DIETARY HYPERCHOLESTEROLEMIA IN RELATION TO CHOLESTEROL AND FAT ABSORPTION IN COCKERELS by Owen Bu r n e t t L i n d s a y , B.S.A. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE i n the Department of P o u l t r y S c ience We accept t h i s t h e s i s as conforming t o the st a n d a r d r e q u i r e d from c a n d i d a t e s f o r the degree of MASTER OF SCIENCE IN AGRICULTURE. Members of the Department of THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1963 In presenting t h i s t h esis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that per-mission for extensive copying of t h i s t h e s i s for s c h o l a r l y purposes may be granted by the Head of my Department or by h i s representatives. I t i s understood that copying or p u b l i -cation of t h i s t h esis for f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department The U n i v e r s i t y of B r i t i s h Columbia,. Vancouver 8, Canada. ABSTRACT D i f f e r e n c e s i n the mean plasma c h o l e s t e r o l l e v e l s of a d u l t White S i n g l e Comb Leghorn c o c k e r e l s were ac c e n t u a t e d when a d i e t c o n t a i n i n g 10% hydrogenated v e g e t a b l e o i l and 1% c h o l e s t e r o l was f e d f o r s i x days. The c h o l e s t e r o l l e v e l s promoted by the d i e t were found t o be n e g a t i v e l y c o r r e l a t e d (p 0.05) w i t h the amount of c h o l e s t e r o l e x c r e t e d by the groups. The d i f f e r e n c e s observed among groups i n the r a t e o f e l i m i n a t i o n o f the excess c h o l e s t e r o l from the c i r c u l a t i o n were not found t o be s t a t i s t i c a l l y s i g n i f i c a n t . V a r i a t i o n i n c h o l e s t e r o l a b s o r p t i o n may t h e r e f o r e be r e s p o n s i b l e , i n l a r g e measure f o r d i f f e r e n c e s i n the degree of h y p e r c h o l e s t e r o l e m i a induced, by f e e d i n g a d i e t h i g h i n c h o l e s t e r o l . A s i g n i f i c a n t c o r r e l a t i o n (p 0.01) between the amounts of c h o l e s t e r o l and s a p o n i f i a b l e l i p i d s e x c r e t e d f o l l o w i n g the f e e d i n g of a h i g h - f a t , h i g h - c h o l e s t e r o l d i e t suggests t h a t the amount o f d i e t a r y f a t absorbed may be a major determinant of the amount of c h o l e s t e r o l absorbed. S u b j e c t i o n o f c o c k e r e l s t o the f e e d i n g o f a h i g h -f a t , h i g h - c h o l e s t e r o l d i e t over a p r o l o n g e d p e r i o d r e s u l t e d i n an i n c r e a s e i n l i p i d a b s o r p t i o n . C o c k e r e l s which s u r v i v e d 410 days of f e e d i n g m a i n t a i n e d throughout the t e s t , a lower mean plasma c h o l e s t e r o l l e v e l vessels of survivors compared evidence of a t h e r o s c l e r o s i s . than nonsurvivors. The blood to nonsurvivors showed l i t t l e ACKNOWLEDGEMENT The author takes t h i s o p p o r t u n i t y t o express h i s g r a t i t u d e t o P r o f e s s o r Jacob B i e l y , Head of the Department of P o u l t r y S c i e n c e f o r p r o v i d i n g the f a c i l i t i e s used i n t h i s study and f o r h i s co n s t a n t i n t e r e s t and encouragement. In a d d i t i o n the author wants t o thank Mrs. B. E. March of the Department of P o u l t r y S c i e n c e f o r her s u p e r v i s i o n and a s s i s t a n c e throughout the course o f the experiments. F u r t h e r he wishes t o express h i s a p p r e c i a t i o n t o Dr. C. W. Roberts o f the Department of P o u l t r y Science f o r h i s v a l u a b l e c r i t i c i s m s . TABLE OF CONTENTS Page INTRODUCTION 1 LITERATURE REVIEW . . . . 4 C h a r a c t e r i s t i c s Which Render F a t s C h o l e s t e r e m i c . 4 The Degree of S a t u r a t i o n 4 The U n s a p o n i f i a b l e Fat Content . . . . . . . . 5 E s s e n t i a l F a t t y A c i d C o n c e n t r a t i o n 6 Carbon Chain Length of G l y c e r i d e F a t t y A c i d s . 7 R e g u l a t i o n of C h o l e s t e r o l L e v e l s by the P r o c e s s e s of A b s o r p t i o n , E x c r e t i o n and S y n t h e s i s . . . . 7 C h o l e s t e r o l A b s o r p t i o n 7 C h o l e s t e r o l E x c r e t i o n 9 C h o l e s t e r o l S y n t h e s i s 10 Mechanisms R e s p o n s i b l e f o r the Promotion o f C h o l e s t e r o l A b s o r p t i o n by D i e t a r y F a t s . . . . 12 EXPERIMENT I 13 E x p e r i m e n t a l 13 S e l e c t i o n o f E x p e r i m e n t a l B i r d s . . . . . . . . 13 Methods 15 A n a l y s i s o f Feces 16 E t h e r - E x t r a c t a b l e L i p i d s 16 U n s a p o n i f i a b l e L i p i d s . . . . . 16 Page T o t a l C h o l e s t e r o l 16 Chromic Oxide 17 Plasma C h o l e s t e r o l . 17 R e s u l t s and D i s c u s s i o n 18 EXPERIMENT I I . . . . . . ' 23 I n t r o d u c t i o n 23 Ex p e r i m e n t a l 23 R e s u l t s and D i s c u s s i o n 24 TABLES (EXPERIMENTS I and I I ) 28 FIGURES (EXPERIMENTS I and I I ) 38 SUMMARY AND CONCLUSIONS 47 BIBLIOGRAPHY 50 1 INTRODUCTION The a s s o c i a t i o n of h y p e r c h o l e s t e r o l e m i a i n man w i t h the l i b e r a l consumption o f the more s a t u r a t e d f a t s has l e d t o an i n t e n s i v e s e a r c h f o r c h a r a c t e r i s t i c s of f a t s which may c o n f e r c h o l e s t e r o l e m i c a c t i v i t y . E v i d e n ce has thus been advanced i n d i c a t i n g t h a t t h i s type of a c t i v i t y may be d e r i v e d from c h a r a c t e r i s t i c s such as the type and c o n c e n t r a t i o n of the c o n s t i t u e n t f a t t y a c i d s , the degree of s a t u r a t i o n of these a c i d s and the n a t u r e of the u n s a p o n i f i a b l e component of the f a t . Because f a t s v a r y as w i d e l y i n these c h a r a c t e r i s t i c s as i n t h e i r c h o l e s t e r o l e m i c p o t e n c i e s these f a c t o r s c o n t i n u e to be of major i n t e r e s t . C h o l e s t e r o l e m i c a c t i v i t y of a f a t cannot, however, always be a t t r i b u t e d e x c l u s i v e l y t o a s i n g l e c h a r a c t e r i s t i c . Hence many workers have now d i r e c t e d t h e i r a t t e n t i o n t o the d i s c o v e r y of c h a r a c t e r i s t i c s which would p r o v i d e a more r e l i a b l e measure of c h o l e s t e r o l e m i c potency. An example of e f f o r t s i n t h i s d i r e c t i o n , i s the advancement of the s o l u b i l i t y t h e o r y which s t a t e s t h a t the l i m i t i n g f a c t o r i n c h o l e s t e r o l a b s o r p t i o n i s the s o l u b i l i t y of c h o l e s t e r o l i n the d i e t a r y f a t . T h i s t h e o r y has however been d i s f a v o u r e d by a number of arguments, the most r e c e n t b e i n g , t h a t among c e r t a i n f a t s l a r g e d i f f e r e n c e s i n c h o l e s t e r o l e m i c e f f e c t s appear d i s p r o p o r t i o n a t e t o the 2 s l i g h t d i f f e r e n c e s i n the s o l v e n t a c t i o n o f these f a t s on c h o l e s t e r o l . The i n c o n s i s t e n t c h o l e s t e r o l e m i c e f f e c t s o f s e v e r a l f a t s when f e d t o v a r i o u s s p e c i e s are a l s o not accounted f o r by t h i s t h e o r y . In view of evidence t h a t c h o l e s t e r o l l e v e l s are r e l a t e d t o the d i e t a r y i n t a k e o f f a t s c o u p l e d w i t h the w e l l r e c o g n i z e d v a r i a t i o n among f a t s i n t h e i r degree of u t i l i z a -t i o n , i t appears t h a t the f a c t o r of d i g e s t i b i l i t y a l s o , deserves important c o n s i d e r a t i o n s i n s t u d i e s concerned w i t h f a t - i n d u c e d c h o l e s t e r o l e m i a . More r e c e n t evidence o f a d i f f e r e n c e i n s u s c e p t i b i l i t y of s t r a i n s of p o u l t r y t o h y p e r c h o l e s t e r o l e m i a , i n d u c e d by a h i g h - f a t , h i g h - c h o l e s t e r o l d i e t has