e r e l s a d m i n i s t e r e d potassium l i t h o c h o l a t e on d i f f e r e n t d i e t s  D i e t f e d when b i l e administered  salt  Basal  Lecithin  supplemented  Plasma DPS Replicate Treatment  396 422  409  434 422  428  131 was  administered  i n s m a l l e r dosages and  consumed when the b i l e s a l t was  as an emulsion  administered.  T h i s important c o n s i d e r a t i o n  of the amount of f a t t h a t should have been p r e s e n t u n a t e l y not taken The  i n t o account  i n the crop was  over the course of the  unfort-  experiment.  r e s u l t s are a l s o open to the i n t e r p r e t a t i o n t h a t because the  more s o l u b l e b i l e s a l t was The  w i t h the f a t b e i n g  administered  the e f f e c t of l e c i t h i n  disappeared.  p o t e n t i a t i n g e f f e c t of s o y a l e c i t h i n on the h y p e r c h o l e s t e r o l e m i a  by l i t h o c h o l i c a c i d f e e d i n g would then be e x p l a i n e d as due i n the s o l u b i l i z a t i o n and  consequently,  induced  to an i n c r e a s e  the a b s o r p t i o n of l i t h o c h o l i c  acid.  T h i s i n t e r p r e t a t i o n does not however e l i m i n a t e the p o s s i b i l i t y t h a t an increase i n b i l e s a l t l e c i t h i n had  a b s o r p t i o n c o u l d have o c c u r r e d i n the presence  t h e r e been the proper r e l a t i o n between s i z e of dosage and amount  o f i n g e s t e d f a t i n the c r o p . oil  present  Furthermore the l a r g e excess  i n the b a s a l d i e t would no doubt have f u l f i l l e d  f o r s o l u b i l i z a t i o n of the s m a l l amount of l i t h o c h o l i c a c i d Higher DPS  l e v e l s were induced  of d i e t a r y c o r n the  r e a d i l y absorbed  than the f r e e a c i d .  of 50 grams per b i r d per day  were used and  93.3  amount r e s u l t e d  diet.  i n b i r d s used i n the p r e s e n t s a l t was  i s assumed f o r the b i r d s used i n the  previous  of l i t h o c h o l i c  In the p r e s e n t experiment, b i r d s o f a comparable gm.  more  I f an average r a t e o f f e e d consumption  experiment then those b i r d s would have consumed 100 mg. d a i l y f o r 12 days.  requirement  i n the  experiment compared to experiment 5, s u g g e s t i n g t h a t the b i l e  here may  of  of b i l e s a l t a d m i n i s t e r e d d a i l y f o r 7 days.  i n h i g h e r plasma s t e r o l  levels.  not n e c e s s a r i l y apply to experiment 5.  acid  age This  Thus the c o n c l u s i o n s drawn The  potassium  a d m i n i s t e r e d because of the ease w i t h which i t can be d i s p e n s e d  salt  was  in solution  132 from a p i p e t t e .  C o n s i d e r a b l e d i f f i c u l t y was  s u i t a b l e emulsion  experienced  u s i n g the f r e e a c i d at the d e s i r e d c o n c e n t r a t i o n .  was  a l s o the f u r t h e r problem of d i s p e n s i n g the emulsion  the  crop. The  i n making a  quantitatively  data f o r f e e d consumption are shown i n T a b l e 6 . I I .  t h a t f o r the f i r s t  day of treatment  consumption of the d i e t which was  t h e r e was  gms./12 h r s . ) .  administered  the l a r g e n e s s o f the dosage.  This e f f e c t Judging  They show  i n the 12 hour i n t e r v a l  i s probably  from the f e e d consumption data f o r effect.  supplemented d i e t at the time when they r e c e i v e d the b i l e s a l t ,  (21.6).  (17.2 The  gms./period/bird)  decrease  121  an i n d i c a t i o n of  d a t a of T a b l e 6.II seem to i n d i c a t e a l s o t h a t b i r d s consuming the  this diet  fol-  (472 vs  the f o l l o w i n g days the b i r d s appeared to have a d j u s t e d to t h i s The  into  a d e p r e s s i o n i n the r a t e o f  lowing t h a t d u r i n g which the b i r d s were t r e a t e d w i t h b i l e s a l t and 496 vs 220  There  lecithin  ate l e s s of  compared to the b i r d s r e c e i v i n g  i t after  i n f e e d consumption appeared to have been compensated  by the consumption of an i n c r e a s e d amount o f the b a s a l d i e t  (27.3  gms.)  which f o l l o w e d . An o b s e r v a t i o n worthy of mention i n the p r e s e n t t h e r e was as was  no v i s i b l e evidence  apparent  of hemolysis  i n the p r e v i o u s experiment.  a d m i n i s t e r i n g the potassium  salt  experiment i s t h a t  of the c i r c u l a t i n g e r y t h r o c y t e s Thus i t would appear t h a t by  i n s t e a d o f the f r e e a c i d the  and h y p e r c h o l e s t e r o l e m i c p r o p e r t i e s of l i t h o c h o l i c a c i d may The  ambiguities  i n the r e s u l t s o b t a i n e d  i n d i c a t e t h a t much refinement  hemolytic  be d i s s o c i a t e d .  i n the p r e s e n t  and m o d i f i c a t i o n o f the technique  should be made i f the t e c h n i q u e  experiment utilized  i s to be used to e v a l u a t e the e f f e c t  of  133  TABLE 6 . I I .  Twelve (12) hour feed consumption o f experimental cockerels  Basal  diet  Treatment 1 Day 1 2 3 4 5 6 7 Total Avg. Avg./b i r d  Replicates 249 278 248 238 241 228 281  223 220 362 286 273 294 289  1763  1947  251.9  278.1  21.0  23.2  Treatment 2  Treatment 472* 498* 610* 524* 514* 522* 570* 3710 530 22.1  Lecithin  Replicates 94 349 301 403 358 371 362  126 351 391 362 368 365 374  2238  2337  319. 7  333.9  26. 7  27.8  supplemented  Total Avg. Avg./bird  50 373 253 296 258 275 281  71 409 252 277 265 270 294  1786  1838  255.1  262.6  21.3  21.9  220** 700** 692** 765** 726** 736** 736** 4575 654 27.3  diet  Treatment 2  Treatment 1 1 2 3 4 5 6 7  Treatment  121** 782** 505** 573** 523** 545** 575** 3624 518 21.6  228 189 220 196 184 192 200  268 147 219 262 200 185 189  1409  1472  201. 3  210.3  16.8  17.5  * d i e t f e d when b i l e s a l t was a d m i n i s t e r e d ** d i e t f e d d u r i n g the 12 hour i n t e r v a l f o l l o w i n g  496* 338* 439* 458* 384* 377* 389* 2881 412 17.2  134 l e c i t h i n on be  clearly  l i t h o c h o l i c acid inconclusive.  absorption.  These r e s u l t s  are  considered  to  135  SUMMARY - PART THREE  Two experiments were undertaken i n an attempt t o d e l i n e a t e the mechanisms r e s p o n s i b l e f o r i n c r e a s i n g  b i l e acid excretion  s i t o s t e r o l and s o y a l e c i t h i n a r e f e d .  I n the f i r s t ,  when b e t a  the e f f e c t o f the two  substances on the c o n c e n t r a t i o n o f d i g i t o n i n - p r e c i p i t a b l e  s t e r o l s i n the  plasma o f the l i t h o c h o l i c a c i d - f e d  R e s u l t s were  o b t a i n e d showing t h a t promoted a f u r t h e r  cockerel  was s t u d i e d .  s o y a l e c i t h i n when a d m i n i s t e r e d as the o n l y supplement  i n c r e a s e o f 507c. i n the l e v e l o f c i r c u l a t i n g s t e r o l s but  t h a t b e t a s i t o s t e r o l was without e f f e c t .  