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Studies on egg yolk myelin figures and granule low-density lipoproteins Garland, Timothy D. 1973

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STUDIES ON EGG YOLK MYELIN FIGURES AND GRANULE LOW-DENSITY LIPOPROTEINS b y TIMOTHY D. GARLAND B.Sc.(Agr), U n i v e r s i t y of B r i t i s h Columbia, 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Food Science We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October, 197 3 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e 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 f o r r e f e r e n c e and s t u d y . I f u r t h e r 1 agree -that p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r 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 the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h C olumbia Vancouver 8, Canada STUDIES ON EGG YOLK MYELIN FIGURES AND GRANULE LOW-DENSITY LIPOPROTEINS TIMOTHY D. GARLAND UNDER THE SUPERVISION OF DR, WILLIAM D. POWRIE The e x c e p t i o n a l e m u l s i f y i n g p o w e r o f egg y o l k h a s y e t t o be f u l l y u n d e r s t o o d . A l t h o u g h i t i s known t h a t c e r t a i n y o l k p r o t e i n s a n d l i p o p r o t e i n c o m p l e x e s h a v e s p e c i f i c e m u l s i -f y i n g p r o p e r t i e s , t h e i r c o n t r i b u t i o n t o e m u l s i o n f o r m a t i o n and s t a b i l i t y h a s n o t b e e n e s t a b l i s h e d . I n d e e d , k n o w l e d g e on t h e s t r u c t u r e a n d c h e m i c a l c o m p o s i t i o n o f some l i p o p r o t e i n s i s l a c k i n g . T h i s s t u d y was i n i t i a t e d t o e x a m i n e t h e s t r u c -t u r a l , c h e m i c a l a n d p h y s i c a l p r o p e r t i e s o f two t y p e s o f l i p o p r o t e i n c o m p l e x e s , t h e m y e l i n f i g u r e (MF) a n d t h e g r a n u l e l o w - d e n s i t y l i p o p r o t e i n ( L D L g ) , a n d t o a s s e s s t h e f u n c t i o n a l b e h a v i o r o f t h e s e c o m p o n e n t s i n s i m p l e o i l and w a t e r e m u l s i o n s . F i v e f r a c t i o n s ( F I t o FV) w e r e . o b t a i n e d by u l t r a -c e n t r i f u g a t i o n o f w a s h e d y o l k g r a n u l e s i n 1.71M N a C l o f two f l o a t i n g f r a c t i o n s , t h e s u b p e l l i c l e ( F I D was f o u n d t o c o n s i s t o f o n l y MF and LDLg . V a r i o u s t e c h n i q u e s w e r e e m p l o y e d t o s e p a r a t e F I I i n t o f r a c t i o n s w h i c h w e r e e x a m i n e d by e l e c t r o n m i c r o s c o p y t o e v a l u a t e t h e h o m o g e n e i t y a n d s t r u c t u r a l i n t e g r i t y o f t h e f r a c t i o n s . U l t r a f i l t r a t i o n was i v , e f f e c t i v e f o r p r e p a r i n g n a t i v e L D L g , b u t c a u s e d d e s t r u c t i o n o f MF. C o m p l e x i n g t h e l o w - d e n s i t y l i p o p r o t e i n s w i t h d e x t r a n s u l f a t e was f o u n d t o be an a d e q u a t e m e t h o d o f s e p a r a t i n g t h e c o m p o n e n t s o f F I I , b u t i n t e r a c t i o n o f MF w i t h d e x t r a n s u l f a t e r e d u c e d t h i s ' m e t h o d ' s e f f e c t i v e n e s s . S e p a r a t i o n by S e p h a r o s e 2B g e l f i l t r a t i o n was t h e most r a p i d a n d e f f e c t i v e m e t h o d f o r s e p a r a t i o n o f t h e two t y p e s o f p a r t i c l e s f r o m F I I . E l e c t r o n m i c r o s c o p y o f MF f r a c t i o n s e p a r a t e d by S e p h a r o s e 2B g e l f i l t r a t i o n r e v e a l e d t h a t t h e m i c e l l e s w e r e composed o f e i t h e r a s i n g l e t h i c k l a m e l l a , o r two o r more somewhat t h i n n e r c o n c e n t r i c l a m e l l a e a b o u t a w e a k l y o s m i o p h i l i c c o r e . T h e s e o u t e r l a m e l l a e v a r i e d i n e l e c t r o n d e n s i t y a f t e r OsO^ f i x a t i o n . M o r e o v e r , d e n s i t y g r a d i e n t s t u d i e s i n d i c a t e d t h a t h e a v y and l i g h t MF s p e c i e s e x i s t . E l e c t r o n m i c r o g r a p h s showed t h a t LDLg a r e g l o b u l a r i n s h a p e , w i t h h i g h l y e l e c t r o n d e n s e s u r f a c e u l t r a p a r t i c l e s . P o s s i b l e s t r u c t u r e s f o r b o t h MF and LDLg p a r t i c l e s a r e d i s c u s s e d . MF a n d LDLg f r a c t i o n s , s e p a r a t e d by S e p h a r o s e 2B g e l f i l t r a t i o n , w e r e a n a l y z e d f o r t o t a l n i t r o g e n a n d p h o s p h o r u s by K j e l d a h l a n d T e c h n i c o n A u t o a n a l y s e r m e t h o d s , r e s p e c t i v e l y . N i t r o g e n c o n t e n t ( 2 . 5 % ) o f t h e f r a c t i o n s was a l m o s t i d e n t i c a l b u t t h e p h o s p h o r u s c o n t e n t s w e r e 1.25% f o r MF a n d 1.05% f o r L D L g . The l i p i d c o m p o n e n t s o f MF a n d LDLg w e r e q u a n t i t a t i v e l y d e t e r m i n e d a f t e r s e p a r a t i o n b y s i l i c a g e l t h i n l a y e r c h r o m o -t o g r a p h y . L i p i d c o n t e n t s o f MF and LDLg were 8 6 a n d 8 4 % , r e s p e c t i v e l y . B o t h t y p e s o f l i p o p r o t e i n w e r e composed o f c h o l e s t e r o l , p h o s p h a t i d y l c h o l i n e , p h o s p h a t i d y l e t h a n o l a m i n e , l y s o p h o s p h a t i d y l e t h a n o l a m i n e , s p h i n g o m y e l i n , a n d t r i g l y c e r i d e s . C o m p a r i s o n o f p a r t i c l e c o m p o s i t i o n r e v e a l e d t h a t a m o unts o f some o f t h e s e c o n s t i t u e n t s d i f f e r e d . The c o m p o s i t i o n o f t h e l i p i d - p r o t e i - n c o m p l e x e s o f b o t h MF a n d LDLg a p p e a r e d t o v a r y w i t h age o f t h e b i r d : y o u n g b i r d s p r o d u c e d egg y o l k s whose MF and LDLg c o m p o n e n t s w e r e s i g n i -f i c a n t l y d i f f e r e n t i n t o t a l n i t r o g e n a n d p h o s p h o r u s c o n t e n t s . E l e c t r o n m i c r o s c o p y was e m p l o y e d t o a s s e s s .the s u r f a c e c h a r a c t e r i z a t i o n o f e m u l s i f i e d o i l d r o p l e t s i n e m u l s i o n s p r e -p a r e d w i t h e i t h e r n a t i v e a n d l y o p h i l i z e d s a m p l e s o f y o l k , MF and LDLg. MF- and LDLg w e r e d i s r u p t e d d u r i n g e m u l s i f i c a t i o n and t h e d i s r u p t e d p a r t i c l e s f o r m e d d i s t i n c t z o n e s a b o u t t h e s u r f a c e o f t h e o i l d r o p l e t s w i t h t h e r e s u l t o f c o n t r i b u t i n g t o t h e s t a b i l i t y o f t h e e m u l s i o n s . T h r e e z o n e s w e r e o b s e r v e d on t h e s u r f a c e s o f d r o p l e t s o f e m u l s i o n s made w i t h n a t i v e MF, n a t i v e L D L g , and l y o p h i l i z e d L D L g , w h e r e a s f o u r z o n e s were p r e s e n t on d r o p l e t s i n l y o p h i l i z e d MF e m u l s i o n s . H o w e v e r , o n l y one o f t h e s e z o n e s was t h e same i n MF and LDLg - e m u l s i o n s . The s t a b i l i z i n g z o n e s i n t h e y o l k e m u l s i o n s w e r e s i m i l a r t o z o n e s i n t h e LDLg e m u l s i o n s . S i n c e N a C l was n o t i n c o r p o r a t e d i n t h e y o l k e m u l s i o n s , y o l k g r a n u l e s r e m a i n e d i n t a c t a n d p l a s m a l o w - d e n s i t y l i p o p r o t e i n s , s i m i l a r i n c o m p o s i t i o n a n d v i . s t r u c t u r e t o L D L g , a c t e d a s e m u l s i f i e r s . S u r f a c e t e n s i o n e x p e r i m e n t s showed t h a t MF and LDLg l o w e r e d t h e s u r f a c e t e n s i o n o f w a t e r t o t h e same l e v e l a s t h a t r e p o r t e d f o r y o l k s o l u t i o n s w i t h t h e same s o l i d s c o n t e n t . N a t i v e LDLg e m u l s i o n s w e r e more s t a b l e t h a n n a t i v e MF and y o l k e m u l s i o n s when t h e y w e re s u b j e c t . e d t o h i g h s p e e d c e n t r i f u g a t i o n . L y o p h i l i z a t i o n however., o f MF and LDLg e m u l s i o n s , i m p r o v e d t h e s t a b i l i t y o f t h e r e s u l t i n g e m u l s i o n s . APPROVED BY: D r . W i l l i a m D. P o w r i e , P r o f e s s o r and C h a i r m a n o f t h e D e p a r t m e n t s o f F o o d S c i e n c e a n d A g r i c u l t u r a l E n g i n e e r i n g . DATE V i i . TABLE OF CONTENTS T I T L E PAGE: S t u d i e s on Egg Y o l k M y e l i n F i g u r e s and G r a n u l e L o w - D e n s i t y L i p o p r o t e i n s i PERMISSION PAGE i i ABSTRACT i i i TABLE OF CONTENTS v i i L I S T OF TABLES i x L I S T OF FIGURES x NOMENCLATURE x i v ACKNOWLEDGEMENTS xv GENERAL INTRODUCTION 1 LITERATURE CITED 6 CHAPTER I : .The I s o l a t i o n o f M y e l i n F i g u r e s and L o w - D e n s i t y L i p o p r o t e i n s f r o m Egg Y o l k G r a n u l e s ABSTRACT . ' 8 INTRODUCTION 9 EXPERIMENTAL 18 A. M a t e r i a l s 18 B. Methods 18 1. G r a n u l e p r e p a r a t i o n 18 2. P r e p a r a t i o n o f l o w - d e n s i t y f r a c t i o n ( F I I ) f r o m g r a n u l e s 19 3. Methods f o r s e p a r a t i o n o f m y e l i n f i g u r e s and l o w - d e n s i t y l i p o p r o t e i n s i n F I I 20 a . U l t r a f i l t r a t i o n 20 b. D e x t r a n s u l f a t e - l i p o p r o t e i n c o m p l e x i n g 2 0 c . G e l f i l t r a t i o n 21 C. E l e c t r o n M i c r o s c o p y 23 RESULTS 26 DISCUSSION. 57 CONCLUSIONS 6H LITERATURE CITED 6 5 v i i i . CHAPTER 2: C h a r a c t e r i z a t i o n o f Egg Y o l k M y e l i n F i g u r e s and G r a n u l e L o w - D e n s i t y L i p o p r o t e i n s ABSTRACT 7 0 INTRODUCTION 7 2 EXPERIMENTAL MATERIALS AND METHODS 7 5 A. M a t e r i a l s 7 5 B. Methods 76 1. P r e p a r a t i o n o f MF and LDLg 7 6 2. A n a l y t i c a l Methods 78 a . L i p i d e x t r a c t i o n 78 b . T h i n l a y e r c h r o m a t o g r a p h y 7 9 c . N i t r o g e n and p h o s p h o r u s d e t e r m i n a -t i o n 82 d . P h o s p h o l i p i d q u a n t i t a t i v e a n a l y s i s 87 e. Q u a n t i t a t i v e c h o l e s t e r o l d e t e r m i n a -t i o n 89 RESULTS AND DISCUSSION 91 CONCLUSIONS 109 LITERATURE CITED 110 CHAPTER 3: F u n c t i o n a l P r o p e r t i e s o f Egg Y o l k M y e l i n F i g u r e s and G r a n u l e L o w - D e n s i t y L i p o p r o t e i n s ABSTRACT 112 INTRODUCTION 113 EXPERIMENTAL 116 A. M a t e r i a l s 116 B. Methods 116 1. MF and LDLg p r e p a r a t i o n 116 2. E m u l s i o n p r e p a r a t i o n 117 3. E m u l s i o n s t a b i l i t y 117 4. S u r f a c e t e n s i o n 118 5. E l e c t r o n m i c r o s c o p y 120 RESULTS 122 DISCUSSION 150 CONCLUSIONS 155 LITERATURE CITED 156 L I S T OF TABLES P e r c e n t o f N i t r o g e n and P h o s p h o r u s o f L y o p h i l i z e d MF and LDLg F r a c t i o n s L i p i d C o m p o s i t i o n o f MF and LDLg L y o p h i l i z e d S a m p l e s C o m p o s i t i o n o f MF and LDLg I s o l a t e d f r o m Y o l k o f 39 Wk O l d Hens P h o s p h o l i p i d C o m p o s i t i o n o f MF and LDLg L i p i d E x t r a c t s CHAPTER I L I S T OF FIGURES FIGURE PAGE E l e c t r o n m i c r o g r a p h s o f F r a c t i o n I I f r o m u l t r a c e n t r i f u g e d d i s p e r s i o n s o f g r a n u l e s i n 1.71 M s o d i u m c h l o r i d e . Mf, m y e l i n f i g u r e ; L D L g , g r a n u l e l o w - d e n s i t y l i p o -p r o t e i n . ( 2 1 , 0 0 0 X ) . 29 E l e c t r o n m i c r o g r a p h o f m y e l i n f i g u r e s e d i m e n t f r o m u l t r a c e n t r i f u g e d F I I i n d i s t i l l e d w a t e r . MF, m y e l i n f i g u r e ; MF,, d i s r u p t e d m y e l i n f i g u r e . ( 5 2 , 0 0 0 X ) . 33 E l e c t r o n m i c r o g r a p h o f u l t r a f i l t r a t i o n r e s i d u e a f t e r one p a s s . MF, m y e l i n f i g u r e ; L D L g , g r a n u l e l o w - d e n s i t y l i p o p r o t e i n . ('62 ,000 X ) . 35 E l e c t r o n m i c r o g r a p h s o f u l t r a f i l t r a t i o n r e s i d u e a f t e r t h r e e t r e a t m e n t s . MF, m y e l i n f i g u r e s ; ( 1 4 2 , 0 0 0 X) MF^, d i s r u p t e d m y e l i n f i g u r e , 37 E l e c t r o n m i c r o g r a p h o f u l t r a f i l t r a t i o n f i l t r a t e a f t e r one t r e a t m e n t . L D L g , g r a n u l e l o w - d e n s i t y l i p o p r o t e i n . ( 1 0 5 , 0 0 0 X ) . 38 E l e c t r o n ' m i c r o g r a p h o f F r a c t i o n I I - c a l c i u m -d e x t r a n s u l f a t e c o m p l e x . C, c o m p l e x . ( 3 3 , 0 0 0 X ) . MF, m y e l i n f i g u r e ; 40 E l e c t r o n m i c r o g r a p h o f m y e l i n - r i c h f r a c t i o n a f t e r one d e x t r a n s u l f a t e t r e a t m e n t . MF, m y e l i n f i g u r e ; L D L g , g r a n u l e l o w - d e n s i t y l i p o p r o t e i n . ( 1 6 , 0 0 0 X ) . 42 E l e c t r o n m i c r o g r a p h o f LDLg s u r r o u n d e d by MF a f t e r one d e x t r a n s u l f a t e t r e a t m e n t . MF , d i s r u p t e d m y e l i n f i g u r e , L D L g , g r a n u l e l o w - d e n s i t y l i p o p r o t e i n . ( 6 8 , 7 0 0 X ) . E l e c t r o n m i c r o g r a p h o f M F - r i c h f r a c t i o n a f t e r t h r e e d e x t r a n s u l f a t e t r e a t m e n t s . MF m y e l i n ; MF, , h e a v y m y e l i n ; m a t e r i a l . V34,000 X ) . 1' l i g h i LDM, l o w - d e n s i t y 43 44 x i . FIGURE PAGE 10 E l e c t r o n m i c r o g r a p h o f L D L g - d e x t r a n s u l f a t e c o m p l e x . ( 3 5 4 , 0 0 0 X ) . 45 11 G e l f i l t r a t i o n e l u t i o n p a t t e r n s o f e g g y o l k F I I . ( E l u t a n t : 1.71 M N a C l , Bed H e i g h t 39 cm, P r e s s u r e 57 cm H 2 0 , 50 d r o p / f r a c t i o n ) . 47 12 E l e c t r o n m i c r o g r a p h o f S e p h a r o s e 6B m y e l i n f r a c t i o n . MF, m y e l i n f i g u r e ; L D L g , g r a n u l e l o w - d e n s i t y l i p o p r o t e i n . ( 3 3 , 0 0 0 X ) . 48 13 E l e c t r o n m i c r o g r a p h o f S e p h a r o s e 6B LDLg f r a c t i o n . MF, m y e l i n f i g u r e ; L D L g , g r a n u l e l o w - d e n s i t y l i p o p r o t e i n . ( 5 9 , 0 0 0 X ) . 50 14 E l e c t r o n m i c r o g r a p h o f S e p h a r o s e 4B MF f r a c t i o n . MF, m y e l i n f i g u r e ; MF,, d i s -r u p t e d m y e l i n f i g u r e ; MF m y e l i n f i g u r e . c e n t e r . ( 3 3 , 0 0 0 X ) . c 5 2 15 E l e c t r o n m i c r o g r a p h o f S e p h a r o s e 4B LDLg f r a c t i o n . UP, u l t r a p a r t i c l e . ( 2 5 3 , 0 0 0 X ) . 53 16 E l e c t r o n m i c r o g r a p h o f S e p h a r o s e 2B MF f r a c t i o n . MF, m y e l i n f i g u r e ; MF , m y e l i n f i g u r e c e n t e r s ; MF,, d i s r u p t e d m y e l i n f i g u r e . ( 5 3 , 0 0 0 X ) . 54 17 E l e c t r o n m i c r o g r a p h o f S e p h a r o s e 2B LDLg f r a c t i o n . L D L g , g r a n u l e l o w - d e n s i t y l i p o -p r o t e i n ; UP, u l t r a p a r t i c l e . ( 1 4 0 , 0 0 0 X ) . 55 CHAPTER I I 1 R e f e r e n c e p h o s p h o l i p i d s : T = t r i o l e i n ( R f . 0 . 7 9 ) ; C = c h o l e s t e r o l ( R f , 0 . 6 1 ) ; PE = p h o s p h a t i d y l e t h a n o l a m i n e ( R f , 0 . 3 4 ) ; PC - p h o s p h a t i d y l c h o l i n e ( R f , 0 . 2 4 ) ; LPE = l y s o p h o s p h a t i d y l e t h a n o l a m i n e ( R f . 0 . 1 9 ) ; S = s p h i n g o m y e l i n ( R f , 0 . 1 4 ) ; V i s u a l i z e d b y e x p o s u r e t o i o d i n e . 99 2 T h i n l a y e r c h r o m a t o g r a p h y o f MF l i p i d s : N = n e u t r a l l i p i d s ( R f , 0 . 7 8 ) ; C = c h o l e s t e r o l ( R f , 0 . 6 2 ) ; PE = p h o s p h a t i d y l e t h a n o l a m i n e ( R f , 0 . 3 7 ) ; PC = p h o s p h a t i d y l c h o l i n e ( R f , 0 . 2 4 ) ; LPE = l y s o p h o s p h a t i d y l e t h a n o l a m i n e (Rf.. 0 . 1 9 ) ; S = s p h i n g o m y e l i n ( R f , 0 . 1 5 ) . V i s u a l i z e d b y e x p o s u r e t o i o d i n e . 100 x i i . FIGURE PAGE 3 T h i n l a y e r c h r o m a t o g r a p h y Of LDLg l i p i d s : N = n e u t r a l l i p i d ( R f , 0 . 7 6 ) ; C = c h o l e s t e r o l ( R f , 0 . 6 2 ) ; PE = p h o s p h a t i d y l e t h a n o l a m i n e ( R f , 0 . 4 0 ) ; PC = p h o s p h a t i d y l c h o l i n e ( R f , 0 . 2 4 ) ; LPE = l y s o p h o s p h a t i d y l e t h a n o l a m i n e ( R f , 0 . 1 8 ) ; S = s p h i n g o m y e l i n ( R f , 0 . 1 4 ) . V i s u a l i z e d by e x p o s u r e t o i o d i n e . 101 4 A b s o r b a n c e s p e c t r a o f c h o l e s t e r o l - f e r r i c c h l o r i d e c o m p l e x : A. T h i n l a y e r c h r o m a t o -g r a p h y s t e r o i d (296 y g ) ; B. R e f e r e n c e c h o l e s t e r o l (218 y g ) . 102 CHAPTER I I I 1 E l e c t r o n m i c r o g r a p h o f l y o p h i l i z e d MF.F: f r e e f a t z o n e s (40,000 X ) . 124 2 E l e c t r o n m i c r o g r a p h o f h y d r a t e d l y o p h i l i z e d MF. (16,000 X ) . 126 3 E l e c t r o n ' m i c r o g r a p h o f l y o p h i l i z e d L D L g . (40,000 X ) . 127 4 E l e c t r o n m i c r o g r a p h o f h y d r a t e d l y o p h i l i z e d L D L g . (58 ,000 X ) . 128 5 E l e c t r o n m i c r o g r a p h o f an o i l / w a t e r e m u l s i o n p r e p a r e d w i t h n a t i v e MF. 1: Zone 1, 2: Zone 2. 3: Zone 3. 4: Zone 4. See t e x t f o r zone c h a r a c t e r i z a t i o n . (13,700 X ) . 130 6 E l e c t r o n m i c r o g r a p h o f an o i l / w a t e r e m u l s i o n p r e p a r e d f r o m l y o p h i l i z e d MF. 1: Z o n e . 1 . 2: Zone 2. 3: Zone 3. 4: Zone 4. See t e x t f o r zone c h a r a c t e r i z a t i o n . (60,000 X ) . 131 7 E l e c t r o n m i c r o g r a p h o f an o i l / w a t e r e m u l s i o n p r e p a r e d w i t h l y o p h i l i z e d MF. Zone 4. See t e x t f o r zone c h a r a c t e r i z a t i o n . (160,000 X).132 8 E l e c t r o n m i c r o g r a p h o f an o i l / w a t e r e m u l s i o n p r e p a r e d w i t h n a t i v e MF. Zone 2. See t e x t f o r zone c h a r a c t e r i z a t i o n . (380,000 X ) . 133 9 E l e c t r o n m i c r o g r a p h o f an o i l d r o p l e t s t a b i l i z e d w i t h n a t i v e L D L g . (46,500 X ) . 135 10 E l e c t r o n m i c r o g r a p h o f o i l d r o p l e t s s t a b i l i z e d w i t h l y o p h i l i z e d L D L g . (24,000 X ) . 136 x i i i . FIGURE PAGE .11 E l e c t r o n m i c r o g r a p h o f an o i l d r o p l e t , s t a b i l i z e d w i t h l y o p h i l i z e d LDLg. ( 1 0 0 , 0 0 0 X) 138 12 E l e c t r o n m i c r o g r a p h o f an o i l / w a t e r e m u l s i o n p r e p a r e d w i t h n a t i v e y o l k . G: g r a n u l e . ( 5 5 , 0 0 0 X ) . 139 13 E l e c t r o n m i c r o g r a p h o f o i l d r o p l e t s s t a b i l i z e d w i t h l y o o h i l i z e d y o l k . G: g r a n u l e . ( 4 0 , 0 0 0 X ) . 140 14 O i l and w a t e r s e p a r a t i o n o f v a r i o u s e m u l s i o n s a f t e r 1 h r a t 35°C and 3 0 m i n a t 20,000 X G. L y o p h i l i z e d s a m p l e s : 1: y o l k , 2: F I I , 3: MF, 4: LDLg. N a t i v e s a m p l e s ; 5: y o l k , 6: F I I , 7: MF, 8: LDLg. 14 2 15 S u r f a c e t e n s i o n o f : a. n a t i v e MF; b. l y o -p h i l i z e d MF; c. l y o p h i l i z e d L D L g ; d. n a t i v e L DLg. A l l s a m p l e s i s o l a t e d f r o m e g g s o f 41 week o l d b i r d s . 146 16' S u r f a c e t e n s i o n o f l y o p h i l i z e d MF ( a ) , F I I ( b ) , and LDLg ( c ) i s o l a t e d f r o m e g g s o f h e n s a b o u t 1..5 y e a r s o l d . 148 xiv. NOMENCLATURE F I I S u b p e l l i c l e , f l o a t i n g f r a c t i o n o f a s u p e r c e n t r i f u g e d 1.71M N a C l d i s p e r s i o n o f y o l k g r a n u l e s . HDL H i g h - d e n s i t y l i p o p r o t e i n s . LDFG G r a n u l e l o w - d e n s i t y f r a c t i o n , c omposed o f LDFG-^ and LDFG2 c o m p o n e n t s . LDL L o w - d e n s i t y l i p o p r o t e i n s . LDL-DS L o w - d e n s i t y l i p o p r o t e i n - d e x t r a n s u l f a t e c o m p l e x . L D L g G r a n u l e l o w - d e n s i t y l i p o p r o t e i n s o f t h e F I I . LDLp P l a s m a l o w - d e n s i t y l i p o p r o t e i n s , composed o f LDL-^ (LPL-j_) a n d L D L 2 ( L P L 2 ) c o m p o n e n t s . LPE L y s o p h o s p h a t i d y l e t h a n o l a m i n e . MF M y e l i n f i g u r e s o f t h e F I I . N N i t r o g e n . P P h o s p h o r u s . PC P h o s p h a t i d y l c h o l i n e . PE P h o s p h a t i d y l e t h a n o l a m i n e . S S p h i n g o m y e l i n . VLDL V e r y l o w - d e n s i t y l i p o p r o t e i n s . ACKNOWLEDGEMENTS The a u t h o r i s e x t r e m e l y g r a t e f u l t o D r . W i l l i a m P o w r i e u n d e r whose s u p e r v i s i o n t h e p r o j e c t and t h e s i s p r e -p a r a t i o n was c a r r i e d o u t . H i s d i r e c t i o n , a s s i s t a n c e , c r i t i c i s m a nd i n s u p e r a b l e p a t i e n c e t h r o u g h o u t t h e c o u r s e o f t h i s s t u d y i s g r a t e f u l l y a p p r e c i a t e d . The a u t h o r a l s o e x p r e s s e s g r a t i t u d e t o t h e members o f t h e s u p e r v i s o r y c o m m i t t e e ; D r . J . F . R i c h a r d s , D r . M.A. Tu n g , D r . S. N a k a i and D r . G.G.S. D u t t o n f o r a d v i c e a n d e n c o u r a g e m e n t , e s p e c i a l l y d u r i n g t h e p r e p a r a t i o n o f t h e m a n u s c r i p t . A s p e c i a l a c k n o w l e d g e m e n t i s e x t e n d e d t o my w i f e , M a u r e e n , f o r h e r e n c o u r a g m e n t , u n d e r s t a n d i n g and h e l p w i t h t h e p r e p a r a t i o n o f t h e m a n u s c r i p t . A p p r e c i a t i o n i s a l s o e x p r e s s e d t o Mr. L e s J o h n s o n f o r h i s a s s i s t a n c e w i t h t h e T e c h n i c o n A u t o a n a l y s e r . 1 . GENERAL INTRODUCTION I n many o f t h e f o o d p r o d u c t s m a n u f a c t u r e d t o d a y , c e r t a i n i n g r e d i e n t s a r e i n c l u d e d t o i m p a r t a v a r i e t y o f f u n c t i o n a l p r o p e r t i e s t o t h e f o o d s y s t e m . H o w e v e r , s i n c e t h e m e c h a n i s m s o f f u n c t i o n a l i t y a r e o f t e n n o t known, o p t i m a l c o n -d i t i o n s may n o t be a c h i e v e d . I n p a r t i c u l a r , egg y o l k i s r e g a r d e d as a f u n c t i o n a l c o m p o n e n t f o r t h e c r e a t i o n a n d s t a b i l i z a t i o n o f f o o d e m u l s i o n s s u c h a s m a y o n n a i s e , s a l a d d r e s s i n g s , i c e . c r e a m and b a k e r y p r o d u c t s . A t t h e p r e s e n t t i m e l i t t l e s c i e n t i f i c i n f o r m a t i o n i s a v a i l a b l e on t h e mode o f ' a c t i o n o f y o l k as a n e m u l s i f i e r . H o w e v e r , a t t e m p t s h a v e b e e n made t o d e t e r m i n e t h e t y p e s o f y o l k c o m p o n e n t s i n v o l v e d i n e m u l s i f i c a t i o n a n d s t a b i l i z a t i o n a n d t o e l u c i d a t e t h e m e c h a n i s m . S n e l l e t a l . ( 1 9 3 5 ) p r o p o s e d t h a t p r o t e i n i n c o n j u n c t i o n w i t h l e c i t h i n , " l e c i t h o - p r o t e i n " , was r e s p o n s i b l e f o r y o l k s ' e m u l s i f y i n g c a p a c i t y r a t h e r t h a n t h e s u r f a c t a n t l e c i t h i n i t s e l f . I n f a c t , a t a l a t e r d a t e , p u r i f i e d l e c i t h i n , a m a j o r y o l k p h o s p h o l i p i d , was f o u n d t o be an i n e f f i c i e n t e m u l s i f i e r ( Y e a d o n e t a l . , 1 9 5 8 ) . The i n f o r m a t i o n r e p o r t e d by H a r k i n s a n d Z o l l m a n ( 1 9 2 6 ) t h a t a d e c r e a s e i n i n t e r f a c i a l t e n s i o n i n c r e a s e d t h e e a s e o f e m u l s i f i c a t i o n , s t i m u l a t e d o t h e r r e s e a r c h e r s t o d e t e r m i n e t h e c o m p o n e n t s o f egg y o l k r e s p o n s i b l e f o r l o w s u r f a c e e n e r g y . P o w r i e ( 1 9 5 5 ) f o u n d t h a t a 2% y o l k l e c i t h i n s o l h a d a s u r f a c e t e n s i o n o f 22.3 d y n e s / c m . T h i s v a l u e was c o n f i r m e d somewhat l a t e r by V i n c e n t e t a l . ( 1 9 6 6 ) . M o s t o f t h e y o l k p h o s p h o -l i p i d i s c o n s i d e r e d t o e x i s t i n a b o u n d s t a t e w i t h i n l i p o -p r o t e i n s . - P r e s u m a b l y , w i t h i n y o l k , a l o w c o n c e n t r a t i o n o f u n b o u n d p h o s p h o l i p i d m o l e c u l e s i s i n d y n a m i c e q u i l i b r i u m w i t h b o u n d m o l e c u l e s i n l i p o p r o t e i n s . V i n c e n t e t a l . ( 1 9 6 6 ) s u g g e s t e d t h a t d u r i n g e m u l s i o n f o r m a t i o n , b ound p h o s p h o l i p i d s may be r e l e a s e d and m i g r a t e t o t h e i n t e r f a c e o f t h e e m u l s i o n d r o p l e t s w i t h t h e r e s u l t o f l o w e r i n g i n t e r f a c i a l e n e r g y a n d p r o m o t i n g . e m u l s i f i c a t i o n . The m i g r a t i o n o f p h o s p h o l i p i d s b o u n d w i t h i n l i p o p r o t e i n s h a s b e e n shown t o o c c u r i n o t h e r systems.: s e r u m l i p o p r o t e i n s ( K u n k e l a n d B e a m , 1954) a n d m i l k f a t membranes ( G r e e n b a c k and P a l l a n s c h , 1 9 6 1 ) . The i n c o r p o r a t i o n , o f a d d i t i o n a l p h o s p h o l i p i d t o y o l k d o e s n o t e n h a n c e e m u l s i f y i n g c a p a c i t y . C o r r a n and L e w i s ( 1 9 2 4 ) w e r e t h e f i r s t t o show t h a t a d d e d l e c i t h i n r e d u c e d m a y o n n a i s e s t a b i l i t y . More r e c e n t l y , V a r a d a r a j u l a and C u n n i n g h a m (197 2a) showed t h a t t h e a d d i t i o n o f 0.0 5% l e c i t h i n s i g n i f i c a n t l y r e d u c e d e m u l s i f y i n g c a p a c i t y o f y o l k . C o r r a n and L e w i s ( 1 9 2 4 ) s u g g e s t e d t h a t c h o l e s t e r o l a nd l e c i t h i n m ust be i n b a l a n c e f o r egg y o l k t o be a n e f f i c i e n t e m u l s i f i e r . L e c i t h i n f a v o r s an o i l - i n - w a t e r e m u l s i o n w h e r e a s c h o l e s t e r o l f a v o r s t h e r e v e r s e . The a d d i t i o n o f e i t h e r one u p s e t s t h e b a l a n c e and r e d u c e s e m u l s i o n s t a b i l i t y . As i n d i c a t e d by V i n c e n t e t a l . ( 1 9 6 6 ) , many c o m p o n e n t s o f egg y o l k b r i n g a b o u t a r e d u c t i o n i n i n t e r f a c i a l t e n s i o n a nd p r e s u m a b l y e n h a n c e e m u l s i f i c a t i o n a nd s t a b i l i t y . D a v e y e t a l . ( 1 9 6 9 ) i s o l a t e d t h r e e c r u d e f r a c t i o n s f r o m egg y o l k ( l i p o v i t e l l i n , l i p o v i t e l l e n i n , a n d l i v e t i n ) a n d c o m p a r e d t h e i r e m u l s i f y i n g p r o p e r t i e s . T h e y d i s c o v e r e d t h a t none o f t h e s e f r a c t i o n s , a l o n e o r i n c o m b i n a t i o n , w e re as e f f i c i e n t as n a t i v e y o l k i n e m u l s i f i c a t i o n . The most s t a b l e e m u l s i o n was p r e p a r e d by u s i n g a c o m b i n a t i o n o f a l l t h r e e f r a c t i o n s . The d i f f e r e n c e b e t w e e n t h i s c o m b i n a t i o n and n a t i v e y o l k c o u l d be a t t r i b u t e d t o p h y s i c a l c h a n g e s o c c u r r i n g d u r i n g i s o l a t i o n p r o c e d u r e s . V a r a d a r a j u l a a n d C u n n i n g h a m ( 1 9 7 2 b ) c o n f i r m e d t h e s e r e s u l t s by c o n d u c t i n g s i m i l a r e x p e r i m e n t s . S e v e r a l f a c t o r s h a v e b e e n shown t o h a v e a n i n f l u e n c e on t h e e m u l s i f y i n g p r o p e r t i e s o f y o l k . G e n e t i c v a r i a t i o n o f h e n s a p p e a r s t o be a n i m p o r t a n t f a c t o r . V a r a d a r a j u l a a n d C u n n i n g h a m ( 1 9 7 2 c ) f o u n d t h a t y o l k s f r o m Brown L e g h o r n s were t w i c e a s e f f e c t i v e i n e m u l s i f y i n g s t a b i l i t y a s were y o l k s f r o m W h i t e L e g h o r n s . T h e s e w o r k e r s a l s o r e p o r t e d a d e c r e a s e i n e m u l s i f y i n g p o w e r w i t h age o f t h e h e n . J o r d o n e t a l . ( 1 9 6 2 ) r e p o r t e d t h a t d i e t s s u p p l e m e n t e d w i t h d i f f e r e n t f a t s d i d n o t a f f e c t e m u l s i f y i n g p r o p e r t i e s s i g n i f i c a n t l y . M o r e o v e r , p r o -c e s s i n g a n d p r e s e r v a t i o n p r a c t i c e s h a v e b e e n shown t o a f f e c t e m u l s i f y i n g p o w e r o f y o l k . W i t h t h e e x c e p t i o n o f h o m o g e n i z a -t i o n a n d t h e i n c o r p o r a t i o n o f c e r t a i n a d d i t i v e s , m o s t p r a c t i c e s , r e d u c e t h e e m u l s i f y i n g power o f y o l k ( C u n n i n g h a m and V a r a d a r a j u l a , 1 9 7 3 ) . The m e c h a n i s m o f e m u l s i f i c a t i o n and s t a b i l i z a t i o n h a s n o t b e e n f u l l y e l u c i a t e d . Chang e t a l . (197 3) showed by e l e c t r o n m i s c r o s c o p y t h a t n o t o n e , a s h a d b e e n g e n e r a l l y b e l i e v e d , b u t r a t h e r f o u r d i s t i n c t z o n e s s u r r o u n d e d d r o p l e t s o f t r i g l y c e r i d e i n m a y o n n a i s e . P r e s u m a b l y t h e s e z o n e s c o n s i s t e d o f a v a r i e t y o f egg y o l k p a r t i c l e s o r c o m p o n e n t s o f d i s r u p t e d p a r t i c l e s . T h i s t h e s i s i s b a s e d on t h e s t u d y o f two t y p e s o f e g g y o l k p a r t i c l e s , t h e m y e l i n f i g u r e (MF) a n d t h e g r a n u l e low^-.density l i p o p r o t e i n ( L D L g ) , w h i c h a r e u n d o u b t e d l y i n v o l v e d i n e m u l s i f i c a t i o n . The e x i s t e n c e o f m y e l i n f i g u r e s was f i r s t shown by Chang ( 1 9 6 9 ) who p o s t u l a t e d t h a t t h e y may be c o mposed m a i n l y o f p h o s p h o l i p i d s b e c a u s e o f t h e i r s i m i -l a r i t y w i t h t h e b r a i n p h o s p h o l i p i d m y e l i n s t r u c t u r e s . P r i o r t o 1 9 6 9 , o n l y g r o s s m y e l i n l e a f l e t s t r u c t u r e s , b e l i e v e d t o be a r t i f a c t s c r e a t e d d u r i n g i s o l a t i o n p r o c e d u r e s , h a d b e e n o b s e r v e d . A l t h o u g h , t h e e x i s t e n c e o f g r a n u l e l o w - d e n s i t y l i p o p r o t e i n f r a c t i o n h a s b e e n known f o r a number o f y e a r s , . v e r y l i t t l e i n f o r m a t i o n h a s b e e n r e p o r t e d on t h e s t r u c t u r e a n d c h e m i c a l c o m p o s i t i o n . T h i s t h e s i s i s composed o f t h r e e c h a p t e r s . C h a p t e r one r e p o r t s t h e i s o l a t i o n t e c h n i q u e s d e v e l o p e d t o p r e p a r e p u r e f r a c t i o n s o f b o t h m y e l i n f i g u r e s and g r a n u l e l o w - d e n s i t y l i p o p r o t e i n s , and e l e c t r o n m i c r o s c o p y o f t h e s e p a r t i c l e s . C h a p t e r two i s c o n c e r n e d w i t h t h e c h e m i c a l c o m p o s i t i o n o f t h e 5. i two p a r t i c l e s . T o t a l n i t r o g e n and p h o s p h o r u s v a l u e s a r e r e p o r t e d a l o n g w i t h a q u a n t i t a t i v e a n a l y s i s o f t h e l i p i d s o f e a c h p a r t i c l e by t h i n l a y e r c h r o m a t o g r a p h y . C h a p t e r t h r e e r e p o r t s t h e s u r f a c e a n d e m u l s i f y i n g p r o p e r t i e s o f t h e two p a r t i c l e s . 