suggested a l s o , t h a t w i t h i n the s p e c i e s t h e r e may be v a r i a t i o n i n the u t i l i z a t i o n of v a r i o u s f a t s , l e a d i n g t o v a r i a t i o n i n Suscep-t i b i l i t y t o h y p e r c h o l e s t e r o l e m i a . T h i s study was t h e r e f o r e undertaken t o determine the extent t o which a d u l t White Leghorn c o c k e r e l s may v a r y i n t h e i r h y p e r c h o l e s t e r o l e m i c response t o the f e e d i n g o f a h i g h - f a t , h i g h - c h o l e s t e r o l d i e t . I n a d d i t i o n , the r e l a t i o n of t h i s type o f v a r i a t i o n t o the u t i l i z a t i o n o f the d i e t a r y f a t and c h o l e s t e r o l was to be i n v e s t i g a t e d . I t was c o n s i d e r e d a p p r o p r i a t e t o use as e x p e r i m e n t a l a n i m a l s , c o c k e r e l s s e l e c t e d from two s t r a i n s shown p r e v i o u s l y 3 to d i f f e r i n s u s c e p t i b i l i t y to dietary hypercholesterolemia. The experimental diet was formulated to contain 10% of a highly saturated f a t (containing f a t t y acids whose chain length did not exceed 18 carbon atoms) and 1% c h o l e s t e r o l . The present study was also concerned with the ef f e c t of long-term feeding of the experimental diet on the ch o l e s t e r o l l e v e l of the blood of cockerels and upon the development of atheros c l e r o s i s i n these b i r d s . 4 LITERATURE REVIEW I C h a r a c t e r i s t i c s which Render F a t s C h o l e r e s t e r o l e m i c a) The Degree of S a t u r a t i o n K i n s e l l and M i c h a e l s (1955) and Ahrens et a l . (1955) were among the f i r s t t o demonstrate t h a t f a t s of v a r y i n g degrees of s a t u r a t i o n are not m e t a b o l i c a l l y e q u i v a l e n t i n r e g u l a t i n g b l o o d c h o l e s t e r o l l e v e l s . K i n s e l l and M i c h a e l s (1955) r e p o r t e d t h a t the f e e d i n g of coconut o i l , a h i g h l y s a t u r a t e d f a t , produced h i g h e r c h o l e s t e r o l l e v e l s i n man than the f e e d i n g of soybean o i l , a h i g h l y u n s a t u r a t e d f a t . Ahrens et a l . (1955) u s i n g s i x d i f f e r e n t d i e t a r y f a t s showed t h a t the c h o l e s t e r o l l e v e l s and the i o d i n e number of the d i e t a r y f a t were i n v e r s e l y r e l a t e d . These f i n d i n g s have been c o n f i r m e d by numerous i n v e s t i g a t o r s (Bronte-Stewart et a l . , 1956; Ahrens et a l . , 1957; Avigan and S t e i n b e r g , 1958; K i n s e l l et a l . , 1958 and o t h e r s ) . The c h o l e s t e r o l e m i c e f f e c t s of c e r t a i n f a t s as observed i n man are not always r e p r o d u c i b l e i n o t h e r s p e c i e s . For i n s t a n c e , when c h i c k e n s were f e d a normal p o u l t r y d i e t supplemented w i t h c o r n o i l they were r e n d e r e d h y p e r c h o l e s t e r o -lemic when the p r o t e i n l e v e l of the d i e t was 20% and normo— c h o l e s t e r o l e m i c when the p r o t e i n l e v e l was r a i s e d to 26%. ( B i e l y and March, 1959). The r e p o r t s h i t h e r t o mentioned 5 d e s c r i b e the e f f e c t s o f c o r n o i l on man as h y p o c h o l e s t e r o -l e m i c . b) The U n s a p o n i f i a b l e Fat Content P e t e r s o n (1951) showed t h a t when c h o l e s t e r o l and mixed p l a n t s t e r o l s (at 1 : 1 r a t i o ) were f e d to c h i c k e n s as 0.5% and 1% of the d i e t , t h e r e was a marked p r o t e c t i o n a g a i n s t h y p e r c h o l e s t e r o l e m i a , h e p a t i c l i p i d o s i s and c h o l e s t e r o l o s i s . P l a n t s t e r o l s have a l s o been shown t o i n h i b i t e x p e r i m e n t a l l y i n d u c e d h y p e r c h o l e s t e r o l e m i a i n r a t s ( S w e l l et a l . , 1954; and S w e l l et a l . , 1956), r a b b i t s ( P o l l a c k , 1958), and man ( P o l l a c k , 1956; Farquhar et a l . , 1956; Farquhar and Sokolow, 1958, and o t h e r s ) . These f i n d i n g s added t o the o b s e r v a t i o n t h a t c o r n o i l r e t a i n s some of i t s h y p o c h o l e s t e r o l e m i c a c t i v i t y f o l l o w -i n g h y d r o g e n a t i o n , have l e d Jones et a l . (1956) and Beveridge et a l . (1957) to a t t r i b u t e p a r t of the c h o l e s t e r o l depressant a c t i o n of v e g e t a b l e o i l s t o t h e i r u n s a p o n i f i a b l e f a t c o n t e n t . Wood (1960) and Wood and B i e l y (1960) have p r e s e n t e d evidence t o show t h a t t h e r e may be some r e l a t i o n s h i p between v i t a m i n A content and h y p o c h o l e s t e r o l e m i c a c t i v i t y of the un— s a p o n i f i a b l e content of c e r t a i n marine o i l s . The i n h i b i t i o n of e x p e r i m e n t a l l y i n d u c e d hyper-c h o l e s t e r o l e m i a by p l a n t s t e r o l s has been shown to be due to the i n h i b i t i o n of c h o l e s t e r o l a b s o r p t i o n (Hernandez and C h a i k o f f , 1954; and Hernandez et a l . , 1953) but the p r e c i s e 6 manner i n which t h i s i s accomplished remains obscure. One e x p l a n a t i o n o f f e r e d was t h a t p l a n t s t e r o l s compete w i t h c h o l e s t e r o l f o r c h o l e s t e r o l e s t e r a s e . ( P e t e r s o n , 1951; Duncan and B est, 1956; S w e l l et a l . , 1954). T h i s p o s t u l a t e d the n e c e s s i t y f o r e s t e r i f i c a t i o n p r i o r to a b s o r p t i o n o f c h o l e s t e r o l . In view of evidence t h a t e s t e r i f i c a t i o n i s not a p r e r e q u i s i t e f o r the passage of c h o l e s t e r o l i n t o the mucosa of the i n t e s t i n a l w a l l ( P i h l , 1955; S w e l l et a l . , 1958 and 1958 b ) , t h i s e x p l a n a t i o n has been d i s c r e d i t e d . Other ex-p l a n a t i o n s are t h a t p l a n t s t e r o l s compete w i t h c h o l e s t e r o l f o r a c c e p t o r s i t e s on the l i p o p r o t e i n s e i t h e r w i t h i n the mucosal c e l l or on i t s c e l l membrane ( C l o v e r and Green, 1957), and t h a t i t b i n d s c h o l e s t e r o l i n the lumen o f the i n t e s t i n e making i t u n a v a i l a b l e f o r a b s o r p t i o n ( P o l l a c k , 1953). c) E s s e n t i a l F a t t y a c i d C o n c e n t r a t i o n . Hegsted et a l . (1957) i m p l i c a t e d t h i s f a c t o r as a determinant of c h o l e s t e r o l l e v e l s . Upon f e e d i n g to r a t s a d i e t c o n t a i n i n g 10 or 20% f a t they demonstrated t h a t the c h o l e s t e r o l l e v e l s reached on the d i e t were i n v e r s e l y r e l a t e d to the product of the percentage of e s s e n t i a l f a t t y a c i d s ( L i n o l e i c and a r a c h i d o n i c ) and the percentage o f s a t u r a t e d f a t t y a c i d s i n the d i e t . K i n s e l l and S i n c l a i r (1957) i n ex-periments w i t h r a t s a l s o r e c o g n i z e d the h y p e r c h o l e s t r o l e m i c e f f e c t of d i e t s low i n e s s e n t i a l f a t t y a c i d s . The i n v e s t i g a t i o n s of Ahrens et a l . (1959) however, c a s t doubts on such a r e g u l a t o r y r o l e of t h e s e a c i d s . These 7 workers have shown t h a t f a t s low i n e s s e n t i a l f a t t y a c i d s (e.g. menhaden o i l ) may depress serum c h o l e s t e r o l l e v e l s t o at l e a s t the same extent as f a t s w i t h a h i g h e s s e n t i a l f a t t y a c i d content (e.g. c o r n o i l ) . d) Carbon Chain Length o f G l y c e r i d e F a t t y A c i d s Beveridge et a l . (1959) and Hashim et a l . (1960) have p r e s e n t e d evidence t o show t h a t i n man s a t u r a t e d f a t t y a c i d s 8 t o 10 carbon atoms i n l e n g t h , produced o n l y s l i g h t changes i n the c h o l e s t e r o l l e v e l o f the b l o o d . S i m i l a r o b s e r v a t i o n s have been made i n the dog by Grande (1962). T h i s