When the supplements were g i v e n  t o g e t h e r the plasma s t e r o l l e v e l f e l l between that  observed when l i t h o c h o l i c  a c i d alone was a d m i n i s t e r e d and t h a t when s o y a l e c i t h i n  i n addition to  l i t h o c h o l i c a c i d was f e d . These r e s u l t s were taken as evidence soyalecithin  that  augmented the a b s o r p t i o n o f l i t h o c h o l i c a c i d and t h a t  l i t h o c h o l i c a c i d suppressed t h e endogenous e x c r e t i o n some substance r e q u i r e d  of cholesterol  and/or  f o r b e t a s i t o s t e r o l t o express i t s h y p o c h o l e s t e r o l e m i c  characteristics. In the second experiment potassium l i t h o c h o l a t e was a d m i n i s t e r e d by p i p e t t e  d a i l y f o r seven days i n t h e amount o f 93.3 mg. t o two groups o f  growing c o c k e r e l s o f t h e same s t a r t i n g weight.  One group o f b i r d s  received  the b i l e s a l t w h i l e consuming a b a s a l d i e t c o n t a i n i n g 8.0% o f c o r n o i l , the  other while r e c e i v i n g  t h i s d i e t supplemented w i t h 3.07» s o y a l e c i t h i n .  The  d i e t s were a l t e r n a t e d  a t 12 hour i n t e r v a l s .  were n o t a f f e c t e d administered.  The plasma s t e r o l l e v e l s  by the d i e t consumed a t the time when the b i l e s a l t was  Because d e f i c i e n c i e s  i n the t e c h n i q u e were l a t e r r e c o g n i z e d  136  no c o n c l u s i o n s were drawn. experiment i s d i s c u s s e d .  The s i g n i f i c a n c e o f the r e s u l t s o f the f i r s t  137  GENERAL SUMMARY  The m e s e n t e r i c  s m a l l i n t e s t i n e o f the c h i c k e n was i n v e s t i g a t e d  f o r the a b i l i t y t o v a r y the plasma s t e r o l l e v e l through i t s a b s o r p t i v e capacity f o r b i l e s a l t . i n v i v o technique  Absorptive  c a p a c i t y was s t u d i e d w i t h the use o f an  which was developed f o r a c c o m p l i s h i n g  the i n v e s t i g a t i o n  and was determined by measuring the amount o f b i l e s a l t absorbed from e i t h e r open segments o r c l o s e d sacs o f i n t e s t i n e . t h i s technique  I t was demonstrated u s i n g  t h a t along the l e n g t h o f the m e s e n t e r i c  small  intestine,  a g r a d i e n t e x i s t s i n a b s o r p t i v e c a p a c i t y f o r b i l e s a l t w i t h the g r e a t e s t c a p a c i t y f o r a b s o r p t i o n , r e s i d i n g i n the t e r m i n a l p o r t i o n . g r a d i e n t and c a p a c i t y was e x h i b i t e d when t a u r o c h o l a t e studied.  The same  and g l y c o c h o l a t e were  When the c o n c e n t r a t i o n o f d i g i t o n i n - p r e c i p i t a b l e s t e r o l s  i n the  plasma o f mature c o c k e r e l s was s t u d i e d i n c o n j u n c t i o n w i t h the a b s o r p t i v e c a p a c i t y f o r b i l e s a l t , a t two c o n c e n t r a t i o n s  of b i l e s a l t  (0.15 and 0.757«)  i t was found t h a t the two v a r i a b l e s were independent o f each o t h e r .  Since  there  should  i s convincing  evidence  determine t o a l a r g e extent  t h a t the amount o f b i l e s a l t reabsorbed  the l e v e l o f c i r c u l a t i n g s t e r o l s , one i n t e r -  p r e t a t i o n which was p l a c e d on these r e s u l t s was t h a t some o f the components of the d i e t o r o f substances e x c r e t e d of b i l e  endogenously, determine the amount  s a l t s made a v a i l a b l e f o r r e a b s o r p t i o n and were i n d i r e c t l y  f o r e s t a b l i s h i n g the l e v e l o f c i r c u l a t i n g s t e r o l s  responsible  i n the b i r d s s t u d i e d .  