6 . LITERATURE CITED C h a n g , C M . 1 9 6 9 . S t u d i e s o n egg y o l k . Ph.D. T h e s i s , U n i v . o f W i s . , M a d i s o n , Wis.-C h a n g , C M . , W.D. P o w r i e a n d 0. Fennema. 197 2. E l e c t r o n m i c r o s c o p y o f m a y o n n a i s e . Can. I n s t . F o o d S c i . T e c h n o l . J . S_, 134. C o r r a n , J.W. a n d W.C.N. L e w i s . 1924. L e c i t h i n a n d c h o l e s t r o l i n r e l a t i o n t o t h e p h y s i c a l n a t u r e o f c e l l membranes, B i o c h e m i c a l J . 1_8 , 1364 . C u n n i n g h a m , F.E. and P. V a r a d a r a j u l a . 1 9 7 3 . A r e v i e w o f t h e f a c t o r s a f f e c t i n g e m u l s i f y i n g c h a r a c t e r i s t i c s o f h e n ' s egg y o l k . W o r l d ' s P o u l t . S c i . J . 29_, 1 3 2 . D a v e y , E.M., M.E. Z a b i k a n d L.E. Dawson. 1 9 6 9 . F r e s h a n d f r o z e n e g g y o l k p r o t e i n f r a c t i o n s : E m u l s i o n s t a b i l i z i n g p o w e r s , v i s c o s i t y and e l e c t r o p h o r e s i s p a t t e r n s . P o u l t r y S c i . 48., 2 5 1 . G r e e n b a n k , G.R. and M.J. P a l l a n s c h . 1 9 6 1 . M i g r a t i o n o f p h o s p h a t i d e s i n p r o c e s s i n g d a i r y p r o d u c t s . J . D a i r y S c i . 44-, 1 5 9 7 . H a r k i n s , W.A. and N. Z o l l m a n . 1 9 26. I n t e r f a c i a l t e n s i o n a n d e m u l s i f i c a t i o n . I . The e f f e c t s o f b a s e s , s a l t s a n d a c i d s u p o n t h e i n t e r f a c i a l t e n s i o n b e t w e e n a q u e o u s s o d i u m o l e a t e s o l u t i o n a n d b e n z e n e . J . Am. Chem. S o c . 4_8, 69. J o r d a n , R., G.E. V a i l , S . C R o g l e r a n d W.J. S t a d e l m a n . 1 9 6 2 . F u r t h e r s t u d i e s on e g g s f r o m hens on d i e t s d i f f e r -i n g i n f a t c o n t e n t . F o o d T e c h n o l . 16_, 118. K u n k e l , H.G. and A.G. B e a r n . 1 954. P h o s p h o l i p i d s t u d i e s o f d i f f e r e n t s e r u m l i p o p r o t e i n s e m p l o y i n g P3 2. P r o c . S o c . E x p t l . B i o l . Med. 8_6, 887 . P o w r i e , W.D. 1 9 5 5 . A s t u d y o f t h e g e l a t i o n o f h e n ' s e gg y o l k . Ph.D. T h e s i s . U n i v . o f M a s s a c h u s e t t s , A m h e r s t , M a s s . S n e l l , H.M., A.G. O l s e n a n d R.E. K r e m a r s . 1 9 3 5 . L e c i t h o -p r o t e i n . The e m u l s i f y i n g i n g r e d i e n t i n egg y o l k . I n d . E n g . Chem. 27, 1 2 2 2 . V a r a d a r a j u l a , P. and F.E. C u n n i n g h a m . 197 2a. A s t u d y o f s e l e c t e d c h a r a c t e r i s t i c s o f h e n ' s e g g y o l k . I . I n f l u e n c e o f a l b u m e n and s e l e c t e d a d d i t i v e s . P o u l t r y S c i . 5 1 , 542. V a r a d a r a j u l a , P. and F.E. C u n n i n g h a m . 1972b. A s t u d y o f s e l e c t e d c h a r a c t e r i s t i c s o f h e n ' s egg y o l k . I I . I n f l u e n c e o f p r o c e s s i n g p r o c e d u r e s , p a s t e r i -z a t i o n a n d y o l k f r a c t i o n a t i o n . P o u l t r y S c i . 5 1 , 941 . V a r a d a r a j u l a , P. and F.E. C u n n i n g h a m . 19 7 2 c . A s t u d y o f s e l e c t e d c h a r a c t e r i s t i c s o f h e n ' s egg y o l k . I I I . I n f l u e n c e o f b r e e d , s t r a i n a n d age o f l a y e r , P o u l t r y S c i . 5 1 , 1 3 2 7 . V i n c e n t , R., W.D. P o w r i e and 0. Fennema. 1966. S u r f a c e a c t i v i t y o f y o l k , p l a s m a a n d d i s p e r s i o n s o f y o l k f r a c t i o n s . J . F o o d S c i . 3_1, 643. Y e a d o n , D.A., L.A. G o l d b l a t t a n d A.M. A l t s c h u l . 1 9 5 8 . L e c i t h i n i n o i l - i n - w a t e r e m u l s i o n s . J . Am. O i l Chem. S o c . 3 5 , 4 3 5 . 8 . ABSTRACT The g r a n u l e s o f egg y o l k w e r e d i s r u p t e d w i t h 1.71 M s o d i u m c h l o r i d e a n d s e p a r a t e d i n t o two f l o a t i n g and t h r e e s e d i m e n t i n g f r a c t i o n s on u l t r a c e n t r i f u g a t i o n . The p e l l i c l e . ( F I ) a nd s u b p e l l i c l e ( F I I ) f r a c t i o n s c o n t a i n e d m o s t o f t h e g r a n u l a r l o w - d e n s i t y m a t e r i a l . A m i n o r amount o f l o w - d e n s i t y l i p o p r o t e i n s was t r a p p e d p h y s i c a l l y w i t h i n t h e h i g h - d e n s i t y m a t r i x o f t h e s e d i m e n t i n g f r a c t i o n s . The s u b p e l l i c l e ( F I I ) was p a r t i c u l a r l y r i c h i n m y e l i n f i g u r e s (MF) and u n i f o r m l y - s i z e d g r a n u l e l o w - d e n s i t y l i p o p r o t e i n s ( L D L g ) . U l t r a f i l t r a t i o n , d e x t r a n s u l f a t e -l i p o p r o t e i n c o m p l e x i n g a n d g e l f i l t r a t i o n t e c h n i q u e s w e r e e v a l u a t e d f o r t h e i r a b i l i t y t o s e p a r a t e t h e s e two F I I c o m p o n e n t s i n t h e p u r i f i e d f o r m . E l e c t r o n m i c r o s c o p y was u s e d t o a s s e s s f r a c t i o n p u r i t y a n d t h e s t r u c t u r a l i n t e g r i t y o f t h e p a r t i c l e s . U l t r a f i l t r a t i o n was c o n s i d e r e d e f f e c t i v e i n p r e -p a r i n g p u r e LDLg b u t was d e s t r u c t i v e t o MF. The d e x t r a n s u l f a t e m e t h o d was v i e w e d a s a d e q u a t e f o r s e p a r a t i o n o f b o t h c o m p o n e n t s b u t p o s s i b l e i n t e r a c t i o n o f d e x t r a n s u l f a t e w i t h MF a s w e l l a s LDLg d e c r e a s e d i t s e f f e c t i v e n e s s . S e p h a r o s e 2B g e l f i l t r a t i o n was c o n s i d e r e d t h e b e s t m e t h o d o f i s o l a t i o n . MF a n d LDLg w e r e s e p a r a t e d r a p i d l y a n d t h e f r a c t i o n s w e r e c o n s i d e r e d t o be i n t h e p u r i f i e d f o r m . A d i s c u s s i o n o f s t r u c t u r e s f o r b o t h MF a n d LDLg p a r t i c l e s was u n d e r t a k e n i n t h i s c h a p t e r . 9. CHAPTER I THE ISOLATION OF MYELIN FIGURES AND LOW-DENSITY LIPOPROTEINS FROM EGG YOLK GRANULES INTRODUCTION Egg y o l k i s u s e d i n a v a r i e t y o f f o o d p r o d u c t s p r i m a r i l y b e c a u s e o f i t s e x c e p t i o n a l l y h i g h e m u l s i f y i n g c a p a b i l i t y . I t n o t o n l y f u n c t i o n s e f f i c i e n t l y t o c r e a t e an e m u l s i o n b u t a l s o c o n t r i b u t e s t o e m u l s i o n s t a b i l i t y . I n t h e c a s e o f m a y o n n a i s e , egg y o l k p r o m o t e s t h e f o r m a t i o n o f v e r y s m a l l o i l d r o p l e t s by l o w e r i n g s u r f a c e e n e r g i e s and p r o l o n g s s h e l f - l i f e by f o r m i n g c o m p l e x z o n e s on t h e s u r f a c e o f o i l d r o p l e t s t o p r e v e n t d r o p l e t - d r o p l e t c o a l e s c e n c e ( C h a n g , 1 9 6 9 ; Chang e t a l . , 1 9 7 2 ) . Y o l k c o n s i s t s o f a c o m p l e x m i x t u r e o f many t y p e s o f m o l e c u l e s and p a r t i c l e s w h i c h c o n t r i b u t e t o i t s e m u l s i f y i n g p o w e r . T h e s e c o m p o n e n t s o f e g g y o l k do n o t f u n c t i o n a s e f f i c i e n t l y s e p a r a t e l y a s when t h e y a r e e x i s t i n g t o g e t h e r i n n a t i v e y o l k f o r t h e f o r -m a t i o n a n d s t a b i l i z a t i o n o f an e m u l s i o n ( D a v e y e t a l . , 1 9 6 8 ; V a r a d a r a j u l a e t a l . , 1 9 7 2 b ; Y e a d o n ' e t a l . , 1 9 5 8 ) . As a c o m p l e x s y s t e m , y o l k p r e s e n t s t h e r e s e a r c h e r w i t h a n i n t e r e s t i n g i f n o t f r u s t r a t i n g p u z z l e . R e c e n t l y t h e e l e c t r o n m i c r o s c o p e h a s b e e n u s e d t o e x p l o r e t h e u l t r a -s t r u c t u r e o f y o l k p a r t i c l e s and a s s i s t i n a s s i g n m e n t o f l o c a l i t i e s w h e r e s p e c i f i c c h e m i c a l compounds r e s i d e . E m p i r i c a l l y , y o l k c o n s i s t s , o f a p p r o x i m a t e l y 50% w a t e r , 10 . 33% l i p i d , 16% p r o t e i n and about 1% a s h . S i n c e l e s s t h a n 2% o f t h e y o l k s o l i d s a r e d i a l y s a b l e , Cook and M a r t i n (1962) s u g g e s t e d t h a t y o l k s o l i d s must e x i s t i n a g g r e g a t e d o r macro-m o l e c u l a r forms. T h i s c o n c e p t has now been s u b s t a n t i a t e d by a number o f a u t h o r s ( B e l l a i r s , 1961; Chang, 1969; G r o d z i n s k i , 19 51; N i c h o l s e t a l . , 19 6 9 ) . N a t i v e y o l k i s composed o f two p h y s i c a l l y -s e p a r a b l e f r a c t i o n s : t h e g r a n u l e f r a c t i o n , w h i c h can be sed i m e n t e d by s u p e r c e n t r i f u g i n g y o l k , a n d p l a s m a , t h e low-d e n s i t y f r a c t i o n . The g r a n u l e s a r e composed o f t h e h i g h -d e n s i t y l i p o p r o t e i n s , l o w - d e n s i t y l i p o p r o t e i n s , p h o s v i t i n s and m y e l i n f i g u r e s ; t h e plasma f r a c t i o n i s made up e s s e n -t i a l l y o f t h e l o w - d e n s i t y l i p o p r o t e i n s a l o n g w i t h seme m y e l i n f i g u r e s , a few y o l k spheres and w a t e r s o l u b l e l i v e t i n s . A c c o r d i n g t o Chang ( 1 9 6 9 ) , g r a n u l e s t e n d t o be c i r c u l a r and have d i a m e t e r s up t o 1.7um. G r a n u l e s w i t h d i a m e t e r s between 1.0 and 1.3 urn, were t h e most predominant. Chang ( 1 9 6 9 ) , w i t h t h e s u p p o r t o f e l e c t r o n m i c r o g r a p h s , s u g g e s t e d t h a t g r a n u l e s were made up o f e l e c t r o n dense sub-u n i t s h a v i n g f l u f f y s u r f a c e s . When y o l k i s d i l u t e d w i t h 1.71M N a C l , t h e g r a n u l e s a r e d i s r u p t e d w i t h t h e appearance o f m y e l i n f i g u r e s (MF) l o w - d e n s i t y l i p o p r o t e i n s (LDLg) and e l e c t r o n - d e n s e p a t c h e s i n t h e m i c r o g r a p h s . • A c c o r d i n g t o B u r l e y e t a l . ( 1 9 6 1 ) , g r a n u l e s c o n s i s t o f t h r e e main p r o t e i n f r a c t i o n s : 7 0% l i p o v i t e l l i n , a h i g h - d e n s i t y l i p o p r o t e i n ; 12% l o w - d e n s i t y m a t e r i a l 11. c o m p o s e d m o s t l y o f l o w - d e n s i t y l i p o p r o t e i n ( L D L g ) ; and 1 6 % p h o s v i t i n . When Chang ( 1 9 6 9 ) u l t r a c e n t r i f u g e d w a s h ed y o l k g r a n u l e s d i s s o l v e d i n 1.71M s o d i u m c h l o r i d e , two f l o a t i n g f r a c t i o n s and t h r e e s e d i m e n t i n g f r a c t i o n s w e r e p r o d u c e d . The u p p e r m o s t f l o a t i n g f r a c t i o n ( F I ) c o n s i s t e d o f a number o f l o w - d e n s i t y p a r t i c l e s : l o w - d e n s i t y l i p o p r o t e i n p a r t i c l e s ( L D L g ) s i m i l a r i n s t r u c t u r e t o p l a s m a LDL b u t v a r y i n g w i d e l y i n d i a m e t e r (300 t o 1000 R) , f r e e - l i p i d d r o p l e t s o f v a r y i n g s i z e s , g l o b u l e - c o n t a i n i n g c l u m p s o f unknown c o m p o s i t i o n a n d many m y e l i n f i g u r e s ( M F ) . The F I I f l o a t i n g f r a c t i o n c o n s i s t e d o f m y e l i n f i g u r e s and u n i f o r m l y - s i z e d LDL ( . a p p r o x i m a t e l y 27 0 K i n d i a m e t e r ) . The l o w - d e n s i t y m a t e r i a l ( c o m b i n a t i o n o f F I and F I I ) o f g r a n u l e s h a s a s l i g h t l y h i g h e r p h o s p h a t e c o n t e n t t h a n t h e p l a s m a l o w - d e n s i t y m a t e r i a l a l t h o u g h t h e l i p i d c o n t e n t i s a b o u t t h e same ( C o o k , 1 9 6 8 ) . Cook ( 1 9 6 8 ) b e l i e v e d t h i s d i f f e r e n c e i n p h o s p h a t e c o n t e n t was t h e r e s u l t o f p h o s v i t i n c o n t a m i n a t i o n . S a i t o e t a l . ( 1 9 6 5 ) s e p a r a t e d t h e g r a n u l a r l o w - d e n s i t y m a t e r i a l (LDFG) i n t o two c o m p o n e n t s by u l t r a -c e n t r i f u g a t i o n b u t t h e r e s o l u t i o n was p o o r . G o r n a l l and K u k s i s ( 1 9 7 1 a ) r e s o l v e d t h e LDFG i n t o two f r a c t i o n s , LDFG-1 an d LDFG-2, by h y d r o x y l a p a t i t e t h i n l a y e r c h r o m a t o g r a p h y . G o r n a l l a n d K u k s i s ( 1 9 7 1 b ) a l s o c o m p a r e d p l a s m a LDL and g r a n u l e LDL l i p i d c o m p o s i t i o n b u t f o u n d no m a j o r d i f f e r e n c e s . Some m y e l i n f i g u r e s (MF) a r e f o u n d i n p l a s m a b u t t h e m a j o r i t y a r e p r e s e n t i n g r a n u l e s . T h e s e MF, a b o u t 1000 R i n l e n g t h , c o n s i s t o f one o r more e l e c t r o n - d e n s e l a m e l l a e ( f i x e d w i t h osmium t e t r o x i d e ) and a l e s s e l e c t r o n - d e n s e c o r e . The l a m e l l a e a r e e q u i d i s t a n t f r o m one a n o t h e r w i t h a r e p e a t b a n d p e r i o d o f a b o u t 45 % ( C h a n g , 1 9 6 9 ) . T h i s s t r u c t u r e i s s i m i l a r t o b r a i n p h o s p h o l i p i d m y e l i n f i g u r e s o b s e r v e d by S t o e c k e n i u s ( 1 9 5 8 , 1 9 6 2 ) . R e c e n t l y B a i n and H a l l ( 1 9 7 0 ) , s t u d y i n g s t o r a g e d i s o r d e r s o f e g g s , o b s e r v e d numerous " m y e l i n i c f i g u r e s " i n b o t h n o r m a l and a f f e c t e d egg p l a s m a and s p h e r e f l u i d . T h e s e a u t h o r s c o n c l u d e d t h a t r a t h e r t h a n an i n t e g r a l p a r t o f egg y o l k m i c r o s t r u c t u r e , t h e m i c e l l e s w e r e a r t i f a c t s f o r m e d d u r i n g f i x a t i o n p r o c e d u r e s , by m o d i f i c a t i o n o f m y e l i n l e a f l e t s f i r s t r e p o r t e d by G r o d z i n s k i ( 1 9 4 6 , 1 9 5 1 ) . A c c o r d i n g t o Chang ( 1 9 6 9 ) t h e s e d i m e n t i n g f r a c t i o n s • ( F i l l , F I V , FV) c o n s i s t e d o f f l u f f y m a s s e s composed o f l o n g s t r a n d s w i t h a t t a c h e d m i c r o p a r t i c l e s ( a p p r o x i m a t e l y 100 X). W i t h i n F I V , h o w e v e r , e n t r a p p e d MF and LDL w e r e u s u a l l y f o u n d . Chang ( 1 9 6 9 ) h a s p r o p o s e d t h a t t h e m i c r o p a r t i c l e s o f F i l l , F I V and FV a r e t h e h i g h - d e n s i t y l i p o v i t e l l i n s and t h e s t r a n d s a r e p h o s v i t i n m o l e c u l e s o r a g g r e g a t e s o f p h o s v i t i n . J o u b e r t and C o o k , ( 1 9 5 8 a , 1 958b) c a l c u l a t e d t h e f r i c t i o n a l r a t i o s o f t h e s e p r o t e i n s and c o n c l u d e d t h a t t h e l i p o v i t e l l i n m i c e l l e s a r e s p h e r i c a l and t h e p h o s v i t i n m o l e c u l e s a r e e l o n g a t e d . S i n c e R a d o m s k i and Cook ( 1 9 6 4 a , 1 9 6 4 b ) showed t h a t p h o s v i t i n -and l i p o v i t e l l i n h a d a n a f f i n i t y f o r e a c h o t h e r , Chang ( 1 9 6 9 ) c o n c l u d e d t h a t t h e o b s e r v e d l o n g s t r a n d s w i t h a t t a c h e d m i c r o -p a r t i c l e s w e r e i n d e e d p h o s v i t i n - l i p o v i t e l l i n c o m p l e x e s . The c o m p o s i t i o n o f t h e a-and 3 - l i p o v i t e l l i n s , f i r s t s e p a r a t e d by B e r n a r d ! and Cook ( I 9 6 0 ) , h a s b e e n r e v i e w e d by Cook ( 1 9 6 8 ) and P o w r i e ( 1 9 7 3 ) . The i s o l a t i o n a n d c h a r a c t e r i z a t i o n o f t h e two c o m p o n e n t s o f p h o s v i t i n a r e p r e s e n t e d by C l a r k ( 1 9 7 0 ) . The m a j o r c o m p o n e n t s o f t h e p l a s m a l o w - d e n s i t y m a t e r i a l a r e t h e l o w - d e n s i t y l i p o p r o t e i n s w h i c h - a r e s m a l l s p h e r i c a l p a r t i c l e s ( B e l l a i r s , 1 9 6 1 ; C h a n g , 1 9 6 9 ; M a r t i n e t a l . , 1 9 6 4 ; N i c h o l s e t a l . , 1 9 6 9 ) . S a a r i e t a l . ( 1 9 6 4 ) r e p o r t e d t h a t t h e p l a s m a l o w - d e n s i t y l i p o p r o t e i n s ( L D L ) c o n s i s t e d o f 66% o f t h e y o l k s o l i d s . T h e s e r e s e a r c h e r s d e v i s e d a d i f f e r e n t i a l f l o t a t i o n p r o c e d u r e f o r s e p a r a t i n g l o w - d e n s i t y l i p o p r o t e i n s i n t o two f r a c t i o n s , L P L ^ a n d LPL2• LPL-j^ c o m p r i s i n g a b o u t one f i f t h o f t h e t o t a l LDL-j_ i s s l i g h t l y h i g h e r i n l i p i d c o n t e n t and s l i g h t l y l o w e r i n p r o t e i n c o n t e n t t h a n t h e LPL2. A c c o r d i n g t o M a r t i n e t a l . (19 6 4 ) , t h e gram m o l e c u l a r w e i g h t s o f t h e LPL-^ a n d L P L ^ w e r e o n t h e a v e r a g e 10.3 X 1 0 6 and 3.3 X 1 0 6 r e s p e c t i v e l y . The c h e m i c a l c o m p o s i t i o n a n d p h y s i c a l p r o p e r t i e s o f t h e two f r a c t i o n s h a v e b e e n r e v i e w e d by Cook ( 1 9 6 8 ) , P a r k i n s o n ( 1 9 6 6 ) and P o w r i e ( 1 9 7 3 ) . Some i n f o r m a t i o n h a s b e e n r e p o r t e d o n t h e m i c r o -s t r u c t u r e o f t h e s e l i p o p r o t e i n s . S e v e r a l a u t h o r s h a v e p o s t u l a t e d t h a t p l a s m a LDL an d g r a n u l e LDL a r e c o n s t r u c t e d w i t h a c o r e o f n e u t r a l l i p i d s s u r r o u n d e d by a t h i n s t a b i l i z i n g 14 . i f i l m o f p r o t e i n , p h o s p h o l i p i d s a n d c h o l e s t e r o l ( C o o k and M a r t i n , 1 9 6 2 ; G u r d , 1 9 6 0 ; Kamat and L a w r e n c e , 1 9 7 2 ) . The m i c r o g r a p h s o f Chang ( 1 9 6 9 ) show q u i t e c l e a r l y t h a t e l e c t r o n - , d e n s e u l t r a p a r t i c l e s o f a b o u t 45 X d i a m e t e r r e s i d e on t h e s u r f a c e o f LDL m i c e l l e s . B a s e d on t h e r e p o r t o f Chang ( 1 9 6 9 ) and s e v e r a l o t h e r p u b l i c a t i o n s , a m o d e l o f t h e s t r u c t u r e o f LDL m i c e l l e s c a n be p r o p o s e d . B a s i c a l l y t h e n e u t r a l t r i g l y c e r i d e s c a n be r e g a r d e d as t h e c o r e u p o n w h i c h p h o s p h o l i p i d s , c h o l e s t e r o l and p r o t e i n s a r e a t t a c h e d . A t t h e p e r i p h e r y o f t h e c o r e , p h o s p h o l i p i d s and c h o l e s t e r o l w o u l d f o r m a c o n t i n u o u s p r o -t e c t i v e l a y e r . I m m e d i a t e l y a b o v e t h i s z o n e , a l a y e r o f p r o t e i n may be b o u n d e l e c t r o s t a t i c a l l y t o t h e p o l a r g r o u p s o f t h e i n t e r n a l p h o s p h o l i p i d s and p e r h a p s a l s o b o und d i r e c t l y t o t h e n e u t r a l l i p i d s by h y d r o p h o b i c i n t e r a c t i o n . On t h e s u r f a c e o f t h e p r o t e i n l a y e r , p h o s p h o l i p i d u l t r a p a r t i c l e s c o u l d r e s i d e t h r o u g h e l e c t r o s t a t i c i n t e r a c t i o n . Chang ( 1 9 6 9 ) b e l i e v e d t h i s s o r t o f l i n k a g e was p o s s i b l e . The f o l l o w i n g d a t a s u b s t a n t i a t e t h i s c o n c e p t : 1. A c c o r d i n g t o A u g u s t y n i a k e t a l . ( 1 9 6 4 ) i n t a c t p l a s m a LDL p o s s e s s o n l y e n o u g h p r o t e i n t o c o v e r 2/3 o f t h e i r s u r f a c e a r e a . T h e r e i s , h o w e v e r , e n o u g h p r o t e i n t o f o r m an e x t e n s i v e c o v e r i n g a b o u t t h e t r i g l y c e r i d e c o r e o f LDL. An LDL p a r t i c l e o f 250 % d i a m e t e r 5 o2 w o u l d h a v e a s u r f a c e a r e a o f a b o u t 2.0 X 10 A ; t h e LDL d e v o i d o f i t s u l t r a p a r t i c l e w o u l d be a b o u t 150 8 i n d i a m e t e r a n d a b o u t 7.0 X 1 0 ^ 9? i n s u r f a c e a r e a . The f a c t t h a t p e p s i n h y d r o l y s i s c o m p l e t e l y l i b e r a t e s u l t r a p a r t i c l e s f r o m t h e c o r e ' s s u r f a c e c o r r o b o r a t e s t h i s p o s s i b l e p r o t e i n s i t e ( C h a n g 1 9 6 9 ) . M o r e o v e r , s u r f a c e z o n e s f i x e d w i t h o smium t e t r o x i d e become v e r y e l e c t r o n - d e n s e i n d i c a t i n g t h e p r e s e n c e o f s u r f a c e p h o s p h o l i p i d s i n t h e f o r m o f u l t r a p a r t i c l e s (Chapman a n d F l u c k , 1966 ; H a y e s e t a l . j 1963 ; S t o e c k e n i u s , 1958 ) . 2. The p l a s m a LDL c o n t a i n a p p r o x i m a t e l y 2 3 % p h o s p h o l i p i d s , e n o u g h t o f o r m u l t r a p a r t i c l e s a t t h e s u r f a c e a n d t o p r o v i d e ' t h e s t a b i l i z i n g z o n e a r o u n d t h e LDL c o r e = H y d r o l y s i s o f LDL p h o s p h o l i p i d by p h o s p h o l i p a s e C r e s u l t s i n r e m o v a l o f m o s t ( 9 5 % ) o f t h e p h o s p h o l i p i d ( B u r l e y e t a l . , 1 9 6 3 ) . The p h o s p h o l i p i d m u s t be a c c e s s i b l e t o t h e enzyme and. t h e r e f o r e n e a r t h e LDL s u r f a c e . The r e m a i n i n g 5% o f t h e LDL p h o s p h o l i p i d m i g h t r e s i d e a t t h e p e r i p h e r y o f t h e n e u t r a l t r i -g l y c e r i d e c o r e , p r o t e c t e d f r o m h y d r o l y s i s by t h e p r o t e i n l a y e r . 3. S t e e r e t a l . ( 1 9 6 8 ) r e p o r t t h a t , when a LDL p r e p a r a t i o n i s t r e a t e d w i t h e i t h e r p r o n a s e o r t r y p s i n , 2 0 % o f t h e p r o t e i n r e m a i n s u n h y d r o l y z e d . T h i s w o u l d s u g g e s t t h a t t h i s p r o t e i n i s b o u n d h y d r o p h o b i c a l l y t o t h e n e u t r a l l i p i d c o r e a nd e x i s t s i n a p r o t e c t e d , i n a c c e s s i b l e c o n f i g u r a t i o n . I t i s i n t e r e s t i n g t o n o t e t h a t w i t h p a r t i a l o r c o m p l e t e r e m o v a l o f t h e l i p i d p r i o r t o p r o t e o l y s i s , t h e p r o t e i n becomes a c c e s s i b l e a nd i s c o m p l e t e l y d i g e s t e d by p r o n a s e ( S t e e r e t a l . , 1968 ) . 4. S c h n e i d e r a nd T a t t r i e ( 1 9 6 8 ) s t a t e t h a t p h o s p h o l i p i d i s v i r t u a l l y i n s o l u b l e i n n e u t r a l l i p i d . T h i s s t a t e -ment p r o v i d e s a d d i t i o n a l e v i d e n c e t h a t p h o s p h o l i p i d s 'must r e s i d e a t t h e s u r f a c e o f t h e l i p i d c o r e . The l i v e t i n s c a n be e a s i l y s e p a r a t e d f r o m t h e p l a s m a l o w - d e n s i t y l i p o p r o t e i n s ( L D L p ) by c e n t r i f u g a t i o n o f y o l k p l a s m a d i l u t e d w i t h 1.OM N a C l w h e r e u p o n t h e LDL m i c e l l e s f l o a t t o t h e t o p . L i v e t i n s w e r e f i r s t i s o l a t e d i n a u n d e -n a t u r e d f o r m by Kay and M a r s h a l l ( 1 9 2 8 ) . S h e p a r d a n d H o t t l e ( 1 9 4 9 ) showed t h a t t h i s w a t e r - s o l u b l e p r o t e i n f r a c t i o n c o n s i s -t e d o f a t l e a s t t h r e e e l e c t r o p h o r e t i c c o m p o n e n t s . L a t e r M a r t i n e t a l . ( 1 9 5 7 ) s u b s t a n t i a t e d t h e s e r e s u l t s by e l e c t r o -p h o r e c t i c a l l y s e p a r a t i n g a - , 6- and v - l i v e t i n . W i l l i a m s ( 1 9 6 2 ) r e p o r t e d a n t i g e n s f o r t h e t h r e e l i v e t i n s w e r e p r e s e n t i n h e n ' s b l o o d s e r u m and t h a t t h e a - , 8- and y - l i v e t i n s w e r e s i m i l a r t o t h e s e r u m p r o t e i n s , a l b u m i n , o ^ - g l y c o p r o t e i n , a nd y - g l o b u l i n r e s p e c t i v e l y . H u i and Common ( 1 9 6 6 ) h a v e shown by-g e l e l e c t r o p h o r e s i s t h a t t h e t h r e e l i v e t i n s , c o m p r i s i n g 1 0 % o f e g g y o l k s o l i d s , w e r e composed o f 1.6 e l e c t r o p h o r e c t i c a l l y d i f f e r e n t c o m p o n e n t s . 17. As t h i s b r i e f o v e r v i e w o f egg y o l k m i c r o s t r u c t u r e a n d c o m p o s i t i o n i n d i c a t e s , v e r y l i t t l e i s known a b o u t t h e m y e l i n f i g u r e a n d g r a n u l e l o w - d e n s i t y l i p o p r o t e i n p a r t i c l e s , n o r h a v e t h e s e p a r t i c l e s b e e n i s o l a t e d a n d s t u d i e d i n p u r e f o r m . T h e r e f o r e t h e o b j e c t i v e o f t h i s s t u d y was t o e v a l u a t e m e t h o d s f o r s e p a r a t i n g m y e l i n f i g u r e s a n d l o w - d e n s i t y l i p o p r o t e i n s i n t h e F I I f l o a t i n g f r a c t i o n o f g r a n u l e s . E l e c t r o n m i c r o s c o p y was u s e d t o a s s e s s t h e p u r i t y o f t h e f r a c t i o n s a n d t h e s t r u c t u r a l i n t e g r i t y o f t h e p a r t i c l e s . The f o l l o w i n g m e t h o d s w e r e s e l e c t e d o n t h e b a s i s t h a t n a t i v e s t r u c t u r e s o f t h e p a r t i c l e s w o u l d n o t be a l t e r e d d u r i n g s e p a r a t i o n : u l t r a -f i l t r a t i o n , d e x t r a n s u l f a t e c o m p l e x i n g a nd g e l f i l t r a t i o n . EXPERIMENTAL A. M a t e r i a l s V J h i t e L e g h o r n e g g s w e r e o b t a i n e d f r o m t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a P o u l t r y R e s e a r c h S t a t i o n . The h e n s h a d b e e n f u r n i s h e d w i t h a s t a n d a r d l a y i n g r a t i o n . A l l e g g s w e r e i n f e r t i l e , l e s s t h a n 24 h o u r s o l d , r a n d o m l y c o l l e c t e d a n d p o o l e d . T y p e 55-S d e x t r a n s u l f a t e , a s i t s s o d i u m s a l t , was o b t a i n e d f r o m S i g m a C h e m i c a l C o . , S t . L o u i s , M i s s . The a v e r a g e m o l e c u l a r w e i g h t o f t h e d e x t r a n was 5 0 0 , 0 0 0 . S e p h a r o s e 2B, 4B, and 6B g e l s w e r e p u r c h a s e d f r o m P h a r m a c i a F i n e C h e m i c a l s , M o n t r e a l Que. T h e s e g e l s c o n t a i n e d 0.02% s o d i u m a z i d e a s a b a c t e r i o s t a t i c a g e n t . when n o t i n u s e t h e s e g e l s w e r e p r e s e r v e d w i t h s o d i u m a z i d e a n d s t o r e d a t 6°C i n s e a l e d c o n t a i n e r s . I n a l l e x p e r i m e n t s , a n a l y t i c a l g r a d e c h e m i c a l s a n d d i s t i l l e d w a t e r w e r e u s e d . B. M e t h o d s 1. G r a n u l e p r e p a r a t i o n ' : ; . E a c h i n t a c t y o l k , s e p a r a t e d f r o m t h e a l b u m e n , was r o l l e d o n w e t t e d a b s o r b e n t - p a p e r t o w e l s t o r e m o v e r e s i d u a l e g g w h i t e and t h e n on d r y p a p e r t o w e l s t o r e m o v e s u r f a c e m o i s t u r e a n d r e m a i n i n g t r a c e s o f a l b u m e n . The y o l k was p l a c e d i n a s m a l l p l a s t i c , c o n i c a l c u p w i t h a s m a l l 1/2" d i a m e t e r h o l e i n i t s b a s e . A f t e r t h e cup was p o s i t i o n e d a b o v e a c o l l e c t i n g b e a k e r , t h e p r o t r u d i n g v i t e l l i n e membrane was b r o k e : w i t h a s c a l p e l . The i n t a c t v i t e l l i n e membrance r e s i d e d i n t h e cup a f t e r d r a i n a g e . E a c h b a t c h o f g r a n u l e s was p r e p a r e d f r o m 3 00g o f p o o l e d y o l k s a nd s e p a r a t e d f r o m p l a s m a a c c o r d i n g t o t h e method o f Chang ( 1 9 6 9 ) w i t h s l i g h t m o d i f i c a t i o n . L i q u i d y o l k was c e n t r i f u g e d i n a Beckman M o d e l L2-6 5B u l t r a c e n t r i f u g e a t 30,000 rpm o r 78,500 X G a t R a v > i n a t y p e 30 r o t o r f o r a b o u t 16 h r a t 15°C i n o r d e r t o s e d i m e n t y o l k g r a n u l e s . The s u p e r n a t a n t ( p l a s m a ) w a s r e m o v e d by d e c a n t i n g a nd p i p e t t i n g . S e d i m e n t e d g r a n u l e s w e r e d i s p e r s e d i n 12 v o l u m e s o f d i s t i l l e d w a t e r a nd s t i r r e d v e r y s l o w l y f o r 1 h r i n a c o v e r e d E r l e n m e y e r f l a s k i n a 5°C c o l d r oom t o r e d u c e m i c r o b i a l g r o w t h . The g r a n u l e s u s p e n s i o n was c e n t r i f u g e d a t 15°C i n a S o r v a l l S u p e r -s p e e d RC2-B a u t o m a t i c r e f r i g e r a t e d c e n t r i f u g e a t a p p r o x i m a t e l y 2000 X G f o r 1 h r . A d i s p e r s i o n o f one p a r t wet g r a n u l e s t o 12 p a r t s d i s t i l l e d w a t e r was s l o w l y s t i r r e d f o r 30 m i n a t o 5 C a n d s u b s e q u e n t l y c e n t r i f u g e d a t a b o u t 2000 X G f o r 30 m i n . T h i s w a s h i n g p r o c e d u r e was r e p e a t e d t w i c e more. 2. P r e p a r a t i o n o f l o w - d e n s i t y f r a c t i o n ( F I I ) f r o m G r a n u l e s Wet g r a n u l e s , w e re d i s p e r s e d i n 4.25 v o l u m e s o f 1.71M N a C l and c e n t r i f u g e d a t 7 8,500 X G f o r 16 h r a t 15°C. Two f l o a t i n g a n d t h r e e s e d i m e n t a r y f r a c t i o n s w e r e f o r m e d . The f l o a t i n g p e l l i c l e ( F I ) o f t h e l o w - d e n s i t y l a y e r was r e m o v e d f r o m t h e c e n t r i f u g e t u b e w a l l w i t h a s m a l l m e t a l s p a t u l a and t h e s u b p e l l i c l e f r a c t i o n ( F I I ) was r e m o v e d by 2 P a s t e u r p i p e t f o r f r a c t i o n a t i o n . 