i n v e s t i g a t o r f e d t h r e e groups o f f a t s v a r y i n g g r e a t l y i n the carbon c h a i n l e n g t h o f t h e i r c o n s t i t u e n t f a t t y a c i d s . When the predominant f a t t y a c i d s o f the group were 12 t o 14 carbon atoms i n l e n g t h the f a t s were found t o e x e r t t h e i r most pronounced 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 . When they were 8 t o 10 and 16 to 18 carbon atoms i n l e n g t h 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 was s l i g h t and i n t e r m e d i a t e r e s p e c t i v e l y . I I R e g u l a t i o n o f C h o l e s t e r o l L e v e l s by the Pr o c e s s of A b s o r p t i o n , E x c r e t i o n and S y n t h e s i s a) C h o l e s t e r o l A b s o r p t i o n Dubach and H i l l (1946) and Bollman (1951) who worked w i t h r a b b i t s " a n d r a t s r e s p e c t i v e l y i n d i c a t e d t h a t the i n c o r -p o r a t i o n o f f a t i n t o an e s s e n t i a l l y f a t - f r e e d i e t promoted no f u r t h e r i n c r e a s e s i n the a b s o r p t i o n o f c h o l e s t e r o l beyond t h a t o bserved on the f a t - f r e e d i e t . Blomstrand and Ahrens (1958) 8 i n v e s t i g a t e d the a b s o r p t i o n o f l a b e l l e d c h o l e s t e r o l i n a p a t i e n t w i t h c h y l u r i a who shunted 40% of absorbed f a t i n t o her u r i n e . These workers observed t h a t 20% of the adminis-t e r e d c h o l e s t e r o l was absorbed i n f i f t e e n hours whether g i v e n s i n g l y or i n combination w i t h f a t . The peak of l a b e l -l e d c h o l e s t e r o l was found t o occur at n i n e hours compared t o s i x hours f o r the f a t . Hellman et a l . (1958) s t u d i e d the a b s o r p t i o n o f r a d i o c h o l e s t e r o l i n man, d i r e c t l y , by cannu-l a t i o n o f the l e f t t h o r a c i c duct. A l t h o u g h f a t was not a c o n s t i t u e n t of t h e i r t e s t - m e a l they found t h a t the l e v e l o f e s t e r i f i e d c h o l e s t e r o l i n the lymph i n c r e a s e d , r e a c h i n g a peak i n s i x hours. Sano (1924), and Cook (1936, 1938) and Cook and McCullagh (1939) have demonstrated ( i n r a t s and dogs) t h a t the presence of f a t i n the i n t e s t i n e s i s o b l i g a t o r y to c h o l e s t e r o l a b s o r p t i o n . These workers have c o n s e q u e n t l y advanced the view t h a t f a t s p l a y an important r o l e i n c h o l e s t e r o l a b s o r p t i o n . T h i s view i s i n c o n t r a s t t o t h a t e xpressed by the p r e v i o u s l y mentioned i n v e s t i g a t o r s . I t i s w e l l supported by evidence t h a t d i e t a r y f a t s enhance c h o l e s -t e r o l a b s o r p t i o n i n the r a t ( A l b r i n k et a l . , 1955; M o r r i s , 1954; D a s k a l a k i s , 1955, and B r o k e t t et a l . , 1934), and the c h i c k e n ( B i e l y and March, 1959; and Wood and B i e l y , 1960). The r e l a t i o n s h i p between f a t and c h o l e s t e r o l a b s o r p t i o n becomes c l e a r e r when the l i p i d c o n t e n t o f the f e c e s of e x p e r i m e n t a l animals ( i n s t e a d o f the b l o o d c h o l e s -t e r o l l e v e l ) i s used as a measure of c h o l e s t e r o l a b s o r p t i o n . 9 For example, when Grande (1962) f e d to dogs a h i g h - f a t , h i g h - e h o l e s t e r o l d i e t he observed t h a t an i n c r e a s e i n the e x c r e t i o n of s t e r o i d substances accompanied an i n c r e a s e i n the e x c r e t i o n of f a t . These r e s u l t s are i n a c c o r d w i t h those of Cheng and S t a n l e y (1956) who, u s i n g r a t s , demonstrated a d i r e c t r e l a t i o n s h i p between the d i e t a r y f a t i n t a k e and the amount of c h o l e s t e r o l s e c r e t e d , r e a b s o r b e d and e x c r e t e d . The r e s u l t s of numerous experiments seem t o i n d i c a t e t h a t f r e e f a t t y a c i d s p o t e n t i a t e c h o l e s t e r o l a b s o r p t i o n to a g r e a t e r extent than g l y c e r i d e s (Kim and Ivy, 1952; P i h l , 1955; S w e l l , 1954; and S w e l l et a l . , 1955). The l o n g . c h a i n s a t u r a t e d f a t t y a c i d s have been r e p o r t e d however, to i n h i b i t c h o l e s t e r o l a b s o r p t i o n i n the r a t ( L i n et a l . , 1955), the cebus monkey (Partman et a l . , 1957), and the c h i c k e n (Stamler et a l . , 1957). b) C h o l e s t e r o l E x c r e t i o n W i l s o n and S i p e r s t e i n (1958) a d m i n i s t e r e d c h o l e s t e r o l i n t r a v e n o u s l y to t h r e e groups of r a t s , p r e f e d d i e t s c o n t a i n i n g 30% c o r n o i l , 30% l a r d and e s s e n t i a l l y no f a t r e s p e c t i v e l y . They observed l a r g e i n c r e a s e s i n the alpha-hydroxy, n e u t r a l s t e r o l s i n the f e c e s o f those r a t s which were f e d c o r n o i l . These s t e r o l s accounted f o r 17 to 25% of the a d m i n i s t e r e d r a d i o a c t i v i t y . Hellman et a l . (1957) i n a s i m i l a r experiment 14 i n j e c t e d c h o l e s t e r o l - C i n t r a v e n o u s l y i n t o humans p r e f e d d i e t s c o n t a i n i n g b u t t e r or c o r n o i l . The q u a n t i t y o f 10 l a b e l l e d s t e r o l s i n the f e c e s were found to i n c r e a s e when the serum c h o l e s t e r o l c o n c e n t r a t i o n s decreased w i t h c o r n o i l f e e d i n g . The o b s e r v a t i o n t h a t t h e more s a t u r a t e d f a t s do not a f f e c t the e x c r e t i o n of c i r c u l a t i n g c h o l e s t e r o l t o any great extent (Coleman and Bauman, 1957), i s a l s o i n a c c o r d w i t h the f o r e g o i n g f i n d i n g s . That u n s a t u r a t e d f a t s a c c e l e r a t e the e l i m i n a t i o n of c i r c u l a t i n g c h o l e s t e r o l i s a l s o shown i n the decrease i n plasma c h o l e s t e r o l l e v e l of r a t s f o l l o w i n g the i n t r a p e r i -t o n e a l i n j e c t i o n of c o d l i v e r o i l . (Gran and N i c o l a y s e n , 1 9 6 1 ) . When the e x r e t i o n of b i l e a c i d s i s used as a measure of c h o l e s t e r o l e x c r e t i o n , r e s u l t s s i m i l a r t o those p r e v i o u s l y d e s c r i b e d have been o b t a i n e d . Thus Byers and Friedman (1958) have observed g r e a t e r e x c r e t i o n s of b i l e a c i d s and b i l e c h o l e s t e r o l i n r a t s f e d c h o l e s t e r o l and walnut o i l than i n r a t s f e d c h o l e s t e r o l and coconut o i l . These r e -s u l t s agree w i t h those of Haust and Beveridge (1958) and Bronte-Stewart et a l . (1956). c) C h o l e s t e r o l S y n t h e s i s Wood and M i g i c o v s k y (1958) observed t h a t s y n t h e s i s of c h o l e s t e r o l i n v i v o and i n v i t r o was enhanced by f e e d i n g d i e t s c o n t a i n i n g c o r n o i l , r a p e s e e d o i l , e r u c i c a c i d and o l e i c a c i d w h i l e s y n t h e s i s i n v i v o was depressed by coconut o i l and l a u r i c a c i d . 11 On the o t h e r hand H i l l et a l . (1958) and L i n a z o s o r o et a l . (1958) have demonstrated t h a t t h e r e was not any appa-r e n t d i f f e r e n c e i n the r a t e of s y n t h e s i s of c h o l e s t e r o l between r a t s f e d 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 . They a l s o showed t h a t d i e t a r y f a t s enhance c h o l e s t e r o l s y n t h e s i s . T h i s i s an e s s e n t i a l agreement w i t h the f i n d i n g s of Ahrens (1957). Murkherjee and A l f i n - S l a t e r (1958) i n v e s t i g a t e d the e f f e c t s of f e e d i n g r a t s d i e t s c o n t a i n i n g 15% c o t t o n s e e d o i l o r 30% hydrogenated c o t t o n s e e d o i l . They observed a g r e a t e r i n c r e a s e i n the r a t e of s y n t h e s i s i n animals f e d c o t t o n s e e d o i l . When c h o l e s t e r o l i s added to t h e d i e t of r a t s marked s u p r e s s i o n of h e p a t i c s y n t h e s i s has been observed (Gould and T a y l o r , 1950; F r a n t z et a l . , 1954; and M o r r i s et a l . , 1957). Gould (1951) has a l s o advanced evidence to show th a t the degree of s u p r e s s i o n i s d i r e c t l y r e l a t e d t o the l e v e l of d i e t a r y c h o l e s t e r o l . There i s thus l i t t l e e vidence i n d i c a t i n g t h a t c h o l e s t e r o l l e v e l s i n d u c e d by f e e d i n g d i f f e r e n t types and q u a n t i t y of f a t s are r e l a t e d t o changes i n the r a t e of c h o l e s t e r o l s y n t h e s i s . 