A s e r i e s o f experiments was t h e r e f o r e undertaken t o assess the a b i l i t y o f some c l a s s e s o f f a t t o e l i c i t e x c r e t i o n o f b i l e a c i d s and c o n c o m i t a n t l y  a change i n the r a t e o f f e c a l modify the l e v e l o f plasma s t e r o l s .  138 In the f i r s t  experiment o f the s e r i e s , d i e t s formulated  t o c o n t a i n 15% o f  supplementary t r i g l y c e r i d e s o f v a r y i n g degree o f u n s a t u r a t i o n mature c o c k e r e l s  i n exchange f o r a b a s a l l o w - f a t  diet.  were f e d t o  I t was found  that  when a c o r n o i l supplemented d i e t was s u b s t i t u t e d f o r .the b a s a l d i e t the plasma s t e r o l l e v e l remained unchanged but that when h e r r i n g o i l and coconut oil  supplemented d i e t s were s u b s t i t u t e d , the l e v e l i n c r e a s e d by 9.2 and  45.37. r e s p e c t i v e l y . received  The amount o f b i l e a c i d s e x c r e t e d  d a i l y when the b i r d s  the f a t - e n r i c h e d d i e t s was i n v a r i a b l y l e s s than when they r e c e i v e d  the b a s a l d i e t .  The magnitude o f the change i n e x c r e t i o n r a t e s brought  about by a d o p t i o n o f the f e e d i n g procedure d e s c r i b e d of unsaturation  o f the f a t s u b s t i t u t e d .  depended on the degree  The amounts o f change o b t a i n e d  were  7.3%, 19.5% and 38.17o when the b a s a l d i e t was r e p l a c e d by one c o n t a i n i n g added c o r n o i l , served  h e r r i n g o i l and coconut o i l r e s p e c t i v e l y .  to c l a r i f y  These r e s u l t s  the e f f e c t o f f a t s o f d i f f e r i n g u n s a t u r a t i o n  s t e r o l l e v e l o f the c h i c k e n cholesterol-lowering  on the plasma  and support the h y p o t h e s i s t h a t the plasma  c h a r a c t e r i s t i c o f u n s a t u r a t e d f a t s i s due a t l e a s t  i n part to t h e i r greater  a b i l i t y t o promote the f e c a l e l i m i n a t i o n o f b i l e  acids. In a s e p a r a t e experiment, s o y a l e c i t h i n and b e t a s i t o s t e r o l were f e d at l e v e l s o f 2.5 and 0.5% r e s p e c t i v e l y to growing c o c k e r e l s .  The r e s u l t s o f  t h i s experiment showed t h a t the two substances tended t o lower the l e v e l o f c i r c u l a t i n g s t e r o l s and t h a t they markedly i n c r e a s e d excretion of b i l e a c i d s .  the r a t e o f f e c a l  I t was found t h a t the a d m i n i s t r a t i o n o f b e t a  s i t o s t e r o l was accompanied by an i n c r e a s e o f 30% i n the f e c a l output o f b i l e acids.  This  i n c r e a s e bordered on s i g n i f i c a n c e a t the 57o l e v e l o f s i g n i f i c a n c e .  139 Soyalecithin  administration  o f 1007o i n t h i s output. Both a f f e c t e d but  promoted a s i g n i f i c a n t i n c r e a s e  (P < 0.01)  The e f f e c t o f t h e two supplements was  additive.  the r e l a t i v e amounts o f major and minor b i l e a c i d s  t h i s e f f e c t was g r e a t e r w i t h s o y a l e c i t h i n *  excreted  When b e t a s i t o s t e r o l and  s o y a l e c i t h i n were f e d s e p a r a t e l y the r a t i o o f t h e percentage i n c r e a s e i n minor b i l e a c i d s was  also  to that  found t h a t  i n t h e major was 1.79 and 3.65 r e s p e c t i v e l y .  