3. M e t h o d s f o r s e p a r a t i o n o f m y e l i n f i g u r e s a nd l o w - d e n s i t y l i p o p r o t e i n s i n F I I a. U l t r a f i l t r a t i o n . S e p a r a t i o n o f MF f r o m LDLg w a s • a c c o m p l i s h e d by c e n t r i f u g i n g a d i l u t e ( 1 : 1 0 ) F I I p r e p a r a t i o n i n I . E . C . f i l t e r f u g e c e n t r i f u g e t u b e s e a c h w i t h a M i l l i p o r e f i l t e r . E a c h l o w e r r e s e r v o i r t u b e was f i t t e d n e a r t h e mouth w i t h a c o a r s e w i r e - m e s h s u p p o r t g r i d , a p h o t o e t c h e d s t a i n l e s s s t e e l o ° f i l t e r s u p p o r t s c r e e n and one 500 A + 30 A p o r e - s i z e d M i l l i p o r e f i l t e r . A f t e r t h e t o p o p e n - e n d e d r e s e r v o i r t u b e was s c r e w e d i n t o t h e l o w e r r e s e r v o i r t u b e , a n 11 m l s a m p l e was p o u r e d i n t o t h e t o p t u b e and a c a p was s c r e w e d i n t o t h e u p p e r mouth t o p r e v e n t l e a k a g e . The p r e p a r a t i o n s w e re c e n -t r i f u g e d a t a p p r o x i m a t e l y 4000 X G f o r 5 h r . I n some c a s e s , t h e m y e l i n r e s i d u e on t h e M i l l i p o r e . f i l t e r was r e s u s p e n d e d w i t h 1.71M N a C l and r e c e n t r i f u g e d ; a p r o c e d u r e t h a t was r e p e a t e d one o r more t i m e s . The LDLg was c o n c e n t r a t e d by c e n t r i f u g i n g t h e u l t r a f i l t r a t e a t 78,500 X G f o r 8 h r t o f o r m a f l o a t i n g p e l l i c l e . S a m p l e s o f t h e m y e l i n r e s i d u e and t h e c o n c e n t r a t e d LDLg w e r e embedded i n an a g a r - g l y c e r o l s o l u t i o n i n p r e p a r a t i o n f o r e l e c t r o n m i c r o s c o p y . b. D e x t r a n s u l f a t e - l i p o p r o t e i n c o m p l e x i n g A 1 0 % s o l u t i o n o f d e x t r a n s u l f a t e was p r e p a r e d w i t h d i s t i l l e d w a t e r , pH 6.7. Two p a r t s o f 1 0 % d e x t r a n s u l f a t e w i t h one. p a r t F I I f r a c t i o n was s l o w l y m i x e d w i t h a m a g n e t i c s t i r r e r f o r 15 m i n . The m i x t u r e was s t o r e d i n a s e a l e d c e n t r i f u g e t u b e a t 5°C f o r 24 h r and t h e n c e n t r i f u g e d i n t h e S o r v a l l c e n t r i f u g e a t a p p r o x i m a t e l y 8000 X G f o r 1 h r a t 5°C t o f o r m a m y e l i n - r i c h f l o a t i n g p e l l i c l e a nd an L D L g - r i c h s u b n a t a n t . The r e s u l t i n g m y e l i n - r i c h f l o a t i n g f r a c t i o n was r e m o v e d by P a s t e u r p i p e t a n d m i x e d w i t h an e q u a l v o l u m e o f 0.86 M ( 5 % ) N a C l i n s t e a d o f 1.71M ( 1 0 % ) N a C l t o r e d u c e m o n o v a l e n t c a t i o n f l o o d i n g o f t h e p o l a r g r o u p s and t h u s p r o m o t e e l e c t r o s t a t i c i n t e r a c t i o n b e t w e e n t h e d e x t r a n s u l f a t e and t h e r e m a i n i n g l i p o p r o t e i n . The d i l u t e d p r e p a r a t i o n was m i x e d w i t h two p a r t s o f 10% d e x t r a n s u l f a t e , s t o r e d a t 5°C f o r 4 h r a n d c e n t r i f u g e d a t a p p r o x i m a t e l y 8000 X G f o r 1 h r . T h i s p r o c e d u r e was r e p e a t e d o n c e more. The f l o a t i n g p e l l i c l e was d i s s o l v e d i n f o u r p a r t s 1.71M N a C l and c e n t r i f u g e d a t 78,500 X G f o r 8 h r t o c o n c e n t r a t e t h e MF f r a c t i o n . T h i s p r o c e d u r e was r e p e a t e d o n c e m o re. S a m p l e s o f MF f r a c t i o n a n d l o w - d e n s i t y l i p o p r o t e i n d e x t r a n s u l f a t e c o m p l e x i n t h e s u b n a t a n t w e re m i x e d w i t h a g a r - g l y c e r o l s o l u t i o n s i n p r e p a r a t i o n f o r e l e c t r o n m i c r o s c o p y c. G e l f i l t r a t i o n A S e p h a d e x 5R25-45 c h r o m a t o g r a p h i c c o l u m n f i t t e d w i t h a b o t t o m end a d a p t o r was f i l l e d w i t h h y d r a t e d S e p h a r o s e 2B, 4B, o r 6B t o a h e i g h t o f 39 cm. S e p h a r o s e was i n i t i a l l y s u s p e n d e d i n 10 v o l u m e s o f 1.71M N a C l and s l o w l y p a c k e d i n t o t h e c o l u m n . C a r b o n d i o x i d e b u b b l e s , w h i c h a c c o r d i n g t o P h a r m a c i a F i n e C h e m i c a l s i n s t r u c t i o n s h e e t c a n be a t t r i b u t e d t o d i e t h y l p y r o c a r b o n a t e added t o t h e g e l as a s t e r i l i z a t i o n a g e n t , f o r m e d i m m e d i a t e l y i n e a c h o f t h e g e l s b u t d i s a p p e a r e d a f t e r two d a y s o f e l u t i o n . A s o l u t i o n o f 1.71M N a C l was a l l o w e d t o f l o w c o n t i n u o u s l y i n t o t h e c o l u m n t o e n s u r e g e l bed e q u i l i b r i u m a nd c o m p l e t e r e m o v a l o f s o d i u m a z i d e , an added p r e s e r v i n g a g e n t . A p r e s s u r e o f 57.0 cm o f w a t e r was m a i n t a i n e d on e a c h g e l f o r a l l r u n s . A t t h i s p r e s s u r e , t h e f l o w r a t e s f o r e a c h o f t h e t h r e e g e l s , 2B, 4B and 6B were a p p r o x i m a t e l y 0.6 ml/ m i n , 1.1 m l / m i n and 1.2 m l / m i n , r e s p e c t i v e l y . Two m l o f F I I f r a c t i o n w i t h a t o t a l d r y s o l i d s c o n -t e n t o f a b o u t 11% was c a r e f u l l y l a y e r e d by P a s t e u r p i p e t o n t o t h e S e p h a r o s e g e l s u r f a c e and e l u t i o n was c a r r i e d o u t w i t h 1.71M N a C l s o l u t i o n . A LKB u l t r o - R a c d r o p f r a c t i o n c o l l e c t o r was u t i l i z e d t o c o l l e c t 5 0 - d r o p f r a c t i o n s . T h i s 5 0 - d r o p a l i q u o t c o r r e s p o n d e d t o 3.2 ml f o r t h e 1.71M N a C l e l u a n t and a p p r o x i m a t e l y 2.3 ml f o r b o t h MF and LDLg f r a c t i o n s . Volume d i f f e r e n c e s c a n be a t t r i b u t e d t o v a r i a t i o n i n w a t e r s u r f a c e t e n s i o n p r o p e r t i e s . An I s c o M o d e l UA-2 u l t r a v i o l e t a n a l y z e r c o u p l e d w i t h a V a r i c o r d M o d e l 4 2-B v a r i a b l e r e s p o n s e s t r i p c h a r t r e c o r d e r m o n i t o r e d t h e a b s o r b a n c e a t 280 nm. W i t h t h e S e p h a r o s e 2B g e l , f r a c t i o n numbers 16 t o 25 were p o o l e d t o c o l l e c t t h e m y e l i n f i g u r e s and f r a c t i o n numbers 42 t o 69 were p o o l e d t o c o l l e c t t h e LDLg f r a c t i o n . The a b o v e m e n t i o n e d g r a n u l e c o m p o n e n t s were c o l l e c t e d by p o o l i n g f r a c t i o n numbers 20 t o 26 and 36 t o 52 f r o m t h e 4B g e l a n d f r a c t i o n n umbers 22 t o 25 and 27 t o 30 f r o m the. 6B g e l . E a c h p o o l e d s a m p l e was c e n t r i f u g e d a t 78,500 X G f o r 8 h r t o c o n c e n t r a t e e a c h o f t h e f r a c t i o n s . C. E l e c t r o n M i c r o s c o p y F o r e l e c t r o n m i c r o s c o p y , an A E I C o r i n t h 27 5 e l e c t r o n m i c r o s c o p e a t 60 kV was u s e d . A g f a 70 mm r o l l f i l m was e m p l o y e d t o r e c o r d t h e m i c r o s c o p i c i m a g e . S a m p l e s s u b j e c t e d t o e l e c t r o n m i c r o s c o p y i n c l u d e d : F I I f r a c t i o n , LDLg u l t r a f i l t r a t e , m y e l i n f i g u r e r e s i d u e s a f t e r two p a s s e s and t h r e e p a s s e s t h r o u g h t h e M i l l i p o r e f i l t e r s , d e x t r a n s u l f a t e - l i p o p r o t e i n c o m p l e x , m y e l i n f i g u r e f r a c t i o n s a f t e r one and t h r e e d e x t r a n s u l f a t e t r e a t m e n t s , and s a m p l e s o f MF and LDLg e l u t e d f r o m e a c h o f t h e t h r e e t y p e s o f S e p h a r o s e g e l s . A l l s a m p l e s w e r e embedded i n 5 v o l u m e s o f warm 2.3% (w/v) D i f c o n u t r i e n t a g a r - 1 % Cv/v) g l y c e r o l s o l u t i o n a t a p p r o x i m a t e l y 40°C. A t l e a s t 5 v q l u m e s were r e q u i r e d t o f o r m a f i r m g e l b u t more t h a n 5 v o l u m e s was d i s a d v a n t a g e o u s b e c a u s e o f s a m p l e d i l u t i o n . A f t e r s e t t i n g , t h e a g a r p l u g was p l a c e d i n a p e t r i d i s h c o n t a i n i n g a b o u t 5mm o f c o l d f i x a t i v e c o n -s i s t i n g o f 2.5% g l u t a r a l d e h y d e - 0 . 1 5 M p h o s p h a t e b u f f e r pH 7.2 s o l u t i o n . S m a l l c u b e s , 1 mm o r l e s s , w e r e p r e p a r e d u s i n g a r a z o r b l a d e and t r a n s f e r r e d a l o n g w i t h f i x a t i v e b y p i p e t t o a 3 d r a m g l a s s v i a l ( 2 1 by 50 mm). The g l u t a r a l d e h y d e f i x a t i v e s o l u t i o n t r a n s f e r r e d w i t h t h e c u b e s t o t h e v i a l was r e p l a c e d i m m e d i a t e l y w i t h f r e s h s o l u t i o n and m a i n t a i n e d a t a c o n s t a n t l e v e l o f 3 mm. A l l f u r t h e r o p e r a t i o n s t o o k p l a c e w i t h i n t h e s e v i a l s and t h e l e v e l o f t h e v a r i o u s s o l u t i o n s was a l w a y s o m a i n t a i n e d a t 3 mm. A f t e r p r i m a r y f i x a t i o n a t 6 C f o r 1 h r , t h e c u b e s w e r e washed t h r e e t i m e s w i t h 0.15M p h o s p h a t e b u f f e r pH 7.2 and p o s t f i x e d w i t h 1% osmium t e t r o x i d e - p h o s p h a t e b u f f e r pH 7.2 s o l u t i o n f o r 30 t o 45 m i n a t room t e m p e r a t u r e . A f t e r t h r e e w a s h i n g s w i t h p h o s p h a t e b u f f e r , t h e c u b e s w e r e d e h y d r a t e d f o r 10 m i n p e r i o d s ' w i t h s u c c e s s i v e e t h a n o l - w a t e r s o l u t i o n s o f 30, 5 0 , 70 and 90% ( v / v ) . The c u b e s w e r e t h e n i m m e r s e d f o r 30 m i n p e r i o d s i n t h r e e c h a n g e s o f 1 0 0 % e t h a n o l a t r o o m t e m p e r a t u r e . To f a c i l i t a t e E p o n 812 i n f i l t r a t i o n , a n i n t e r m e d i a t e p r o p y l e n e o x i d e i n f i l t r a t i o n s t e p was r e q u i r e d . The d e h y -d r a t e d c u b e s w e r e p a s s e d t h r o u g h a s e r i e s o f e t h a n o l - p r o p y l e n e o x i d e s o l u t i o n s w i t h p r o p o r t i o n s o f 5:1, 2:1, 1:1, 1:2 and 1:5 ( v / v ) f o r 30 m i n p e r i o d s e a c h . The c u b e s w ere t h e n i m m e r s e d f o r 30 m i n p e r i o d s , i n two c h a n g e s o f 1 0 0 % p r o p y l e n e o x i d e . I n f i l t r a t i o n w i t h . E p on 812 was c o n d u c t e d i n a s i m i l a r m a n n e r t o e t h a n o l - p r o p y l e n e o x i d e i n f i l t r a t i o n . E p o x y i n f i l t r a t e d c u b e s , p l a c e d i n a p l a s t i c c a p s u l e c o n t a i n i n g f r e s h e p o x y , w ere c u r e d a t 4 0 ° C f o r 12 h r and f u r t h e r c u r e d a t 6 0 ° C f o r 2 2 t o 2 8 h r . o S e c t i o n s o f a b o u t 500 t o 900 A i n t h i c k n e s s w e r e c u t w i t h a g l a s s k n i f e on a R e i c h e r t Om U3 u l t r a m i c r o t o m e . S e c t i o n s w e r e p l a c e d o n c o l l o d i o n a nd c a r b o n - c o v e r e d c o p p e r 200-mesh g r i d s a n d s u b s e q u e n t l y s t a i n e d w i t h f r e s h 0.7 5% Cw/v) a q u e o u s u r a n y l a c e t a t e f o r 15 t o 30 m i n and R e y n o l d ' s ( 1 9 6 3 ) l e a d c i t r a t e s o l u t i o n f o r 5 t o 2 0 m i n . RESULTS U l t r a c e n t r i f u g a t i o n o f w a s h e d egg y o l k g r a n u l e s d i s p e r s e d i n 1.71 M s o d i u m c h l o r i d e was u s e d t o p r e p a r e t h e s u b p e l l i c l e F I I a s t h e s t a r t i n g m a t e r i a l f o r t h e i s o l a t i o n o f m y e l i n f i g u r e s (MF) and g r a n u l e l o w - d e n s i t y l i p o p r o t e i n s ( L D L g ) by t h e t e c h n i q u e s o f : u l t r a f i l t r a t i o n , d e x t r a n s u l f a t e - l i p o p r o t e i n c o m p l e x i n g and m o l e c u l a r s i e v e c h r o m a -t o g r a p h y ( g e l f i l t r a t i o n ) . To o p t i m i z e t h e e l e c t r o n m i c r o -s c o p e v i s u a l i z a t i o n o f t h e two p a r t i c l e s w i t h i n t h e F I I and o t h e r i s o l a t e s , v a r i o u s m e t h o d s o f p r e p a r a t i o n w e r e e v a l u a t e d , p r i m a r i l y i n r e g a r d t o t h e i r a b i l i t y t o p r o v i d e o p t i m a l c o n t r a s t w h i l e m a i n t a i n i n g s t r u c t u r a l i n t e g r i t y . Two b a s i c m e t h o d s w e r e e v a l u a t e d f o r d i s t r i b u t i n g p a r t i c l e s i n a f i r m e m b e d d i n g g e l : 1. a g a r e m b e d d i n g and 2. a g a r - g l y c e r o l e m b e d d i n g . F o r b o t h m e t h o d s , i t was f o u n d t h a t , a t a d i l u t i o n o f 1 p a r t s a m p l e t o 5 p a r t s e m b e d d i n g s o l u t i o n , an i n i t i a l s o l u t i o n o f a g a r o f a t l e a s t 2%.was r e q u i r e d t o p r o d u c e a g e l f i r m e n o u g h f o r c u t t i n g i n t o s m a l l c u b e s and f i r m e n o u g h so t h a t t h e c u b e s w o u l d n o t d i s i n t e -g r a t e i n a n y o f t h e s u b s e q u e n t p r e p a r a t i v e s o l u t i o n s . A 2.3% a g a r s o l u t i o n was f o u n d t o be o p t i m a l ; h i g h e r a g a r c o n c e n t r a t i o n s i n c r e a s e d f i x a t i o n t i m e . The u s e o f g l y c e r o l w i t h a g a r ( a s shown by C h a n g , 1969) was f o u n d t o be s u p e r i o r i n p r e s e r v i n g s t r u c t u r a l i n t e g r i t y a s c o m p a r e d t o a g a r a l o n e ) . F o u r m e t h o d s o f f i x a t i o n w e r e a p p r a i s e d : 1. d i r e c t 1.0% osmium t e t r o x i d e (OsO^) f i x a t i o n , 2. d i r e c t 1.5% p o t a s s i u m p e r m a n g a n a t e (KMnO^) f i x a t i o n , 3. d o u b l e f i x a t i o n b y 2.5% g l u t a r a l d e h y d e f o l l o w e d by 1.0% OsO^ a n d 4. d o u b l e f i x a t i o n by 2.5% g l u t a r a l d e h y d e - 2 . 5 % a c r o l e i n f o l l o w e d by 1.0% OsO^. E a c h m e t h o d was f u r t h e r e v a l u a t e d w i t h r e s p e c t t o t h e e f f e c t s o f two b u f f e r s : 1. 0.15 M p h o s p h a t e b u f f e r a t pH 7.2 and 2. P a l a d e ' s ( 1 9 5 2 ) . v e r o n a l a c e t a t e b u f f e r a t pH 7.2. No b e n e f i t s w e r e d e r i v e d by t h e i n c o r p o r a t i o n o f a c r o l e i i n t h e g l u t a r a l d e h y d e s o l u t i o n a n d no d i f f e r e n c e s w e r e n o t e d b e t w e e n P a l a d e ' s a nd p h o s p h a t e b u f f e r . D o u b l e f i x a t i o n w i t h g l u t a r a l d e h y d e a n d OsO^ was c o n s i d e r e d s u p e r i o r t o t h e d i r e c t f i x a t i o n m e t h o d s . KMnC^ f i x a t i o n was p a r t i c u l a r l y d i s r u p t i v e t o b o t h MF and LDLg s t r u c t u r e s . S t o e c k e n i u s ( 1 9 5 8 ) f o u n d t h a t KMnO^ f i x a t i o n d i s r u p t e d human b r a i n m y e l i n s t r u c t u r e s and r e s u l t e d i n c o n s i d e r a b l e v a r i a t i o n i n c o n t r a s t . H ayes e t a l . ( 1 9 6 5 ) f o u n d t h a t s e r u m l i p o p r o t e i n s w e r e s u b s t a n t i a l l y c h a n g e d , b o t h i n s h a p e and s i z e , by KMnO^ f i x a t i o n . Two d e h y d r a t i o n m e t h o d s were a l s o a s s e s s e d : 1. t h e u s e o f a s e r i e s o f a q u e o u s e t h a n o l s o l u t i o n s o f i n c r e a s i n g e t h a n o l c o n t e n t , e n d i n g w i t h 1 0 0 % e t h a n o l and 2. an i d e n t i c a l s e r i e s o f a q u e o u s a c e t o n e s o l u t i o n s . S i n c e no d i f f e r e n c e s i n t h e q u a l i t y o f t h e e l e c t r o n m i c r o g r a p h s c o u l d be d e t e c t e d w i t h t h e two m e t h o d s , e t h a n o l was a r b i t r a r i l y c h o s e n a s t h e d e h y d r a t i o n medium. 28, Epon 812 was u s e d as t h e embedding m a t e r i a l b e c a u s e i t i s v e r y s i m i l a r t o t h e e x c e l l e n t embedding medium A r a l d i t e 6005 b u t r e q u i r e s much l e s s i n f i l t r a t i o n t i m e ( S j o s t r a n d , 1967 ) . Two p o s t s t a i n i n g t e c h n i q u e s t o enhance c o n t r a s t were e v a l u a t e d : 1. R e y n o l d ' s (19 63) l e a d c i t r a t e and 2. u r a n y l a c e t a t e . L e a d c i t r a t e was more e f f e c t i v e i n i m p r o v i n g c o n t r a s t t h a n u r a n y l a c e t a t e . The e f f e c t s were a d d i t i v e , h o w e v e r , and t h e u s e o f b o t h l e a d c i t r a t e and u r a n y l a c e t a t e p r o d u c e d optimum c o n t r a s t . As a r e s u l t o f t h e s e e v a l u a t i o n s o f t h e v a r i o u s p r e -p a r a t o r y m e t h o d s , t h e f o l l o w i n g p r o c e d u r e s were s e l e c t e d f o r t h i s s t u d y : a g a r - g l y c e r o l e m b e d d i n g , d o u b l e f i x a t i o n w i t h g l u t a r a l d e h y d e and OsO^ ( p h o s p h a t e b u f f e r ) , e t h a n o l i c d e h y d r a t i o n , Epon 812 embedding and l e a d c i t r a t e and u r a n y l a c e t a t e p o s t s t a i n i n g . F i g u r e 1 p r o v i d e s a t y p i c a l v i e w o f t h e F I I f r a c t i o n . T h i s e l e c t r o n m i c r o g r a p h shows t h e F I I f r a c t i o n c o n s i s t s o f r e l a t i v e l y l a r g e o s m o p h i l i c MF m i c e l l e s ( a b o u t 1000 8 i n l e n g t h ) and s m a l l e r LDLg p a r t i c l e s o f l e s s e r e l e c t r o n d e n s i t y . The MF m i c e l l e s a r e v e r y s i m i l a r t o t h o s e o b s e r v e d by Chang ( 1 9 6 9 ) ; t h e y show m o r p h o l o g i c a l v a r i a t i o n ( e l l i p s o i d , o v o i d o r s p h e r i c a l s h a p e s ) and some a n g u l a r i t y i n o u t e r w a l l l a m e l l a r s t r u c t u r e . The e l e c t r o n m i c r o g r a p h s o f Chang (1969) show, on o c c a s i o n , . ; a! h i g h l y o r i e n t e d m y e l i n s t r u c t u r e 29 F i g u r e 1. E l e c t r o n m i c r o g r a p h s o f F r a c t i o n I I f r o m u l t r a c e n t r i f u g e d d i s p e r s i o n s o f g r a n u l e s i n 1.71 M s o d i u m c h l o r i d e . MF, m y e l i n f i g u r e ; L D L g , g r a n u l e l o w - d e n s i t y l i p o -p r o t e i n . ( 2 1 , 0 0 0 X ) . 30. e x h i b i t i n g up t o 5 o r 6 c o n c e n t r i c l a m e l l a e e q u i d i s t a n t f r o m o e a c h o t h e r , w i t h a r e p e a t b a n d p e r i o d o f a b o u t 45 A. H o w e v e r , t h e g r e a t e r m a j o r i t y o f t h e MF a r e composed o f e i t h e r a s i n g l e t h i c k b a n d o r two somewhat t h i n n e r l a m e l l a e s i m i l a r t o t h o s e o b s e r v e d by B a i n and H a l l ( 1 9 7 0 ) . Chang ( 1 9 6 9 ) s u g g e s t e d t h a t t h e p o l y l a m e l l a r m y e l i n s t r u c t u r e s may be b i m o l e c u l a r l a y e r s o f p h o s p h o l i p i d m o l e c u l e s . C e r t a i n l y t h e o . . . 40 A c e n t e r t o c e n t e r b a n d d i s t a n c e f o r m y e l i n p h o s p h o l i p i d b a n d s r e p o r t e d e a r l i e r by S t o e c k e n i u s ( 1 9 5 8 ) i s v e r y c l o s e t o t h a t c a l c u l a t e d by Chang ( 1 9 6 9 ) f o r egg y o l k o r i e n t e d MF. H o w e v e r , r e s e a r c h d a t a t o be r e p o r t e d i n a l a t e r c h a p t e r , ' i n d i c a t e d t h e MF c o n s i s t e d o f a h i g h p e r c e n t a g e o f p r o t e i n , c h o l e s t e r o l and n e u t r a l l i p i d , s u g g e s t i n g a much more com-p l i c a t e d s t r u c t u r e t h a n j u s t a b i m o l e c u l a r a r r a n g e m e n t o f p h o s p h o l i p i d s . Chang ( 1 9 6 9 ) d o e s n o t d i f f e r e n t i a t e b e t w e e n t h e mono, d i and p o l y l a m e l l a r MF. N o t a s i n g l e o c c u r r e n c e o f t h e p o l y l a m e l l a r m y e l i n f i g u r e was o b s e r v e d i n m i c r o g r a p h s o f t h i s p r e s e n t r e s e a r c h . C h a n g ' s ( 1 9 6 9 ) m i c r o g r a p h s o f t h e F I I showed no s u c h s t r u c t u r e s b u t w e r e o n l y f o u n d i n t h e m i c r o g r a p h s o f y o l k p l a s m a , o r s l i g h t l y d i s r u p t e d g r a n u l e s . As n o t e d p r e v i o u s l y , MF v a r y i n s i z e and s h a p e . The MF i n F i g u r e 1 a l s o show t h i s v a r i a b i l i t y . I t must be b o r n e i n m i n d t h a t i t i s i m p o s s i b l e , when s e c t i o n i n g t o c u t s m a l l p a r t i c l e s a l l t h r o u g h t h e same p l a n e . S i m i l a r l y , a r e a s o f e l e c t r o n - d e n s e m a t e r i a l i n e a c h MF w i l l d e p e n d u p o n t h e l o c a t i o n o f t h e c u t t h r o u g h t h e p a r t i c l e . MF d i m e n s i o n s o f 48 0 t o 110 0 R i n w i d t h and 560 t o 1300 A i n l e n g t h a s r e p o r t e d by Chang (196 3) a r e s i m i l a r t o v a l u e s o b t a i n e d d u r i n g t h i s c u r r e n t r e s e a r c h . • • The LDLg m i c e l l e s o f t h e F I I ( F i g u r e 1) a r e s i m i l a r i n s i z e , s h a p e and s t r u c t u r e t o t h o s e o b s e r v e d by Chang ( 1 9 6 9 ) . H o w e v e r , t h e f i n e s t r u c t u r e o f t h e LDLg was n o t a s w e l l v i s u a l i z e d a s i t was i n some o f Chang's (1969) m i c r o g r a p h s . The m a j o r r e a s o n f o r t h i s was t h e d i f f e r e n c e i n r e s o l v i n g power o f t h e m i c r o s c o p e s u s e d . The JEM 7A m i c r o s c o p e u s e d by Chang had a b e t t e r r e s o l v i n g power t h a n t h e A E I C o r i n t h 275 e l e c t r o n m i c r o s c o p e . The t h i c k n e s s o f a s p e c i m e n was a s e c o n d a r y f a c t o r . S i n c e s e c t i o n s were on t h e a v e r a g e o 700 A i n t h i c k n e s s , s u p e r p o s i t i o n e f f e c t s a r e p o s s i b l e w i t h p a r t i c l e s a b o u t 270 X i n d i a m e t e r . On c l o s e e x a m i n a t i o n o f F i g u r e 1, t h e s p h e r i c a l s h a p e o f t h e LDLg m i c e l l e s car. be v i s u a l i z e d . S u r f a c e e l e c t r o n - d e n s e u l t r a p a r t i c l e s r e s i d e on t h o s e p a r t i c l e s t h a t a r e d i s c e r n i b l e . U l t r a p a r t i c l e s a r e p r e s e n t i n random f a s h i o n t h r o u g h o u t t h e l o w - d e n s i t y l i p o -p r o t e i n m a t r i x o f t h e F I I ( F i g u r e 1) p r e s u m a b l y as a r e s u l t o f t h e s u p e r p o s i t i o n e f f e c t , b u t some f r e e u l t r a p a r t i c l e s may be p r e s e n t . The mean d i a m e t e r s o f LDLg and u l t r a p a r t i c l e s were c a l c u l a t e d by Chang (1969) t o be 270 A* and 45 A r e s p e c t i v e l y w h i c h a r e s i m i l a r t o t h e v a l u e s o b t a i n e d i n t h i s p r e s e n t r e s e a r c h . LDLg m i c e l l e s a r e t h o u g h t t o be s i m i l a r t o p l a s m a LDL m i c e l l e s . The d i a m e t e r o f t h e p l a s m a LDL as r e p o r t e d by Kamat and L a w r e nce (197 2) and N i c h o l s (19 69) i s c o n s i s t e n t w i t h v a l u e s r e p o r t e d by Chang (1969) f o r g r a n u l e LDL. However, t h e f o r m e r a u t h o r s , u s i n g e l e c t r o n m i c r o s c o p y n e g a t i v e s t a i n i n g t e c h n i q u e s ( p h o s p h o t u n g s t i c a c i d ) , f o u n d no d i s c e r n -i b l e f i n e s t r u c t u r a l d e t a i l s a l t h o u g h t h e i r m i c r o g r a p h s i n d i c a t e a m o t t l e d s u r f a c e o f t h e LDL m i c e l l e s , w h e r e as Chang (1969) f o u n d t h e s u r f a c e s o f b o t h t y p e s o f LDL c o v e r e d w i t h s m a l l u l t r a p a r t i c l e s , w h e t h e r v i s u a l i z e d by n o r m a l s t a i n i n g o r shadow c a s t i n g t e c h n i q u e s . U l t r a f i l t r a t i o n C e n t r i f u g a t i o n i s a means o f i s o l a t i n g components f r o m a s u s p e n s i o n o r f o r s e p a r a t i n g a m i x t u r e o f p a r t i c l e s o f d i f f e r e n t d e n s i t i e s b e c a u s e o f t h e e a s e and e f f i c i e n c y o f t h e method; more s o p h i s t i c a t e d methods w o u l d be e m p l o y e d i f t h e f o r m e r were i m p r a c t i c a l . An aqueous s u s p e n s i o n o f t h e F I I w i l l r e s u l t , upon c e n t r i f u g a t i o n , i n t h e f o r m a t i o n o f a LDLg r i c h f l o a t i n g p e l l i c l e and a s e d i m e n t o f MF. H o w ever, t h i s method has a c e r t a i n d i s a d v a n t a g e . F I I i s i s o l a t e d f r o m g r a n u l e s w i t h 1.71 M s o d i u m c h l o r i d e , a s o l v e n t w h i c h c a u s e d no a p p a r e n t s t r u c t u r a l a l t e r a t i o n o f e i t h e r o f t h e F I I com-p o n e n t s b u t r e d u c i n g t h e c o n c e n t r a t i o n o f s o d i u m c h l o r i d e by d i l u t i o n w i t h d i s t i l l e d w a t e r t o b e l o w 0.1 M N a C l b r o u g h t n o t i c e a b l e d i s r u p t i o n o f some n a t i v e s t r u c t u r e s ( F i g u r e 2 ) . F i g u r e 2. E l e c t r o n m i c r o g r a p h o f m y e l i n f i g u r e s e d i m e n t f r o m u l t r a c e n t r i f u g e d F I I i n d i s t i l l e d w a t e r . MF, m y e l i n f i g u r e ; MF,, d i s r u p t e d m y e l i n f i g u r e . ( 5 2 , 0 0 0 X ) . a M o r e o v e r , d i s r u p t e d MF m i g h t l i b e r a t e l o w - d e n s i t y m a t e r i a l w h i c h w o u l d c o n t a m i n a t e t h e LDLg f r a c t i o n u p o n c e n t r i f u g a t i o n . P e r h a p s t h e f o r c e s o f h i g h s p e e d c e n t r i f u g a t i o n a l s o c o n t r i -b u t e d t o t h i s d i s r u p t i v e e f f e c t . A n o t h e r c o m p l i c a t i n g f a c t o r i s t h e f a c t t h a t t h e MF v a r y i n d e n s i t y . A c o n c e n t r a t i o n o f l e s s t h a n 3% s o d i u m c h l o r i d e i s r e q u i r e d t o s e d i m e n t them a l l so s u b s t a n t i a l d i l u t i o n i s n e e d e d . When t h e MF a r e d i s p e r s e d i n 0.32 M s u c r o s e i n t h e b o t t o m h a l f o f a c e n t r i f u g e t u b e , w i t h 0.1 M s u c r o s e l a y e r e d a b o v e , two d i s p e r s e z o n e s a r e f o r m e d u p on c e n t r i f u g a t i o n . A s i m i l a r e x p e r i m e n t w i t h a s a l t e n v i r o n m e n t r e s u l t s i n a s i m i l a r s e p a r a t i o n . I t i s i n t e r e s t i n g t o n o t e t h a t A u t i l i o e t a l . ( 1 9 6 4 ) f o u n d t h a t p u r i f i e d m y e l i n f r o m t h e c e n t r a l n e r v o u s s y s t e m e x i s t e d a s " l i g h t " a nd " h e a v y " c o m p o n e n t s . I n t h e p r e s e n t s t u d y , i s o l a t i o n b y d e n s i t y g r a d i e n t t e c h n i q u e s was n o t p u r s u e d f u r t h e r b e c a u s e o f t h e f o l l o w i n g u n d e s i r a b l e f a c t o r s : 1. s u c r o s e c a u s e d MF d i s r u p t i o n , 2. s t a b l e s o d i u m c h l o r i d e g r a d i e n t s w e re d i f f i c u l t t o m a i n t a i n , 3. t h e method was t i m e c o n s u m i n g and 4. y i e l d s were r e l a t i v e l y l o w . A m e t h o d was d e s i r e d w h i c h w o u l d o f f e r a r a p i d , e f f i c i e n t s e p a r a t i o n s i n c e l a r g e q u a n t i t i e s o f b o t h p a r t i c l e s w e r e r e q u i r e d f o r f u r t h e r a n a l y s i s . F i g u r e 3 r e p r e s e n t s a t y p i c a l v i e w o f t h e r e s i d u e r e m a i n i n g o n t h e s u r f a c e o f a 500 8 M i l l i p o r e f i l t e r a f t e r a s i n g l e p a s s d u r i n g c e n t r i f u g a t i o n . A s i g n i f i c a n t p o r t i o n F i g u r e 3. E l e c t r o n m i c r o g r a p h o f u l t r a f i l t r a t i o n r e s i d u e a f t e r one p a s s . MF, m y e l i n f i g u r e ; L D L g , g r a n u l e l o w - d e n s i t y l i p o p r o t e i n . (62,000X) o f t h e l o w - d e n s i t y m a t e r i a l p a s s e d t h r o u g h t h e f i l t e r . Upon s u b j e c t i n g a g a i n t h e r e s u s p e n d e d r e s i d u e ( i n 1.71 M N a C l ) t o u l t r a f i l t r a t i o n , a h i g h c o n c e n t r a t i o n o f MF w i t h l i t t l e L DLg c o n t a m i n a t i o n was o b t a i n e d on t h e f i l t e r . R e p e t i t i o n o f t h i s t r e a t m e n t r e s u l t e d i n d i s r u p t i o n o f t h e MF, a s shown i n . t h e h i g h m a g n i f i c a t i o n m i c r o g r a p h ( F i g u r e 4) o f a 3X u l t r a f i l t e r e d s a m p l e ; MF l a m e l l a e a r e d i s r u p t e d and e x p a n d e d i n t o l o n g membranous l e a f l e t s . W i t h t h e l o s s o f t h e n a t i v e s t r u c t u r e o f MF, t h e u l t r a f i l t r a t i o n t e c h n i q u e was n o t c o n -s i d e r e d d e s i r a b l e . H o w e v e r , u l t r a f i l t r a t i o n i s a n e x c e l l e n t m e t h o d f o r p r e p a r i n g a p u r i f i e d LDLg f r a c t i o n . The p u r i t y o f t h e LDLg i n t h e s i n g l e p a s s u l t r a f i l t r a t e i s d e m o n s t r a t e d i n F i g u r e 5. M o r e o v e r , LDLg s t r u c t u r e d o e s n o t a p p e a r t o be a f f e c t e d by p a s s a g e t h r o u g h t h e f i l t e r ( 1 5 0 u i n t h i c k n e s s ) . A l t h o u g h a p u r i f i e d ( M F - f r e e ) LDLg f r a c t i o n c a n be p r e p a r e d by t h i s m e t h o d , some LDLg was r e t a i n e d i n t h e f i l t e r w i t h - M F . R e t e n t i o n may be e x p l a i n e d by p o r e p l u g g i n g by MF and e n t r a p m e n t o f LDLg by e x p a n d e d MF s t r u c t u r e s . D e x t r a n S u l f a t e - L i p o p r o t e i n C o m p l e x i n g . The f o r m a t i o n o f e i t h e r s o l u b l e o r i n s o l u b l e com-p l e x e s o f l i p o p r o t e i n s w i t h s u l f a t e d p o l y s a c c h a r i d e s ( s u c h a s d e x t r a n s u l f a t e ) h a s b e e n d e s c r i b e d by many a u t h o r s ( C o g a n and N i s h i d a , 1 9 6 9 ; C o r n w e l l and K r u g e r , 1 9 6 1 ; H i l l a n d D v o r n i k , 1 9 6 9 ; N i s h i d a , 1 9 6 8 ; N i s h i d a and C o g a n , 1 9 7 0 ; O n c l e y e t a l . , 1 9 5 6 ; R i c k e t s , 1 9 5 1 ) . The f a c t o r s w h i c h 38. F i g u r e 5. E l e c t r o n m i c r o g r a p h o f u l t r a f i l t r a t i o n f i l t r a t e a f t e r one t r e a t m e n t . L D L g , g r a n u l e l o w - d e n s i t y l i p o p r o t e i n . ( 1 0 5 , 0 0 0 X ) . 