12 I I I Mechanisms R e s p o n s i b l e f o r the Promotion of C h o l e s t e r o l A b s o r p t i o n by D i e t a r y F a t s S w e l l et a l . (1955) and Vahouny (1957) have shown t h a t the presence o f exogenous f a t s i s not an adjunct to c h o l e s t e r o l a b s o r p t i o n i f adequate q u a n t i t i e s of b i l e s a l t s are i n c l u d e d i n the d i e t . T h i s has been shown t o be due to the a c t i v a t i o n of c h o l e s t e r o l e s t e r a s e by b i l e s a l t s ( S w e l l et a l . , 1953 a, 1953 b ) . Sw e l l and F l i c k (1955) have conse-q u e n t l y suggested t h a t the manner i n which exogenous f a t s augment the a b s o r p t i o n of c h o l e s t e r o l i s by s t i m u l a t i n g the f l o w of b i l e which i n t u r n w i l l i n c r e a s e the a c t i v i t y o f c h o l e s t e r o l e s t e r a s e . P i h l (1955) has however expressed h i s r e s e r v a t i o n s c o n c e r n i n g the importance of t h i s mechanism when h i g h l e v e l s o f f a t are f e d . Wilkens et a l . (1962) has a t t r i b u t e d the c h o l e s -t e r o l e m i c potency of f a t s to t h e i r s o l v e n t a c t i o n on c h o l e s -t e r o l . They o b t a i n e d a h i g h c o r r e l a t i o n (+ 0.982) between the c h o l e s t e r o l s o l u b i l i t i e s of the v a r i o u s f a t s and t h e i r serum c h o l e s t e r o l e f f e c t s i n man. 13 EXPERIMENT I Ex p e r i m e n t a l S e l e c t i o n of E x p e r i m e n t a l B i r d s Twenty-four a d u l t t h i r d g e n e r a t i o n c o c k e r e l s were s e l e c t e d from each of two s t r a i n s of S i n g l e Comb, White Leghorns p r e v i o u s l y shown to d i f f e r i n s u s c e p t i b i l i t y t o r e n a l d i s o r d e r s ( B i e l y and March, 1958) and d i e t a r y i n d u c e d h y p e r c h o l e s t e r o l e m i a ( B i e l y and March, 1960). The c o c k e r e l s from which s e l e c t i o n s were made (35 from s t r a i n A and 40 from s t r a i n B) subsequent to h a t c h i n g were f e d s i m i l a r s t a r t i n g and growing d i e t s . They were kept i n b a t t e r y cages d u r i n g the s t a r t i n g and growing p e r i o d s and a t the end of the growing p e r i o d were t r a n s f e r r e d t o f l o o r pens and f e d the b a s a l d i e t of Tabl e I. S e l e c t i o n o f the e x p e r i m e n t a l b i r d s was c a r r i e d out as f o l l o w s . The s e v e n t y - f i v e b i r d s o f s t r a i n s A and B were f e d ad l i b i t u m , the b a s a l d i e t of T a b l e I. The plasma c h o l e s t e r o l l e v e l s o f these b i r d s were determined f o l l o w i n g t h r e e days f e e d i n g o f t h i s d i e t . These l e v e l s were a g a i n determined f o l l o w i n g s i x days of f e e d i n g . Repeat determina-t i o n s (at s i x days) were made i n order t o i n c r e a s e the r e -l i a b i l i t y of the c h o l e s t e r o l l e v e l s which were t o be used as the b a s i s f o r s e l e c t i n g and gro u p i n g the e x p e r i m e n t a l b i r d s . 14 From each s t r a i n t w enty-four b i r d s ( i n c l u d i n g those w i t h the extreme and i n t e r m e d i a t e c h o l e s t e r o l l e v e l s f o r the s t r a i n ) were s e l e c t e d . The c h o l e s t e r o l l e v e l s o f these b i r d s are g i v e n i n T a b l e s I I and I I I . C h o l e s t e r o l l e v e l s o f b i r d s from s t r a i n A ranged from 126 — 194 mg %• and those of b i r d s from s t r a i n B, from 112 - 185 mg %. The range i n the l e v e l s o f both groups c o u l d have been made s i m i l a r but o n l y a f t e r the e l i m i n a t i o n of b i r d s w i t h the h i g h e s t and lowest c h o l e s t e r o l l e v e l s . These were r e g a r d e d as b e i n g v a l u a b l e i n t h i s study and were t h e r e f o r e i n c l u d e d . The b i r d s from each s t r a i n were then a s s i g n e d i n t o s i x l o t s c o n t a i n i n g an equal number of b i r d s w i t h f a i r l y s i m i l a r c h o l e s t e r o l l e v e l s . Due to a shortage of t h i r d g e n e r a t i o n c o c k e r e l s the r e p l i c a t i o n o f l o t s was not p o s s i b l e . The g rouping of b i r d s i s a l s o shown i n T a b l e s I I and I I I and are as f o l l o w s . Lot 1 2 3 4 5 6 Mean Pretreatment S t r a i n A 133.7 149.0 156.5 165.8 175.3 187.0 C h o l e s t e r o l L e v e l ( m g . % ) S t r a i n B 121.0 0 156.5 158.2 162.5 169.8 181.3 The l o t s were then p l a c e d i n t o f o u r i n d i v i d u a l cage u n i t s . Each u n i t c o n s i s t e d of t h r e e t i e r s , each 15 capable of ho u s i n g f o u r b i r d s , such t h a t w i t h one t i e r a l l o c a t e d t o one l o t , b i r d s from each s t r a i n were housed i n t o two complete cage u n i t s . The mean pretreatment c h o l e s -t e r o l l e v e l s of c o r r e s p o n d i n g l o t s (e.g. l o t s A 1 and B 1) were i n a few cases not as s i m i l a r as was d e s i r a b l e . None-t h e l e s s i t was c o n s i d e r e d advantageous t o compare the hyper— c h o l e s t e r o l e m i c responses of thes e l o t s under c o n d i t i o n s t h a t were as u n i f o r m as p o s s i b l e . T h i s was accomplished by randomizing the l o t s from one s t r a i n and then d i s t r i b u t i n g the r e m a i n i n g l o t s i n such a manner t h a t c o r r e s p o n d i n g l o t s were ad j a c e n t t o each o t h e r . The r e s u l t o f t h i s arrangement was t h a t the s t r a i n s a l t e r n a t e d i n f o u r cage u n i t s p l a c e d i n a s i n g l e row. F o l l o w i n g the assignment o f l o t s the b i r d s were f e d the b a s a l d i e t o f Table I supplemented w i t h 10% hydrogenated v e g e t a b l e o i l , 1% c h o l e s t e r o l and 1% chromic oxide (a marker). They were a l l o w e d f r e e access t o f e e d and water. Methods On the t h i r d and s i x t h day, f e c e s were c o l l e c t e d and plasma c h o l e s t e r o l l e v e l s determined-by the method of Rosenthal et a l . (1957). The f e c e s were c o l l e c t e d from each b i r d over a twenty-four hour p e r i o d , p o o l e d f o r each l o t and f r e e z e - d r i e d . At the end of the s i x t h day c h o l e s t e r o l was withdrawn from the d i e t and a f t e r f o u r and seven days of w i t h -drawal f e c e s were a g a i n c o l l e c t e d , p o o l e d by l o t s and f r e e z e -d r i e d . Plasma c h o l e s t e r o l d e t e r m i n a t i o n s were a l s o made a g a i n 16 at these times. A n a l y s i s o f Feces a) E t h e r - E x t r a c t a b l e L i p i d s E x t r a c t i o n of f e c a l l i p i d s w i t h d i e t h y l e t h e r was c a r r i e d out i n a s o x h l e t apparatus f o r s i x t e e n hours. The e x t r a c t was f i l t e r e d u s i n g Whatman number 42 f i l t e r paper and the e t h e r e vaporated on a 50°C water bath. The r e s i d u e was d r i e d at 85°C f o r one and a h a l f hours and then c o o l e d i n a d e s s i c a t o r p r i o r t o weighing. b) U n s a p o n i f i a b l e L i p i d s These were determined by the procedure d e s c r i b e d i n the " O f f i c i a l Methods of A n a l y s i s " o f the A.O.A.C. w i t h the f o l l o w i n g m o d i f i c a t i o n . The e t h e r s o l u t i o n c o n t a i n i n g t h e j u n s a p o n i f i a b l e l i p i d s was d r i e d w i t h anhydrous sodium s u l f a t e and the l i p i d s r e c o v e r e d i n a manner s i m i l a r t o t h a t p r e v i o u s l y d e s c r i b e d f o r the e t h e r - e x t r a c t a b l e l i p i d s . c) T o t a l C h o l e s t e r o l The f r e e z e - d r i e d samples of f e c e s were e x t r a c t e d w i t h a mixture o f a l c o h o l acetone ( 1 : 1 ) u n t i l e x t r a c t s gave a n e g a t i v e t e s t f o r c h o l e s t e r o l w i t h the r a p i d p r o c e -dure of R o s e n t h a l et a l . (1957). T o t a l c h o l e s t e r o l was determined a c c o r d i n g t o the r e v i s e d S p e r r y Webb (1950) procedure i n which the p r e c i p i t a t e d d i g i t o n i d e i s r e a c t e d w i t h a s u l f u r i c a c i d - a c e t i c anhydride (1 : 20) c o l o r r e a g e n t . 