the i n c o r p o r a t i o n  of soyalecithin  i n t o the d i e t  a s i g n i f i c a n t i n c r e a s e i n t h e r a t e o f growth o f t h e c o c k e r e l s In attempting t o e l u c i d a t e noted i n b i l e a c i d e x c r e t i o n ,  the mechanism(s) i n v o l v e d  It  caused  (P <. 0.01). i n t h e changes  plasma s t e r o l l e v e l s were measured i n growing  c o c k e r e l s s u b j e c t e d t o a low c h o l e s t e r o l ,  l i t h o c h o l i c acid  supplemented  d i e t and t h i s d i e t supplemented w i t h b e t a s i t o s t e r o l and s o y a l e c i t h i n . When a d m i n i s t e r e d as t h e o n l y supplement b e t a s i t o s t e r o l d i d not a l t e r t h e s e v e r i t y o f t h e l i t h o c h o l i c a c i d induced h y p e r c h o l e s t e r o l e m i a . aggravated t h e h y p e r c h o l e s t e r o l e m i a by c a u s i n g a f u r t h e r i n the l e v e l o f d i g i t o n i n - p r e c i p i t a b l e  sterols.  Soyalecithin  i n c r e a s e o f 507o  When the two substances  were s i m u l t a n e o u s l y a d m i n i s t e r e d the plasma s t e r o l l e v e l s f e l l between those observed on the two treatments mentioned. evidence t h a t cholesterol action  l i t h o c h o l i c a c i d suppressed the endogenous e x c r e t i o n o f  o r some agent r e q u i r e d  i n the gut and t h a t  lithocholic acid.  f o r b e t a s i t o s t e r o l t o e x e r t an e f f e c t i v e  soyalecithin  On the b a s i s  increase i n b i l e acid excretion net  These r e s u l t s were taken as  enhanced the a b s o r p t i o n o f  o f these r e s u l t s  i t was concluded t h a t t h e  observed when s o y a l e c i t h i n was f e d , was the  r e s u l t o f two opposing e f f e c t s on b i l e a c i d e x c r e t i o n .  consisted  i n increasing  the r e a b s o r p t i o n o f b i l e a c i d s ,  One e f f e c t  the o t h e r p r o b a b l y  140 of augmenting  the b i l i a r y output o f b i l e a c i d s and/or promoting  a c t i o n i n the d i g e s t i v e  tract.  Dietary triglycerides,  b e t a s i t o s t e r o l and s o y a l e c i t h i n  t h e r e f o r e i n f l u e n c e the l e v e l of c i r c u l a t i n g acid  excretion.  bacterial  sterols  may  through e f f e c t s  on b i l e  141  SUGGESTIONS FOR FUTURE WORK  There i s some s u g g e s t i o n i n the r e s u l t s o f experiment 5 t h a t the h y p o c h o l e s t e r o l e m i c e f f e c t o f b e t a s i t o s t e r o l may be l e c i t h i n dependent. The  administration  o f t h i s s t e r o l alone f a i l e d t o r e l i e v e the h y p e r s t e r -  olemia induced by the f e e d i n g  of l i t h o c h o l i c acid.  When the s t e r o l was  a d m i n i s t e r e d s i m u l t a n e o u s l y w i t h s o y a l e c i t h i n , plasma s t e r o l l e v e l s were lowered.  The i n e f f e c t i v e n e s s o f b e t a s i t o s t e r o l when a d m i n i s t e r e d  singly  i n the presence o f l i t h o c h o l i c a c i d was r a t i o n a l i z e d w i t h the v i e w t h a t l i t h o c h o l i c a c i d suppressed the endogenous e x c r e t i o n  of c h o l e s t e r o l  some substance r e q u i r e d  an e f f e c t i v e a c t i o n  f o r beta s i t o s t e r o l to exert  and/or  i n the g u t . There i s some evidence s u g g e s t i n g t h a t have i n f a c t b l o c k e d the endogenous e x c r e t i o n and  Sauberlich  l i t h o c h o l i c acid  of c h o l e s t e r o l .  