39. a f f e c t t h e l i p o p r o t e i n - d e x t r a n s u l f a t e (LDL-DS) i n t e r a c t i o n h a v e b e e n r e v i e w e d b y C o r n w e l l a n d K r u g e r ( 1 9 6 1 ) . A l t h o u g h t h e r e i s some l i t e r a t u r e a v a i l a b l e on t h e d e x t r a n s u l f a t e i n t e r a c t i o n o f egg y o l k h i g h - d e n s i t y l i p o p r o t e i n s , no i n f o r m a t i o n i s a v a i l a b l e c o n c e r n i n g d e x t r a n s u l f a t e i n t e r -a c t i o n w i t h e g g y o l k l o w - d e n s i t y l i p o p r o t e i n s . I n i t i a l l y a number o f e x p e r i m e n t s w e r e p e r f o r m e d t o e s t a b l i s h c o n d i t i o n s w h i c h m i g h t l e a d t o a n i d e a l s e p a r a t i o n o f t h e LDLg f r o m t h e MF. P r e l i m i n a r y e x p e r i m e n t s w e re b a s e d on t h e B u r s t e i n a n d S a m a i l l e ( 1 9 5 7 ) m e t h o d o f c o m p l e x i n g . D i v a l e n t c a l c i u m i o n s w e r e a d d e d t o p r o m o t e t h e f o r m a t i o n o f an i n s o l u b l e c o m p l e x by p r o m o t i n g t h e e l e c t r o s t a t i c i n t e r -a c t i o n b e t w e e n t h e DS and LDLg p a r t i c l e s b u t t h i s a d d i t i o n was f o u n d u n d e s i r a b l e s i n c e t h e r e s u l t i n g e x t e n s i v e n e t w o r k e n t r a p p e d a c o n s i d e r a b l e number o f MF ( F i g u r e 6 ) . An i n s o l u b l e c o m p l e x was d e s i r e d a s a means o f e a s i l y s e p a r a t i n g t h e two p a r t i c l e s . The u s e o f 1.71 M N a C l s o l u t i o n i n w h i c h t h e MF an d LDLg m a i n t a i n s n a t i v e i n t e g r i t y d i d n o t b r i n g a b o u t a n i n s o l u b l e c o m p l e x f o r m a t i o n , e v e n w i t h t h e a d d i t i o n o f c a l c i u m . I t was d i s c o v e r e d t h a t when d e x t r a n s u l f a t e was a d d e d t o t h e F I I ( r e g a r d l e s s o f c o n c e n t r a t i o n ) two p h a s e s w e r e f o r m e d u p on s t a n d i n g o r c e n t r i f u g a t i o n : a M F - r i c h u p p e r p h a s e a n d a LDLg-DS s o l u b l e l o w e r p h a s e . The LDLg--DS c o m p l e x c a n t h e n be p r e c i p i t a t e d f r o m a d i l u t e d s o l u t i o n by e i t h e r a c i d i f i c a t i o n o r c a l c i u m a d d i t i o n . F i g u r e 6. E l e c t r o n m i c r o g r a p h o f F r a c t i o n I I - c a l c i u m -d e x t r a n s u l f a t e c o m p l e x . MF, m y e l i n f i g u r e ; C, c o m p l e x . ( 3 3 , 0 0 0 X ) . F i g u r e 7 i s a r e p r e s e n t a t i v e e l e c t r o n m i c r o g r a p h o f t h e M . F - r i c h s u p e r n a t a n t a f t e r a s i n g l e DS t r e a t m e n t . I t i s e v i d e n t t h a t some LDLg c o n t a m i n a t e d t h e MF f r a c t i o n t h r o u g h e n t r a p m e n t by membraneous w a l l s . T h i s f e a t u r e i s b e s t shown i n F i g u r e 8. N i s h i d a and Cogan ( 1 9 7 0 ) h a v e shown t h a t d e x t r a n s u l f a t e r e a c t s w i t h b o t h p r o t e i n and p h o s p h o l i p i d m o i t i e s o f l i p o p r o t e i n s . P e r h a p s t h e o u t e r l a m e l l a o f t h e MF m i g h t be e a s i l y d i s r u p t e d by d e x t r a n s u l f a t e a c t i o n . S o n i c v i b r a t i o n ( M e t t l e r U l t r a s o n i c C l e a n e r , M o d e l ME-55 a t 20 k H z ) was e f f e c t i v e i n d i s r u p t i n g t h e s e ' p o c k e t s o f LDLg. F i g u r e 9 shows a M F - r i c h f r a c t i o n a f t e r t h r e e d e x t r a n s u l f a t e and a s i n g l e s o n i c v i b r a t i o n t r e a t m e n t ( a f t e r f i r s t a d d i t i o n o f D S ) . P o c k e t s o f LDLg w e r e e l i m i n a t e d , a l t h o u g h some l o w -d e n s i t y m a t e r i a l r e m a i n e d . I t i s o f i n t e r e s t t o n o t e i n F i g u r e 9 t h a t t h e l a m e l l a e o f MF v a r y i n t h i c k n e s s and e l e c t r o n d e n s i t y . T h o s e MF w i t h t h i c k l a m a l l a e m i g h t be r e s i s t a n t t o d i s r u p t i o n b u t t h o s e w i t h t h i n n e r w a l l s m i g h t be b r o k e n more e a s i l y w i t h t h e l i b e r a t i o n o f i n t e r n a l c o m p o n e n t s . The m i c r o g r a p h o f t h e i n f r a n a t e a f t e r a s i n g l e DS t r e a t m e n t o f F I I i s shown i n F i g u r e 10. T h i s f r a c t i o n , o n l y r a r e l y c o n t a m i n a t e d w i t h a MF, c a n be v i s u a l i z e d as h i g h l y a g g r e g a t e d LDLg p a r t i c l e s . I r r e g u l a r c e l l s w i t h e l e c t r o n -d e n s e w a l l s c a n be s e e n . G e l F i l t r a t i o n M o l e c u l a r ( p a r t i c l e ) s i e v e c h r o m a t o g r a p h y o r g e l f i l t r a t i o n was a m e t h o d w h i c h seemed p r o m i s i n g f o r i s o l a t i n g 42 . Figure 7. E l e c t r o n micrograph of m y e l i n - r i c h f r a c t i o n a f t e r one dextran s u l f a t e treatment. MF, myelin f i g u r e ; LDLg, granule low-density l i p o p r o t e i n . (16,000X). 4 3 . F i g u r e 8. E l e c t r o n m i c r o g r a p h o f LDLg s u r r o u n d e d by MF a f t e r one d e x t r a n s u l f a t e t r e a t m e n t . MF^, d i s r u p t e d m y e l i n f i g u r e , L D L g , g r a n u l e l o w - d e n s i t y l i p o p r o t e i n . ( 7 8 , 7 0 0 X ) . 44 . F i g u r e 9. E l e c t r o n m i c r o g r a p h o f M F - r i c h f r a c t i o n a f t e r t h r e e d e x t r a n s u l f a t e t r e a t m e n t s . MFn, l i g h t m y e l i n ; M F n , h e a v y m y e l i n ; LDM, l o w - d e n s i t y m a t e r i a l . ( 3 4 , 0 0 0 X ) . F i g u r e 10. E l e c t r o n m i c r o g r a p h o f L D L g - d e x t r a n s u l f a t e c o m p l e x . C 3 5 4 , 0 0 0 X ) . MF and LDLg p a r t i c l e s o n t h e b a s i s o f s i z e d i f f e r e n c e s . P o i s o n ( 1 9 6 1 ) was f i r s t t o e x t e n d t h e f r a c t i o n a l m o l e c u l a r w e i g h t r a n g e o f g e l f i l t r a t i o n t e c h n i q u e s by i n t r o d u c i n g g r a n u l a r a g a r g e l s . A g a r , h o w e v e r , i s a c o m p l e x o f a g a r o s e and a g a r o p e c t i n and t h e a g a r o p e c t i n m o i e t y i m p a r t s u n d e s i r -a b l e i o n e x c h a n g e e f f e c t s t h r o u g h i t s s u l f a t e and c a r b o x y l g r o u p s ( A r a k i , 19 3 7 ) . G e l s composed o f o n l y t h e a g a r o s e c o m p o n e n t w e r e f i r s t p r e p a r e d by H j e r t e n ( 1 9 6 2 ) who l a t e r ( H j e r t e n , 1964) p e r f e c t e d t h e t e c h n i q u e by p r e p a r i n g t h e g e l i n b e a d f o r m . Of t h e g e l s a v a i l a b l e t o d a y , P h a r m a c i a ' s S e p h a r o s e 2B, 4B a n d 6B g e l s o f f e r e d a m o l e c u l a r w e i g h t f r a c t i o n a t i o n l i m i t o f , 40 X 10' , 20 X 10 and 4 X 1 0 6 r e s p e c t i v e l y . The 6B g e l was n o t e x p e c t e d t o e f f e c t a c o m p l e t e s e p a r a t i o n s i n c e t h e m o l e c u l a r w e i g h t o f t h e LDLg p a r t i c l e i s p r e s u m a b l y i n t h e v i c i n i t y o f 3 t o 10 X 1 0 6 . M a r t i n e t a l . ( 1 9 6 4 ) p r e p a r e d f r o m p l a s m a LDL-^ c o n s t i t u t i n g one f i f t h o f t h e t o t a l l o w - d e n s i t y f r a c t i o n , a nd t h e LDL^ w i t h m o l e c u l a r w e i g h t s 10.3 X 10 a n d 3.3 X 10 r e s p e c t i v e l y . F i g u r e 1 1 a d e p i c t s t h e e l u t i o n p a t t e r n o f F I I d u r i n g p a s s a g e t h r o u g h t h e 6B g e l ; o n l y p a r t i a l s e p a r a t i o n was o b t a i n e d w i t h b o t h p e a k s e l u t i n g n e a r t h e g e l ' . s v o i d v o l u m e . The a v e r a g e m o l e c u l a r w e i g h t o f t h e LDLg m u s t , t h e r e f o r e , be n e a r t h e e x c l u s i o n l i m i t o f t h e g e l . F i g u r e 12 i s a n e l e c t r o n m i c r o g r a p h o f t h e m a t e r i a l 47 0.6 0.4 0.2 e c O co C N 0.6 ^ 0 . 4 LU U Z ^ 0 . 2 on o to CD < 0 0.6 0.4 0.2 F i g u r e 11, S E P H A R O S E 6 B a S E P H A R O S E 4 B S E P H A R O S E 2B 5 0 F R A C T I O N N U M B E R 100 G e l f i l t r a t i o n e l u t i o n p a t t e r n s o f egg y o l k F I I . ( E l u t a n t : 1.71 M N a C l , Bed H e i g h t 39 cm, P r e s s u r e 57 cm H 2 0 , 50 d r o p / f r a c t i o n ) . F i g u r e 12. E l e c t r o n m i c r o g r a p h o f S e p h a r o s e 6B m y e l i n f r a c t i o n . MF, m y e l i n f i g u r e ; L D L g , g r a n u l e l o w - d e n s i t y l i p o p r o t e i n . ( 3 3 , 0 0 0 X ) . f o u n d i n t h e f i r s t p e a k f r a c t i o n e l u t e d f r o m t h e 6B g e l (s h o w n i n F i g u r e 1 1 a ) . The f r a c t i o n i s composed o f m a i n l y u n d i s r u p t e d MF w i t h some LDLg c o n t a m i n a t i o n . T h i s c o n t a m i n a t i o n w o u l d be e x p e c t e d s i n c e t h e two p e a k s w e r e e l u t e d n e a r t h e g e l v o i d v o l u m e a nd o v e r l a p p e d one a n o t h e r . The s e c o n d p e a k f r a c t i o n ( F i g u r e 13) was composed l a r g e J L y o f L D L g , b u t was c o n t a m i n a t e d w i t h MF. U l t r a p a r t i c l e s , o b s e r v e d a s v e r y s m a l l e l e c t r o n - d e n s e s p o t s , a p p e a r t o be d e t a c h e d f r o m t h e LDLg p a r t i c l e . D u r i n g p a s s a g e o f LDLg m i c e l l e s t h r o u g h t h e c o l u m n , t h e e x t e n s i v e g e l s u r f a c e a r e a may h a v e e n c o u r a g e d u l t r a p a r t i c l e d e t a c h m e n t . The e l u t i o n p a t t e r n s o f t h e 4B and 2B g e l s ( F i g u r e l i b a n d 1 1 c ) s h o w . t h e s e p a r a t i o n o f t h e p a r t i c l e s i n t o two d i s t i n c t p e a k s w i t h MF e l u t i n g a t t h e g e l v o i d v o l u m e a n d LDLg e l u t i n g s u b s e q u e n t l y . Zone b r o a d e n i n g o f t h e s e c o n d p e a k s i n t h e e l u t i o n p a t t e r n s o f b o t h I B a n d 2B g e l s c a n be a t t r i b u t e d t o v a r i a t i o n i n m o l e c u l a r s i z e o f LDLg r a t h e r t h a n t h e i n f l u e n c e o f s o l v e n t f l o w r a t e o r p a r t i c l e g e l s i z e The r e s o l u t i o n o f t h e LDLg i n t o two f r a c t i o n s o r s p e c i e s was n o t e v i d e n t . P l a s m a l o w - d e n s i t y l i p o p r o t e i n s e x i s t a s two s p e c i e s ( M a r t i n e t a l . , 1 9 6 4 ; S a a r i e t a l . , 1 9 6 4 ) . S a i t o e t a l . ( 1 9 6 5 ) a n d G o r n a l l and K u k s i s ( 1 9 7 1 a ) r e s o l v e d t h e g r a n u l a r l o w - d e n s i t y m a t e r i a l i n t o two c o m p o n e n t s , L D L g ^ an d LDLg2« T h e i r s e p a r a t i o n may h a v e b e e n s e p a r a t i o n o f MF an d LDLg. H o w e v e r i t must be r e m e m b e r e d , t h a t t h e F I I d o e s F i g u r e 13. E l e c t r o n m i c r o g r a p h o f S e p h a r o s e 6B LDLg f r a c t i o n . MF, m y e l i n f i g u r e ; L D L g , g r a n u l e l o w - d e n s i t y l i p o p r o t e i n . ( 5 9 , 0 0 0 X ) . 5 1 . n o t c o n t a i n a l l t h e g r a n u l e l o w - d e n s i t y l i p o p r o t e i n b u t t h e F I f r a c t i o n c o n t a i n s a s i g n i f i c a n t amount. M o r e o v e r , LDLg o f t h e F I I a r e v e r y u n i f o r m i n s i z e c o m p a r e d t o t h e e x t r e m e s i z e v a r i a b i l i t y o f t h e F I ( C h a n g , 1 9 6 9 ) . O n l y a s i n g l e s p e c i e s o f LDLg was e x p e c t e d on g e l f i l t r a t i o n o f t h e F I I . H o w e v e r , i n t h e e l u t i o n ' p a t t e r n o f t h e S e p h a r o s e 2B g e l , t h e e x i s t e n c e o f a m i n o r t r a i l i n g p e a k was e v i d e n t . Some l o w - d e n s i t y m a t e r i a l was p r e s e n t i n t h e MF-r i c h f r a c t i o n c o l l e c t e d f r o m t h e 4B g e l ( F i g u r e 14) b u t t h i s m a t e r i a l i s n o t t h o u g h t t o be LDLg c o n t a m i n a t i o n . T h i s m a t e r i a l e x i s t s as l a m a l l a - f r e e b o d i e s s i m i l a r i n s i z e t o t h e MF, and may be r e g a r d e d a s t h e l o w e l e c t r o n - d e n s e m a t e r i a l ( i n c e n t e r o f MF m i c e l l e ) w h i c h h a d b e e n l i b e r a t e d f r o m d i s r u p t e d MF. E x p a n d e d a nd b r o k e n MF a r e a l s o e v i d e n t i n t h i s m i c r o g r a p h ( F i g u r e 1 4 ) . F i g u r e 15 shows t h e m a t e r i a l f o u n d w i t h i n t h e s e c o n d p e a k f r a c t i o n ( L D L g ) e l u t e d f r o m t h e 4B g e l . I t was f o u n d t o be f r e e f r o m MF. W e l l - d e f i n e d LDLg s t r u c t u r e was n o t a p p a r e n t i n m i c r o g r a p h s o f t h i s f r a c t i o n . An e l e c t r o n m i c r o g r a p h ( F i g u r e 16) o f t h e 2B MF f r a c t i o n shows t h a t i t was v e r y s i m i l a r . t o t h e MF f r a c t i o n e l u t e d f r o m t h e 4B g e l ( F i g u r e 1 5 ) . H o w e v e r , a n o t a b l e d i f f e r e n c e was a p p a r e n t i n t h a t t h e 2B-MF m i c e l l e s w e r e more i n t a c t t h a n t h e 4B-MF. The LDLg f r a c t i o n e l u t e d f r o m t h e 2B g e l ( F i g u r e 17) a l s o a p p e a r e d t o be s t r u c t u r a l l y 52. F i g u r e 14. E l e c t r o n m i c r o g r a p h o f S e p h a r o s e 4B MF f r a c t i o n . MF, m y e l i n f i g u r e ; M F d , d i s -r u p t e d m y e l i n f i g u r e ; MF , m y e l i n f i g u r e c e n t e r . ( 3 3 , 0 0 0 X ) . lOOOA F i g u r e 15. E l e c t r o n m i c r o g r a p h o f S e p h a r o s e 4B LDLg f r a c t i o n . UP, u l t r a p a r t i c l e . ( 2 5 3 , 0 0 0 X ) . 54 . F i g u r e 16. E l e c t r o n m i c r o g r a p h o f S e p h a r o s e 2B MF f r a c t i o n . MF, m y e l i n f i g u r e ; MF , m y e l i n f i g u r e c e n t e r s ; M F d , d i s r u p t e d m y e l i n f i g u r e . ( 5 3 , 0 0 0 X ) . 55. F i g u r e 17. E l e c t r o n m i c r o g r a p h o f S e p h a r o s e 2B LDLg f r a c t i o n . L D L g , g r a n u l e l o w - d e n s i t y l i p o -p r o t e i n ; UP, u l t r a p a r t i c l e . ( 1 U 0 , 0 0 0 X ) . more i n t a c t t h a n t h e LDLg e l u t e d f r o m t h e 4B g e l ( F i g u r e 1 6 ) . The 2B g e l i n t h e l a r g e p o r e s i z e w o u l d be e x p e c t e d t o h a v e a l e s s d i s r u p t i v e e f f e c t on t h e LDLg m i c e l l e s . U l t r a -p a r t i c l e s , a s shown i n F i g u r e 16 and F i g u r e 17 h a d a b o u t t h e same d i m e n s i o n s a s t h o s e r e p o r t e d by Chang ( 1 9 6 9 ) . 57 . DISCUSSION The r e s u l t s o f t h i s s t u d y i n d i c a t e t h a t m y e l i n f i g u r e s .(MF) ;and l o w - d e n s i t y l i p o p r o t e i n s ( L D L g ) c a n be i s o l a t e d f r o m g r a n u l e s by a v a r i e t y o f m e t h o d s , b u t t h e p u r i t y and s t r u c t u r a l i n t e g r i t y o f e a c h f r a c t i o n a r e d e p e n -d e n t on t h e m e t h o d o f o p e r a t i o n . The m a i n t e n a n c e o f t h e i n t a c t l a m e l l a e i n MF d u r i n g i s o l a t i o n p r o c e d u r e s i s i m p e r a t i v e . The r e t e n t i o n o f t h e n a t i v e s t r u c t u r e i s d e p e n d e n t on a v o i d a n c e o f h y d r o p h o b i c s u r f a c e s ( s u c h a s o i l and a i r s u r f a c e s ) , d e h y d r a t i o n o r f r e e z i n g , a n d h i g h c o n c e n t r a t i o n s o f p o l y h y d r o x y compounds. The m i x i n g o f h y d r o p h o b i c t r i g l y c e r i d e o i l w i t h MF c a u s e s a d e s t a b i l i z a t i o n . o f t h e i n t a c t l a m e l l a e . E l e c t r o n m i c r o -g r a p h s o f d i s t o r t e d l a m e l l a e o f MF i n t h e p r e s e n c e o f o i l d r o p l e t s a r e shown i n C h a p t e r 3 o f t h i s t h e s i s . I n C hang's ( 1 9 6 9 ) m i c r o g r a p h s on f r o z e n - t h a w e d and f r e e z e - d r i e d y o l k , no i n t a c t MF p a r t i c l e s w e r e n o t e d . I n d e e d , l o n g e l e c t r o n -d e n s e m e m b r a n e - l i k e r i b b o n s w e re e v i d e n t i n t h e f r e e z e - d r i e d y o l k . P r e s u m a b l y t h e s e r i b b o n s w e r e . d e r i v e d f r o m d e s t a b i l i -z e d l a m e l l a e . I n t h e e l e c t r o n m i c r o g r a p h s o f f r e e z e - d r i e d MF ( s h o w n i n c h a p t e r 3 ) , s i m i l a r e l e c t r o n - d e n s e membrane-l i k e r i b b o n s w e re e v i d e n t . I n t h e p r e s e n t s t u d y o n g r a d i e n t d e n s i t y s e p a r a t i o n o f MF f r o m L D L g , h i g h c o n c e n t r a t i o n o f s u c r o s e c a u s e d e x t e n s i v e s w e l l i n g o f t h e MF. I t i s q u i t e a p p a r e n t f r o m t h e a b o v e i n f o r m a t i o n t h a t p h y s i c a l t e c h n i q u e s 58 . f o r MF s e p a r a t i o n must n o t i n c l u d e any e x p o s u r e o f t h e MF f r a c t i o n t o h y d r o p h o b i c c o n t a c t s u r f a c e s and m ust c i r c u m v e n t any s t e p s w h i c h w o u l d b r i n g a b o u t a l o s s o f s t r u c t u r e d w a t e r ( p r o t e c t i v e i c e - l i k e s h e a t h a r o u n d t h e p a r t i c l e s ) . LDLg m i c e l l e s may be a l t e r e d d u r i n g i s o l a t i o n p r o -c e d u r e s by t h e l o s s o f u l t r a p a r t i c l e s , p r o t e i n d i s a s s o c i a t i o n and c o a l e s c e n c e o f m i c e l l e l i p i d c o r e s . P r e s u m a b l y t h e u l t r a p a r t i c l e s a r e a t t a c h e d t o t h e t r i g l y c e r i d e c o r e t h r o u g h e l e c t r o s t a t i c a n d h y d r o g e n b o n d i n g w i t h s u r f a c e p r o t e i n s . T hus an i n c r e a s e i n t h e c o n c e n t r a t i o n o f c h a r g e d m o l e c u l e s o r i o n s may p r o m o t e d i s a s s o c i a t i o n o f t h e u l t r a p a r t i c l e s . M o r e o v e r , t h e p r e s e n c e o f e x t e n s i v e h y d r o p h o b i c s u r f a c e s ( o i l d r o p l e t s ) w i l l e n h a n c e u l t r a p a r t i c l e l o s s a n d c o a l e s -c e n s e ( C h a n g , 19 6 9 ) . A c c o r d i n g t o Chang ( 1 9 6 9 ) f r e e z i n g a n d f r e e z e - d r y i n g o f p l a s m a c a u s e c o a l e s c e n c e o f t h e LDL m i c e l l e s , p r e s u m a b l y a f t e r t h e p r o t e c t i v e l a y e r s o f u l t r a -p a r t i c l e s and p r o t e i n s h a v e b e e n s t r i p p e d f r o m t h e t r i g l y c e r i d e s u r f a c e s . U l t r a f i l t r a t i o n , one o f t h e p h y s i c a l t e c h n i q u e s u s e d i n t h i s s t u d y f o r MF a n d LDLg s e p a r a t i o n , c a n be r e g a r d e d a s t h e o r e t i c a l l y a c c e p t a b l e on t h e b a s i s t h a t t h e a b o v e m e n t i o n e d c r i t e r i a a r e s a t i s f i e d . The M i l l i p o r e f i l t e r , t o w h i c h t h e p a r t i c l e s i n F I I a r e s u b j e c t e d , d u r i n g t h e u l t r a f i l t r a t i o n p r o c e s s , a r e c o m p o s e d o f c e l l u l o s e e s t e r s w h i c h i s h i g h l y h y d r a t e d i n t h e p r e s e n c e o f w a t e r . The h y d r a t e d f i l t e r p o r e s a r e c o n s i d e r e d t o be a l m o s t e n t i r e l y f r e e o f i n t e r n a l c r o s s -l i n k i n g and t h u s a l l o w u n h i n d e r e d p a s s a g e o f s m a l l p a r t i c l e s t h r o u g h t h e 15Qum l e n g t h p a t h o f e a c h p o r e . The u n i f o r m i t y i n p o r e d i a m e t e r (500 R + 30 8) i n t h e M i l l i p o r e f i l t e r i s a d v a n t a g e o u s f o r s e p a r a t i n g t h e LDLg m i c e l l e s w i t h d i a m e t e r s o f a b o u t 27 0 R f r o m t h e MF p a r t i c l e s w i t h d i a m e t e r s o f a p p r o x i m a t e l y 1000 R. Some m i n o r d i s a d v a n t a g e s o f t h e u l t r a f i l t r a t i o n m e t h o d w e r e n o t e d d u r i n g e x p e r i m e n t a t i o n w i t h t h e F I I f r a c t i o n . I n t h e f i r s t p l a c e , t h e f l o w r a t e o f t h e l i q u i d p h a s e was s l o w e v e n a t h i g h s p e e d s o f c e n t r i f u g a t i o n a n d t h u s t h e a c q u i s i t i o n o f l a r g e q u a n t i t i e s o f MF a n d LDLg f o r f u r t h e r s t u d i e s w o u l d t a k e a c o n s i d e r a b l e p e r i o d o f t i m e w i t h t h e p o s s i b i l i t y o f m i c r o b i a l d e c o m p o s i t i o n a n d a u t o x i -d a t i o n o f l i p i d s . A n o t h e r u n d e s i r a b l e f e a t u r e i s t h e t e n d e n c y o f t h e MF p a r t i c l e s t o o b s t r u c t t h e p o r e e n t r a n c e s and r e d u c e e f f i c i e n t f l o w - t h r o u g h o f t h e LDLg m i c e l l e s . M o r e o v e r , a f t e r s e v e r a l r e p e t i t i o n s o f r e s u s p e n d i n g a n d r e u l t r a f i l t e r i n g o f t h e f i l t e r r e s i d u e t o e l i m i n a t e t h e LDLg some MF l a m e l l a e f r a g m e n t a t i o n was e v i d e n t ( F i g u r e 4-) . F i n a l l y , t h e f l o a t a t i o n o f LDLg d u r i n g t h e c e n t r i f u g a t i o n -u l t r a f i l t r a t i o n p r o c e s s r e d u c e d t h e e f f e c t i v e n e s s o f t h e t e c h n i q u e f o r s e p a r a t i o n . U l t r a f i l t r a t i o n i s a n e f f e c t i v e m e t h o d f o r i s o l a t i n p u r i f i e d LDLg f r o m F I I f r a c t i o n w i t h o u t a n y a p p a r e n t d i s -a s s o c i a t i o n o f t h e u l t r a p a r t i c l e s f r o m t h e s u r f a c e s o f t h e 60 . m i c e l l e s . A p p a r e n t l y t h e h y d r a t e d LDLg m i c e l l e s w i t h d i a m e t e r s i n t h e v i c i n i t y o f 300 A5 p a s s e d t h r o u g h t h e h y d r a t e d p o r e c h a n n e l s w i t h o u t a p p r e c i a b l e s u r f a c e s h e a r i n g . The d e x t r a n s u l f a t e m e t h o d was c o n s i d e r e d t o be a n o t h e r p o s s i b l e a p p r o a c h f o r s e p a r a t i o n by c o m p l e x i n g LDLg w i t h a h i g h m o l e c u l a r w e i g h t s u l f a t e d p o l y s a c c h a r i d e t o f o r m a p r e c i p i t a t e w h i l e t h e MF p a r t i c l e s w o u l d r e m a i n i n t h e u n c o m p l e x e d s t a t e . U n t i l j u s t r e c e n t l y , t h e m e c h a n i s m o f l o w - d e n s i t y l i p o p r o t e i n - s u l f a t e d p o l y s a c c h a r i d e c o m p l e x f o r m a t i o n was c o n s i d e r e d t o be t h e e l e c t r o s t a t i c i n t e r a c t i o n o f t h e n e g a t i v e l y c h a r g e d d e x t r a n s u l f a t e g r o u p s w i t h t h e p o s i t i v e c h a r g e s o f t h e p h o s p h o l i p i d s on t h e LDL s u r f a c e s . T h i s c o n c e p t was b a s e d p r i m a r i l y on t h e i n f o r m a t i o n t h a t e n z y m a t i c h y d r o l y s i s o f p a r t o f t h e LDL s u r f a c e p r o t e i n d i d n o t i n f l u e n c e t h e a b i l i t y o f t h e LDL t o r e a c t w i t h a m y l o p e c t i n s u l f a t e ( C o r n w e l l and K r u g e r , 1 9 6 1 ) . H o w e v e r , r e c e n t e v i d e n c e , p r e s e n t e d b y N i s h i d a and Cogan ( 1 9 7 0 ) , i n d i c a t e d t h a t t h e p r o t e i n o f human p l a s m a LDL p l a y s t h e m a j o r r o l e a n d p h o s p h o l i p i d s t h e m i n o r r o l e i n c o m p l e x f o r m a t i o n . C o m p l e x f o r m a t i o n c a n be s u b s t a n t i a l l y r e d u c e d by a c y l a t i o n o f t h e f r e e a m i n o g r o u p s o f t h e LDL p r o t e i n w i t h a c e t i c a n d s u c c i n i c a n h y d r i d e . N i s h i d a a n d Cogan ( 1 9 7 0 ) a l s o r e p o r t e d t h a t t h e e x t e n t o f c o m p l e x f o r m a t i o n i s n o t s i g n i f i c a n t l y c h a n g e d a f t e r a s much a s 5 6% o f t h e p h o s p h o l i p i d o f LDL h a d .been h y d r o l y z e d by p h o s p h o l i p a s e C. 6 1 . T h e s e r e s u l t s a p p e a r t o c o n f l i c t w i t h t h e p r o p o s a l t h a t t h e s u r f a c e o f a n LDL p a r t i c l e i s p h o s p h o l i p i d i n n a t u r e . P e r -h a p s p h o s p h o l i p i d u l t r a p a r t i c l e s on t h e s u r f a c e s o f LDL m i c e l l e s m i g h t be e a s i l y d i s a s s o c i a t e d when t h e h i g h l y -c h a r g e d s u l f a t e p o l y s a c c h a r i d e s come i n c o n t a c t w i t h s u r f a c e p r o t e i n s . When t h e DS s o l u t i o n was a d d e d t o t h e F I I s u s p e n s i o n , t h e i n t e r a c t i o n o f DS m o l e c u l e s w i t h LDL m i c e l l e s b r o u g h t a b o u t t h e f o r m a t i o n o f s m a l l a g g r e g a t e s w h i c h w e r e v i s u a l i z e d b y e l e c t r o n m i c r o s c o p y ( F i g u r e 10) b u t no p r e c i p i t a t e was f o r m e d . The MF p a r t i c l e s d i d n o t a g g r e g a t e b u t a s shown i n F i g u r e s 7, 8 a n d 9 some MF l a m e l l a e a p p e a r t o be d i s r u p t e d . The a d d i t i o n o f c a l c i u m i o n s t o t h e LDLg-DS c o m p l e x i n 1.71 M N a C l d i d n o t b r i n g a b o u t i n s o l u b i l i z a t i o n . D i s s o c i a t i o n o f LDLg f r o m t h e DS-LDLg c o m p l e x was n o t a t t e m p t e d s i n c e s t r u c t u r a l c h a n g e s w e r e deemed p r o b a b l e a n d o t h e r s i m p l e r i s o l a t i o n m e t h o d s w e r e a v a i l a b l e . G e l f i l t r a t i o n h a s b e e n u s e d i n many s t u d i e s f o r t h e s e p a r a t i o n o f l a b i l e b i o p o l y m e r s . T h i s m e t h o d a f f o r d s a r a p i d s i m p l e means o f i s o l a t i n g p a r t i c u l a t e m a t t e r . S e p h a r o s e i s c o m p o s e d o f a g a r o s e , a l i n e a r p o l y m e r o f a l t e r n a t i n g D - g a l a c t o s e a n d 3, 6 a n h y d r o - L - g a l a c t o s e r e s i d u e s . I t h a s v e r y f e w c h a r g e d g r o u p s w h i c h m i g h t c a u s e p a r t i c l e d i s r u p t i o n and i s h i g h l y h y d r a t e d i n t h e p r e s e n c e o f w a t e r . The m o s t c r i t i c a l f a c t o r i n v o l v e d w i t h t h e i s o l a t i o n o f p a r t i c l e s p e c i e s b y g e l f i l t r a t i o n i s t h e c h o i c e o f a g e l w i t h a n a p p r o p r i a t e p o r e s i z e . A p o r e w i t h a d i a m e t e r s l i g h t l y s m a l l e r t h a n t h a t o f LDLg (3 t o 10 m i l l i o n m o l e c u l a r w e i g h t ) may c a u s e a d i s a s s o c i a t i o n o f u l t r a p a r t i c l e s f r o m t h e s u r f a c e s . I n d e e d , t h e d i s a s s o c i a t i o n o f u l t r a p a r t i c l e s f r o m t h e LDLg s u r f a c e w i t h S e p h a r o s e 6B ( F i g u r e 13) may be a t t r i b u t e d t o t h e l a r g e number o f p a r t i a l i n s e r t i o n s o f LDLg m i c e l l e s i n t o t h e p o r e s . The p o r e d i a m e t e r i n S e p h a r o s e 4B g e l b e a d s ( w i t h m o l e c u l a r w e i g h t f r a c t i o n a l l i m i t o f g 20 X 10 ) i s s l i g h t l y l a r g e r t h a n t h e d i a m e t e r o f L D L g . D i s a s s o c i a t i o n o f u l t r a p a r t i c l e s w o u l d be e x p e c t e d a s t h e p a r t i c l e s s q u e e z e t h r o u g h t h e p o r e s . D i s a s s o c i a t e d u l t r a -p a r t i c l e s w e r e no'ted i n t h e S e p h a r o s e 4 B - t r e a t e d LDLg ( F i g u r e 1 5 ) . When t h e F I I f r a c t i o n was p a s s e d t h r o u g h a c o l u m n o f S e p h a r o s e 2B ( m o l e c u l a r w e i g h t f r a c t i o n a l l i m i t o f 40 X 10 ) w i t h p o r e d i a m e t e r s much l a r g e r t h a n t h o s e o f LDLg m i c e l l e s , no a p p r e c i a b l y , d i s a s s o c i a t e d u l t r a p a r t i c l e s w e r e n o t e d ( F i g u r e 1 7 ) . The MF f r a c t i o n e l u t i n g a t e a c h o f t h e g e l ' s v o i d v o l u m e s showed v e r y l i t t l e d i s r u p t i o n ( F i g u r e s 1 2 , 14 and 1 6 ) , b e c a u s e MF p a r t i c l e s a r e so l a r g e t h a t t h e y a r e u n a b l e t o e n t e r t h e p o r e s o f t h e g e l b e a d s . O v e r a l l , g e l f i l t r a t i o n w i t h S e p h a r o s e 2B was c o n s i d e r e d t h e b e s t i s o l a t i o n m e t h o d b e c a u s e . ! , o f i t s a b i l i t y t o s e p a r a t e l a r g e q u a n t i t i e s o f b o t h f r a c t i o n s i n a s h o r t p e r i o d o f t i m e , 2. s i m p l i c i t y o f m e t h o d and 3. m i n i m a l d i s r u p t i v e e f f e c t s . The g e l f i l t r a t i o n t e c h n i q u e seems e x t r e m e l y p r o m i s i n g f o r f u r t h e r s t u d i e s - o n t h e r e s o l u t i o n o f v a r i o u s s p e c i e s o f L D L g . W i t h t h e a v a i l a b i l i t y o f g e l s w i t h l a r g e r p o r e s i z e s , t h e s e p a r a t i o n o f MF s p e c i e s i n v a r i o u s f r a c t i o n s seems p o s s i b l e . W i t h r e s p e c t t o t h e MF s t r u c t u r e , i t i s d i f f i c u l t t o p r o p o s e a m o d e l s i n c e d e t a i l e d i n f o r m a t i o n o n t h e f i n e s t r u c t u r e and c h e m i c a l c o m p o s i t i o n i s n o t a v a i l a b l e . MF m i c e l l e s a r e e n v i s i o n e d a s l a r g e p a r t i c l e s c o n s i s t i n g o f a n i n n e r n e u t r a l l i p i d c o r e s t a b i l i z e d w i t h one o r more l a m e l l a o f p h o s p h o l i p i d , c h o l e s t e r o l a n d p r o t e i n . S i n c e osmium t e t r o x i d e i n t e r a c t s s t r o n g l y w i t h p h o s p h o l i p i d s t o f o r m an e l e c t r o n - d e n s e a r e a , i t may be a s s u m e d t h a t t h e e l e c t r o n -d e n s e l a m e l l a e c o n s i s t , i n p a r t , o f p h o s p h o l i p i d s . C h a p t e r 2 r e p o r t s on t h e p h o s p h o l i p i d a n d l i p i d c o m p o s i t i o n o f MF. I n t e r n a l r e g i o n s o f MF a r e o n l y w e a k l y o s m i o p h i l i c , i n d i c a t i n g t h e p r e s e n c e o f n e u t r a l l i p i d s . P e p s i n t r e a t m e n t o f a s u s p e n s i o n o f w a s h e d g r a n u l e s o r p l a s m a , a l t h o u g h e f f e c t i v e i n d i s r u p t i n g t h e g r a n u l e s a n d p l a s m a L D L , d o e s n o t a f f e c t t h e l a m a l l a e o f t h e m y e l i n f i g u r e s f o u n d w i t h i n the g r a n u l e a n d p l a s m a ( C h a n g , 1 9 6 9 ) . P e r h a p s p r o t e i n i s i n t i m a t e l y b o u n d w i t h s u r f a c e p h o s p h o l i p i d s a n d e x i s t s i n a p r o t e c t e d c o n f i g u r a t i o n . The r e s u l t s o f t h i s p r e s e n t r e s e a r c h show t h a t two m y e l i n s p e c i e s , " l i g h t " Clow e l e c t r o n -d e n s e l a m e l l a e ) and " h e a v y " ( h i g h e l e c t r o n - d e n s e l a m e l l a e ) may e x i s t . The d i f f e r e n c e s i n e l e c t r o n d e n s i t y may be a t t r i b u t e d t o t h e amount o f p r o t e i n i n t h e l a m e l l a e o f t h e MF m i c e l l e s . The m i c r o g r a p h s o f t h i s c h a p t e r i n d i c a t e v a r i a t i o n i n o u t e r w a l l t h i c k n e s s . CONCLUSIONS I n o r d e r t o i s o l a t e MF a n d LDLg i n l a r g e q u a n t i t i e s f o r f u r t h e r s t u d i e s , v a r i o u s s e p a r a t i o n p r o c e d u r e s w e r e s t u d i e d . D e n s i t y c e n t r i f u g a t i o n and d e n s i t y g r a d i e n t p r e -p a r a t i o n m e t h o d s w e r e c o n s i d e r e d i n a d e q u a t e . U l t r a f i l t r a t i o n was c o n s i d e r e d an' i d e a l m e t h o d f o r p r e p a r i n g L D L g . D e x t r a n s u l f a t e - l i p o p r o t e i n c o m p l e x i n g was a c c e p t e d a s an a d e q u a t e m e t h o d o f i s o l a t i n g b o t h MF and L D L g . S e p h a r o s e 2B g e l f i l t r a t i o n was j u d g e d t h e b e s t m e t h o d o f i s o l a t i n g b o t h f r a c t i o n s . S e p h a r o s e 4B was a c l o s e s e c o n d and 6B was n o t p r a c t i c a l . MF v a r y i n d e n s i t y and e x i s t a s h e a v y and l i g h t s p e c i e s . A l s o , h i g h l y o s m i o p h i l i c and m o d e r a t e l y o s m i o p h i l i c s p e c i e s o f MF o c c u r . LITERATURE CITED A r a k i , C. 1 9 3 7 . A g a r - a g a r . . I l l A c e t y l a t i o n o f t h e a g a r -l i k e s u b s t a n c e o f G e l i d u i n v a m a r i s i i . . J . Chem. S o c . J a p a n 5_8_, 1338T" ~ : A u g u s t y n i a k , J . , W.G. M a r t i n a n d 17.H. Cook. 1 9 6 4 . C h a r a c t e r i z a t i o n o f l i p o v i t e l l e n i n c o m p o n e n t s a n d t h e i r r e l a t i o n t o l o w - d e n s i t y l i p o p r o t e i n s t r u c t u r e s . B i o c h i m . B i o p h y s . A c t a 8_4, 7 2 1 . A u t i l i o , L.A., W.T. N o r t o n a n d R.D. T e r r y . 