17 The i n t e n s i t y o f the c o l o r produced was determined i n a model B Beckman spectrophotometer at 625 m i l l i m i c r o n s . d) Chromic Oxide The procedure o f Schurch et a l . (1950) was f o l l o w e d . Samples of f e c e s (and f e e d ) were ashed and then f u s e d w i t h sodium p e r o x i d e . The c o l o r i n t e n s i t y o f d i s t i l l e d water ex-t r a c t s of the product o b t a i n e d was determined i n a model B Beckman spectrophotometer at 550 m i l l i m i c r o n s . Plasma C h o l e s t e r o l Plasma c h o l e s t e r o l d e t e r m i n a t i o n s were made on the t h i r d , s i x t h , t e n t h ( f o u r days a f t e r withdrawal of d i e t a r y c h o l e s t e r o l ) and t h i r t e e n t h (seven days a f t e r withdrawal of d i e t a r y c h o l e s t e r o l ) days of the experiment. D e t e r m i n a t i o n s were made on 0.1 ml. samples of h e p a r i n i z e d plasma. The plasma was o b t a i n e d from two ml. samples o f b l o o d drawn from the wing v e i n i n t o h e p a r i n i z e d t u b es. T o t a l c h o l e s t e r o l was determined s p e c t r o p h o t o m e t r i c a l l y u s i n g the procedure o f Z l a t k i s et a l . (1953) as m o d i f i e d by Rose n t h a l et a l . (1957). The c o l o r reagent used was made up as f o l l o w s . I r o n Stock S o l u t i o n - 2.25 grams F e C l ^ 6 RY,0 d i s s o l v e d i n 100 mis. 87% ph o s p h o r i c a c i d . C o l o r Reagent - 8.0 mis. i r o n s t o c k s o l u t i o n d i l u t e d to 100 mis. w i t h con-c e n t r a t e d s u l f u r i c a c i d . 18 The r e a c t i o n mixture c o n s i s t e d of 0.1 mis. plasma, 3 mis. g l a c i a l a c e t i c a c i d and 2 mis. c o l o r r e a g e n t . The c o l o r i n t e n s i t y was determined s p e c t r o p h o t o m e t r i c a l l y at 560 m i l l i m i c r o n s . R e s u l t s and D i s c u s s i o n The i n c r e a s e s i n plasma c h o l e s t e r o l l e v e l s o f i n d i v i d u a l c o c k e r e l s r e s u l t i n g from f e e d i n g the b a s a l d i e t o f T a b l e I supplemented w i t h 1% c h o l e s t e r o l and 10% hydro-genated v e g e t a b l e o i l f o r t h r e e and s i x days are p r e s e n t e d i n T a b l e s I I and I I I . The data show t h a t h y p e r c h o l e s t e r o -l e m i a was induc e d i n a l l but two c o c k e r e l s , both of which belonged t o l o t 1 of s t r a i n B. The a d d i t i o n a l changes i n c h o l e s t e r o l l e v e l s brought about by withdrawal of the d i e t a r y c h o l e s t e r o l f o l l o w i n g s i x days of c h o l e s t e r o l -f e e d i n g are a l s o p r e s e n t e d i n Ta b l e s I I and I I I . The hyper-c h o l e s t e r o l e m i c response of the v a r i o u s groups (as i n d i c a t e d by the mean plasma c h o l e s t e r o l l e v e l s ) t o these d i e t a r y changes i s g r a p h i c a l l y shown i n F i g u r e s 1 ( s t r a i n A), 2 ( s t r a i n B), 3 and 4 ( s t r a i n s A and B). The mean plasma c h o l e s t e r o l l e v e l o f c h o l e s t e r o l -f e d c o c k e r e l s of s t r a i n A at t h r e e and s i x days i n c r e a s e d from a pre-treatment l e v e l of 161 mg.% t o 299 and 486 mg.% r e s p e c t i v e l y whereas t h a t of c o c k e r e l s from s t r a i n B changed from 159 mg.% t o c o r r e s p o n d i n g l e v e l s of 278 and 399 mg.%. A s i g n i f i c a n t c o r r e l a t i o n (p 0.01) between p r e -and p o s t - t r e a t m e n t c h o l e s t e r o l l e v e l s was demonstrated f o r 19 individual birds but when strain distinctions were made these levels were significantly related only for strain B birds. The regression of post- on pretreatment levels i s shown in Figure 5. It was observed that birds previously thought to be similar on the basis of their pretreatment levels were not always so when fed the cholesterol-rich diet. The variation in response of birds within a lot appeared to be more marked in birds from strain A compared to strain B. This probably accounted for the absence of a significant correlation he-tween pre- and post treatment levels of birds of strain A . The quantities of li p i d s (cholesterol, ether-extractable l i p i d s and saponifiable l i p i d s ) excreted by i n -dividual lots following six days of cholesterol feeding are given in Table V. These results when analysed (Table VIII) showed that the mean peak cholesterol level and cholesterol excreted were significantly correlated (p 0.05). A s i g n i f i -cant correlation between the fecal cholesterol and the fecal saponifiable l i p i d s (p 0.01) and the fecal cholesterol and the percentage saponifiable l i p i d s in the fecal ether extract (p 0.01) was also demonstrated. The regression of the peak cholesterol level on the fecal cholesterol, the fecal choles-terol on the fecal saponifiable l i p i d s and on the percent saponifiable l i p i d s i n the fecal ether extract are shown in Figures 6, 7, and 8. 20 These r e s u l t s i n d i c a t e d a three-way i n t e r -r e l a t i o n s h i p between the degree of h y p e r c h o l e s t e r o l e m i a induced and the amount o f d i e t a r y f a t and c h o l e s t e r o l absorbed. The mean q u a n t i t i e s of c h o l e s t e r o l , s a p o n i f i a b l e l i p i d s and e t h e r e x t r a c t a b l e l i p i d s e x c r e t e d by b i r d s of each s t r a i n were as f o l l o w s : Grams L i p i d s per 10 Kilograms D i e t E t h e r S t r a i n C h o l e s t e r o l v S a p o n i f i a b l e E x t r a c t a b l e L i p i d s L i p i d s A 48.8 187.9 259.2 B 58.7 322.2 404.4 The d a t a show t h a t g r e a t e r amounts o f l i p i d s were r e t a i n e d by b i r d s of s t r a i n A. T h i s would p r o b a b l y e x p l a i n the h i g h e r c h o l e s t e r o l l e v e l s t h a t were promoted i n these b i r d s . The d ata s u b s t a n t i a t e the view p r e v i o u s l y e x p r e s s e d by B i e l y and March (1960) t h a t a s t r a i n d i f f e r e n c e i n c h o l e s t e r o l absorp-t i o n was p r o b a b l y r e s p o n s i b l e f o r the d i f f e r e n c e i n s u s c e p t i -b i l i t y of the s t r a i n s t o d i e t a r y i n d u c e d h y p e r c h o l e s t e r o l e m i a . That the degree o f u t i l i z a t i o n of the d i e t a r y f a t may have been a determinant of the peak c h o l e s t e r o l l e v e l was w e l l i l l u s t r a t e d by b i r d s p r e s e n t i n l o t s w i t h the lowest p r e -treatment c h o l e s t e r o l l e v e l s . T h i s i s p a r t i c u l a r l y t r u e i n the ease of b i r d s b e l o n g i n g to s t r a i n B. T a b l e s I I and I I I show th a t the o n l y b i r d s r e f r a c t o r y to c h o l e s t e r o l - f e e d i n g , as 21 i n d i c a t e d by the s t a b i l i t y o f t h e i r c h o l e s t e r o l l e v e l s w h i l e on t e s t were p r e s e n t i n l o t B 1. The b i r d s w i t h i n l o t T B l ex-c r e t e d f a r g r e a t e r amounts of c h o l e s t e r o l and f a t than was expected on the b a s i s of amounts e x c r e t e d by the r e m a i n i n g l o t s . The assumption t h a t the amount of c h o l e s t e r o l absorbed i s determined t o a g r e a t e x t e n t by the amount of f a t absorbed i s w e l l supported by evidence (reviewed) i n -d i c a t i n g t h a t d i e t a r y f a t s enhance c h o l e s t e r o l a b s o r p t i o n . The marked v a r i a t i o n i n the amount of d i e t a r y l i p i d s u t i l i z e d i n d i c a t e s t h a t i n d i v i d u a l v a r i a t i o n may be a l s o marked. Marked i n d i v i d u a l v a r i a t i o n i n the u t i l i z a t i o n of d i e t a r y l i p i d s has been p r e v i o u s l y r e p o r t e d f o r r a t s (Squibb et a l . , 1958). The changes i n the mean plasma c h o l e s t e r o l l e v e l s of the v a r i o u s l o t s which o c c u r r e d upon withdrawing the d i e t a r y c h o l e s t e r o l f o r f o u r and seven days and the amount of c i r c u l a t i n g c h o l e s t e r o l e l i m i n a t e d at these, times are shown i n T a b l e s I I , I I I and IV and summarized below. S t r a i n Excess C h o l e s - Excess (mg.