could  Leveille  (1966) have p r e s e n t e d evidence showing t h a t d i e t a r y  litho-  c h o l i c a c i d has an u n f a v o u r a b l e e f f e c t on the removal o f c h o l e s t e r o l  from  the  c i r c u l a t i n g l i p o p r o t e i n s o f the c h i c k e n .  that  the  serum o f the c h o l e s t e r o l - f e d  c h i c k , when i n j e c t e d i n t o the c i r c u l a t i o n  of mice (which are not s u s c e p t i b l e l i t h o c h o l i c administration, i n the h e p a t i c synthesis chick and  These workers r e p o r t e d  to h y p e r c h o l e s t e r o l e m i a  induced by  Beher et_ al_., 1963) caused a s i g n i f i c a n t  increase  uptake o f c h o l e s t e r o l and depressed the r a t e o f h e p a t i c  o f c h o l e s t e r o l but t h a t the serum o f the l i t h o c h o l i c a c i d - f e d  though h i g h e r i n c h o l e s t e r o l d i d not produce these e f f e c t s .  Sauberlich  c o n s e q u e n t l y advanced the v i e w t h a t  an a l t e r a t i o n i n the s t r u c t u r e l i p o p r o t e i n s a r e the v e h i c l e s  l i t h o c h o l i c a c i d caused  o f the c i r c u l a t i n g l i p o p r o t e i n s . of transport  Leveille  Since  f o r b o t h c h o l e s t e r o l and  142 phospholipids,  i t i s r e a s o n a b l e t o suggest  t h a t the mechanism(s) which  o p e r a t e a g a i n s t the removal o f c h o l e s t e r o l , by the l i v e r ,  from the c i r -  c u l a t i o n o f the l i t h o c h o l i c a c i d - f e d c h i c k a r e a l s o f u n c t i o n a l a g a i n s t the t r a n s f e r o f c h o l e s t e r o l and p h o s p h o l i p i d s i n t o the g a l l b l a d d e r and a c r o s s the i n t e s t i n a l w a l l . a c t i o n of beta s i t o s t e r o l  Thus, i f some agent were e s s e n t i a l f o r the  i n the g u t , and was reduced  l i t h o c h o l i c a c i d was a d m i n i s t e r e d , t h i s agent pholipid.  i n amount when  c o u l d v e r y l i k e l y be phos-  The e f f e c t i v e n e s s o f b e t a s i t o s t e r o l  i n the presence  of l e c i t h i n  i n l o w e r i n g the plasma s t e r o l l e v e l o f the l i t h o c h o l i c a c i d - f e d c h i c k i s compatible w i t h t h i s i n f e r e n c e . An  i n t e r e s t i n g study would t h e r e f o r e be t o measure the amount o f  c h o l e s t e r o l and p h o s p h o l i p i d s i n the f e c e s o f b i r d s t r e a t e d as i n experiment 5.  I n such a study the plasma s t e r o l l e v e l s should a l s o be measured along  w i t h the h e m a t o c r i t v a l u e o f b l o o d from the e x p e r i m e n t a l b i r d s .  The l a t t e r  would g i v e a good i n d i c a t i o n o f the amount o f b i l e a c i d absorbed  s i n c e the  b i l e a c i d was found  t o induce hemolysis  at the 0.2% l e v e l which was f e d .  There s h o u l d be a h i g h c o r r e l a t i o n between h e m a t o c r i t v a l u e s and plasma sterol levels  i f the s t e r o l  l e v e l s a r e due p r i n c i p a l l y t o b i l e  a b s o r p t i o n and not t o c h o l e s t e r o l a b s o r p t i o n .  acid  143 BIBLIOGRAPHY  Adamson, L. F., G. K. Leeper and E. Ross, 1961. 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