1 9 6 4 . The p r e p a r a t i o n a n d some p r o p e r t i e s o f p u r i f i e d m y e l i n f r o m t h e c e n t r a l n e r v o u s s y s t e m . J . N e u r o c h e m . 1 1 , 1 7. B a i n , J.M. a n d J.M. H a l l . 1 9 7 0 . The e f f e c t o f d i e t a r y c y c l o p r o p e n e f a t t y a c i d s a n d t h e s t r u c t u r e o f new-l a i d a n d s t o r e d h e n ' s e g g s : a n o p t i c a l a n d e l e c t r o n m i c r o s c o p e s t u d y . A u s t . J . B i o l . S c i . 23 , 657. B e l l a i r s , R. 1 9 6 1 . The s t r u c t u r e o f ' t h e y o l k o f t h e h e n ' s eg g a s s t u d i e d b y e l e c t r o n m i c r o s c o p y . I . The y o l k o f t h e u n i n c u b a t e d e g g . J . B i o p h y s . B i o c h e m . C y t o l - l i , 2 07. B e r n a r d i , G. and W.H. Cook. 1 9 6 0 . An e l e c t r o p h o r e t i c a n d u l t r a c e n t r i f u g a l s t u d y o n t h e p r o t e i n s o f t h e h i g h d e n s i t y f r a c t i o n o f egg y o l k . B i o c h i m . B i o p h y s . A c t a 44_, 86. B u r l e y , R.W. a n d W.H. Coo k . 1 9 6 1 . I s o l a t i o n a n d c o m p o s i t i o n o f a v i a n e g g y o l k g r a n u l e s a n d t h e i r c o n s t i t u e n t a + 8 l i p o p r o t e i n s . C a n . J . B i o c h e m . P h y s i o l . 3 9 , 1295. B u r l e y , R.W. a n d D.J. K u s h n e r . 19 6 3.- The a c t i o n o f C l o s t r i d i u m ' p e r f i n g e n s p h o s p h a t i d a s e on l i p o v i t e l l i n and o t h e r y o l k c o n s t i t u e n t s . C a n . J . B i o c h e m . P h y s i o l . 4 1 , 4 0 9 . B u r s t e i n , M. a n d J . S a m a i l l e . 1 9 5 7 . J . P h y s i o l . ( P a r i s ) 4 9 , 83, as r e v i e w e d by C o r n w e l l a n d K r u g e r ( 1 9 6 1 ) . Chang, CM. 1 9 6 9 . S t u d i e s o n e g g y o l k . 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P o i s o n , A. 1 9 6 1 . F r a c t i o n a t i o n o f p r o t e i n m i x t u r e s on c o l u m n s o f g r a n u l a t e d agar.' B i o c h i m . B i o p h y s . A c t a 50_, 565. P o w r i e , W.D. 197 3. C h e m i s t r y o f eg g s a nd egg p r o d u c t s . I n Egg S c i e n c e a n d T e c h n o l o g y . E d s . , W.J. S t a d e l m a n and O.J. C o t t e r i l l . P u b . , A v i P u b l i s h i n g C o ., W e s t p o r t , Conn. R a d o m s k i , M.W. and W.H. Cook. 1 9 6 4 a . F r a c t i o n a t i o n a n d d i s a s s o c i a t i o n o f t h e a v i a n l i p o v i t e l l i n s and t h e i r i n t e r a c t i o n w i t h p h o s v i t i n . C an. J . B i o c h e m . 4 2 , 395 . R a d o m s k i , M.W. and W.H. Cook. 1964b. C h r o m a t o g r a p h i c s e p a r a t i o n o f p h o s v i t i n , a £ B - l i p o v i t e l l i n o f egg y o l k g r a n u l e s i n T E A E - c e l l u l o s e . Can. J . B i o c h e m . 4 2 , 120 3. R e y n o l d ' , E.S. 1963 . The u s e o f l e a d c i t r a t e a t h i g h pH a s a n e l e c t r o n - o p a q u e s t a i n i n e l e c t r o n m i c r o s c o p y . J . C e l l . B i o l . 17_, 208 . R i c k e t s , C.R. 19 51. D e x t r a n s u l f a t e -- a s y n t h e t i c a n a l o g u e o f h e p a r i n . B i o c h e m 5_1, 1 2 9 . S a a r i , A . L . , W.D. P o w r i e and 0. Fennema. 1 9 6 4 . I s o l a t i o n a n d c h a r a c t e r i z a t i o n o f l o w - d e n s i t y l i p o p r o t e i n s i n n a t i v e e g g y o l k p l a s m a . J . Fo o d S c i . 2 9., ;3-Q7 . S a i t o , Z., W.G. M a r t i n a n d W.H. Cook'. 1965 . C h a n g e s i n t h e m a j o r m a c r o m o l e c u l a r f r a c t i o n s o f e g g y o l k d u r i n g e m b r y o g e n i s e s . Can. J . B i o c h e m . 4 3 , 17 55. S c h n e i d e r , H. and N.H. T a t t r i e . 1968. M u t u a l s o l u b i l i t y o f t h e l i p i d c o m p o n e n t s o f egg y o l k l o w - d e n s i t y l i p o p r o t e i n . C a n . J . Biochem._ 4_6, 979 . S h e p a r d , C.C. and G. A. H ' o t t l e . 1949. S t u d i e s on t h e c o m p o s i t i o n o f t h e l i v e t i n f r a c t i o n o f t h e y o l k o f h e n e g g s w i t h t h e u s e o f e l e c t r o p h o r e t i c a n a l y s i s . J . B i o l . Chem. 1 7 9 , 349. S j o s t r a n d , F.S. 1967. E m b e d d i n g o f c h e m i c a l l y f i x e d m a t e r i a l . E l e c t r o n m i c r o s c o p y o f c e l l s and t i s s u e s . P u b . , A c a d e m i c P r e s s I n c . ( L o n d o n ) L t d . S t e e r , O.C., W.G. M a r t i n a n d W.H. Cook. 1 9 6 8 . S t r u c t u r a l i n v e s t i g a t i o n o f t h e - l o w - d e n s i t y l i p o p r o t e i n o f h e n ' s egg y o l k u s i n g p r o t e o l y s i s . B i o c h e m . 9_, 3309 . S l o e c k e n i u s , W. 1 958. An e l e c t r o n m i c r o s c o p e s t u d y o f m y e l i n f i g u r e s . J . B i o p h y s . and B i o c h e m . C y t o l . 5_, 4 9 1 . S t o e c k e m i u s , W. 1 9 6 2 . S t r u c t u r e o f t h e p l a s m a membrane. C i r c u l a t i o n ' '26_, 1 0 6 6 . W i l l i a m s , J . -1962. Serum p r o t e i n s and l i v e t i n s o f h e n ' s egg y o l k . B i o c h e m . J . ' 8_3_, 346. V a r a d a r a j u l a , P. a n d F.E. C u n n i n g h a m . 1 9 7 2 . A s t u d y o f s e l e c t e d c h a r a c t e r i s t i c s o f h e n ' s egg y o l k . I I . I n f l u e n c e o f p r o c e s s i n g p r o c e d u r e s , p a s t e u r i z a t i o n a n d y o l k f r a c t i o n i z a t i o n . P o u l t r y S c i . 51_, 941. Y e a d o n , D.A. L.A. G o l d b l a t t a nd A.M.. A l t s c h u l . 1958 . L e c i t h i n i n o i l - i n - w a t e r e m u l s i o n s . J . Am. O i l C h e m i s t s S o c . '35 , 435 . 70. CHAPTER 2 CHARACTERIZATION OF EGG YOLK MYELIN FIGURES AND GRANULE LOW-DENSITY LIPOPROTEINS ABSTRACT M y e l i n f i g u r e s (MF) and g r a n u l e l o w - d e n s i t y l i p o -p r o t e i n s ( L D L g ) o f t h e F I I , t h e s u b p e l l i c l e f r a c t i o n o f a 10% N a C l d i s r u p t e d , s u p e r c e n t r i f u g e d g r a n u l e d i s p e r s i o n , w e r e s e p a r a t e d by S e p h a r o s e 2B g e l f i l t r a t i o n . D i a l y z e d , l y o p h i l i z e d s a m p l e s o f e a c h s p e c i e s o f p a r t i c l e w e r e a n a l y z e d f o r t o t a l n i t r o g e n a nd p h o s p h o r u s by K j e l d a h l a n d T e c h n i c o n A u t o a n a l y s e r m e t h o d s . N o n - l i p i d r e s i d u e s w e re a n a l y z e d f o r n i t r o g e n a nd p h o s p h o r u s by t h e same t e c h n i q u e s . L i p i d c l a s s e s w e r e r e s o l v e d by c h r o m a t o g r a p h y on t h i n l a y e r s o f s i l i c a g e l . F u r t h e r , l i p i d c o m p o n e n t s were i d e n t i f i e d and q u a n t i t a t i v e l y d e t e r m i n e d by m e t h o d s d e s c r i b e d w i t h i n t h e e x p e r i m e n t a l s e c t i o n . MF were f o u n d t o c o n s i s t o f c o m p o n e n t s s i m i l a r t o t h o s e o f L D L g , a l t h o u g h c o n c e n t r a t i o n s o f t h e v a r i o u s compon-e n t s v a r i e d . S i m i l a r l y , g r a n u l e and p l a s m a l o w - d e n s i t y l i p o p r o t e i n s ( L D L p ) w e r e f o u n d t o be composed o f s i m i l a r c o n s t i t u e n t s . MF c o n t a i n e d a b o u t 8 6% l i p i d w h e r e a s LDLg w e r e a b o u t 84% l i p i d . As a p r o p o r t i o n o f t h e t o t a l l i p i d , MF w ere f o u n d t o be p a r t i c u l a r l y r i c h i n c h o l e s t e r o l , a b o u t 1 1 . 5 % . O t h e r s t r u c t u r e s o f egg y o l k a r e known t o be much l o w e r i n c h o l e s t e r o l c o n c e n t r a t i o n . LDLg c o n t a i n e d a b o u t 3.7% c h o l e s t e r o l , s i m i l a r t o v a l u e s r e p o r t e d f o r LDLp. T o t a l p h o s p h o l i p i d o f MF and LDLg were f o u n d t o be a p p r o x i m a t e l y 30% a n d 2 6 % , r e s p e c t i v e l y . As a p e r c e n t a g e o f t h e t o t a l p h o s p h o l i p i d MF a n d LDLg c o n t a i n e d s i m i l a r a m o unts o f p h o s p h a t i d y c h o l i n e ( a b o u t 81%) a n d p h o s p h a t i d y l e t h a n o l a -m i n e ( a b o u t 1 6 % ) . LDLg c o n t a i n e d a l m o s t i d e n t i c a l a m o u nts o f l y s o p h o s p h a . t i d y l e t h a n o l a m i n e a n d s p h i n g o m y e l i n ( a p p r o x i m a t e l y 1.7% o f t h e t o t a l p h o s p h o l i p i d ) w h e r e a s MF c o n s i s t e d o f a b o u t 1.3% l y s o p h o s p h a t i d y l e t h a n o l a m i n e a nd 2.0% s p h i n g o m y e l i n . P e r c e n t n i t r o g e n was a b o u t 2.5% f o r b o t h MF and LDLg;' p e r c e n t p h o s p h o r u s a p p r o x i m a t e l y 1.25% f o r MF and 1.0 5% f o r LDLg. INTRODUCTION When egg y o l k i s c e n t r i f u g e d a t h i g h s p e e d s , g r a n u l e s a r e s e d i m e n t e d a n d a c l e a r f l u i d s u p e r n a t a n t , c a l l e d p l a s m a , i s f o r m e d . G r a n u l e s make up a b o u t 19 t o 23% o f t h e y o l k s o l i d s ( B u r l e y and Co o k , 1 9 6 1 ; S a a r i e t a l . , 1 9 6 4 ; S a i t o e t a l . , 1 9 6 5 ) . A c c o r d i n g t o B u r l e y a n d Cook ( 1 9 6 1 ) , g r a n u l e s c o n s i s t o f 70% a- and 6 - l i p o v i t e l l i n s , 1 6 % p h o s v i t i n a n d 1 2 % l o w - d e n s i t y l i p o p r o t e i n . The l o w - d e n s i t y l i p o p r o t e i n ( L D L g ) i n g r a n u l e s c a n be i s o l a t e d by a d d i n g 1.71 M N a C l t o w a t e r - w a s h e d , s e d i m e n t e d g r a n u l e s a n d c e n t r i f u g i n g t h e d i s p e r s i o n . As shown by e l e c t r o n m i c r o s c o p y , t h e two f l o a t i n g f r a c t i o n s ( F I . a n d F I I ) c o n t a i n e d m o s t o f t h e LDLg a l o n g w i t h some m y e l i n f i g u r e s (MF) ( C h a n g , 1 9 6 9 ) . The s e d i m e n t i n g f r a c t i o n s ( F i l l , F I V an d FV) c o n s i s t e d p r i m a r i l y o f f l u f f y m a s s e s made up o f l o n g p r o t e i n s t r a n d s w i t h e l e c t r o n - d e n s e m i c r o p a r t i c l e s a t t a c h e d . A c c o r d i n g t o Chang ( 1 9 6 9 ) t h e s t r a n d s w e r e p h o s v i t i n c o m p l e x e s and t h e a t t a c h e d m i c r o p a r t i c l e s w e r e l i p o v i t e l l i n s . A m a j o r p r o p o r t i o n o f LDLg a n d MF a r e l o c a t e d i n t h e F I I f l o a t i n g f r a c t i o n . : LDLg m i c e l l e s a r e c o n s i d e r e d t o be s i m i l a r i n s t r u c t u r e , c o m p o s i t i o n and p h y s i c a l p r o p e r t i e s t o p l a s m a l o w -d e n s i t y l i p o p r o t e i n C L D L p ) . On a s t r u c t u r a l b a s i s , Chang ( 1 9 6 9 ) h a s shown t h a t LDLg and LDLp a r e q u i t e s i m i l a r i n s h a p e , s i z e a n d p r e s e n c e o f e l e c t r o n - d e n s e u l t r a p a r t i c l e s 73. ( a p p r o x i m a t e l y 45 R d i a m e t e r ) on t h e o u t e r s u r f a c e s . G r a n u l e l o w - d e n s i t y l i p o p r o t e i n s ( L D L g ) a r e c o n -s i d e r e d t o be s i m i l a r i n c o m p o s i t i o n and p h y s i c a l p r o p e r t i e s t o p l a s m a l o w - d e n s i t y l i p o p r o t e i n s ( L D L p ) . Cook ( 1 9 6 8 ) h a s r e p o r t e d t h a t LDLg d i f f e r f r o m LDLp o n l y i n t h e i r h i g h e r p h o s p h o r u s c o n t e n t , p r e s u m a b l y t h e r e s u l t o f p h o s v i t i n c o n t a m i n a t i o n . T a t t r i e a n d Cook ( 1 9 6 9 ) s t a t e t h a t LDLg m i c e l l e s a r e i n d i s t i n g u i s h a b l e f r o m t h e m a j o r t y p e o f LDLp m i c e l l e s b u t r e s u l t s t o s u b s t a n t i a t e t h e i r p r o p o s a l s w e r e n o t p r e s e n t e d . R e c e n t l y G o r n a l l and K u k s i s ( 1 9 7 1 a and b) r e p o r t e d d e t a i l e d i n f o r m a t i o n on t h e c o m p o s i t i o n o f LDFG and two s e p a r a t e f r a c t i o n s (LDFG-1 and LDFG-2) o f LDFG. The l i p i d c o n t e n t s o f LDFG, LDFG-1 a n d LDFG-2 w e r e 88 , 85 and 92 p e r c e n t , r e s p e c t i v e l y ( G o r n a l l and K u k s i s , 1 9 7 1 a ) . The r e m a i n i n g p e r c e n t a g e s w e r e p r o t e i n . A c c o r d i n g t o G o r n a l l a n d K u k s i s ( 1 9 7 1 b ) , t h e l i p i d c o m p o s i t i o n o f LDLg was 7.3% c h o l e s t e r o l , 22.6% p h o s p h o l i p i d and 7 0 . 2 % t r i -g l y c e r i d e . A l t h o u g h t h e LDFG-2 f r a c t i o n h a d a l i p i d c o m p o s i t i o n s i m i l a r t o t h a t o f LDFG, LDFG-1, w h i c h r e m a i n e d a t t h e o r i g i n d u r i n g t h i n l a y e r c h r o m a t o g r a p h i c s e p a r a t i o n o f t h e two f r a c t i o n s , c o n t a i n e d l e s s t r i g l y c e r i d e ( 5 7 . 0 % ) a n d more p h o s p h o l i p i d ( 2 9 . 7 % ) a n d c h o l e s t e r o l ( 1 3 . 3 % ) . The m y e l i n f i g u r e s (MF) i n F I I h a v e b e e n i s o l a t e d i n a p u r i f i e d f o r m by g e l f i l t r a t i o n a s o u t l i n e d i n C h a p t e r I , b u t no c o m p o s i t i o n a l s t u d i e s h a v e b e e n c a r r i e d o u t . Two t y p e s o f MF p a r t i c l e s h a v e b e e n o b s e r v e d ; l o w e l e c t r o n - d e n s e l a m e l l a e MF, and h i g h e l e c t r o n - d e n s e l a m e l l a e MF. The d i f f e r e n c e i n e l e c t r o n d e n s i t y o f t h e l a m e l l a e may be a t t r i b u t a b l e • t o t h e p r o p o r t i o n o f p h o s p h o l i p i d and p r o t e i n . So f a r , no m e t h o d h a s b e e n r e p o r t e d f o r t h e s e p a r a t i o n o f t h e s e MF t y p e s . The c e n t e r o f e a c h MF p a r t i c l e t r e a t e d w i t h OsO^ a p p e a r s i n t h e e l e c t r o n m i c r o g r a p h s ( C h a p t e r I.) t o be g r a y , a n i n d i c a t i o n o f n e u t r a l l i p i d . The p u r p o s e o f t h i s s t u d y was t o e x a m i n e t h e c h e m i c a l c o m p o s i t i o n o f LDLg and MF i s o l a t e d by t h e S e p h a r o s e 2B g e l f i l t r a t i o n t e c h n i q u e o u t l i n e d i n C h a p t e r I . • EXPERIMENTAL MATERIALS AND METHODS A. M a t e r i a l s E g g s f r o m W h i t e L e g h o r n s , l e s s t h a n 24 h r o l d w e r e o b t a i n e d f r o m t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a P o u l t r y R e s e a r c h S t a t i o n . E g g s w e r e t a k e n f r o m two f l o c k s o f b i r d s . When t h e f i r s t f l o c k became o l d a n d u n p r o d u c t i v e , e g g s were o b t a i n e d f r o m a s e c o n d f l o c k . E g g s w e r e p o o l e d f o r r e p r e s e n t a t i v e s a m p l i n g . A l l e g g s w i t h b l o o d s p o t s w e r e d i s c a r d e d . S e a m l e s s c e l l u l o s e d i a l y s i s t u b i n g was o b t a i n e d f r o m F i s h e r S c i e n t i f i c Co. L t d . The t u b i n g was p e r m e a b l e t o w a t e r and l o w m o l e c u l a r w e i g h t compounds (M.W. < 1 2 , 0 0 0 ) . A v e r a g e p o r e s i z e was a b o u t 48 R. P o l y g r a m S i l N-HR p r e c o a t e d p l a s t i c t h i n l a y e r c h r o m a t o g r a p h y s h e e t s w e r e p u r c h a s e d f r o m B r i n k m a n I n s t r u m e n t s ( C a n a d a ) L t d . The 20 X 20 cm s h e e t s c o n s i s t e d o f a 0.2 mm l a y e r o f s i l i c a g e l a d s o r b e n t , w i t h o u t b i n d e r , on a p l a s t i c b a c k i n g . The s u f f i x "HR" d e n o t e d a n e x c e p t i o n a l l y p u r e a d s o r b e n t p r e p a r a t i o n . The f o l l o w i n g r e f e r e n c e s t a n d a r d s a nd l i p i d d e t e c t i o n r e a g e n t s w e r e o b t a i n e d f r o m 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 , I n c . , S t a t e C o l l e g e , P e n n . : egg l e c i t h i n ( p h o s p h a t i d y l c h o l i n e ) , b o v i n e p h o s p h a t i d y l e t h a n o l a m i n e , e g g l y s o l e c i t h i n , b o v i n e l y s o p h o p h a t i d y l e t h a n o l a m i n e , b o v i n e p h o s p h a t i d y l s e r i n e , p l a n t p h o s p h a t i d y l i n o s i t o l , a n d b o v i n e s p h i n g o m y e l i n , m o lybdenum b l u e , n i n h y d r i n , p o t a s s i u m d i c h r o m a t e - s u l f u r i c a c i d and 2 , 7 - d i c h l o r o f l u o r e s c e i n . C h o l e s t e r o l s t a n d a r d was p u r c h a s e d f r o m A l d r i c h C h e m i c a l Co. I n c . , M i l w a u k e e , O h i o and t r i o l e i n r e f e r e n c e t r i g l y c e r i d e f r o m N u t r i t i o n a l B i o c h e m i c a l C o r p . , C l e v e l a n d , O h i o . A n a l y t i c a l g r a d e s o l v e n t s a n d r e a g e n t s w e r e u s e d t h r o u g h o u t t h i s s t u d y . B. M e t h o d s 1. P r e p a r a t i o n o f MF and LDLg Y o l k was s e p a r a t e d f r o m t h e egg by t h e m e t h o d d e s c r i b e d i n C h a p t e r 1. F r a c t i o n I I ( F I I ) was i s o l a t e d f r o m w a s h ed y o l k g r a n u l e s b y the. m e t h o d o f Chang (1969 ) w i t h a s l i g h t m o d i f i c a t i o n . A 300 g p o r t i o n o f t h e y o l k p o o l e d f r o m 6 d o z eggs was c e n t r i f u g e d i n a Beckman M o d e l L2-65B u l t r a c e n t r i f u g e a t 30,000 rpm o r 78,500 X G a t R a v f o r a b o u t 16 h r a t 15°C t o s e d i m e n t i n s o l u b l e y o l k g r a n u l e s . The p l a s m a was r e m o v e d •and t h e y o l k g r a n u l e s d i s p e r s e d i n 12 v o l u m e s o f d i s t i l l e d w a t e r . The d i s p e r s i o n was s l o w l y m i x e d w i t h a m a g n e t i c s t i r r e r i n a s e a l e d E r l e n m e y e r f l a s k a t 5°C f o r 1 h r . Washed g r a n u l e s w e r e c e n t r i f u g e d i n a S o r v a l l s u p e r s p e e d RC2-B a u t o m a t i c r e f r i g e r a t e d c e n t r i f u g e a t a p p r o x i m a t e l y 2,000 X G f o r 1 h r a t 15°C. T h i s w a s h i n g p r o c e d u r e was r e p e a t e d t h r e e a d d i t i o n a l t i m e s , w i t h a s p i n n i n g t i m e o f 1 h r f o r t h e f i r s t r u n and 3 0 m i n f o r s u b s e q u e n t r u n s . C l e a n we t g r a n u l e s w e re t h e n d i s r u p t e d i n a p p r o x i m a t e l y 4.25 v o l u m e s o f 1.71 M s o d i u m c h l o r i d e t o p r o v i d e 300 m l o f d i s p e r s i o n (max. c a p a c i t y o f t h e c e n t r i f u g e ) , and c e n t r i f u g e d a t 78,500 X G f o r a b o u t 16 h r a t 15°C. The d i s p e r s i o n was f r a c t i o n a t e d i n t o two f l o a t i n g f r a c t i o n s ( F I , F I I ) and t h r e e s e d i m e n t i n g f r a c t i o n s ( F i l l , F I V , F V ) . The s e m i - s o l i d p e l l i c l e was p u n c t u r e d , p e e l e d b a c k a nd p r e s s e d a g a i n s t t h e c e n t r i f u g e t u b e w a l l w i t h a s m a l l p o i n t e d m e t a l s p a t u l a . The e x p o s e d F I I was r e m o v e d by P a s t e u r p i p e t and p l a c e d i n a s e a l e d f l a s k u n d e r n i t r o g e n a n d s t o r e d a t 5°C u n t i l r e q u i r e d . Maximum s t o r a g e t i m e was 4 d a y s . S e p a r a t i o n o f t h e F I I i n t o MF and LDLg f r a c t i o n s was a c c o m p l i s h e d by S e p h a r o s e 2B g e l f i l t r a t i o n a s d e s c r i b e d i n C h a p t e r 1. C o l l e c t e d and p o o l e d s a m p l e s w e r e d i a l y z e d ( a p p r o x i m a t e l y 25 m l / t u b e ) a g a i n s t 5 l i t e r s o f d i s t i l l e d w a t e r f o r 36 h r a t 5°C t o remove t h e N a C l . The w a t e r was s t i r r e d c o n t i n u o u s l y a n d was f r e q u e n t l y c h a n g e d . D e s a l t e d s a m p l e s w e r e f r o z e n a t -20°C and s u b s e q u e n t l y l y o p h i l i z e d w i t h a V i r t i s , M o d e l 10-145-MRBA, f r e e z e d r i e r . D r i e d m a t e r i a l was s t o r e d u n d e r n i t r o g e n i n s e a l e d 6 o z b r o w n g l a s s b o t t l e s a t 5°C u n t i l r e q u i r e d f o r f u r t h e r a n a l y s i s . M o i s t u r e c o n t e n t o f l y o p h i l i z e d s a m p l e s was d e t e r m i n e d b y m o d i f i e d AOAC: 13,002 m e t h o d (5 h r , 25 i n Hg, 7 0 ° C ) . M o i s t u r e c o n t e n t o f MF and LDLg d i d n o t e x c e e d 78 . 0 . 0 8 7 % ; t h e a v e r a g e c o n t e n t was a p p r o x i m a t e l y 0.04% f o r a l l s a m p l e s . 2. A n a l y t i c a l M e t h o d s a. L i p i d e x t r a c t i o n A p p r o x i m a t e l y 100 mg o f l y o p h i l i z e d p o w d e r w e r e a c c u r a t e l y w e i g h e d , p l a c e d i n a p o r c e l a i n m o r t a r and ground-w i t h s u c c e s s i v e 2 m l p o r t i o n s o f a n a p p r o p r i a t e l i p i d s o l v e n t . F o r t o t a l l i p i d e x t r a c t i o n o f t h e MF s a m p l e s , s t e p s 1. t o 3. o f t h e f o l l o w i n g s o l v e n t s e q u e n c e were a d h e r e d t o and f o r t h e LDLg f r a c t i o n , s t e p s 1. t o 4. were f o l l o w e d . 1. c h l o r o f o r m - m e t h a n o l ( 2 : 1 ) , 10 m l 2. c h l o r o f o r m - m e t h a n o l ( 2 : 1 ) , 10 m l 3. c h l o r o f o r m - m e t h a n o l ( 9 : 1 ) , 10 m l 4. e t h a n o l , 10 ml.' P r o v i d i n g t h e m a t e r i a l was f i n e l y g r o u n d , t h e s e s o l v e n t s e q u e n c e s w e r e f o u n d t o be e f f e c t i v e , a s c o n f i r m e d by t h i n l a y e r c h r o m a t o g r a p h y , i n r e m o v i n g a l l t r a c e s o f l i p i d . A f t e r e a c h g r i n d i n g w i t h 2 m l o f s o l v e n t , t h e p r e -p a r a t i o n was a l l o w e d t o s t a n d f o r a few m i n u t e s w h i l e i n s o l u b l e m a t e r i a l s e d i m e n t e d . The s u p e r n a t a n t was r e m o v e d by p i p e t t o a c e n t r i f u g e t u b e . The e x t r a c t i o n p r o c e d u r e was r e p e a t e d u n t i l t h e 10 m l o f s o l v e n t h a d b e e n u s e d . The c o m b i n e d s o l v e n t e x t r a c t s was c e n t r i f u g e d a t a p p r o x i m a t e l y 10,000 X G f o r 30 m i n t o e l i m i n a t e p a r t i c u l a t e m a t t e r b y s e d i m e n t a t i o n . The c l e a r , p a l e y e l l o w s u p e r n a t a n t was c a r e -f u l l y r e m o v e d f r o m t h e s e d i m e n t a n d s t o r e d u n d e r n i t r o g e n . The s e d i m e n t i n t h e c e n t r i f u g e t u b e was s u s p e n d e d i n 2 m l o f t h e n e x t s o l v e n t i n t h e l i p i d e x t r a c t i o n s e q u e n c e and a d d e d t o t h e r e s i d u e i n t h e m o r t a r a nd t h e g r i n d i n g p r o -c e d u r e c o n t i n u e d . F u r t h e r a 2 m l p o r t i o n was u s e d t o wash o u t t h e c e n t r i f u g e t u b e a nd t h e n p o u r e d i n t o t h e m o r t a r . The a b o v e s e q u e n c e o f e v e n t s was. r e p e a t e d u n t i l a l l t h e s o l v e n t s h a d b e e n u s e d . The s u p e r n a t a n t f o r e a c h s o l v e n t e x t r a c t i o n was t h e n f i l t e r e d t h r o u g h Whatman No.52 f i l t e r p a p e r on a B u c h n e r f u n n e l u n d e r s l i g h t s u c t i o n . The c o l l e c t i o n f l a s k a n d f i l t e r p a p e r w ere washed w i t h 3 s u c c e s s i v e 4 m l p o r t i o n s o f c h l o r o f o r m - m e t h a n o l ( 2 : 1 ) . The v o l u m e o f e a c h e x t r a c t was r e d u c e d by n i t r o g e n e v a p o r a t i o n . The c o n c e n -t r a t e d s o l u t i o n was a d d e d t o a 10 m l v o l u m e t r i c f l a s k a l o n g w i t h 3 w a s h i n g s ( c h l o r o f o r m - m e t h a n o l , 2:1) o f t h e B u c h n e r f l a s k . Volume was r e d u c e d by n i t r o g e n e v a p o r a t i o n a s r e q u i r e d . N i t r o g e n was u s e d t o e v a p o r a t e 2 m l a l i q u o t s t o d r y n e s s i n p r e w e i g h e d 2 m l v o l u m e t r i c f l a s k s . P e r c e n t t o t a l l i p i d was t h e n c a l c u l a t e d and t h e p e r c e n t n o n - l i p i d r e s i d u e was o b t a i n e d b y d i f f e r e n c e . T o t a l l i p i d a n a l y s i s o f MF and LDLg f r a c t i o n was c o n d u c t e d on eggs o b t a i n e d f r o m b i r d s o f t h e s e c o n d f l o c k , 39 a n d 41 weeks o l d . Two s e p a r a t e l i p i d e x t r a c t i o n s ' w e r e p e r f o r m e d f o r e a c h g r o u p o f h e n s a nd t h e d e t e r m i n a t i o n o f t o t a l l i p i d was p e r f o r m e d 4 t i m e s f o r e a c h l i p i d e x t r a c t . b. T h i n l a y e r c h r o m a t o g r a p h y R e c t a n g u l a r t r o u g h g l a s s c h a m b e r s ( 2 1 X 21 X 9 cm) l i n e d w i t h Whatman No.1 f i l t e r p a p e r w e r e u s e d t h r o u g h o u t 80. t h i s s t u d y f o r t h e a s c e n d i n g d e v e l o p m e n t o f t h i n l a y e r s h e e t s . P r i o r t o u s e , t h e t h i n l a y e r s h e e t s w e re washed w i t h c h l o r o -f o r m / m e t h a n o l / a m m o n i u m h y d r o x i d e ( 6 5 : 3 5 : 5 ) by a s c e n d i n g s o l v e n t d e v e l o p m e n t t o remove any g e l c o n t a m i n a t i o n . An i r r e g u l a r d a r k z o n e was a l w a y s p r e s e n t a t t h e t o p o f t h e s o l v e n t washed s h e e t when s p r a y e d w i t h s u l f u r i c a c i d - p o t a s s i u m d i c h r o m a t e a nd c h a r r e d i n an o v e n a t 120°C. The c o n t a m i n a t e d s e c t i o n ( a b o u t 3 cm) was c u t o f f and d i s c a r d e d . The f o l l o w i n g m e thod was u s e d t o s e p a r a t e t h e m a j o r l i p i d s o f b o t h MF and LDLg e x t r a c t s . W i t h i t s l o n g e s t s i d e (20 cm) v e r t i c a l , t h e TLC s h e e t was s p o t t e d w i t h 400 t o 700 yg o f l i p i d a t t h e b o t t o m r i g h t h a n d c o r n e r , 2 cm f r o m e a c h e d g e . A 10 u l m i c r o p i p e t was u s e d t o s p o t t h e p l a t e and n i t r o g e n was u s e d t o d r y t h e s p o t . P l a t e s w e r e d e v e l o p e d w i t h c h l o r o -f o r m / m e t h a n o l / a m m o n i u m h y d r o x i d e ( 6 5 : 3 5 : 5 ) t o a d i s t a n c e 11 cm a b o v e t h e o r i g i n , a t o t a l o f 13 cm. The p l a t e was r e m o v e d , d r i e d , a n d f u r t h e r d e v e l o p e d i n t h e same d i r e c t i o n w i t h h e x a n e / e t h e r ( 4 : 1 ) t o a d i s t a n c e o f 17.5 cm a b o v e t h e o r i g i n . T h i s p r o c e d u r e f a c i l i t a t e d t h e s e p a r a t i o n o f p o l a r l i p i d s f r o m n o n - p o l a r l i p i d s . W hereas t h e c h o l e s t e r o l and n e u t r a l t r i -g l y c e r i d e l i p i d s w e r e c o m p l e t e l y s e p a r a t e d f r o m e a c h o t h e r , t h e p h o s p h o l i p i d s p o t s o v e r l a p p e d . A f t e r d r y i n g , t h e c h r o m a -t o g r a m was c u t t o s e g r e g a t e t h e p o l a r l i p i d s f r o m t h e n o n -p o l a r l i p i d s . The s h e e t c o n t a i n i n g t h e p o l a r l i p i d s was t h e n d e v e l o p e d a t r i g h t a n g l e s t o t h e p r e v i o u s s o l v e n t d i r e c t i o n 8 1 . (17 cm s i d e up) w i t h n - b u t a n o l / g l a c i a l a c e t i c a c i d / w a t e r ( 6 0 : 2 0 : 2 0 ) t o a h e i g h t o f 8 cm a b o v e t h e o r i g i n t o s e p a r a t e t h e p h o s p h o l i p i d s . A s a s t e p t o c o n f i r m a t i o n , of. t h e p r e s e n c e o f o n l y c h o l e s t e r o l a n d t r i g l y c e r i d e s t h e s h e e t s w i t h t h e n o n - p o l a r s p o t s was a l s o d e v e l o p e d w i t h h e x a n e / e t h e r / g l a c i a l a c e t i c a c i d ( 9 0 : 1 0 : 1 ) a t r i g h t a n g l e s t o t h e p r e v i o u s s o l v e n t d i r e c t i o n . The c h r o m a t o g r a m m e d l i p i d s w e r e d e t e c t e d by t h r e e d i f f e r e n t m e t h o d s : 1. e x p o s u r e t o i o d i n e v a p o r i n a c l c s e d g l a s s t a n k , 2. s p r a y i n g w i t h a s o l u t i o n o f 5 5 % (w/v) s u l f u r i c a c i d a n d 0.6% (w/v) p o t a s s i u m d i c h r o m a t e w i t h s u b -s e q u e n t c h a r r i n g i n a n o v e n a t 120°C, a n d 3. s p r a y i n g w i t h 0.2% 2 , 7 - d i c h l o r o f l u o r e s c e i n e t h a n o l i c s o l u t i o n w i t h s u b s e q u e n t e x p o s u r e t o UV l i g h t . I d e n t i f i c a t i o n o f t h e s e p a r a t e d l i p i d s was a c c o m -p l i s h e d u s i n g a number o f m e t h o d s : 1. R e f e r e n c e s t a n d a r d s . R e f e r e n c e l i p i d s w e r e s p o t t e d a n d d e v e l o p e d i n a n i d e n t i c a l manner t o t h e unknown l i p i d s . S t a n d a r d s w e r e e m p l o y e d s i n g l y , a s m i x t u r e s , o r w e r e a d d e d t o t h e u nknown l i p i d s a m p l e s . 2. P h o s p h o r i c a c i d s p r a y . A 1 5 % p h o s p h o r i c a c i d s o l u t i o n was h e a v i l y s p r a y e d o n t h e TLC s h e e t s u n t i l t h e y w e r e s a t u r a t e d and v i s i b l y t r a n s l u c e n t ( W a l d i , 1 9 6 5 a ) . P l a t e s w e r e h e a t e d a t 1 2 0 C C f o r a p p r o x i m a t e l y 10 m i n a n d s u b s e q u e n t l y o b s e r v e d u n d e r UV l i g h t ( 3 6 5 nm). W i t h t h i s 82. m e t h o d t h e v a r i o u s s t e r o i d s h a v e s p e c i f i c c o l o r s . The o b s e r v e d p i n k i s h r e d c o l o r was s p e c i f i c f o r c h o l e s t e r o l and i t s e s t e r s . 