%) Excess (mg.%) t e r o l p r e s e n t E l i m i n a t e d at E l i m i n a t e d at at 6 days 4 days 7 days amt % amt % A 328.6 227 72 314 97 B 241.0 202 86 239 100 The d a t a show t h a t b i r d s from s t r a i n A e l i m i n a t e d more c i r -c u l a t i n g c h o l e s t e r o l per u n i t time than b i r d s from S t r a i n B 22 but i n r e l a t i o n t o the amount of excess pre s e n t they e l i m i n a t e d l e s s e r amounts. When the d i f f e r e n c e i n r a t e o f e l i m i n a t i o n o f excess c h o l e s t e r o l was at i t s maximum ( i . e . , at 4 days) i t was found t o be n o n s i g n i f i c a n t at the 5% l e v e l of s i g n i f i c a n c e . T h i s d i f f e r e n c e t h e r e f o r e d i d not appear t o be g r e a t l y r e s p o n s i b l e f o r the s t r a i n d i f f e r e n c e i n peak c h o l e s t e r o l l e v e l s . The da t a o f T a b l e IV suggest t h a t the a b s o l u t e amount of c i r c u l a t i n g c h o l e s t e r o l e l i m i -n a t ed per u n i t time was d i r e c t l y r e l a t e d t o the amount of excess p r e s e n t . T h i s r e l a t i o n s h i p has been p r e v i o u s l y observed by H o r l i c k et a l . (1948) who r e n d e r e d b i r d s hyper-c h o l e s t e r o l e m i a b y - i n t r a v e n o u s i n j e c t i o n o f h y p e r l i p e m i c plasma o b t a i n e d from b i r d s f e d a h i g h - c h o l e s t e r o l d i e t . H o r l i c k and a s s o c i a t e s however r e p o r t e d a r e t u r n t o normal l e v e l s i n twelve t o twenty-four hours. S i n c e t h i s time i s at v a r i a n c e w i t h t h a t noted i n the p r e s e n t study i t has been assumed t h a t h y p e r c h o l e s t e r o l e m i a i n d u c e d by i n j e c t i o n o f h y p e r c h o l e s t e r o l e m i c plasma does not s i m u l a t e t h a t produced by d i e t a r y means. 23 EXPERIMENT I I I n t r o d u c t i o n In experiment I a r e l a t i o n s h i p between p r e - and post treatment c h o l e s t e r o l l e v e l s of c h o l e s t e r o l — f e d c o c k e r e l s was demonstrated. I t was t h e r e f o r e c o n s i d e r e d of i n t e r e s t t o make a p r e l i m i n a r y i n v e s t i g a t i o n i n t o the r e l a t i o n s h i p between pretreatment c h o l e s t e r o l l e v e l and s u s c e p t i b i l i t y of c h o l e s -t e r o l - f e d c o c k e r e l s t o a t h e r o s c l e r o s i s . B i r d s not p r e v i o u s l y s u b j e c t e d to c h o l e s t e r o l - f e e d i n g were to be s e l e c t e d from s t r a i n A (the more s u s c e p t i b l e t o h y p e r c h o l e s t e r o l e m i a ) and f e d a c h o l e s t e r o l - r i c h d i e t f o r s e v e r a l months. The purpose of experiment I I was a l s o t o observe the e f f e c t s of. long-term f e e d i n g of the e x p e r i m e n t a l d i e t (10% f a t and a l t e r n a t i n g l e v e l s of 1 and 2% c h o l e s t e r o l ) on the plasma c h o l e s t e r o l l e v e l s of the b i r d s used and upon t h e i r u t i l i z a t i o n of the d i e t a r y f a t and c h o l e s t e r o l . E x p e r i m e n t a l S i x c o c k e r e l s w i t h c h o l e s t e r o l l e v e l s r a n g i n g from 175 - 195 mg.% were s e l e c t e d from s t r a i n A and p l a c e d i n i n -d i v i d u a l cages w i t h f r e e a ccess t o the e x p e r i m e n t a l d i e t and water. The e x p e r i m e n t a l d i e t was s i m i l a r i n c o m p o s i t i o n to t h a t used i n experiment I except f o r p e r i o d i c changes i n the l e v e l of c h o l e s t e r o l . The d u r a t i o n of f e e d i n g on each 24 regimen was as f o l l o w s : C h o l e s t e r o l Content D u r a t i o n of Feeding-1% 7 days (days 1 - 7 ) 2% 13 " ( "• 8 - 20) l°/o 390 " ( " 21 - 410) Feces were c o l l e c t e d from i n d i v i d u a l b i r d s , s t o r e d and a n a l y s e d as p r e v i o u s l y i n d i c a t e d i n experiment I. Plasma c h o l e s t e r o l l e v e l s were a l s o determined by methods used i n experiment I. R e s u l t s and D i s c u s s i o n The changes i n plasma c h o l e s t e r o l l e v e l s which accompanied the f e e d i n g of the b a s a l d i e t of experiment I supplemented w i t h 10% hydrogenated v e g e t a b l e o i l and a l t e r -n a t i n g l e v e l s of 1 and 2% c h o l e s t e r o l f o r 410 days are shown i n Table VI and F i g u r e 9. An i n c r e a s e i n c h o l e s t e r o l l e v e l s was observed to 88 days. Beyond t h i s p e r i o d t h e r e was a g e n e r a l d e p r e s s i o n i n these l e v e l s . As i n d i c a t e d i n T a b l e VI mean c h o l e s t e r o l l e v e l s f e l l d u r i n g t h i s l a t t e r p e r i o d from 1171 mg.% to 440 mg.%. I t i s assumed t h a t t h i s d e c l i n e r e s u l t e d from an i n -c r e a s e i n t h y r o i d a c t i v i t y brought on by w i n t e r c o n d i t i o n s . T h i s assumption r e s t s on the f i n d i n g s t h a t h y p e r t h y r o i d i s m i s more p r e v a l e n t i n the domestic fowl d u r i n g the w i n t e r months (Cruikshank, 1930; Winchester, 1940) and t h a t an i n -c r e a s e i n t h y r o i d a c t i v i t y produces a h y p o c h o l e s t e r o l e m i c 25 e f f e c t (Byers et a l . , 1958). A s i g n i f i c a n t c o r r e l a t i o n between pretreatment and peak c h o l e s t e r o l l e v e l s at twenty days c o u l d not be shown but the c o r r e l a t i o n c o e f f i c i e n t c a l c u l a t e d f o r these l e v e l s at t h a t time ( r = +0.785) c l o s e l y approached s i g n i f i c a n c e ( r = +0.811, p 0.05). These r e s u l t s i n d i c a t e d t h a t a con-s i d e r a b l e p a r t of the v a r i a b i l i t y i n c h o l e s t e r o l l e v e l s of b i r d s f e d c h o l e s t e r o l f o r twenty days was accounted f o r by v a r i a b i l i t y i n the pretreatment c h o l e s t e r o l l e v e l s . T able V I I shows q u a n t i t i e s of l i p i d s e x c r e t e d by i n d i v i d u a l b i r d s i n r e l a t i o n to t h e i r plasma c h o l e s t e r o l l e v e l s at v a r i o u s t i m e s . A n a l y s i s of these data r e v e a l e d a s i g n i f i c a n t c o r r e l a t i o n (p 0.05) between plasma c h o l e s -t e r o l l e v e l s and the amount of e t h e r e x t r a c t i n the f e c e s d u r i n g the p e r i o d between 134 and 170 days. Data r e g a r d i n g c h o l e s t e r o l e x c r e t i o n were not c o l l e c t e d f o r t h i s p e r i o d . Consequently the e x i s t e n c e of a three-way i n t e r r e l a t i o n s h i p between c h o l e s t e r o l l e v e l s and the amounts of c h o l e s t e r o l and s a p o n i f i a b l e l i p i d s i n the f e c e s c o u l d not be demonstrated as i n experiment I. S i n c e t h e r e was a s i g n i f i c a n t c o r r e l a t i o n between plasma c h o l e s t e r o l l e v e l s and amount of e t h e r e x t r a c t e x c r e t e d d u r i n g the p e r i o d i n q u e s t i o n (134 — 170 days) i t i s r e a s o n a b l e t o assume t h a t the t h r e e way i n t e r r e l a t i o n s h i p was a l s o i n e x i s t e n c e at t h i s time. D u r i n g the p e r i o d between 357 and 363 days t h e r e was no i n d i c a t i o n of an i n t e r r e l a t i o n -s h i p between c h o l e s t e r o l l e v e l s and the amounts of c h o l e s t e r o l and s a p o n i f i a b l e l i p i d s i n the f e c e s . 