3. N i n h y d r i n s p r a y . A s o l u t i o n o f 0.3 g n i n h y d r i n d i s s o l v e d i n 100 m l n - b u t a n o l a n d m i x e d w i t h 3 m l g l a c i a l a c e t i c a c i d was l i g h t l y s p r a y e d o v e r t h e c h r o m a t o g r a m s . TLC s h e e t s w e r e h e a t e d i n a n o v e n a t 110°C f o r 10 m i n . P u r p l e s p o t s w e r e i n d i c a t i v e o f f r e e a m i n o g r o u p s o r a m i n o p h o s p h o -l i p i d s ( W a l d i , 1 9 6 5 b ) . 4. Mo l y b d e n u m b l u e s p r a y . A s o l u t i o n o f 5 ml 6 0% p e r c h l o r i c a c i d , 10 m l 1 N h y d r o c h l o r i c a c i d and 25 m l o f 4% (w/v.) ammonium m o l y b d a t e was made up t o 100 m l w i t h d i s -t i l l e d w a t e r a n d s p r a y e d l i g h t l y o v e r t h e c h r o m a t o g r a m , w h i c h was t h e n h e a t e d i n an o v e n a t 110°C f o r 5 m i n . B l u e s p o t s i n d i c a t e d p h o s p h o r u s c o n t a i n i n g l i p i d s ( J o h n s t o n , 1 9 7 1 ) . 5. S p e c t r o p h o t o m e t r i c c o m p a r i s o n . The a b s o r p t i o n s p e c t r a ( b e t w e e n 450 and 850 nm) o f p u r e c h o l e s t e r o l w e r e c o m p a r e d w i t h t h e s p e c t r a o f t h e s t e r o i d i s o l a t e d b y t h i n l a y e r c h r o m a t o g r a p h y . •' c. N i t r o g e n a n d p h o s p h o r u s d e t e r m i n a t i o n P e r c e n t n i t r o g e n a n d p e r c e n t p h o s p h o r u s o f b o t h l y o p h i l i z e d MF a n d LDLg i s o l a t e s a n d t h e n o n - l i p i d r e s i d u e s o f t h e s e f r a c t i o n s w e r e d e t e r m i n e d w i t h t h e a i d o f a T e c h n i c o n A u t o a n a l y s e r I I . N i t r o g e n was a l s o d e t e r m i n e d b y m i c r o - K j e l d a h l , o f f i c i a l AOAC - 38-012 m e t h o d . 83. S i n c e t h e T e c h n i c o n ' s c o n t i n u o u s d i g e s t e r u n i t was o n l y e f f e c t i v e f o r a d i s s o l v e d sample,.MF and LDLg p r e p a r a t i o n s w e r e p r e d i g e s t e d . A c c u r a t e l y w e i g h e d l y o p h i l i z e d s a m p l e s o f a b o u t 100 mg w e r e h y d r a t e d f o r 5 m i n i n 5 m l o f d i s t i l l e d w a t e r i n a 12 5 m l E r l e n m e y e r f l a s k , a f t e r w h i c h 5 m l o f 7 2 % p e r c h l o r i c a c i d was a d d e d . The m i x t u r e was h e a t e d f o r a b o u t 1 h r w i t h a p l a t e h e a t e r a d j u s t e d t o p r o v i d e a g e n t l e r e f l u x i n g . An a d d i t i o n a l 5 m l o f 7 2% p e r c h l o r i c a c i d was a d d e d t o c o m p l e t e t h e d i g e s t i o n a f t e r a f u r t h e r 20 m i n o f h e a t i n g . The s o l u t i o n was t r a n s f e r r e d t o a 50 m l v o l u m e t r i c f l a s k , a l o n g w i t h f i v e a q u e o u s w a s h i n g s o f t h e d i g e s t i o n f l a s k , and b r o u g h t up t o v o l u m e , A 10 m l a l i q u o t o f t h i s s t o c k s o l u t i o n was d i l u t e d w i t h d i s t i l l e d w a t e r a n d b r o u g h t up t o v o l u m e i n a 10 0 m l v o l u m e t r i c f l a s k . A t t h i s d i l u t i o n , t h e p e r c h l o r i c a c i d d i d n o t i n t e r f e r e w i t h t h e d e v e l o p m e n t o f t h e c h r o m o p h o r e s . T h i s l a c k o f i n t e r f e r e n c e was c o n f i r m e d by c o m p a r i n g a q u e o u s s t a n d a r d s o l u t i o n s (ammonium s u l f a t e a n d p o t a s s i u m o r t h o p h o s p h a t e ) w i t h d i g e s t e d s t a n d a r d s o l u t i o n s ; b o t h g a v e i d e n t i c a l r e a d i n g s . .. : A l i q u o t s o f d i g e s t e d s a m p l e , b l a n k s a n d s t a n d a r d s o l u t i o n s w e r e d i g e s t e d i n t h e T e c h n i c o n c o n t i n u o u s d i g e s t e r u n i t w i t h a c o n c e n t r a t e d s u l f u r i c a c i d , p e r c h l o r i c a c i d a n d s e l e n i u m c a t a l y s t m i x t u r e . The d i g e s t i o n s o l u t i o n was p r e p a r e d by d i s s o l v i n g 1 2 g o f s e l e n i u m i n 200 m l o f d i s t i l l e d w a t e r w i t h 50 ml o f 84 . 60% p e r c h l o r i c a c i d . C o n c e n t r a t e d s u l f u r i c a c i d was a d d e d t o 4 1 a n d , u p o n c o o l i n g , t h e d i g e s t a n t was b r o u g h t up t o 4 1 a g a i n 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 . T h i s d i g e s t i o n m i x t u r e e n s u r e d t h e c o n v e r s i o n o f o r g a n i c n i t r o g e n t o ammonium i o n s and o r g a n i c p h o s p h o r u s t o o r t h o p h o s p h a t e . The d i g e s t e d s a m p l e f r o m t h e c o n t i n u o u s d i g e s t e r was a u t o m a t i c a l l y c o o l e d , d i l u t e d a nd d i v i d e d i n t o two s t r e a m s f o r s i m u l t a n e o u s a n a l y s i s o f ammonium and o r t h o -p h o s p h a t e . Ammonia was c o l o r i m e t r i c a l l y d e t e c t e d by means o f t h e B e r t h l o t r e a c t i o n i n w h i c h a b l u e i n d o p h e n o l c o m p l e x f o r m s when ammonia r e a c t s w i t h s o d i u m p h e n a t e and s o d i u m h y p o c h l o r i t e . C o l o r was i n t e n s i f i e d by t h e a d d i t i o n o f t h e c a t a l y s t m anganese s u l f a t e . P o t a s s i u m t a r t r a t e was r e q u i r e d t o p r e v e n t p r e c i p i t a t i o n o f manganese h y d r o x i d e . The r e a g e n t s f o r t h i s p r o c e d u r e w e r e p r e p a r e d a s f o l l o w s : A l k a l i n e t a r t r a t e a nd M n + + W h i l e s t i r r i n g , 20 m l o f 1% manganese s u l f a t e was s l o w l y a d d e d t o 1 l i t e r o f 10 N s o d i u m h y d r o x i d e c o n t a i n i n g 5% s o d i u m p o t a s s i u m t a r t r a t e . A l k a l i n e p h e n o l 13 8 m l o f 88% l i q u i d p h e n o l was g r a d u a l l y a d d e d t o 36 2 m l o f 5 N s o d i u m h y d r o x i d e w i t h c o n t i n u o u s s t i r r i n g a n d w a t e r c o o l i n g . 85 S o d i u m h y p o c h l o r i t e . A s o l u t i o n w i t h a b o u t 5% a v a i l a b l e c h l o r i n e was u s e d . The d e t e r m i n a t i o n o f p h o s p h o r u s , d e p e n d s o n t h e f o r m a t i o n o f a p h o s p h o m o l y b d e n u m - a n t i m o n y b l u e c o m p l e x w h i c h r e s u l t s f r o m t h e r e a c t i o n o f ammonium m o l y b d a t e , a n t i m o n y p o t a s s i u m t a r t r a t e and o r t h o p h o s p h a t e . The c o m p l e x i s g e n e r a l l y r e d u c e d t o an i n t e n s e b l u e compound w i t h a s c o r b i c a c i d . H o w e v e r , a s c o r b i c a c i d f o r m e d a p r e c i p i t a t e w i t h s e l e n i u m i n t h e d i g e s t a n t . H y d r a z i n e was e m p l o y e d a s a s u i t a b l e a l t e r n a t i v e t o a s c o r b i c a c i d . R e a g e n t s f o r t h i s p r o c e d u r e were p r e p a r e d a s f o l l o w s + 3 Ammonium m o l y b d a t e a n d Sb W i t h c o n s t a n t s t i r r i n g 20 m l o f 1% a n t i m o n y p o t a s s i u m t a r t r a t e a n d 0.2 5 m l o f L e v o r I V ( T e c h n i c o n w e t t i n g a g e n t ) was s l o w l y a d d e d t o 1.0 1 o f 2% ammonium m o l y b d a t e . H y d r a z i n e . A 0.2% ( v / v ) a q u e o u s s o l u t i o n o f h y d r a -z i n e h y d r a t e was p r e p a r e d . F o r t h e s i m u l t a n e o u s d e t e r m i n a t i o n o f p h o s p h o r u s and n i t r o g e n , 2.9 m l / m i n o f s a m p l e was c o n t i n u o u s l y m i x e d w i t h 1.7 m l / m i n o f d i g e s t i o n s o l u t i o n a n d 2.9 m l / m i n o f d i s -t i l l e d w a t e r . The r e s u l t i n g m i x t u r e was p a s s e d t h r o u g h a t w o - s t a g e d i g e s t e r , s e t a t 320°C and 290°C w h i c h t a k e s a b o u t 2 m i n . The d i g e s t e d s o l u t i o n was t h e n a i r c o o l e d ( w i t h b l o w e r ) a n d d i l u t e d w i t h 10 p a r t s d i s t i l l e d w a t e r . F o r n i t r o g e n a n a l y s i s , 1.6 m l / m i n o f d i g e s t e d 86 . s a m p l e was m i x e d w i t h 1.2 m l / m i n a l k a l i n e t a r t r a t e r e a g e n t , 0.6 m l / m i n a l k a l i n e p h e n o l r e a g e n t , and 0.6 m l / m i n c h l o r i n e r e a g e n t . The r e s u l t i n g s o l u t i o n was c o o l e d ( w a t e r j a c k e t e d c o i l ) and p a s s e d t h r o u g h a c o l o r i m e t e r f i t t e d w i t h a 50 mm f l o w c e l l . A b s o r b a n c e was r e a d a t 630 nm.. The r e c o r d e r was c a l i b r a t e d a g a i n s t s t a n d a r d ammonium s u l f a t e s o l u t i o n s a n d c o r r e c t e d w i t h b l a n k s . C h a r t r e a d o u t was i n mg/1 o f n i t r o g e n . F o r t h e a n a l y s i s o f p h o s p h o r u s , 1.2 m l / m i n o f s a m p l e was c o n t i n u o u s l y m i x e d w i t h 1.2 m l / m i n ammonium m o l y b d a t e r e a g e n t and 0.2 m l / m i n o f h y d r a z i n e r e a g e n t . A b s o r -b a n c e was m e a s u r e d a t 880 nm and t h e r e c o r d e r c a l i b r a t e d a g a i n s t s t a n d a r d p o t a s s i u m o r t h o p h o s p h a t e a n d b l a n k s o l u t i o n s . The a b o v e p r o c e d u r e s a r e b a s e d on T e c h n i c o n m e t h o d s No. 155-71W f o r o r t h o p h o s p h a t e a n d No. 146-71A f o r t o t a l n i t r o g e n . Two s e p a r a t e s a m p l e s o f b o t h MF and LDLg f r a c t i o n s f r o m e g g s o f b i r d s 3 5 wk, 3 6 wk, 3 9 wk, 41 wk and 1.5 y r ( o l d f l o c k ) w e r e a n a l y z e d f o r p h o s p h o r u s a n d n i t r o g e n . E a c h s a m p l e was r u n t w i c e t h r o u g h t h e T e c h n i c o n A n a l y s e r . N i t r o g e n was a l s o d e t e r m i n e d u s i n g a s i m i l a r s e t o f s a m p l e s by AOAC m i c r o - K j e l d a h l m e t h o d . F o r t h e n o n - l i p i . d r e s i d u e s o f b o t h MF and LDLg f r a c t i o n s , y o l k t a k e n f r o m 3 9 wk o l d h e n s was c o n s i d e r e d r e p r e s e n t a t i v e . Two s e p a r a t e s a m p l e s o f e a c h w e r e p r e p a r e d 87 . and d u p l i c a t e s w e r e r u n t h r o u g h t h e T e c h n i c o n A u t o a n a l y s e r t o d e t e r m i n e p e r c e n t n i t r o g e n a n d p e r c e n t p h o s p h o r u s . d. P h o s p h o l i p i d q u a n t i t a t i v e a n a l y s i s . P h o s p h o l i p i d c o m p o n e n t s o f MF a n d LDLg l i p i d e x t r a c t s w e r e i s o l a t e d b y t h i n l a y e r s i l i c a g e l c h r o m a t o g r a p h y a s p r e v i o u s l y d e s c r i b e d . L i p i d s p o t s w e r e d e t e c t e d w i t h i o d i n e v a p o r and c i r c l e d w i t h p e n c i l . B l a n k z o n e s c o r r e s p o n d i n g i n s i z e t o t h e s e p a r a t e d p h o s p h o l i p i d s p o t s , w e r e m a r k e d on t h e same c h r o m a t o g r a m . M o r e o v e r , f o r t h e a n a l y s i s o f t o t a l l i p i d p h o s p h o r u s , a known amount o f l i p i d e x t r a c t a n d p o t a s s i u m o r t h o p h o s p h a t e was' s p o t t e d on t h e d e v e l o p e d c h r o m a t o g r a m a n d was a l s o m a r k e d o f f . A s p i r a t i o n o f s p o t s d i r e c t l y i n t o acidr-washed 30 m l K j e l d a h l f l a s k s was a c c o m p l i s h e d b y f i t t i n g a r u b b e r s t o p p e r w i t h t w o p l a s t i c t u b e s ; one o f t h e t u b e s h a d a p o i n t e d end w h i c h was u s e d a s b o t h a g e l s c r a p e r a n d i n t a k e f o r t h e d i s l o d g e d g e l m a t e r i a l ; t h e s e c o n d t u b e was a t t a c h e d t o a w a t e r t a p a s p i r a t o r t o p r o v i d e s u c t i o n . The o t h e r end o f t h i s a s p i r a t o r t u b e c o n t a i n e d a s m a l l p i e c e o f . t i s s u e p a p e r t o a c t a s a f i l t e r . A b l u n t n e e d l e was u s e d t o p u s h t h e t i s s u e t h r o u g h i n t o t h e b o t t o m o f t h e f l a s k a f t e r a s p i r a t i o n was c o m p l e t e . P r i o r t o a s p i r a t i o n , 1 m l o f 7 2% p e r c h l o r i c a c i d was r o t a t e d a b o u t t h e b o t t c m c f t h e K j e l d a h l f l a s k . T h i s d i g e s t i o n r e a g e n t a c t e d a s a l i q u i d t r a p f o r a s p i r a t e d 88 . The f l a s k c o n t e n t s . ( g e l , p e r c h l o r i c a c i d , t i s s u e p a p e r ) w e r e b r o u g h t t o a b o i l i n a K j e l d a h l h e a t i n g r a c k and t h e h e a t e r s a d j u s t e d t o p r o v i d e a g e n t l e r e f l u x i n g . The f l a s k s w e r e a l l o w e d t o c o o l a f t e r a 1 h r d i g e s t i o n ' p e r i o d . A l l o f t h e p h o s p h o l i p i d p h o s p h o r u s was now c o n v e r t e d t o o r t h o p h o s p h a t e . The c o n t e n t s o f t h e f l a s k a l o n g w i t h 4 c o n s e c u t i v e w a s h i n g s o f t h e f l a s k w e r e p l a c e d i n a c e n t r i f u g e t u b e and s p u n a t a b o u t 5,0 00 X G f o r 20 m i n t o s e d i m e n t t h e s i l i c a g e l . The s u p e r n a t a n t was p l a c e d i n a 25 m l v o l u m e t r i c f l a s k a l o n g w i t h 3 f u r t h e r a q u e o u s w a s h i n g s o f t h e g e l r e s i d u e . The s o l u t i o n was . b r o u g h t up t o v o l u m e w i t h d i s t i l l e d w a t e r . A l l s a m p l e s w e r e a n a l y z e d w i t h no f u r t h e r d i g e s t i o n f o r p h o s p h o r u s w i t h t h e T e c h n i c o n A u t o a n a l y s e r a s p r e v i o u s l y d e s c r i b e d . S a m p l e s w e r e a u t o m a t i c a l l y d i l u t e d w i t h 5 p a r t s w a t e r t o r e d u c e t h e p e r c h l o r i c a c i d c o n c e n t r a t i o n . S a m p l e s f o r a n a l y s i s w e re o b t a i n e d f r o m 3 9 wk o l d b i r d s . One l i p i d e x t r a c t e a c h was o b t a i n e d f r o m s i n g l e MF and LDLg f r a c t i o n s . F o r t h e m a j o r , p h o s p h o l i p i d s , l e c i t h i n ( P C ) and p h o s p h a t i d y l e t h a n o l a m i n e (PE), 4 o r 5 c h r o m a t o -grams were d e v e l o p e d and e a c h s p o t was s e p a r a t e l y a n a l y z e d . H o w e v e r , f o r t h e p h o s p h o l i p i d s l y s o p h o s h a t i d y l e t h a n o l a m i n e ( L P E ) a n d s p h i n g o m y e l i n ( S ) , w h i c h w e r e p r e s e n t o n l y i n m i n o r a m o u n t s , s p o t s f r o m 2 c h r o m a t c g r a m s were c o m b i n e d a n d 4 89 . d e t e r m i n a t i o n s w e re s u b s e q u e n t l y made. M o l a r r a t i o s o f p h o s p h o l i p i d s were o b t a i n e d by e x p r e s s i n g t h e r e s u l t s (mg/1) a s a p e r c e n t o f t h e . t o t a l l i p i d p h o s p h o r u s ( m g / 1 ) . F o r e x p r e s s i o n o f r e s u l t s as a p e r c e n -t a g e o f t h e t o t a l l i p i d , p h o s p h o r u s , v a l u e s w e re m u l t i p l i e d by t h e f o l l o w i n g f a c t o r s : PC 2 5 . 4 , PE 2 5 . 4 , LPE 1 6 . 0 , and S 24.8 . e. Q u a n t i t a t i v e c h o l e s t e r o l d e t e r m i n a t i o n . C h o l e s t e r o l was s e p a r a t e d f r o m MF and LDLg l i p i d e x t r a c t s by t h i n l a y e r c h r o m a t o g r a p h y . The d o u b l e d e v e l o p m e n t t e c h n i q u e p r e v i o u s l y m e n t i o n e d was u s e d . A s p o t t i n g s e q u e n c e was e m p l o y e d t o a s s i s t i n l o c a t i n g i s o l a t e d c h o l e s t e r o l s p o t s . A l i p i d s a m p l e o f known c o n c e n t r a t i o n was s p o t t e d 3 t i m e s 2 cm a b o v e t h e b o t t o m o f t h e p l a t e i n t h r e e s e p a r a t e l o c a t i o n s . The f i r s t s a m p l e was s p o t t e d 2.5 cm f r o m t h e l e f t h a n d s i d e o f t h e p l a t e , t h e second, s a m p l e a t a f u r t h e r 2.5 cm, t h e t h i r d z o n e , a t a. f u r t h e r 2.5 cm was n o t s p o t t e d , a n d t h e f o u r t h zone was s p o t t e d w i t h s a m p l e . Upon d e v e l o p m e n t o f t h e c h r o m a t o g r a m , t h e two o u t e r z o n e s w ere c u t away and h e a t e d a t 110°C a f t e r b e i n g s p r a y e d w i t h 1 5 % p h o s p h o r i c a c i d . The two s t r i p s w e r e t h e n u s e d t o l o c a t e t h e c h o l e s t e r o l s p o t on t h e r e m a i n i n g segment o f t h e u n s p r a y e d c h r o m a t o g r a m . A s e c t i o n o f t h e p l a s t i c s h e e t was c u t away t o i s o l a t e t h e c h o l e s t e r o l s p o t . A c o m p a r a b l e s e c t i o n was c u t away f r o m t h e u n s p o t t e d zone t o a c t a s a 90 . b l a n k . The s i l i c a g e l was s c r a p e d o f f i n t c a c e n t r i f u g e t u b e u s i n g a g l a s s f u n n e l a n d s u s p e n d e d i n 2 m l o f a c e t o n e -e t h a n o l s o l u t i o n ( 1 : 1 ) . The s u s p e n s i o n was c e n t r i f u g e d a t a b o u t 5,00 0 X g f o r 5 m i n a n d t h e s u p e r n a t a n t c o l l e c t e d i n a t e s t t u b e . T h i s p r o c e d u r e was r e p e a t e d 4 more t i m e s on t h e g e l r e s i d u e r e s u l t i n g i n t h e c o m p l e t e e x t r a c t i o n o f c h o l e s t e r o l , a s c o n f i r m e d by t h i n l a y e r c h r o m a t o g r a p h y . The c o m b i n e d e x t r a c t s w e r e e v a p o r a t e d t o d r y n e s s w i t h n i t r o g e n and the; r e s i d u e t r e a t e d a n d a n a l y z e d by t h e method o f Zak e t a l . ( 1 9 5 7 ) . T h i s i n v o l v e d t h e s p e c t r o p h o t o m e t r y a n a l y s i s o f a c o l o r e d f e r r i c c h l o r i d e - c h o l e s t e r o l c o m p l e x . D e t e r m i n a -t i o n o f t h e amount o f e x t r a c t e d c h o l e s t e r o l was a c c o m p l i s h e d w i t h t h e a i d o f a s t a n d a r d c u r v e ; a b s o r b a n c e m e a s u r e d a t 550 nm, c o r r e c t e d w i t h b l a n k s . Known amounts o f p u r e c h o l e s t e r o l w e r e c h r o m a t o -g r a p h e d a n d t r e a t e d i n a s i m i l a r manner. R e c o v e r y was a l w a y s g r e a t e r t h a n 9 8%. Two s e p a r a t e s a m p l e s o f MF and LDLg l i p i d e x t r a c t s i s o l a t e d f r o m 3 9 wk o l d b i r d s w e r e e a c h c h r o m a t o g r a p h e d 5 t i m e s . The s p o t c o n t a i n e d 388 yg MF l i p i d o r 435 yg LDLg l i p i d . RESULTS AND DISCUSSION The o n l y d e t a i l e d r e p o r t on t h e c h e m i c a l c o m p o s i -' t i o n o f l o w - d e n s i t y l i p o p r o t e i n f r o m egg y o l k g r a n u l e s was p r e s e n t e d by G o r n a l l a n d K u k s i s ( 1 9 7 1 b ) . T h e s e r e s e a r c h e r s p r e p a r e d a l o w - d e n s i t y l i p o p r o t e i n f r a c t i o n (LDFG) by l a y e r i n g a 5% N&C1 s o l u t i o n o v e r a g r a n u l e d i s p e r s i o n w i t h 1 0 % N a C l and c e n t r i f u g i n g . J u d g i n g f r o m t h e r e s u l t s o f Chang ( 1 9 6 9 ) t h e i r f l o a t i n g l o w - d e n s i t y f r a c t i o n c o n s i s t e d o f n o t o n l y l o w - d e n s i t y l i p o p r o t e i n b u t a l s o l a r g e g l o b u l e s } g l o b u l e - c o n t a i n i n g c l u m p s and m y e l i n f i g u r e s . I n t h e p r e s e n t s t u d y two f l o a t i n g f r a c t i o n s ( F I and F I I ) w e r e o b t a i n e d b y u l t r a c e n t r i f u g a t i o n b u t o n l y F I I was s e l e c t e d f o r f u r t h e r f r a c t i o n a t i o n i n t o LDLg and MF s i n c e i t d i d n o t c o n t a i n o t h e r p a r t i c l e s s u c h a s l a r g e g l o b u l e s a n d g l o b u l e - c o n t a i n i n g c l u m p s w h i c h w e r e p r e s e n t i n t h e F I a l o n g w i t h LDLg and MF. G o r n a l l a n d K u k s i s (1971a).. w i t h t h e a i d o f t h i n l a y e r c h r o m a -t o g r a p h y o n h y d r o x y l a p a t i t e , w e r e a b l e t o s e p a r a t e t h e i r LDFG i n t o a m a j o r ( 7 7 % ) m i g r a t i n g f r a c t i o n LDFG-2 and a m i n o r n o n - m i g r a t i n g f r a c t i o n LDFG-1. S i n c e t h e t y p e s o f p a r t i c l e s i n t h e s e f r a c t i o n s w e r e n o t d e t e r m i n e d m e a n i n g f u l c o m p a r i s o n s o f t h e i r c o m p o s i t i o n a l d a t a w i t h t h a t f o r LDLg and MF i n t h e p r e s e n t s t u d y a r e i m p o s s i b l e . A s n o t e d p r e v i o u s l y , no r e p o r t s h a v e b e e n p u b l i s h e d on t h e c h e m i c a l c o m p o s i t i o n o f 92'. t h e MF f r a c t i o n o f y o l k . The t o t a l n i t r o g e n (N) and t o t a l p h o s p h o r u s ( P ) w e r e d e t e r m i n e d f o r LDLg and MF f r a c t i o n s t o o b t a i n a p p r o x i -mate v a l u e s o f p r o t e i n and p h o s p h o l i p i d , c o n t e n t s . The p e r c e n t p r o t e i n c o n t e n t o f t h e f r a c t i o n s c a n be c a l c u l a t e d by m u l t i p l y i n g % N by 6.2 5 a n d p e r c e n t p h o s p h o l i p i d by m u l t i p l y i n g %P by 25 on t h e b a s i s t h a t o n l y m i n o r a m o u n t s o f P and N a r e p r e s e n t , r e s p e c t i v e l y i n t h e p r o t e i n s and p h o s p h o -l i p i d s ( S a a r i e t a l . , 1 9 6 4 ) . The p e r c e n t N and P c o n t e n t s o f LDLg and MF f r a c t i o n s f r o m e g g s l a i d b y b i r d s o f v a r i o u s a g e s a r e p r e s e n t e d i n T a b l e I . C o m p a r i s o n s o f t h e % N v a l u e s o b t a i n e d by t h e K j e l d a h l and T e c h n i c o n m e t h o d s r e v e a l e d v e r y s m a l l d i f f e r e n c e s f c r e a c h s a m p l e . F u r t h e r , t h e a v e r a g e d e v i a t i o n s a r e n o t a b l y v e r y l o w v a l u e s . As shown i n T a b l e 1, t h e o l d e r ' b i r d s (39 weeks t o 1.5 y e a r s ) l a i d e g g s w i t h LDLg h a v i n g h i g h e r % N and % P v a l u e s t h a n t h o s e f o r y o u n g e r b i r d s (35 and 36 w e e k s ) . I n t h e e g g s o f o l d e r b i r d s , % N i n LDLg i s a b o u t 33 % h i g h e r and t h e % P i s a b o u t 4 0 % h i g h e r . W i t h r e g a r d t o MF t h e % N f o r t h e o l d e r b i r d s was a b o u t 10C% h i g h e r t h a n t h a t f o r t h e y o u n g e r b i r d s , b u t t h e d i f f e r e n c e i n t h e % P v a l u e s was v e r y s l i g h t . S t e e r e t a l . ( 1 9 6 8 ) f o u n d t h a t LDL o f t h e p l a s m a c o n s i s t e d o f 2.05% t o t a l N and 0.87% t o t a l P w h i c h a r e r o u g h l y c l o s e t o t h e v a l u e s r e p o r t e d 93. TABLE I. PERCENT OF NITROGEN AND PHOSPHORUS OF L Y O P H I L I Z E D MF AND LDLg FRACTIONS Sample' MF F r a c t i o n % N i t r o g e n •* a v . d e v . % P h o s p h o r u s + a v . d e v . K j e l d a h l T e c h n i c o n T e c h n i c o n 3 5 wk-Fk2 1. 26 + 0. 00 1. 27 + 0. 00 1. 13 + 0. 02 36 wk-Fk2 1. 29 + 0 . 00 1. 22 + 0. 03 1. 15 + 0 . 01 3 9 wk-Fk2 2. 52 + 0 . 00 2. 53 + 0. 04 1. 14 + 0. 00 41 wk-Fk2 2 . 50 + 0 . 01 1. 20 + 0. 00 1 y r ( a p p r o x ) - F k l 2 . 44 + 0. 12 1.5 y r ( a p p r o x ) - F k l 2. 39 4 0 . 03 2. 36 + 0 . 02 1. 20 4 0. 01 LDLg F r a c t i o n 3 5 wk-Fk2 1. 9 3 + 0. 0 0 1. 90 + 0 . 01 0 . 72 4- 0. 02 3 6 wk-Fk2 1. 82 + 0. 01 1. 89 + 0 . 01 0 . 7 5 4- 0. 01 3 9 wk-Fk2 2. 51 + 0 . 02 2. 50 + 0 . 02 1. 05 + 0. 01 41 wk-Fk2 2. 54 + 0. 03 1. 01 4- 0. 01 1 y r ( a p p r o x ) - F k l 2. 50 + 0. 01 1.5 y r ( a p p r o x ) - F k l 2. 46 + 0. 01 2 . 39 + 0 . 05 1. 03 4- 0 . 01 a F k l = f l o c k o n e . Fk2 = f l o c k t w o . b a v e r a g e d e v i a t i o n b a s e d on n = 2. c a v e r a g e d e v i a t i o n baised o n n = 2; e a c h n i s a n a v e r a g e o f two T e c h n i c o n e v a l u a t i o n s o f t h e same s a m p l e . i n t h e p r e s e n t s t u d y f o r LDLg f r o m e g g s o f y o u n g b i r d s . V / i t h t h e % N v a l u e s o b t a i n e d by K j e l d a h l m e t h o d , t h e a p p r o x i m a t e % p r o t e i n v a l u e s w e r e c a l c u l a t e d t o be 7.9% a n d 1 5 . 3 % f o r MF f r o m e g g s l a i d by y o u n g b i r d s (.35 and 36 w e e k s ) a n d o l d e r ("39 weeks t o 1.5 y e a r s ) b i r d s , r e s p e c t i v e l y . The % p r o t e i n v a l u e s i n LDLg o f y o u n g and o l d e r b i r d s w e r e a b o u t 11.8 and 15.4 r e s p e c t i v e l y . The a v e r a g e p h o s p h o l i p i d c o n t e n t o f MF was 2 9 . 5 % f o r a l l e g g s C a y e r a g e d s i n c e % P v a l u e s d i f f e r e d o n l y s l i g h t l y ) a n d t h e LDLg o f eggs f r o m y o u n g a n d o l d e r b i r d s c o n t a i n e d 18.3 and 25.8% p h o s p h o l i p i d , r e s p e c t i v e l y . I t i s o f i n t e r e s t t o n o t e t h a t t h e p r o t e i n c o n t e n t s o f MF a n d LDLg f r o m t h e e g g s o f o l d e r b i r d s w e r e a b o u t t h e same b u t t h e p h o s p h o l i p i d c o n t e n t o f MF was s l i g h t l y h i g h e r t h a n t h a t f o r LDLg C29.5% v s . 2 5 . 8 % ) . T a b l e I I p r e s e n t s t h e amount o f l i p i d e x t r a c t f r o m MF a n d LDLg o b t a i n e d f r o m e g g s l a i d by b i r d s 39 and 41 weeks o l d . The MF f r a c t i o n s h a d mean l i p i d v a l u e s i n t h e v i c i n i t y o f 8 6% a n d t h e r e m a i n i n g p e r c e n t a g e , n a m e l y 1 4 % , c a n be c o n s i d e r e d t o be p r o t e i n . T h i s p r o t e i n v a l u e i s somewhat l o w e r t h a n t h e % p r o t e i n v a l u e o f 15.8 c a l c u l a t e d f r o m t h e % N o f MF f r o m e g g s l a i d by b i r d s o f t h e same a g e . The LDLg f r a c t i o n s c o n t a i n e d 83 t o 84% t o t a l . l i p i d . The LDLg p r o t e i n c o n t e n t o b t a i n e d by d i f f e r e n c e w o u l d be 15 t o 1 6 % w h i c h i s c l o s . e t o t h e p r o t e i n v a l u e o f 1 5 . 8 % o b t a i n e d f r o m t h e %N i n 95 . TABLE I I L I P I D COMPOSITION OF MF AND LDLg L Y O P H I L I Z E D SAMPLES S a m p l e P e r c e n t L i p i d 1 mean SD MF - 39 wk 8 6 . 0 1 a 0 . 36 MF - 141 wk 8 5 . 5 9 a 0 .67 LDLg - 39 wk 8 4 . 1 0 b 0.35 LDLg - 4 1 wk 83 . 06° 0.37 number o f o b s e r v a t i o n s = 8 a , b , c : means s h a r i n g t h e same s u p e r s c r i p t do n o t d i f f e r a t P < 0.05. T a b l e I . The l i p i d c o n t e n t o f t h e l o w - d e n s i t y l i p o p r o t e i n f r a c t i o n f r o m g r a n u l e s was r e p o r t e d by G o r n a l l and K u k s i s ( 1 9 7 1 a ) t o be 88% w h e r e a s T a t t r i e and Cook ( 1 9 6 9 ) r e p o r t e d v a l u e s b e t w e e n 84 and 85% u p o n f r a c t i o n a t i o n o f LDFG. G o r n a l l a n d K u k s i s ( 1 9 7 1 a ) o b t a i n e d l i p i d v a l u e s o f 85% f c r LDFG-1 a n d 9 2% f o r LDFG-2. The l i p i d c o n t e n t o f LDLg i s v e r y s i m i l a r t o t h a t f o r t h e l o w - d e n s i t y l i p o p r o t e i n f r a c t i o n s f r o m p l a s m a . The l i p i d c o n t e n t r e p o r t e d by G o r n a l l and K u k s i s ( 1 9 7 1 a ) , S t e e r e t a l . ( 1 9 6 8 ) and T a t t r i e a nd Cook ( 1 9 6 9 ) , f o r p l a s m a l o w -d e n s i t y l i p o p r o t e i n f e l l w i t h i n t h e r a n g e o f 8 6 - 9 0 % . S a a r i e t a l . ( 1 9 6 4 ) i s o l a t e d two u l t r a c e n t r i f u g a l l y - d i f f e r e n t l i p o p r o t e i n s f r o m t h e p l a s m a l o w - d e n s i t y f r a c t i o n , t h e LDL^ a n d t h e LDL^• T o t a l l i p i d was a b o u t 89% f o r LDL^ ( L D L ^ c o n -s i s t e d a b o u t one f i f t h o f t h e t o t a l LDL) and 86% f o r L D L 2 . M a r t i n e t a l . ( 1 9 6 4 ) f o u n d t h a t t h e i r LDL^ c o n s i s t e d o f a b o u t 87% l i p i d a n d L D L 2 a b o u t 83% l i p i d , w i t h m o l e c u l a r w e i g h t s o f 10.3 X 10° a n d 3.3 X 10 , r e s p e c t i v e l y . The n o n - l i p i d r e s i d u e s o f MF a n d LDLg w e r e a n a l y z e d f o r the; N- a n d P c o n t e n t s . The r e s u l t s a r e shown i n T a b l e I I I . The % n i t r o g e n i n t h e n o n - l i p i d r e s i d u e f o r MF and LDLg was f o u n d t o be 11.43 and 1 0 . 3 4 . The v a l u e o f LDLg r e s i d u e was l o w c o m p a r e d t o 1 4 . 8 % N and 1 3 . 7 % N f o r n o n -l i p i d r e s i d u e s o f LDL, a n d LDL„, r e s p e c t i v e l y , f r o m p l a s m a TABLE I I I COMPOSITION OF MF AND LDLg ISOLATED FROM YOLK OF 3 9 WK OLD HENS MF LDLg mean S D a mean SD T o t a l l i p i d , % 86 . 01 0 .36 84. 10 0. 35 N o n - l i p i d r e s i d u e , % 13 . 99 0 .36 15. 90 0 . 35 T o t a l p h o s p h o r u s , % 1. 24 0 .oo b 1. 05 0 . o i b L i p i d p h o s p h o r u s , % 1. 44 0 . 01 1. 23 0. 03 N o n - l i p i d r e s i d u e p h o s p h o r u s , % 0. 14 0 .06 b 0 . 15 0. o i b . T o t a l n i t r o g e n , % £ • 53 0 .04 b 2 . 50 0. 02 b N o n - l i p i d r e s i d u e n i t r o g e n , % 11. 4 3 0 .17 b 10. 34 0. 0 2 b C h o l e s t e r o l , % o f l i p i d 11. 53 0 .20 3 . 73 0 . 11 P h o s p h o l i p i d , % 29. 94 1 .15 25. 68 0 . 36 P h o s p h o l i p i d , % o f l i p i d 34. 81 . 1 .35 30. 58 0. 43 P h o s p h a t i d y l c h o l i n e , % o f p h o s p h o l i p i d 80 . 73 2 .10 80. 95 0. 11 P h o s p h a t i d y l e t h a n o l a m i n e , % o f p h o s p h o l i p i d 15. 97 1 .45 15. 69 o. 21 L y s o p h o s p h a t i d y l e t h a n o l a -m i n e f % o f p h o s p h o l i p i d 1. 29 0 .11 1. 69 0. 05 S p h i n g o m y e l i n , % o f p h o s p h o l i p i d 2. 01 0 .18 1. 67 0. 06 a number o f o b s e r v a t i o n s , n-=-4-10, s e e t e x t , b a v e r a g e d e v i a t i o n ( n = 2). 98.. ( S a a r i e t a l . , 1 9 6 4 ) . The l o w % N v a l u e s o f MF and LDLg may be a t t r i b u t e d t o t h e p r e s e n c e o f l a r g e a mounts o f m i n e r a l m a t t e r a n d c a r b o h y d r a t e s . S i n c e t h e MF and LDLg n o n - l i p i d r e s i d u e s had P c o n t e n t s o f 0.14 a n d 0.15%, i t i s a s sumed t h a t p h o s p h o p r o t e i n s w i t h e s t e r i f i e d p h o s p h a t e g r o u p s were, p r e s e n t i n t h e r e s i d u e . S a a r i . e t a l . ( 1 9 6 4 ) r e p o r t e d t h a t n o n - l i p i d r e s i d u e s o f LDL^ and L D L 2 had % P v a l u e s o f 0.15 and 0.16, r e s p e c t i v e l y . S a a r i e t a l . ( 1 9 6 4 ) . was f i r s t t o show c o n c l u s i v e l y t h a t c h o l e s t e r o l was t h e m a j o r egg y o l k s t e r o i d . Tu e t a l . ( 1 9 7 0 ) c o n f i r m e d t h i s s t a t e m e n t and a l s o showed t h a t egg y o l k c o n t a i n e d many o t h e r s t e r o i d s . i n m i n o r a m o u n t s . A s t e r o i d z o n e was i s o l a t e d by t h i n l a y e r c h r o m a t o g r a p h y ( T L C ) o f b o t h MF a n d LDLg l i p i d e x t r a c t s . T h i s s t e r o i d p o s s e s s e d t h e same R f v a l u e a s p u r e c h o l e s t e r o l . C h r o m a t o g r a m s o f r e f e r e n c e s t a n d a r d s , MF and LDLg l i p i d e x t r a c t s a r e shown i n F i g u r e s 1, 2 and 3, r e s p e c t i v e l y . I n a d d i t i o n , upon s p r a y i n g t h e c h r o m a t o g r a m w i t h 1 5 % p h o s p h o r i c a c i d , a p i n k i s h r e d s p o t d e v e l o p e d , t y p i c a l o f c h o l e s t e r o l a n d i t s e s t e r s . M o r e o v e r , s p e c t r o p h o t o m e t r i c c o m p a r i s o n ( F i g u r e 4) o f t h e i s o l a t e d s t e r o i d a n d p u r e c h o l e s t e r o l , b o t h p r e p a r e d by t h e m e t h o d o f Zac (19 5 7 ) , i n d i c a t e d t h a t t h e s p e c t r a w e r e a l m o s t i d e n t i c a l . I t was t h e r e f o r e c o n c l u d e d t h a t t h e i s o l a t e d s t e r o i d z o n e f r o m b o t h MF and LDLg l i p i d e x t r a c t s was p r i n c i p a l l y c h o l e s t e r o l . 99 . -* ^ P C I * FIGURE 1. R e f e r e n c e p h o s p h o l i p i d s : T = t r i o l e i n ( R f , 0.79); C = c h o l e s t e r o l ( R f , 0.61") ; PE = p h o s p h a t i d y l e t h a n o l a m i n e ( R f , 0.34); PC = p h o s p h a t i d y l c h o l i n e ( R f , 0.24); LPE = l y s o p h o s p h a t i d y 1 e t h a n o l a m i n e ( R f , 0.19); S - s p h i n g o m y e l i n ( R f , 0.14). V i s u a l i z e d by exposure t o i o d i n e . l o o . FIGURE; 2. T h i n l a y e r chromatography o f Mr l i p i d s : N = n e u t r a l l i p i d s ( R f , 0.78); C = c h o l e s t e r o l ( R f , 0.62); PE = p h o s p h a t i d y l e t h a n o l a m i n e ( R f , 0.37); PC - p h o s p h a t i d y l c h o l i n e ( R f , 0.24); LPE = l y s o p h o s p h a t i d y l e t h a n o l a m i n e ( R f , 0.19); S = s p h i n g o m y e l i n ( R f , 0.15). V i s u a l i z e d by exposure t o i o d i n e . 1 0 1 . FIGURE 3. T h i n l a y e r chromatography o f LDLg l i p i d s : N = n e u t r a l l i p i d ( P f , 0.76); C = c h o l e s t e r o l ( R f , 0.62); PE = p h o s p h a t i d y l e t h a n o l a m i n e ( R f , 0.40); FC = p h o s p h a t i d y l c h o l i n e ( R f , 0.24); LFZ = l y s o p h o s p h a t i d y 1 e t h a n o l a m i n e ( R f , 0.18); S = s p h i n g o m y e l i n ( R f , 0.14). V i s u a l i z e d by exposure t o i o d i n e . 450 500 550 600 650 700 750 800 850 W A V E L E N G T H , n m FIGURE 4. A b s o r b a n c e s p e c t r a o f c h o l e s t e r o l - f e r r i c c h l o r i d e c o m p l e x : A. T h i n l a y e r c h r o m a t o g r a p h y s t e r o i d (296 y g ) ; B. R e f e r e n c e c h o l e s t e r o l (218 y g ) . o 1 0 3 . A s shown, i n T a b l e I I I t h e c h o l e s t e r o l c o n t e n t s o f t h e two p a r t i c l e s w e r e m a r k e d l y d i f f e r e n t . The h i g h c h o l e s -t e r o l v a l u e f o r MF ( 1 1 . 5 3 % ) i s u n i q u e i n egg y o l k s i n c e •.other y o l k p a r t i c l e s h a v e v a l u e s i n t h e v i c i n i t y o f 3 t o 4%. I t ' i s i n t e r e s t i n g t o n o t e t h a t s o l u b l e l i p o p r o t e i n s ( c h y l o m i c r o n s , VLDL, LDL, HDL) o f human b l o o d p l a s m a h a v e c o m p o s i t i o n s s i m i l a r t o t h a t o f MF p a r t i c l e s a n d a r e p a r t i c u l a r l y r i c h i n c h o l e s t e r o l ( M a r g o l i s , 1 9 6 9 ; W h i t e e t a l . , 1 9 6 8 ) . Many a u t h o r s hcive d e t e r m i n e d t h e c h o l e s t e r o l c o n t e n t o f LDLp. S a a r i e t a l . ( 1 9 6 4 ) r e p o r t e d a b o u t 3.4% o f t h e t o t a l l i p i d a s c h o l e s t e r o l w h e r e a s M a r t i n e t a l . ( 1 9 6 3 ) r e p o r t e d a somewhat h i g h e r v a l u e o f 4 t o 4.3% o f t h e t c t a l l i p i d . The 3.7 3% v a l u e r e p o r t e d h e r e f o r LDLg c h o l e s t e r o l a p p e a r s a s a n a v e r a g e o f t h e a b o v e r e s u l t s and t h e two p a r t i c l e s w o u l d a p p e a r t o be s i m i l a r on t h i s b a s i s . G o r n a l l a n d K u k s i s (1971b) f o u n d t h a t t h e i r g r a n u l e l o w - d e n s i t y l i p o p r o t e i n (LDFG) c o n t a i n e d a b o u t 7% c h o l e s t e r o l on a l i p i d b a s i s : s i n c e t h e i r f r a c t i o n c o n s i s t e d o f MF and LDLg p a r t i c l e s t h i s v a l u e seems r e a s o n a b l e . I t h a s b e e n known f o r some t i m e t h a t egg y o l k c o n -t a i n s o n l y a few m a j o r p h o s p h o l i p i d s . R h o d e s a n d L e a (19 56) h a v e shown t h a t egg y o l k c o n s i s t s o f t h e f o l l o w i n g p h o s p h o -l i p i d s : p h o s p h a t i d y l c h o l i n e ( l e c i t h i n ) , 7 3 % ; p h o s p h a t i d y l 104 . e t h a n o l a m i n e , 1 5 % ; l y s o p h o s p h a t i d y l , c h o l i n e , 5.8%; s p h i n g o -m y e l i n , 2.5%; l y s o p h o s p h a t i d y l e t h a n o l a m i n e , 2.1%; p l a s m o g e n , 0.9% and p h o s p h a t i d y l i n o s i t o l , 0.6%. T y p i c a l c h r o m a t c g r a m s o f MF, LDLg and r e f e r e n c e l i p i d s a r e shown i n F i g u r e s 1, 2 and 3. C h r o m a t o g r a m s ( F i g u r e s 2 and 3) show t h e p r e s e n c e o f p h o s p h a t i d y l c h o l i n e ( P C ) , p h o s p h a t i d y l e t h a n o l a m i n e ( P E ) , l y s o p h o s p h a t i d y l e t h a n o l a m i n e ( L P E ) .and s p h i n g o m y e l i n ( S ) i n l i p i d f r a c t i o n s o f MF and LDLg. A c c o r d i n g t o G o r n a l l and K u k s i s ( 1 9 7 1 b ) , PC, PE, S a n d l y s o l e c i t h i n w e r e p r e s e n t i n LDFG. The l y s o l e c i t h i n may be c o n s i d e r e d a s a l y s o p h o s p h o l i p i d s i n c e t h e y d i d n o t i n d i c a t e any s t u d i e s on t h e i d e n t i f i c a t i o n o f t h i s c o m p o nent w h i c h was s e p a r a t e d by TLC. F u r t h e r , t h e s e r e s e a r c h e r s r e p o r t e d l y s o l e c i t h i n a s t h e m a j o r l y s o p h o s p h o l i p i d i n egg y o l k , a f i n d i n g w h i c h i s d i v e r g e n t f r o m t h e r e s u l t s o f Rhodes and L e a (19 5 6 ) . S a a r i e t a l . ( 1 9 6 4 ) u s i n g t h i n l a y e r c h r o m o -t o g r a p h y , f o u n d f o u r p h o s p h o l i p i d s i n t h e l i p i d e x t r a c t o f L P L ^ a n d L P L 2 f r o m p l a s m a . The two m a j o r p h o s p h o l i p i d s w e re i d e n t i f i e d a s PC and PE and t h e o t h e r t w o , p r e s e n t i n m i n o r a m o u n t s , w e r e p r e s u m e d t o be s p h i n g o m y e l i n a nd a l y s o p h o s p h o -l i p i d . The a m o u n t s o f t h e p h o s p h o l i p i d s o f MF and LDLg f r a c t i o n s a r e p r e s e n t e d i n T a b l e s I I I and I V . P h o s p h a t i d y l c h o l i n e (PC) was f o u n d t c be t h e most p r e v a l e n t p h o s p h o l i p i d , 105 . TABLE I V . PHOSPHOLIPID COMPOSITION OF MF AND LDLg L I P I D EXTRACTS P h o s p h o l i p i d P, P h o s p h o l i p i d , % P h o s p h o l i p i d . , . % e a % T o t a l L i p i d P o f T o t a l E x t r a c t e d o f T o t a l L i p i d e n mean SD mean SD mean SD MF-PC ( 5 ) 76 . 96 2 . 00 80 . 73 2 .10 28 .10 0 .73 MF-PE ( 5 ) 15 . 22 1. 39 15 . 97 1 .45 5 . 56 0 . 51 MF-LPE ' (4 ) 1. 96 0 . 35 1. 29 0 .11 0 .45 0 . 05 MF-S ( 4 ) 1. 9 6 0 . 17 2 . 01 0 . 18 0 .70 0 . 06 T o t a l 96 . 10 100 . 00 34 .81 M F - L i p i d P ( 2 . 3 0 yg ( 4 ) ;• P) 100 . 00 LDLg-PC ( 5 ) . 79 . 31 0 . 34 80 . 95 0 .35 24 .75 0 .11 L D Lg-PE ( 5 ) 15 . 38 0 . 69 15. 69 0 .69 4 .80 0 . 21 L D L g - L P E ( 4 ) 2. 62 0. 28 1. 69 0 .17 0 .52 0 .05 LDLg-S ( 4 ) 1. 67 0 . 21 1. 67 0 .19 0 . 51 0 .06 T o t a l 98 . 88 100 . 00 30 .58 L D L g - L i p i d P ( 4 ) 100.00 ( 1 . 4 4 " yg P) a 39 wk b i r d s : PC = p h o s p h a t i d y l c h o l i n e PE = p h o s p h a t i d y l e t h a n o l a m i n e LPE = l y s o p h o s p h a t i d y l e t h a n o l a m i n e S = s p h i n g o m y e l i n . b V a l u e s b a s e d on t h e f o l l o w i n g c o n v e r s i o n f a c t o r s : L and PE = 2 5 . 4 , LPE = 1 6 , S = 24.8. c T o t a l p h o s p h o l i p i d e x t r a c t e d : MF = 5 5 . 6 9 y g , L D L g = 3 6 . 0 8 y g d T o t a l l i p i d o f s a m p l e s p o t t e d : MF - 1 6 0 y g , L D L g = 1 1 8 y g e Number o f o b s e r v a t i o n s . 106 . c o m p r i s i n g a b o u t 8 1 % o f t h e t o t a l p h o s p h o l i p i d i n b o t h MF and LDLg. s t r u c t u r e s . A c c o r d i n g t o G o r n a l l and K u k s i s ( 1 9 7 1 b ) t h e p h o s p h a t i d y l c h o l i n e c o n t e n t o f LDLg was 69%. S a a r i e t a l . ( 1 9 6 4 ) f o u n d t h a t p h o s p h a t i d y l c h o l i n e made up 84% o f t h e p l a s m a LDL p h o s p h o l i p i d . T a t t r i e and Cook ( 1 9 6 9 ) r e p o r t e d somewhat l o w e r v a l u e s f o r p l a s m a LDL . p h o s p h a t i d y l c h o l i n e b e t w e e n 71 a n d 76%. P h o s p h a t i d y l e t h a n o l a m i n e ( P E ) , t h e n e x t m a j o r p h o s p h o l i p i d i s o l a t e d f r o m b o t h MF a n d LDLg s a m p l e s , c o m p r i s e d a b o u t 1 6 % o f t h e t o t a l p h o s p h o l i p i d o f e a c h p a r t i c l e . G o r n a l l and K u k s i s ( 1 9 7 1 b ) r e p o r t e d t h a t t h e PE o f LDFG was a b o u t 19 % . The PE c o n t e n t s i n t h e l o w - d e n s i t y l i p o p r o t e i n s i s o l a t e d f r o m p l a s m a b y v a r i o u s i n v e s t i g a t o r s r a n g e b e t w e e n 14 and 23%. S a a r i e t a l . ( 1 9 6 4 ) r e p o r t e d a PE v a l u e o f 1 4 % , T a t t r i e a nd Cook ( 1 9 6 9 ) a somewhat h i g h e r r a n g e o f 16 t o 20% and G o r n a l l and K u k s i s ( 1 9 7 1 b ) t h e h i g h e s t r e p o r t e d v a l u e o f 2 3 % . L y s o p h o s p h a t i d y l e t h a n o l a m i n e ( L P E ) a n d s p h i n g o -m y e l i n ( S ) w e r e i s o l a t e d as m i n o r p h o s p h o l i p i d s i n MF and L DLg. The LPE c o n t e n t ( 1 . 2 9 % ) i n t h e MF p h o s p h o l i p i d f r a c t i o n was somewhat l o w e r t h a n t h e 2.01% S c o n t e n t . W i t h r e g a r d t o t h e LDLg p h o s p h o l i p i d , t h e S and LPE c o n t e n t s w e r e a b o u t t h e same, a p p r o x i m a t e l y 1.7%. The d a t a o f G o r n a l l and K u k s i s ( 1 9 7 1 b ) i n d i c a t e d t h a t LDFG c o n t a i n e d 6% S and 6% l y s o l e c i t h i n . P h o s p h o l i p i d s t e r m e d " s p o t A" a n d " s p o t B" ( p r e s u m a b l y S and 107 . a l y s o p h o s p h o l i p i d ) by S a a r i e t a l . ( 1 9 6 4 ) w ere f o u n d t o be b e t w e e n 1.4 and 1.8% and 1.5 and 1.9%, r e s p e c t i v e l y , o f t h e t o t a l l i p i d _ p h o s p h o r u s . V a l u e s o f 1.69% f o r LPE a n d 1.67% f o r S f o r LDL l i p i d ( T a b l e I I I ) a r e a l m o s t i d e n t i c a l i n v a l u e and r a t i o w i t h t h e s e v a l u e s r e p o r t e d by S a a r i e t a l . ( 1 9 6 4 ) . C hang ( 1 9 6 9 ) p r o p o s e d t h a t m y e l i n f i g u r e s w e re b a s i c a l l y p h o s p h o l i p i d i n n a t u r e . The r e s u l t s o f t h i s s t u d y show t h a t , a l t h o u g h p h o s p h o l i p i d s a r e a m a j o r c o m p o n e n t , t h e y a c c o u n t f o r o n l y a b o u t 30% o f t h e t o t a l MF c o m p o s i t i o n . B a i n a n d H a l l (197 0) b e l i e v e d t h a t t h e MF p a r t i c l e s w e r e a r t i f a c t s o f p h o s p h o l i p i d h y d r a t i o n , b u t s u c h a s u g g e s t i o n d o e s n o t a p p e a r t o be l i k e l y , s i n c e t h e MF c o m p o s i t i o n i s much more c o m p l e x t h a n o r i g i n a l l y ' e n v i s i o n e d . P e r h a p s t h e t e r m " m y e l i n f i g u r e " w h i c h was o r i g i n a l l y c o i n e d t o c l a s s i f y p h o s p h o l i p i d p a r t i c l e s w i t h c o n c e n t r i c b i m o l e c u l a r l e a f l e t f i g u r e s i s a m i s n o m e r . However t h e t e r m m y e l i n h a s b e e n u s e d t o d e n o t e in _ v i v o m u l t i l a y e r e d s t r u c t u r e s , s u c h as b r a i n and n e r v o u s t i s s u e m y e l i n . C o n c e i v a b l y MF p a r t i c l e s c o u l d be c l a s s e d a s l i p o p r o t e i n s s i n c e t h e s e p a r t i c l e s a r e made up o f t r i g l y c e r i d e s , p h o s p h o l i p i d s , c h o l e s t e r o l and p r o t e i n w h i c h a r e i n d e e d t h e c o n s t i t u e n t s o f l o w - d e n s i t y l i p o p r o t e i n s . M o r e o v e r , t h e e l e c t r o n m i c r o g r a p h s shown' i n C h a p t e r I show p h o s p h o l i p i d m o e i t i e s , s u r r o u n d i n g a l o w - e l e c t r o n d e n s e m a t e r i a l , p r e s u m a b l y t r i g l y c e r i d e . 108 . E v e n t h o u g h t h e c o m p o s i t i o n o f t h e LDLg was f o u n d t o be somewhat d i f f e r e n t f r o m t h a t o f L DLp, t h e s e d i f f e r e n c e s a r e n o t o f t h e o r d e r w h i c h w o u l d b r i n g a n y d o u b t t o t h e a c c e p t a b i l i t y o f t h e p r o p o s e d s t r u c t u r e o f egg y o l k l o w -d e n s i t y l i p o p r o t e i n c o m p l e x e s ( C h a p t e r 1 ) . S t r u c t u r a l l y t h e s e l o w - d e n s i t y l i p o p r o t e i n s a r e s i m i l a r (Chang., 1 9 6 9 ) and c o m p o s i t i o n a l l y t h e y h a v e s i m i l a r c o n s t i t u e n t s , a l t h o u g h t h e amounts o f some c o m p o n e n t s do v a r y . D a t a r e p o r t e d f o r LDLp c o m p o s i t i o n v a r i e s c o n s i d e r a b l y . P e r h a p s t h i s i s t h e r e s u l t o f t h e i s o l a t i o n m e t h o d o r e n v i r o n m e n t a l and b i r d v a r i a t i o n . i a a , CONCLUSIONS M y e l i n f i g u r e s a r e l a r g e ( a p p r o x i m a t e l y 1000 A * , g > 40 X 10 m o l e c u l a r w e i g h t -- S e p h a r o s e 2B e x c l u s i o n l i m i t ) p a r t i c u l a t e l i p i d - p r o t e i n c o m p l e x e s composed o f s u b s t a n c e s s p e c i f i c t o l i p o p r o t e i n s y s t e m s . G r a n u l e l o w - d e n s i t y l i p o p r o t e i n s a r e composed o f c o m p o n e n t s i d e n t i c a l t o t h o s e o f m y e l i n f i g u r e s , a l t h o u g h , o n a t o t a l w e i g h t b a s i s , t h e c o n c e n t r a t i o n o f e a c h c o n s t i t u e n t d i f f e r s . The g r o s s c o m p o s i t i o n o f g r a n u l e l o w - d e n s i t y l i p o -p r o t e i n s i s s i m i l a r t o t h a t o f p l a s m a l o w - d e n s i t y l i p o p r o t e i n s a l t h o u g h t h e amounts o f e a c h c o n s t i t u e n t o f t h e LDLg v a r y w i t h r e s p e c t t o t h e amounts r e p o r t e d f o r LDLp. R e p o r t e d v a l u e s f o r LDLp a l s o v a r y c o n s i d e r a b l y . A l t h o u g h p h o s p h a t i d y l i n o s i t a l a n d l y s o p h o s p h a t i d y l c h o l i n e h a v e b e e n r e p o r t e d a s c o m p o n e n t s o f egg y o l k l i p i d s , n e i t h e r o f t h e s e l i p i d c o n s t i t u e n t s w e r e f o u n d i n MF and LDLg p a r t i c l e s . 110. LITERATURE CITED B a i n , J.M. a n d J.M. H a l l . 1970. The e f f e c t o f d i e t a r y c y c l o -p r o p e n e f a t t y a c i d s on t h e s t r u c t u r e o f n e w - l a i d a n d s t o r e d h e n s ' e g g s : an o p t i c a l and e l e c t r o n m i c r o s c o p e s t u d y . A u s t . J . B i o l . S c i . 2_3_, 657. B u r l e y , K.VJ. and W.H. Cook. 1 9 6 1 . I s o l a t i o n and c o m p o s i t i o n o f a v i a n egg y o l k g r a n u l e s and t h e i r c o n s t i t u e n t s a - and g - l i p o v i t e l l i n s . Can. J . B i o c h e m . P h y s i o l . , 1 2 9 5 . C h a n g , C M . 1969. S t u d i e s on egg y o l k . Ph.D. T h e s i s . U n i v . o f W i s . , M a d i s o n , W i s . C o o k , -W.H. 1968. M a c r o m o l e c u l a r c o m p o n e n t s o f egg y o l k . I_n Egg Q u a l i t y , A S t u d y o f t h e Hen's E g g . E d . T.C. C a r t e r , O l i v e r and B o y d , P u b . , E d i n b u r g h , S c o t l a n d . G o r n a l l , D.A. a n d A. K u k s i s . 1 9 7 1 a . R e s o l u t i o n o f egg y o l k l i p o p r o t e i n s by c h r o m a t o g r a p h y on t h i n l a y e r s o f h y d r o x y l a p a t i t e . Can. J'. B i o c h e m . 4_9, 44. G o r n a l l , D.A. and A. K u k s i s . 1 9 7 1 b . M o l e c u l a r s p e c i e s o f g l y c e r o p h o s p h a t i d e s and t r i g l y c e r i d e s o f egg y o l k l i p o p r o t e i n s . C a n . J . B i o c h e m . 4 9 , 51. J o h n s t o n , P.V. 1 9 7 1 . B a s i c l i p i d m e t h o d o l o g y . S p e c i a l Pub. 1 9 , U n i v . o f 1 1 1 . , P u b . , U r b a n a - C h a m p a i g n . M a r g o l i s , S. 1969. S t r u c t u r e o f v e r y l o w and l o w - d e n s i t y l i p o p r o t e i n s . In_ S t r u c t u r a l and F u n c t i o n a l A s p e c t s o f L i p o p r o t e i n s i n L i v i n g S y s t e m s . E d . , E. T r i a a n d A.M. S c a n u . P u b . , A c a d e m i c P r e s s , New Y o r k , N.Y. M a r t i n , W.G., J . A u g u s t y n i a k and W.H. Cook. 1964. F r a c t i o n a -t i o n and c h a r a c t e r i z a t i o n o f t h e l o w - d e n s i t y l i p o -p r o t e i n s o f h e n ' s egg y o l k . B i o c h i m . B i o p h y s . A c t a . 8 4 , 714. M a r t i n , W.G., H.N. T a t t r i e and W.H. Cook. 1 9 6 3 . L i p i d e x t r a c t i o n and d i s t r i b u t i o n s t u d i e s o f egg y o l k l i p o p r o t e i n s . Can. J . B i o c h e m . P h y s i o l . 4_1 , 657. R h o d e s , D.N. and C H . L e a . 1 9 5 6 . P h o s p h o l i p i d s . 4. On t h e c o m p o s i t i o n o f h e n ' s egg p h o s p h o l i p i d s . B i o c h e m J . 6 5 ^ 526. S a a r i , A-., W.D. P o w r i e and 0. Fennema. 1964 . I s o l a t i o n and c h a r a c t e r i z a t i o n o f ' l o w - d e n s i t y l i p o p r o t e i n s i n n a t i v e egg y o l k p l a s m a . J . F o o d S c i . 2 9 , 307. 111. S a i t o , Z., W.G. M a r t i n a n d W.H. Cook. 1 9 6 5 . C h a nges i n t h e m a j o r m a c r o m o l e c u l a r f r a c t i o n s o f egg y o l k d u r i n g e m b r y o g e n e s i s . Can. J . B i o c h e m . 4_3_, 17 55 . S t e e r , D.C., W.G. M a r t i n and W.H. Cook. 1968. S t r u c t u r a l i n v e s t i g a t i o n o f t h e l o w - d e n s i t y l i p o p r o t e i n o f h e n ' s e g g y o l k u s i n g p r o t e o l y s i s . B i o c h e m . 7_, 3309 . T a t t r i e , N.H. and W.H. Cook. 19 69. L i p o p r o t e i n s and l i p i d s o f egg y o l k . S . C . I . M o n o g r a p h No. 32. p. 9 2 - 1 0 7 . T u , C , W.D. P o w r i e a n d 0. Fennema. 1970 . S t e r o i d s i n egg y o l k . J . Food S c i . 3_5, 6 01. W a l d i , D. 19 6 5a. S t e r o i d s . I n T h i n L a y e r C h r o m a t o g r a p h y , A L a b o r a t o r y Handbook. E d . , E. S t a h l , P u b . , A c a d e m i c P r e s s , New Y o r k , N.Y. W a l d i , D. 1 9 6 5 b . S p r a y r e a g e n t s f o r t h i n l a y e r c h r o m a t o g r a p h y . I n T h i n L a y e r C h r o m a t o g r a p h y , A L a b o r a t o r y Handbook. E d . , E. S t a h l , P u b . , A c a d e m i c P r e s s , New Y o r k , N.Y. W h i t e , A., P. H a n d l e r a n d E.L. S m i t h . 1968. P r i n c i p l e s o f B i o c h e m i s t r y , 4 t h ed.. Pub. , M c G r a w - H i l l Book Co., New Y o r k , N.Y. Z a k , B., D.A. L u z and M. F i s h e r . 1 9 5 7 . D e t e r m i n a t i o n o f s e r u m c h o l e s t e r o l . Am. J . Med. T e c h n o l . 23, 283. 1 1 2 . CHAPTER 3 FUNCTIONAL PROPERTIES OF EGG YOLK MYELIN FIGURES AND GRANULE LOW DENSITY LIPOPROTEINS ABSTRACT Oil-.in_.water e m u l s i o n s , p r e p a r e d w i t h n a t i v e and l y o p h i l i z e d MF, LDLg and y o l k as t h e e m u l s i f i e r s . were examined by e l e c t r o n m i c r o s c o p y i n o r d e r t o a s s e s s the n a t u r e o f t h e s u r f a c e s t r u c t u r e o f e m u l s i f i e d o i l d r o p l e t s . Y o l k p a r t i c l e s were d i s r u p t e d d u r i n g t h e e m u l s i f i c a -t i o n p r o c e s s and a number o f d i s t i n c t zones c o n t a i n i n g d i f f e r e n t components were formed about t h e o i l d r o p l e t ' s s u r f a c e . P r e -sumably a l l t h e s e zones c o n t r i b u t e d t o t h e s t a b i l i t y o f t h e e m u l s i o n . S u r f a c e t e n s i o n s t u d i e s showed t h a t aqueous MF and LDLg p r e p a r a t i o n s were e f f e c t i v e s u r f a c t a n t s , l o w e r i n g s u r f a c e t e n s i o n t o the same degree r e p o r t e d f o r y o l k s o l u t i o n s . E m u l s i o n s p r e p a r e d w i t h n a t i v e MF and y o l k were not as s t a b l e as t h o s e p r e p a r e d w i t h n a t i v e LDLg. However, l y o p h i l i z a t i o n improved t h e s t a b i l i t y o f t h e MF and y o l k e m u l s i o n s . 1 1 3 . INTRODUCTION Egg y o l k i s a u n i q u e m u l t i f u n c t i o n a l e m u l s i f i e r 1 e s s e n t i a l t o t h e f o r m a t i o n , s t a b i l i z a t i o n a n d n u t r i t i v e v a l u e o f a l a r g e number o f f o o d p r o d u c t s . No s i n g l e i n g r e d i e n t o r c o m b i n a t i o n o f i n g r e d i e n t s h a s b e e n f o u n d t o be a s f u n c t i o n a l l y e f f i c i e n t a s e g g y o l k . U n f o r t u n a t e l y , t h e m e c h a n i s m by w h i c h egg y o l k c o m p o n e n t s a c t and i n t e r a c t w i t h i n a f o o d s y s t e m i s n o t w e l l u n d e r s t o o d . N o r , a l t h o u g h a g r e a t d e a l i s known ( r e v i e w e d i n C h a p t e r I ) , i s t h e r e a c l e a r u n d e r s t a n d i n g o f egg y o l k c o m p o s i t i o n and m i c r o s t r u c t u r e . The r e l a t i o n s h i p o f y o l k ' s c o m p o n e n t s t o i t s f u n c t i o n a l b e h a v i o r must be c l a r i f i e d i n o r d e r t h a t c r i t e r i a may be e s t a b l i s h e d t o o p t i m i z e i t s p e r f o r m a n c e . S i n c e c o m p o s i t i o n a n d s t r u c t u r a l d i f f e r e n c e s a r e p o s s i b l e b e c a u s e o f g e n e t i c e f f e c t s ( b r e e d a n d s t r a i n ) , b i r d f e e d i n g a n d management p r a c t i c e s a n d egg p r o c e s s i n g o r p r e s e r v a t i v e m e t h o d s , a n u n d e r s t a n d i n g o f y o l k ' s c o m p o n e n t s and t h e i r c o n t r i b u t i o n t o f u n c t i o n a l b e h a v i o r c o u l d be o f v a l u e . F a c t o r s a f f e c t i n g - f u n c t i o n a l p e r f o r m a n c e a r e w e l l r e v i e w e d b y B a l d w i n (197 3) a n d C u n n i n g h a m and V a r a d a r a j u l a ( 1 9 7 3 ) . T h i s b a s i c l a c k o f u n d e r s t a n d i n g r e g a r d i n g y o l k c o m p o n e n t s and t h e i r c o n t r i b u t i o n t o f u n c t i o n a l p e r f o r m a n c e s t e m s f r o m t h e f a c t t h a t e g g y o l k i s a v e r y c o m p l e x m a t e r i a l . The r e v i e w by P o w r i e ( 1 9 7 3 ) c l e a r l y i n d i c a t e d t h e c o m p l e x i t y o f egg y o l k c h e m i s t r y . Many w o r k e r s h a v e p r e s e n t e d d a t a on t h e e m u l s i f y i n g p r o p e r t i e s o f egg y o l k , b u t o n l y r e c e n t l y h a s a m a j o r b r e a k -t h r o u g h b e e n made. T h r o u g h e l e c t r o n m i c r o s c o p y o f mayonnaise., Chang e t a l . (197 2 ) h6:ve shown t h a t y o l k a c t u a l l y f o r m s f o u r z o n e s o f s t a b i l i z a t i o n a b o u t an o i l d r o p l e t , n o t a s i n g l e z o n e as had been, p r e v i o u s l y t h o u g h t . T h i s was t h e f i r s t t i m e i n f o r m a t i o n d i r e c t l y c o r r e l a t i n g y o l k s t r u c t u r e and c o m p o s i t i o n w i t h f u n c t i o n a l b e h a v i o r h a d b e e n r e p o r t e d . T h i s s t u d y a t t e m p t s t o f u r t h e r e l u c i d a t e y o l k ' s e m u l s i f y i n g p o w e r t h r o u g h an e l e c t r o n m i c r o s c o p e s t u d y o f t h e e m u l s i f y i n g c a p a c i t y o f two o f y o l k ' s m i c r o s t r u . c t u r a l c o m p o n e n t s , m y e l i n f i g u r e s (MF) and g r a n u l e l o w - d e n s i t y l i p o p r o t e i n s ( L D L g ) . T h e s e p a r t i c l e s w e r e c h o s e n b e c a u s e b o t h a r e composed o f s u b s t a n c e s ( p h o s p h o l i p i d s , s t e r o i d s , p r o t e i n s ) known t c be e i t h e r e m u l s i f y i n g o r p s e u d o e m u l s i f y i n g a g e n t s and b o t h s p e c i e s w e r e f o u n d t c be p a r t i c u l a r l y r i c h i n s u r f a c t a n t p h o s p h o l i p i d s ( C h a p t e r I I ) . ' V i n c e n t e t a l . ( 1966 ) s t a t e d t h a t t h e s u r f a c e a c t i v e a g e n t s o f egg y o l k w e r e e s s e n t i a l t c i n t e r f a c i a l t e n s i o n r e d u c t i o n , a p r i m a r y s t e p t o w a r d e m u l s i o n f o r m a t i o n . The m y e l i n f i g u r e was a l s o c h o s e n a s i t i s a n e s s e n t i a l l y unknown egg y o l k p a r t i c l e . The g r a n u l e l o w - d e n s i t y l i p o p r o t e i n was c h o s e n on a s i m i l a r b a s i s a n d a l s o b e c a u s e o f i t s r e s e m b l a n c e 115. t o t h e p l a s m a l o w - d e n s i t y l i p o p r o t e i n ( C h a p t e r I I ) . P l a s m a l i p o p r o t e i n s a r e m a j o r c o m p o n e n t s o f t h e y o l k a nd a r e t h o u g h t t o be i n t i m a t e l y i n v o l v e d i n t h e e m u l s i f i c a t i o n p r o c e s s ( C h a n g e t a l . , 1 9 7 2 ) . C o n c o m i t a n t w i t h e l e c t r o n m i c r o s c o p y o f n a t u r a l and l y o p h i l i z e d MF and LDLg p a r t i c l e s , s t u d i e s o f e m u l s i o n s t a b i l i t y a n d s u r f a c e t e n s i o n w e r e c o n d u c t e d . 116 . EXPERIMENTAL A. M a t e r i a l s A l l e g g s were o b t a i n e d f r o m W h i t e L e g h o r n h e n s a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a P o u l t r y R e s e a r c h S t a t i o n . A t l e a s t 6 d o z e g g s w e r e p o o l e d e a c h t i m e t o p r o v i d e r e p r e s e n t a t i v e s a m p l i n g . Y o l k s w e r e o b t a i n e d f r o m e g g s s t o r e d f o r n o t more t h a n 24 h r a f t e r l a y i n g . S e a m l e s s c e l l u l o s e d i a l y s i s t u b i n g was o b t a i n e d f r o m F i s h e r S c i e n t i f i c Co. L t d . The membrane, o f a n a v e r a g e p o r e . s i z e . o f 48 8, was p e r m e a b l e t o l o w m o l e c u l a r w e i g h t compounds (M.W. < 1 2 , 0 0 0 ) . R e f i n e d s o y b e a n o i l was p u r c h a s e d f r o m a l o c a l s u p e r -m a r k e t . A l l c h e m i c a l s a n d r e a g e n t s f o r e l e c t r o n m i c r o s c o p y w e re a n a l y t i c a l g r a d e . B. M e t h o d s 1. MF a n d LDLg p r e p a r a t i o n MF and LDLg were i s o l a t e d f r o m y o l k g r a n u l e s by m e t h o d s p r e v i o u s l y d e s c r i b e d ( C h a p t e r I ) . A f t e r s e p a r a t i o n by S e p h a r o s e 2B g e l c h r o m a t o g r a p h y , MF a n d LDLg i s o l a t e s were d i a l y z e d a g a i n s t f i v e c h a n g e s o f 5 , 0 1 o f d i s t i l l e d w a t e r a t 5°C f o r a t o t a l o f 36 h r . S a m p l e s o f e a c h s p e c i e s w e re e i t h e r c o n c e n t r a t e d o r l y o p h i l i z e d . D e s a l t e d s a m p l e s w ere c o n c e n t r a t e d by b l o w i n g a i r a c r o s s h a n g i n g d i a l y s i s t u b e s o f s a m p l e . 117 . Moisture content of the concentrate was determined by A.O.A.C -- 13.002 method and water was added i f necessary to prepare a 10% dispersion. Water was a l s o added to pure yolk to ob t a i n a.10% s o l i d s dispersion. Desalted samples and egg yol k were a l s o f r o z e n at -20°F and l y o p h i l i z e d i n a V i r t i s , model 10-145-MRBA f r e e z e dryer. The moisture content of the d r i e d samples was considered n e g l i g i b l e ; moisture d i d not exceed 0.087% and the average was about 0.04%. A l l samples were stored i n sealed brown j a r s , under n i t r o g e n , at 5°C. 2. Emulsion p r e p a r a t i o n A S o r v a l l micro-homogenizer, 5 ml c a p a c i t y , was used t c prepare emulsions w i t h e i t h e r n a t i v e y o l k , l y o p h i l i z e d y o l k , F I I , MF or LDLg as the e m u l s i f i e r . The p r e p a r a t i o n of small emulsions was n e c e s s i t a t e d by the co n s i d e r a b l e time i n v o l v e d i n p r e p a r i n g l a r g e q u a n t i t i e s of MF and LDLg f r a c t i o n s and because storage time f o r these f r a c t i o n s was kept to a minimum to reduce the chance of any degradative e f f e c t s r e s u l t i n g from a u t o - o x i d a t i o n or m i c r o b i a l contamination. Each emulsion was prepared by adding o i l , a few drops at a time, to a 10% aqueous s o l u t i o n of each e m u l s i f i e r and mixing at a constant S o r v a l l micro-homogenizer speed of 7. The f i n i s h e d emulsion was composed of 60% o i l and 40% e m u l s i f i e r s o l u t i o n (10% s o l i d s ) . 3. Emulsion s t a b i l i t y Emulsion s t a b i l i t y was determined by heating each 118 . e m u l s i o n i n c e l l u l o s e n i t r a t e c e n t r i f u g e t u b e s a t 35°C f o r 1 h r and s u b s e q u e n t l y c e n t r i f u g i n g a t a b o u t 2 0 , 0 0 0 X G f o r 30 m i n . The e m u l s i o n s w e r e e v a l u a t e d i n t e r m s o f v o l u m e s o f t h e o i l and w a t e r p h a s e s . The f r e e o i l was r e m o v e d by P a s t e u r p i p e t t e and t h e o i l w e i g h t l o s s was d e t e r m i n e d by w e i g h t d i f f e r e n c e . A l l e m u l s i o n s w e re s t o r e d a t 5°C f o r 4 d a y s i n s e a l e d c e n t r i f u g e t u b e s p r i o r t o s t a b i l i t y d e t e r m i n a t i o n . 4. S u r f a c e t e n s i o n S u r f a c e t e n s i o n m e a s u r e m e n t s were made w i t h a F i s h e r S e m i - a u t o m a t i c S u r f a c e T e n s i o m a t ( M o d e l 21) e q u i p p e d w i t h a p l a t i n u m - i r i d i u m r i n g o f 6 . 