26 The f o l l o w i n g t a b l e shows t h a t the amount of l i p i d s absorbed by i n d i v i d u a l c o c k e r e l s on the h i g h - f a t , h i g h - c h o l e s t e r o l d i e t was i n c r e a s e d as f e e d i n g was prolonged. B i r d Grams E t h e r - E x t r a c t a b l e L i p i d s E x c r e t e d per Number 10 kg. D i e t 170 days 357 days 1 163.9 106.5 2 197.3 108.8 3 168.1 101.4 4 221.6 110.1 200.1 Mean 190.2 106.7 I t can be seen a l s o t h a t extended f e e d i n g of the d i e t l e d to a r e d u c t i o n i n i n d i v i d u a l v a r i a t i o n i n the amount of l i p i d s absorbed.Whereas at 170 days the amount of l i p i d s e x c r e t e d ranged from 164 - 221 grams per 10 k i l o g r a m s of d i e t at 357 days i t ranged from 101 — 110 grams per 10 k i l o -grams of d i e t . The decrease i n amount of l i p i d s e x c r e t e d can best be e x p l a i n e d by the f i n d i n g of Murthy et a l . (1961). These workers have shown t h a t the c a p a c i t y o f the i n t e s t i n e s o f r a t s to absorb l i p i d s was i n c r e a s e d by con t i n u o u s f e e d i n g o f a c h o l e s t e r o l - r i c h d i e t . They showed t h a t the e s t e r i f y i n g a c t i -v i t y o f the mucosal enzymes of the i n t e s t i n e s was c o n s i d e r a b l y i n c r e a s e d f o r c h o l e s t e r o l when r a t s were f e d a d i e t c o n t a i n i n g 1% c h o l e s t e r o l f o r f i v e weeks. T h i s i n c r e a s e i n a c t i v i t y was g r e a t e r w i t h r e s p e c t t o the s a t u r a t e d f a t t y a c i d s as compared 27 to the u n s a t u r a t e d ones. The s u r v i v a l time of i n d i v i d u a l c o c k e r e l s i s shown i n T a b l e VI. Postmortem examination of n o n s u r v i v o r s r e v e a l e d t h a t c o r o n a r y o c c l u s i o n was the cause of death i n every i n s t a n c e . The c o r o n a r y v e s s e l s of these b i r d s con-t a i n e d heavy d e p o s i t s of substances which gave a p o s i t i v e t e s t f o r c h o l e s t e r o l , w i t h the method of R o s e n t h a l et a l . (1957) p r e v i o u s l y d e s c r i b e d . With one e x c e p t i o n ( b i r d #4) the amount of sub-stances d e p o s i t e d i n the b l o o d v e s s e l s of s u r v i v o r s was c o m p a r a t i v e l y s m a l l . These s m a l l q u a n t i t i e s were a s s o c i a t e d w i t h the lowest c h o l e s t e r o l l e v e l s throughout the t e s t . The b i r d s which s u r v i v e d m a i n t a i n e d a lower mean plasma c h o l e s t e r o l l e v e l throughout the experiment than those which f a i l e d t o s u r v i v e . These r e s u l t s agree w i t h those r e -p o r t e d by F i s h e r et a l . (1960) who f e d to Leghorn p u l l e t s f o r t h r e e y e a r s a c h o l e s t e r o l — f r e e d i e t c o n t a i n i n g 10% animal f a t . The d i s t r i b u t i o n of substances d e p o s i t e d i n the b l o o d v e s s e l s i s a l s o i n agreement w i t h t h a t observed by F i s h e r : g r e a t e s t amounts were p r e s e n t i n the abdominal seg-ment of the a o r t a . TABLE I COMPOSITION OF BASAL DIET Ingredients Per 100 l b s d i e t - l b s gms Ground -wheat 60.00. Oats 10.00 Corn 5.00 Soybean meal 16.50 Dicalcium phosphate 1.75 D i s t i l l e r s dried solubles .2.00 Ground limestone 3.90 Iodized s a l t 0.35 ^Feeding o i l 0.50 Vitamin B^^ 0.54 Vitamin B 2 0.10 Manganese sulfate 5.68 100.00 6.32 * 3000 U . S . P . units vitamin A per gram and 300 International chick units vitamin D3 per gram. TABLE II PLASMA CHOLESTEROL LEVELS (mg %) OF COCKERELS OF STRAIN A IN EXPERIMENT 1 Lot Pretreat- Average Days Choles- Days Choles-ment l e v e l P.T. t e r o l t e r o l l e v e l fed •with-drawn (i) ( i i ) 3 6 4 7 1 123 129 126 324 538 172 135 14-1 129 135 246 204 130 132 130 131 131 225 400 224 150 150 136 143 253 390 210 140 Mean 136 131 134 262 383 I84 139 -2 152 140 146 m^ 141 140 141 - — - — 145 146 146 484 678 410 210 159 145 152 154 398 152 143 Mean 152 146 149 319 538 281. 177 3 158 152 155 295 350 .200 158 159 153 156 344 488 152 145 158 154 156 300 30.2 194 134 158 159 159 304 294 186 209 Mean 158 155 157 311 359 183 162 4 168 160 164 .297 470 190 157 160 165 163 282 462 194 138 179 166 173 205 250 210 180 155 170 163 494 800 420 220 Mean 166 165 166 320 496 254 174 5 185 180 183 448 800 44O 205 165 175 170 171 578 310 169 173 175 174 187 666 400 223 1.7.2 175 174 300 782 440 272 Mean 174 176 175 277 707 398 217 (Continued) 30 Lot Pretreat- " Average Days Choles- Days Choles-ment l e v e l P.T. t e r o l t e r o l l e v e l fed with-drawn (i) ( i i ) 3 6 4 7 6 190 183 187 310 466 292 199 182 182 182 370 398 2 2 4 164 198 189 194 331 592 316 170 181 189 185 210 374 280 205 Mean 188 186 187 305 458 278 185 Grand Mean 162 160 161 299 490 263 176 3 1 TABLE I I I PLASMA CHOLESTEROL LEVELS (mg %) OF COCKERELS OF STRAIN B IN EXPERIMENT 1 Lot Pretreat- Average Days Choles- Days Choles-ment l e v e l P.T. t e r o l t e r o l l e v e l fed with-drawn (1) ( i i ) 3 6 4 7 1 102 122 112 124 140 100 125 110 125 118 105 114 94 101 114 131 123 131 124 110 108 129 133 131 240 394 198 134 Mean 114 128 121 150 193 126 117 2 160 14? 154 368 432 I64 143 150 151 151 282 350 172 142 158 158 158 388 494 160 143 169 157 163 238 356 174 154 Mean 159 154 157 319 408 168 146 3 158 157 158 378 476 162 154 155 159 157 187 476 204 160 163 158 161 200 346 174 168 155 159 157 156 140 148 134 Mean 158 158 158 230 360 172 154 4 155 160 158 172 454 198 169 168 163 166 260 372 152 148 151 165 158 246 476 184 179 168 168 168 441 520 198 158 Mean 161 I64 163 280 456 183 164 5 156 172 I64 255 384 218 169 162 174 168 388 610 250 173 174 177 176 330 354 240 195 160 182 171 429 560 278 183 Mean 163 176 170 351 477 247 180 (Continued) 32 Lot Pretreat-ment l e v e l Average P.T. l e v e l Days Choles-t e r o l fed Days Choles-t e r o l •with-drawn (i) ( i i ) 3 6 4 7 6 180 172 170 1.80 185 185 186 189 183 179 178 185 198 300 446 400 320 350 820 520 190 186 388 390 200 190 185 227 Mean 176 186 181 336 503 289 201 Grand Mean 155 161 . 158 278 399 197 160 33 TABLE IV QUANTITIES OF EXCESS CIRCULATING CHOLESTEROL (mg %) ELIMINATED FOLLOWING CHOLESTEROL WITHDRAWAL FROM THE DIET OF COCKERELS IN EXPERIMENT 1 S t r a i n A Lot Total Excess Amount Eliminated At Present 4 days 7 days 1 249.3 199.0 (79.8) 243.7 (97.8) 2 389.0 257.0 (66.1) 361.5 (92.9) 3 202.0 175.5 (86.9) 197.0 (97.5) 4 329.7 242.0 (73.4) 321.7 (97.6) 5 531.2 309.0 (58.2) 489.2 (92.1) 6 ,270.5 179.5 (66.4) 273.0 (101.0) Mean 328.6 227.0 (71.8) 314.3 (96.5) S t r a i n B 1 72.0 67.5 (93.8) 76.0 (105.6) 2 251.5 240.5 (95.6) 262.5 (104-4) 3 201.3 187.5 (93.1) 205.5 (102.1) 4 293.0 272.5 (93.0) 292.0 ( 9 9 . 7 ) 5 307.2 230.5 (75.0) 297.0 (96.7 > 6 321.2 214.0 (66.6) 302.0 ( 94.0) Mean 241.0 202.1 (86.2) 239.2 (100.4) ( ) % of excess eliminated 34 TABLE V LIPIDS (gm/10 k g diet) IN FECES OF BIRDS OF STRAINS A AND B, FED HIGH FAT, HIGH CHOLESTEROL DIET IN EXPERIMENT 1 S t r a i n A Lot Cholesterol Ether Saponifiable % Saponifiable extractable l i p i d s l i p i d s i n l i p i d s ether extract 1 49.6 306.4 231.5 75.6 2 51.9 307.8 231.2 75.1 3 52.1 274.5 201.5 73.4 4 49.5 290.2 223.3 77.0 5 42.6 187.3 122.2 65.3 6 47.3 189.2 118.0 62.4 Mean 48.8 259.2 187.9 71.5 S t r a i n B 1 77.9 779.1 674.8 86.6 2 61.0 337.7 253.2 75.0 3 52.1 274.5 201.5 73.4 4 57.3 240.8 175.5 72.9 5 42.5 217.0 155.5 71.7 6 59.8 504.5 406.0 80.5 Mean 58.7 404.4 322.1 77.3 c<-\ TABLE VI PLASMA CHOLESTEROL LEVELS (mg %) OF COCKERELS OF STRAIN A, FED HIGH FATy HIGH CHOLESTEROL DIET IN EXPERIMENT 2 B i r d P r e t r e a t - Days of no. ment Days d i e t fed sur v i v a l cholesterol l e v e l 7* 14** ,20** 35*- 88* 134* 275* 345* 363* 370* 1 189 4-60 10 76 1372 10 82 1111 808 858 550 670 670 410 2 175 320 734 1045 806 795 757 343 250 330 340 410 3 180 - 556 936 704 678 505 353 300 500 500 410 Mean (S) 181.3 390.0 788.6 1117.6 864.O 861.3 690.0 518.0 366.6 500.0 503.3 410 4 195 676 1270 2059 1898 1750 1616 20 7 490 1050 1100 384 5 194 580 900 1329 1020 70 6 195 640 1300 2371 14-69 1520 1414 250 Mean (NS) 194.6 632.0 11.56.6 1919.6 14.62.3 1635 1515 207 490 1050 1100 235 Mean (S+NS) 188.0 535.2 972.6 1518.6 1163.2 1170.8 1020.0 44O.3 397.5 637.5 652.5 322 * 1% d i e t a r y cholesterol ** 2% d i e t a r y cholesterol S survivors NS nonsurvivors TABLE VII PLASMA CHOLESTEROL LEVELS (mg %) IN RELATION TO LIPIDS EXCRETED (gm/lO kg diet) BY COCKERELS OF STRAIN A IN EXPERIMENT 2 B i r d Pretreatment Plasma cholesterol Ether extractable Fecal No. cholesterol l e v e l at l i p i d s at cholesterol l e v e l s at 357 days 134- days 363 days 170 days 357 days 1 189 808 670 163.9 106.5 34.9 2 175 757 330 197.3 108.8 49.2 3 180 505 500 168.1 101.4. 55.6 4 195 1616 1050 221.6 110.1 48.2 5 195 141-4- — .200.1 — -Mean 186.8 1020.0 637.5 190.2 106.7 47.0 TABLE VIII SUMMARY OF CALCULATIONS OF REGRESSION AND CORRELATION COEFFICIENTS Experiment 1 S t r a i n X Y X* Y b r df p A + B Pretreatment cholesterol l e v e l Peak cholesterol l e v e l 160.3 444* 5 4.30 +0.473 44 0.01 n f e c a l choles-t e r o l H 53.7 444* 5 -9.11 -0.710 10 0.05 ti f e c a l saponi-f i a b l e l i p i d s f e c a l cholesterol 255.0 53.7 0.05 +0.856 10 0.01 tt % f e c a l saponifiable l i p i d s tt 74.4 53.7 1.19 +0.782 10 0.01 Experiment 2 A Pretreatment cholesterol l e v e l Peak cholesterol l e v e l at 20 days 188.0 1519.0 52.40 +0.785 4 0.05* B Ether extract at 170 days Plasma choleste r o l at 134- days 190.2 1020.0 16.29 +0.830 4 0.05 * Approaching si g n i f i c a n c e 38 F i g . 1. Mean plasma c h o l e s t e r o l l e v e l s of l o t s of c o c k e r e l s of s t r a i n A, i n experiment 1. 39 F i g . 2 . Mean plasma cholesterol levels of lots of cockerels of strain B, in experiment 1. 800-j 70CH 40 Fig. 3. Mean plasma cholesterol levels of cockerels of strains A and B, in experiment 1. 50O "i A1-6 AOO-\ SooJ zoo 150 A i r I a 3 4 5 BAYS Of CHOLESTEROL FEEDING- DAYS OP CHOLESTEROL WITHDRAWAL 41 Fig. 4. Degree of hypercholesterolemia induced in lots of cockerels of strains A and B fed a high-fat, high-cholesterol diet in experiment 1. QOO-i 7oo--J Ul > LU 50o-440-o ui H (0 ui - J O u < —i Q. loo-100-)*:»j\XNCRlA$>fi IH CHOLESjEWot LEVEL Pf?eT«6ATME/VT A 4 &4 n Fig. 5 . Regression of peak cholesterol on pretreatment cholesterol levels of individual cockerels of strains A and B in experiment 1. 4 3 Fig. 6. Regression of mean peak cholesterol levels on the amount of cholesterol in feces of cockerels fed a high-fat, high-cholesterol diet in experiment 1. 8oo - i Fig. 7. Regression of cholesterol on the saponifiable lipids in the feces of cockerels fed a high-fat, high-cholesterol diet in experiment 1. G R A M S S A P O N I F I A B L E LIPIDS IN FECES PER IO K I L O & K A M S OF DIET 45 Fig. 8. Regression of the amount of cholesterol excreted on the percent of sponifiable lipids in the ether extract of feces of cockerels fed a high-fat, high-cholesterol diet in experiment 1. Q I r 1— T 1 "i 6 0 66 70 70 SO 8 5 90 PERCENT 9 A P O N I F / A B U LIPIDS IN ETHER EXTRACT OF FECES & Fig. 9. Plasma cholesterol levels of cockerels of strain A, fed a high-fat, high-cholesterol diet in experiment II. D A Y S ON T E S T '47 SUMMARY AND CONCLUSIONS Two experiments were conducted w i t h a d u l t White Leghorn c o c k e r e l s t o determine whether plasma c h o l e s t e r o l l e v e l s promoted by a d i e t c o n t a i n i n g 10% of a hydrogenated v e g e t a b l e o i l and 1% c h o l e s t e r o l were r e l a t e d t o the degree of u t i l i z a t i o n o f these two d i e t a r y components. In a d d i t i o n the experiments attempted t o determine (1) the manner i n which the above r e l a t i o n s h i p may be a f f e c t e d by extended f e e d i n g o f the d i e t and (2) the e f f e c t o f t h i s type of f e e d -i n g on the development of a t h e r o s c l e r o s i s i n a d u l t c o c k e r e l s . In the s h o r t - t e r m experiment (experiment I) the ex p e r i m e n t a l b i r d s were s e l e c t e d from two s t r a i n s p r e v i o u s l y shown t o d i f f e r i n s u s c e p t i b i l i t y t o d i e t a r y i n d u c e d hyper-c h o l e s t e r o l e m i a . The b i r d s were s e l e c t e d i n o r d e r t o ensure a wide range i n pretreatment c h o l e s t e r o l l e v e l s and were a s s i g n e d i n t o s m a l l l o t s on the b a s i s of these l e v e l s . Each l o t c o n t a i n e d b i r d s w i t h s m a l l d i f f e r e n c e s i n t h e i r c h o l e s -t e r o l l e v e l s . C h o l e s t e r o l and hydrogenated v e g e t a b l e o i l were f e d at l e v e l s o f 1% and 10% r e s p e c t i v e l y f o r s i x days. At the end of t h i s time marked d i f f e r e n c e s i n the mean plasma c h o l e s t e r o l l e v e l s were observed among l o t s and between the two groups of s t r a i n s A and B. 48 When c h o l e s t e r o l was withdrawn from the d i e t plasma c h o l e s t e r o l r e t u r n e d t o the pretreatment l e v e l s . The l e v e l s observed a f t e r f o u r and seven days withdrawal i n d i c a t e d t h a t the d i f f e r e n c e s i n the degree of d i e t a r y h y p e r c h o l e s t e r o l e m i a were not p r i m a r i l y due t o d i f f e r e n c e s i n the r a t e of e l i m i n a t i o n of excess c h o l e s t e r o l from the c i r c u l a t i o n . The amount of c h o l e s t e r o l e x c r e t e d by the v a r i o u s l o t s f o l l o w i n g s i x days f e e d i n g of the h i g h - f a t , h i g h -c h o l e s t e r o l d i e t v a r i e d g r e a t l y and was found t o be s i g n i -f i c a n t l y c o r r e l a t e d w i t h (1) the plasma c h o l e s t e r o l l e v e l s (p 0.05) and (2) the amount of s a p o n i f i a b l e l i p i d s e x c r e t e d (p 0.01). These r e s u l t s suggest t h a t d i f f e r e n c e s i n c h o l e s -t e r o l a b s o r p t i o n may a r i s e from d i f f e r e n c e s i n f a t a b s o r p t i o n . D i f f e r e n c e s i n s u s c e p t i b i l i t y of c o c k e r e l s t o d i e t a r y hyper-c h o l e s t e r o l e m i a may i n consequence be at l e a s t p a r t l y a t t r i b u t a b l e t o d i f f e r e n c e s i n f a t a b s o r p t i o n . In experiment I I c o c k e r e l s were f e d a h i g h - f a t , h i g h - c h o l e s t e r o l d i e t f o r an extended p e r i o d of time. T h i s i s i n c o n t r a s t t o experiment I which was a s h o r t - t e r m t e s t . In the second experiment the r e l a t i o n s h i p between hyper-c h o l e s t e r o l e m i a and d i e t a r y l i p i d u t i l i z a t i o n was not c l e a r l y e v i d e n t . In bo t h experiments however the degree of induced h y p e r c h o l e s t e r o l e m i a and pretreatment c h o l e s t e r o l l e v e l s were found t o be s i g n i f i c a n t l y c o r r e l a t e d . It was observed that birds which survived 410 days on a high-fat, high-cholesterol d i e t , maintained throughout the t e s t a lower mean plasma c h o l e s t e r o l l e v e l than nonsurvivors. The blood vessels of survivors compared to nonsurvivors showed l i t t l e evidence of a t h e r o s c l e r o s i s . 50 BIBLIOGRAPHY Ahrens, E. H., T. T. 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