0 0 cm mean c i r c u m f e r e n c e . The r a t i o o f t h e r a d i u s o f t h e r i n g t o t h e r a d i u s o f t h e w i r e was '53 . 2 . The T e n s i o m a t was i n i t i a l l y c a l i b r a t e d w i t h a 599.998 g w e i g h t t o m e a s u r e a p p a r e n t s u r f a c e t e n s i o n i n d y n e s / c m . Con-v e r s i o n t o a b s o l u t e u n i t s was a c c o m p l i s h e d by d e t e r m i n i n g a c o r r e c t i o n f a c t o r by r e f e r e n c e t o a c o r r e c t i o n f a c t o r c h a r t i s s u e d by F i s h e r S c i e n t i f i c Co, The c h a r t i s b a s e d on t h e r e l a t i o n s h i p b e t w e e n a p p a r e n t and a b s o l u t e u n i t s a s i n d i c a t e d by t h e f o l l o w i n g f o r m u l a : F = O . 7 2 5 0 V ; 0 1 1 1 5 2 - ! + 0.04534 - 1 - 6 7 9 " VC (D-d) R w h e r e : F = t h e c o r r e c t i o n f a c t o r P = t h e a p p a r e n t s u r f a c e t e n s i o n , d y n e s / c m 119 . C =' t h e c i r c u m f e r e n c e o f t h e r i n g , cm 3 D = t h e d e n s i t y o f t h e l o w e r p h a s e ( H ^ O ) , g/cm 3 d = t h e d e n s i t y o f t h e u p p e r p h a s e ( a i r ) , g/cm r = t h e r a d i u s o f t h e w i r e o f t h e r i n g , cm R = t h e r a d i u s o f t h e r i n g , cm. The d i l u t e e m u l s i f i e r s o l u t i o n s w e r e s i m i l a r i n d e n s i t y t o t h e d e n s i t y o f w a t e r w h i c h was t a k e n a t 1 g / c c . M e a s u r e m e n t s w e r e made o n a l l s a m p l e s m a i n t a i n e d a t 25°C i n a w a t e r b a t h p r i o r t o s u r f a c e t e n s i o n e v a l u a t i o n . The r i n g was i m m e r s e d i n t h e e x p e r i m e n t a l s o l u t i o n f o r a f e w m i n u t e s b e f o r e s u r f a c e t e n s i o n d e t e r m i n a t i o n . The T e n s i o m a t was o p e r a t e d i n a c l o s e d room f r e e f r o m d r a f t s . C a r e f u l c l e a n i n g p r o c e d u r e s w e r e e m p l o y e d p r i o r t o s u r f a c e t e n s i o n d e t e r m i n a t i o n s . The p l a t i n u m - i r i d i u m r i n g was s u c c e s s i v e l y w a s h e d i n b e n z e n e , m e t h y l e t h y l k e t o n e , w a t e r a n d d i s t i l l e d w a t e r a f t e r w h i c h i t was f l a m e d w i t h a Bunsen b u r n e r . The s a m p l e c o n t a i n e r , a c e r a m i c d i s h 6 8 mm i n d i a m e t e r . a n d 13 mm h i g h , was l e f t i n p o t a s s i u m d i c h r o m a t e s o l u t i o n o v e r n i g h t a f t e r w h i c h i t was washed i n a s i m i l a r manner t o t h e r i n g . B e t w e e n s a m p l e s , b o t h r i n g a nd d i s h w e r e washed i n s u c c e s s i v e d i l u t i o n s o f 1 0 % s o d i u m c h l o r i d e , w a t e r and d i s t i l l e d w a t e r . The d i s h was p l a c e d o n p a p e r t o w e l l i n g t o d r y and t h e r i n g was' f l a m e d . The T e n s i o m a t was s t a n d a r d i z e d a g a i n s t d i s t i l l e d w a t e r w i t h a known s u r f a c e t e n s i o n o f 71.97 d y n e s / c m a t 25°C. 120 . The; v a l u e s o b t a i n e d f o r d i s t i l l e d w a t e r w ere a l w a y s f r o m 0.3 t o 0.8 dyne/cm l o w e r i n a b s o l u t e v a l u e . The s u r f a c e t e n s i o n o f d i s t i l l e d w a t e r was d e t e r m i n e d b e f o r e . a n d a f t e r a r u n o f 7 t o 8 s a m p l e s . The d i f f e r e n c e i n a b s o l u t e s u r f a c e t e n s i o n b e t w e e n t h e known and o b s e r v e d v a l u e s f o r 25°C d i s t i l l e d w a t e r was c a l c u l a t e d and a d d e d t o t h e c a l c u l a t e d a b s o l u t e v a l u e o b t a i n e d f o r t h e s a m p l e s . . Ten s u r f a c e t e n s i o n v a l u e s w e r e a v e r a g e d f o r e a c h o f t h e d i l u t e d ( 0 . 0 0 5 t o 1.0%) MF, LDLg and F I I s a m p l e s . D i f f e r e n t s a m p l e s w e r e p r e p a r e d and t e s t e d . The maximum a v e r a g e d e v i a t i o n o b s e r v e d b e t w e e n d u p l i c a t e r u n s was-1.42 d y n e s / c m ; v a l u e s w e re g e n e r a l l y much l o w e r t h a n t h i s . L y o p h i l i z e d m a t e r i a l was h y d r a t e d a nd m i x e d w i t h a F i s h e r m i r i - s h a k e r ' p r i o r - t c s u r f a c e t e n s i o n d e t e r m i n a t i o n . 5. E l e c t r o n m i c r o s c o p y An A E I C o r i n t h 27 5 e l e c t r o n m i c r o s c o p e a t 6 0 kV was u s e d f o r e l e c t r o n m i c r o s c o p y o f l y o p h i l i z e d MF and LDLg s p e c i -mens a n d e m u l s i o n s p r e p a r e d w i t h y o l k , MF and LDLg p r e p a r a t i o n s . S a m p l e s ( a l l ' e m u l s i o n s and a h y d r a t e d l y o p h i l i z e d MF p r e p a r a -t i o n ) w e r e i n i t i a l l y embedded, p r i o r t o f i x a t i o n , w i t h 5 p a r t s o f a warm ( a b o u t 4 0°C) 2.3% a g a r - 1.0% g l y c e r o l s o l u t i o n . The c o o l e d , g e l l e d p l u g was p l a c e d i n a p e t r i p l a t e c o n t a i n i n g a 2.5% g l u t e r a l d e h y d e - p h o s p h a t e b u f f e r (pH 7.2) s o l u t i o n a n d 3 s m a l l c u b e s , 1 mm o r l e s s , w e r e c u t away. The c u b e s w e r e t r a n s f e r r e d t o s m a l l 3 dram g l a s s v i a l s c o n t a i n i n g f r e s h 1 2 1 . f i x a t i v e t o a h e i g h t o f a b o u t 3 mm.. A l l f u r t h e r o p e r a t i o n s t o o k p l a c e w i t h i n t h e s e v i a l s . L y o p h i l i z e d s a m p l e s w e r e d i r e c t l y f i x e d w i t h o u t w a t e r d i l u t i o n o r a g a r e m b e d d i n g . P o s t f i x a t i o n was a c c o m p l i s h e d by i m m e r s i n g s a m p l e s i n a p h o s p h a t e b u f f e r e d (pH 7.2) 1% s o l u t i o n o f osmium t e t r o x i d e f o r 2 h r . S a m p l e s were t h e n w a s h ed w i t h p h o s p h a t e b u f f e r and s u b s e q u e n t l y d e h y d r a t e d w i t h s u c c e s s i v e a q u e o u s e t h a n o l s o l u t i o n s ' o f 30, 5 0 , 70 and 90% e t h a n o l f o r 10 m i n p e r i o d s , f o l l o w e d by t h r e e c h a n g e s o f 100% e t h a n o l , 30 m i n e a c h . A f t e r d e h y d r a t i o n i n t h e 70% e t h a n o l s o l u t i o n , t h e s a m p l e s w e r e p l a c e d i n a f i l t e r e d 7 0% e t h a n o l s o l u t i o n s a t u r a t e d w i t h , u r a n y l a c e t a t e f o r 3 h r . The d e h y d r a t i o n s e r i e s was t h e n convpleted..' D e h y d r a t e d s a m p l e s w ere embedded i n S p u r r l o w v i s c o s i t y r e s i n . S a m p l e s w ere p a s s e d t h r o u g h a n e t h a n o l - r e s i n s e r i e s o f 5:1, 3:1, 1:1, 1:3, and 1:5 f o r 30 m i n e a c h . Two c h a n g e s o f 1 0 0 % r e s i n f o l l o w e d , e a c h o f 30 m i n d u r a t i o n . Embedded s a m p l e s w ere p o l y m e r i z e d i n p l a s t i c c a p s u l e s a.t 70°C f o r 5 t o 7 h r . S e c t i o n s o f a b o u t 500 t o 900 R i n t h i c k n e s s w e re c u t w i t h a g l a s s k n i f e o n a R e i c h e r t 0MU3 u l t r a m i c r o t o m e and w e re p l a c e d o n c l e a n u n c o a t e d 400-mesh c o p p e r g r i d s a n d s t a i n e d w i t h R e y n o l d ' s ( 1 9 6 3 ) l e a d c i t r a t e f o r 5 t o 20 m i n . 1 2 2 . RESULTS I n t h i s s t u d y , e l e c t r o n m i c r o s c o p y was v a l u a b l e i n a s s e s s i n g t h e c o n t r i b u t i o n o f MF and LDLg p a r t i c l e s i n s t a b i l i -z i n g o i l - i n - w a t e r (o/w) e m u l s i o n s . A t t h e b e g i n n i n g , some d i f f i c u l t y was e n c o u n t e r e d i n e m b e d d i n g e m u l s i o n s s o t h a t a u n i f o r m h a r d n e s s , e s s e n t i a l t o s u c c e s s f u l u l t r a m i c r o t o m y , r e s u l t e d . I n n umerous t r i a l s w i t h E p on 812, embedded s a m p l e s ( p r e p a r e d by t h e m e t h o d o u t l i n e d i n C h a p t e r I ) , t h i n s e c t i o n s d i s p l a y e d w h a t i s commonly known as s p e c i m e n c h a t t e r . The r e s i n m a t e r i a l w i t h i n a n o i l d r o p l e t was s o f t e r t h a n t h e r e s i n e n c l o s i n g t h e o i l d r o p l e t . O i l d r o p l e t c o m p r e s s i o n and d e f o r m a t i o n w e r e e v i d e n t . P r e s u m a b l y , t h e e m u l s i f i e r c o m p l e x s u r r o u n d i n g e a c h o i l d r o p l e t a c t e d a s a b a r r i e r t o i n f i l t r a t i o n by E p on 8 1 2 , a v i s c o u s e p o x y r e s i n . A c c o r d i n g t c S p u r r ( 1 9 6 9 ) v i s c o u s e p o x y r e s i n s c a n n o t r e a d i l y p e n e t r a t e membrane w a l l s and o t h e r d e n s e t i s s u e s t r u c t u r e s . T h i s p r o b l e m was o v e r c o m e t o a c o n s i d e r a b l e e x t e n t by e m b e d d i n g s p e c i m e n s w i t h S p u r r l o w v i s c o s i t y r e s i n w h i c h i n f i l t r a t e d a t a more r a p i d r a t e : r e s i n p e n e t r a t i o n was b e t t e r w i t h S p u r r ' s medium. C h a t t e r was n o t c o m p l e t e l y e l i m i n a t e d b u t was r e d u c e d s i g n i f i c a n t l y . To e n h a n c e t h e c o n t r a s t , s p e c i m e n s w e r e p r e s t a i n e d w i t h u r a n y l a c e t a t e d u r i n g d e h y d r a t i o n . U s e d a s a g r i d p o s t -s t a i n , u r a n y l a c e t a t e d o e s n o t p e n e t r a t e u n i f o r m l y i n t o t h i n 123. s e c t i o n s . V a r i a t i o n i n c o n t r a s t was a l w a y s e v i d e n t w i t h t h e p o s t - s t a i n i n g t e c h n i q u e . S p h e r i c a l i n t a c t o i l d r o p ^ l e t s c a n be s e e n i n t h e r e p r e s e n t a t i v e m i c r o g r a p h s ( F i g u r e s 5, 9 and 1C) w i t h no e v i d e n c e o f c o a l e s c e n c e . S i n c e no d i s r u p t e d o r d e f o r m e d o i l d r o p l e t s w e r e p r e s e n t , i t was a s s u m e d t h a t s t r u c t u r a . l i n t e g r i t y was r e t a i n e d d u r i n g t h e p r e p a r a t i o n p r o c e d u r e . • Chang ( 1 9 6 9 ) h a s shown t h a t f r e e z e d r y i n g was d i s -r u p t i v e t o t h e m i c r o s t r u c t u r e o f y o l k p a r t i c l e s . Many r e s e a r -c h e r s h a v e r e p o r t e d t h a t d e h y d r a t i o n ( d r u m , spray', f o a m , f r e e z e d r y i n g ) was d e t r i m e n t a l t c y o l k e m u l s i f y i n g c a p a c i t y ( R o l f e s e t a l . , 1 9 5 5 ; S c h u l t z e t a l . , 1966; V a r a d a r a j u l a and C u n n i n g h a m , 1 9 7 2 ; and Z a b i k , 1 9 6 9 ) . S i n c e d e h y d r a t e d y o l k i s u s e d e x t e n s i v e l y i n t h e f o o d i n d u s t r y , i t was o f i n t e r e s t i n t h e p r e s e n t s t u d y t o a s s e s s t h e e m u l s i f y i n g c a p a b i l i t y o f l y o p h i l i z e d s a m p l e s o f MF a n d LDLg a n d t o a t t e m p t t o a s c e r t a i n t h e c a u s e s f o r a n y f u n c t i o n a l c h a n g e s b r o u g h t a b o u t by the. d e h y d r a t i o n p r o c e s s . F i g u r e 1 i s a t y p i c a l e l e c t r o n m i c r o g r a p h o f a l y o p h i l i z e d MF p r e p a r a t i o n . The l o s s o f t h e s t r u c t u r a l i n t e g r i t y o f MF by f r e e z e d r y i n g i s e v i d e n t . B a s e d on t h e p r o p o s e d MF m i c r o s t r u c t u r e ( C h a p t e r I ) , i t a p p e a r s t h a t e l e c t r o n -d e n s e l a m e l l a e ( p o s s i b l y p r o t e i n s and a s s o c i a t e d p h o s p h o l i p i d s ) h a v e b e e n d i s l o d g e d and c o m b i n e d t o f o r m l o n g t h i c k , h i g h l y o s m i o p h i l i c m e m b r a n e - l i k e s t r a n d s . F r e e f a t d r o p l e t s a r e shown 124 . .FIGURE 1. E l e c t r o n m i c r o g r a p h o f l y o p h i l i z e d MF • F: f r e e f a t z o n e s . ( 4 0 , 000 X ) . 1 2 5 . i n F i g u r e 1 a s g r e y a r e a s , w h i c h p r e s u m a b l y w e r e b r o u g h t a b o u t by c o a l e s c e n c e o f t h e n e u t r a l l i p i d c o r e s o f MF p a r t i c l e s . As m e n t i o n e d i n C h a p t e r I I , a b o u t 45% n e u t r a l l i p i d was f o u n d i n t h e MF f r a c t i o n . As shown i n F i g u r e 1, meisses o f e l e c t r o n -d e n s e s m a l l p a r t i c l e s ( m a j o r i t y a b o u t 100 A5) v a r y i n g i n s i z e w e r e p r e d o m i n a n t . P r e s u m a b l y , t h e s e s m a l l p a r t i c l e s a r e made up o f p h o s p h o l i p i d s s i n c e t h e y a r e h i g h l y o s m i o p h i l i c . A p p a r e n t l y , w a t e r i s e s s e n t i a l f o r m a i n t a i n i n g t h e s t r u c t u r a l i n t e g r i t y o f n a t i v e MF and any l o s s o f s t r u c t u r a l w a t e r ( w h i c h i s i n t h e i m m e d i a t e v i c i n i t y o f t h e p a r t i c l e s ) w i l l l e a d t o d i s a s s o c i a t i o n o f l a m e l l a e . The p r o c e s s o f f r e e z e d r y i n g c a u s e d s t r u c t u r a l c h a n g e s o f MF. When a l y o p h i l i z e d MF s a m p l e was h y d r a t e d ( 1 0 % s o l u t i o n ) p r i o r t o p r e p a r a t i o n f o r e l e c t r o n m i c r o s c o p y , t h e MF p a r t i c l e s w e r e n o t r e f o r m e d t o t h e i r o r i g i n a l s t r u c t u r e ( F i g u r e 2 ) . Chang ( 1 S 6 9 ) who p r e s e n t e d m i c r o g r a p h s o f f r e e z e -d r i e d y o l k , g r a n u l e a n d p l a s m a p r e p a r a t i o n s , c o n c l u d e d t h a t f r e e z e d r y i n g was h a r m f u l t o LDLg a nd LDLp s t r u c t u r e s . L a r g e f r e e f a t z o n e s a n d d i s r u p t e d LDL s t r u c t u r e s w e r e p r e v a l e n t . F i g u r e 3 shows t h e e f f e c t s o f f r e e z e d r y i n g t h e LDLg f r a c t i o n . L o n g s t r a n d s ( p e r h a p s p r o t e i n ) w i t h a t t a c h e d e l e c t r o n - d e n s e s m a l l p a r t i c l e s ( p r e s u m a b l y p h o s p h o l i p i d u l t r a p a r t i c l e s ) w e r e f o r m e d . Some LDLg p a r t i c l e s r e m a i n e d i n t h e i n t a c t s t a t e . As shown i n F i g u r e 4 , LDLg were n o t r e f o r m e d u p o n h y d r a t i o n 126 . • FIGURE 2. E l e c t r o n m i c r o g r a p h o f h v d r a t e d l y o p h i l i z e d MF. (16,0 00 X ) . FIGURE 3. E l e c t r o n m i c r o g r a p h o f l y o p h i l i z e d LBL ( 4 0 , 0 0 0 X ) . 128 . 129. ( 1 0 % s o l u t i o n ) . The o b s e r v e d i n t a c t LDLg w e r e p r o b a b l y t h o s e t h a t r e m a i n e d u n c h a n g e d d u r i n g d e h y d r a t i o n ( F i g u r e 3 ) . F i g u r e s 5 and 6 a r e r e p r e s e n t a t i v e m i c r o g r a p h s o f t h e s u r f a c e a r e a o f o i l d r o p l e t s s t a b i l i z e d w i t h n a t i v e a nd l y o p h i l i z e d MF p r e p a r a t i o n s . I t i s a p p a r e n t t h a t b o t h l y o p h i l i z e d a n d n a t i v e MF p a r t i c l e s w e r e d i s r u p t e d c o n s i d e r a b l y b u t t h e l a m e l l a e o f some p a r t i c l e s r e m a i n e d i n t a c t a l t h o u g h t h e l i p i d c o r e was a p p a r e n t l y a b s e n t . A s shown i n F i g u r e s 5 and 6, f o u r d i s t i n c t z o n e s i n t h e e m u l s i o n s a p p e a r t o be i n v o l v e d i n s t a b i l i z a t i o n : Zone 1 t h e i n t e r d r o p l e t r e g i o n w i t h , m o d e r a t e l y e l e c t r o n - d e n s e s t r a n d s h a v i n g a t t a c h e d h i g h l y e l e c t r o n - d e n s e m i c r o p a r t i c l e s ; Zone 2 t h e s u r f a c e r e g i o n w i t h m u l t i l a y e r e l e c t r o n - d e n s e b a n d s and l a r g e a g g r e g a t e s ; Zone 3 t h e s u b - s u r f a c e r e g i o n w i t h l a r g e , h i g h l y e l e c t r o n - d e n s e b o d i e s a b o u t 200 t o 600 8 i n l e n g t h a n d Zone 4 t h e i n t e r f a c i a l r e g i o n w i t h t h i n m o d e r a t e l y e l e c t r o n - d e n s e l a y e r s ( v i e w e d b e s t i n F i g u r e 7 ) . F i g u r e . 8 i s a h i g h l y m a g n i f i e d e l e c t r o n m i c r o -g r a p h o f t h e s u r f a c e Zone 2 o f a n o i l d r o p l e t s t a b i l i z e d w i t h l a m e l l a - c o n t a i n i n g p a r t i c l e s . A l t h o u g h a l l f o u r z o n e s w e r e f o u n d i n n a t i v e MF e m u l s i o n s , Z o n e s 2 and 3 a r e n o t a s w e l l f c r m e d a nd MF p a r t i c l e s r e m a i n more i n t a c t . One w o u l d t h e n e x p e c t t h a t t h e more e x t e n s i v e p a r t i c u l a t e c o v e r a g e , o b s e r v e d i n e m u l s i o n s w i t h l y o p h i l i z e d MF w o u l d c o n f e r g r e a t e r s t a b i l i t y a g a i n s t c o a l e s c e n c e . T h i s was f o u n d t o be t h e c a s e . FIGURE 5. E l e c t r o n m i c r o g r a p h o f an o i l / w a t e r e m u l s i o n p r e p a r e d w i t h n a t i v e MF. 1: Zone 1, 2: Zone 2, 3: Zone 3, 4: Zone 4. See t e x t f o r z o n e c h a r a c t e r i z a t i o n . ( 1 3 , 7 0 0 X ) . 4-* I • 5 0 0 0 A V* FIGURE 6. E l e c t r o n m i c r o g r a p h o f an o i l / w a t e r e m u l s i o n p r e p a r e d f r e m l y o p h i l i z e d MF. 1: Zone 1, 2: Zone 2, 3: Zone 3, 4: Zone 4. See t e x t f o r z o n e c h a r a c t e r i z a t i o n . ( 6 0 , 0 0 0 X ) . 1 3 2 . FIGURE 7. E l e c t r o n m i c r o g r a p h o f an o i l / w a t e r e m u l s i o n p r e p a r e d w i t h l y o p h i l i z e d MF. 4: Zone 4. See t e x t f o r z o n e c h a r a c t e r i z a t i o n . ( 160 , 000 X) . 1 3 3 . FIGURE 8. E l e c t r o n m i c r o g r a p h o f a n o i l / w a t e r e m u l s i o n p r e p a r e d w i t h n a t i v e MF. 2: Zone 2. See t e x t f o r z o n e c h a r a c t e r i z a t i o n . ( 3 8 0 , 0 0 0 X ) . 134 . A l t h o u g h i t i s d i f f i c u l t t o p o s t u l a t e t h e c o m p o s i t i o n o f t h e s e s t a b i l i z a t i o n z o n e s , t h e o s m i o p h i l i c n a t u r e a n d s t r u c t u r a l c h a r a c t e r i s t i c s p r o v i d e some i n f o r m a t i o n . Zone u-c o u l d be a n i n t e r f a c i a l l a y e r o f p r o t e i n s w h i c h a r e c a p a b l e o f f o r m i n g c o n t i n u o u s m e m b r a n e - l i k e l a y e r s a n d w h i c h a r e m o d e r a t e l y o s m i o p h i l i c . I n Zone 1, t h e m o d e r a t e l y e l e c t r o n - d e n s e s t r a n d s a r e m o s t l i k e l y p r o t e i n s . B e i n g h i g h l y o s m i o p h i l i c , t h e a t t a c h e d s m a l l p a r t i c l e s on t h e p r o t e i n s t r a n d s w o u l d be p h o s p h o l i p i d r - c o n t a i n i n g p a r t i c l e s . The c h o l e s t e r o l i n MF p a r t i c l e s (> 1 1 . 0 % o f t o t a l MF l i p i d ) i s p r e s u m a b l y a s s o c i a t e d w i t h p h o s p h o l i p i d s i n t h e c o r e - f r e e l a m e l l a e a n d l a m e l l a e f r a g m e n t s i n Z o n e s 2 a n d 3, r e s p e c t i v e l y . C o r r a n and L e w i s ( 1 9 2 4 ) r e p o r t e d t h a t a l e c i t h i n - c h o l e s t e r o l r a t i o o f l e s s t h a n 8:1 r e s u l t s i n a w/o e m u l s i o n . T h i s r a t i o was f o u n d t c be a b o u t 3:1 MF ( C h a p t e r I I ) . H o w e v e r o n l y o/w e m u l s i o n s w e re f o r m e d i n t h i s p r e s e n t s t u d y . I t c a n be p r e s u m e d t h a t t h e o t h e r c o m p o n e n t s o f t h e MF a n d t h e c h o l e s t e r o l - p h o s p h o l i p i d o r i e n t a t i o n i n t h e l a m e l l a e and f r a g m e n t s o f f s e t t h e a n t a -g o n i s t i c p r o p e r t y o f c h o l e s t e r o l . The l i b e r a t e d " n e u t r a l l i p i d c o r e s o f MF were, p r o b a b l y d e p o s i t e d o n t h e s u r f a c e o f o i l d r o p l e t s d u r i n g e m u l s i f i c a t i o n . E l e c t r o n m i c r o s c o p y o f e m u l s i o n s p r e p a r e d w i t h n a t i v e ( F i g u r e 9) a n d l y o p h i l i z e d ( F i g u r e 10) LDLg p a r t i c l e s r e v e a l e d t h a t t h e s t a b i l i z i n g z o n e s r e s i d e d a t t h e i n t e r f a c e o r i n t h e i n t e r - d r o p l e t r e g i o n . P a r t i c l e s w e r e r a r e l y f o u n d 135 . 5000A FIGURE £, E l e c t r o n m i c r o g r a p h o f an o i l d r o p l e t s t a b i l i z e d w i t h n a t i v e LDLg. (46,500 X ) . FIGURE 1 0 . E l e c t r o n m i c r o g r a p h o f o i l d r o p l e t s s t a b i l i z e d w i t h l y o p h i l i z e d LDLg. (24,000 X ) . 137 . b e l o w t h e o i l d r o p l e t ' s s u r f a c e . The z o n e s a p p e a r e d t h e same f o r b o t h t y p e s o f e m u l s i o n . S h e a r i n g d u r i n g e m u l s i f i c a t i o n p r o b a b l y c a u s e d t h e l o s s o f u l t r a p a r t i c l e s f r o m LDLg m i c e l l e s and c o a l e s c e n c e o f t r i g l y c e r i d e c o r e s . •'As i n t h e MF e m u l s i o n s , a n i n t e r f a c i a l " g r e y " z o n e was o b s e r v e d f o r b o t h n a t i v e and l y o p h i l i z e d LDLg s t a b i l i z e d e m u l s i o n s . Many o f t h e s m a l l e l e c t r o n - d e n s e p a r t i c l e s a b s o r b e d a t t h e s u r f a c e o r f o u n d i n t h e i n t e r d r o p l e t r e g i o n a r e p r o b a b l y d i s l o d g e d phospholipid., u l t r a p a r t i c l e s o r a g g r e g a t e s o f t h e s e u l t r a p a r t i c l e s . I n c o n t r a s t t o t h e m i c r o g r a p h s o f MF e m u l s i o n s , i n t e r d r o p l e t s t r a n d s o r s u r f a c e l a m e l l a e w e r e r a r e l y e v i d e n t . The n e u t r a l l i p i d p o r t i o n o f a d i s r u p t e d LDLg p a r t i c l e must merge w i t h t h e ' o i l p h a s e o f t h e e m u l s i o n s i n c e no n e u t r a l l i p i d p a r t i c l e s a r e p r e s e n t i n t h e i n t e r d r o p l e t r e g i o n . N e u t r a l l i p i d c o u l d r e m a i n e n t r a p p e d w i t h i n t h e e m u l s i f i e r c o m p l e x a s e v i d e n t i n F i g u r e 9. A h i g h m a g n i f i c a t i o n m i c r o g r a p h ( F i g u r e 11) c l e a r l y shows t h e i n t e r f a c i a l z one o f a n e m u l s i o n p r e p a r e d w i t h l y o p h i l i z e d LDLg. M i c r o g r a p h s f o r the' n a t i v e ' LDLg were s i m i l a r . The t h i n m o d e r a t e l y o s m i o p h i l i c z o n e o f F i g u r e . 11 i s a b o u t 15C K i n w i d t h , b u t s i n c e t h i s a p p a r e n t w i d t h d e p e n d s on t h e t h i c k n e s s o f t h e t h i n s e c t i o n a n d t h e c u r v a t u r e o f t h e o i l d r o p l e t , i t i s p r o b a b l y n a r r o w e r . F i g u r e s 12 and 13 a r e t y p i c a l m i c r o g r a p h s o f e m u l s i o n s p r e p a r e d w i t h n a t i v e a n d l y o p h i l i z e d y o l k , r e s p e c t i v e l y . L a r g e 138 . FIGURE 1 1 . E l e c t r o n m i c r o g r a p h o f an o i l d r o p l e t s t a b i l i z e d w i t h l y o p h i l i z e d LDLg. (100,0 00 X ) . 139 . FIGURE 12. E l e c t r o n micrograph o f an oi]/water emulsion prepared with n a t i v e y o l k . G: granule. ( 5 5 , 0 0 0 X ) . 140. FIGURE 13. E l e c t r o n m i c r o g r a p h o f o i l d r o p l e t s s t a b i l i z e d w i t h l y o p h i l i z e d y o l k . G: g r a n u l e . . (40 ,000 X) . 141 e l e c t r o n - d e n s e c l u s t e r s r a n g i n g i n s i z e f r o m a b o u t 300C A* t o 2'ym v?ere f o u n d . T h e s e c l u s t e r s w i t h e l e c t r o n - d e n s e p a r t i c l e s a r e p r o b a b l y p a r t i a l l y d i s r u p t e d g r a n u l e s . S a l t , commonly a d d e d t o m a y o n n a i s e - t y p e p r o d u c t s as a g r a n u l e d i s r u p t i n g a g e n t , was n o t i n c l u d e d i n t h e e m u l s i o n f o r m u l a t i o n and t h i s c o m p l e t e d i s r u p t i o n o f g r a n u l e s w o u l d n o t be e x p e c t e d . . The s t a b i l i z i n g z o n e s a p p e a r e d v e r y s i m i l a r t o t h o s e o f t h e LDLg e m u l s i o n s . A t h i n i n t e r f a c i a l z o n e a n d s u r f a c e and i n t e r d r o p l e t z o n e s composed o f s m a l l p a r t i c l e s a n d p a r t i c l e a g g r e g a t e s w e r e o b s e r v e d . Some m y e l i n f o r m s w e r e a l s o p r e s e n t . The m a j o r i t y o f t h e MF may s t i l l be e n t r a p p e d w i t h i n t h e p a r t i a l l y d i s r u p t e d g r a n u l e . A t y p i c a l MF Zone 1 i s n o t p r e s e n t . A most s t r i k i n g f e a . t u r e o f b o t h e m u l s i o n s was t h e l o c a t i o n o f e m u l s i f i e d p a r t i c l e s ( F i g u r e s 12 and 1 3 ) . None were f o u n d t o r e s i d e b e l o w t h e o i l d r o p l e t s ' s u r f a c e . Chang e t a l (197 2) p u b l i s h e d e l e c t r o n m i c r o g r a p h s o f m a y o n n a i s e e m u l s i o n s w h i c h c l e a r l y showed t h a t some p a r t i c l e s o f y o l k w e r e l o c a t e d w e l l b e l o w t h e o i l d r o p l e t s ' s u r f a c e . B a s e d on t h e r e s u l t s o f t h e - p r e s e n t s t u d y , t h e s e . s u b s u r f a c e p a r t i c l e s ' • a r e f r o m d i s r u p t e d g r a n u l e s s i n c e MI' and LDLg and t h e o r e t i c a l l y LDLp l o c a t e o n l y a t o r a b o v e t h e o i l d r o p l e t s ' s u r f a c e . I n t h e s t u d y o f Chang e t a l . ( 1 9 7 2 ) , e m u l s i o n s c o n s i s t e d o f s a l t • ( i n c l u d e d t o d i s r u p t g r a n u l e s ) s u g a r and v i n e g a r , a l ] o f w h i c h may h a v e a f f e c t e d s p e c i f i c c o m p o n e n t l o c a t i o n s . The s t a b i l i t i e s o f a l l t h e e m u l s i o n s were, a s s e s s e d by t h e c e n t r i f u g a t i o n m e t h o d ( F i g u r e 1 4 ) . A l l o f t h e e m u l s i o n s 6. i a O i l and w a t e r s e p a r a t i o n o f v a r i o u s e m u l s i o n s a f t e r 1 h r a t 35°C and 30 m i n at. 20 ,000 X g. L y o p h i l i z e d s a m p l e s : 1: y o l k . , 2: F I I , 3: MF, 4: L D L g . N a t i v e s a m p l e s ; 5: y o l k , 6: F T I , 7: MF 8: LDLg. h a d w a t e r s e p a r a t i o n upon c e n t r i f u g a t i o n b u t o n l y two showed o i l s e p a r a t i o n . W a t e r s e p a r a t i o n i s n o t a n i n d i c a t i o n o f c i l c o a l e s c e n c e b u t r a t h e r o f c r e a m i n g . M o s t c o m m e r c i a l m a y o n n a i s e s a r e s u b j e c t t o c r e a m i n g ( V i n c e n t , 1 9 6 4 ) . C r e a m i n g i s , h o w e v e r , a m e a s u r e o f e m u l s i o n i n s t a b i l i t y and may e v e n -t u a l l y l e a d t o e m u l s i o n c o a l e s c e n c e . C r e a m i n g i s b a s i c a l l y r e l a t e d t o l a r g e o i l d r o p l e t s i z e and i n d i r e c t l y t o t h e e m u l s i f i e r ' s e f f i c i e n c y i n p r o m o t i n g l o w s u r f a c e e n e r g i e s w i t h c o n s e q u e n t s m a l l d r o p l e t s i z e ( B e c h e r , 1 9 5 7 ) . A l l e m u l s i o n s d i s p l a y e d a s i m i l a r amount o f w a t e r s e p a r a t i o n a nd s u r f a c e t e n s i o n s o f t h e v a r i o u s e m u l s i f i e r s o l u t i o n s were o f t h e same o r d e r ( C h a p t e r I I I , V i n c e n t e t a l . 1 9 6 6 ) . The d e g r e e o f o i l s e p a r a t i o n i s d i r e c t l y r e l a t e d t o t h e e f f i c i e n c y o f t h e e m u l s i f i e r and t h u s t h e s t a b i l i t y o f t h e e m u l s i o n ( R o l f e s e t a l . , 1 9 5 5 ; V i n c e n t e t a l . , 1 9 6 4 ) . None o f t h e l y o p h i l i z e d s a m p l e s showed a n y s i g n o f o i l s e p a r a -t i o n . I t w o u l d ap-pear t h a t t h e p r o c e s s o f f r e e z e d r y i n g c a u s e d p a r t i c l e b r e a k d o w n and u l t i m a t e l y p r o v i d e d a more e x t e n s i v e p a r t i c u l a t e c o v e r a g e on o i l d r o p l e t s . Of t h e n a t i v e s a m p l e s o f y o l k , F I I , MF and L D L g , o n l y t h e e m u l s i o n s made w i t h MF a n d y o l k w e r e d e s t a b i l i z e d by c e n t r i f u g a t i o n ( b o t h a b o u t 1 % ) . LDLg and F I I e m u l s i o n s showed no o i l s e p a r a t i o n . B u r l e y a n d Cook (1961.) f o u n d t h a t y o l k c o n t a i n s a l a r g e p r o p o r t i o n o f g r a n u l e s ( a p p r o x i m a t e l y 23% o f y o l k s o l i d s ) . T h e s e g r a n u l e s ? i n t h e p r e s e n c e , o f s a l t , a r e known t o be 144 . e f f i c i e n t e m u l s i f i e r s ( V i n c e n t , 1 9 4 4 ) , a l t h o u g h i n t h e d i s r u p t e d s t a t e t h e y do c o n t r i b u t e t o t h e s t a b i l i t y o f t h e f i n i s h e d p r o d u c t . I n t h i s s t u d y , g r a n u l e s w e r e n o t d i s r u p t e d b e c a u s e s a l t was n o t a d d e d , and t h u s a s i g n i f i c a n t p r o p o r t i o n o f t h e y o l k p r o b a b l y c o n t r i b u t e d l i t t l e t o t h e s t a b i l i t y o f t h e e m u l s i o n . The l y o p h i l i z e d y o l k e m u l s i o n l i k e l y showed no s i g n s o f o i l s e p a r a t i o n b e c a u s e o f t h e l i b e r a t i o n , o n f r e e z e d r y i n g , o f l o w - d e n s i t y m a t e r i a l ( L D L g ) w h i c h p r o v i d e s e x t r a e m u l s i f i e r ( C h a n g , 1 9 6 9 ) . B o t h n a t i v e a nd l y o p h i l i z e d LDLg e m u l s i o n s w e r e s t a b l e . The i n s t a b i l i t y o f t h e n a t i v e MF e m u l s i o n m i g h t be r e l a t e d t o t h e phenomenon o f e m u l s i o n i n v e r s i o n . C o r r a n a nd L e w i s ( 1 9 2 4 ) showed t h a t i n v e r s i o n was c o n n e c t e d w i t h t h e s o -c a l l e d " l e c i t h i n c h o l e s t e r o l a n t a g o n i s m " w h e r e l e c i t h i n f a v o r s a n o/w e m u l s i o n a nd c h o l e s t e r o l f a v o r s t h e r e v e r s e . The r a t i o f o r a n MF p r e p a r a t i o n i s a b o u t 3:1, s t r o n g l y i n d i c a t i n g t h e f o r m a t i o n o f a w/o e m u l s i o n . H o w e v e r , o n l y o/w e m u l s i o n s w e r e p r e p a r e d w i t h t h e MF. E v i d e n t l y o t h e r c o m p o n e n t s o f t h e MF o r t h e manner i n w h i c h c h o l e s t e r o l i s b o u n d w i t h i n t h e MF o f f s e t t h e e f f e c t o f c h o l e s t e r o l . P o s s i b l y , t h o u g h , t h e r e was a t e n d e n c y t o i n v e r t a n d i n v e r s i o n o c c u r r e d t o a l i m i t e d e x t e n t . The l e c i t h i n - c h o l e s t e r o l r a t i o ( 8 : 1 ) w i t h t h e LDLg was more f a v o r a b l e and t h e p r o p e n s i t y t o i n v e r s i o n r e d u c e d . The F I I e m u l s i o n was s t a b l e , p r o b a b l y b e c a u s e i t c o n s i s t e d o f a h i g h p r o p o r t i o n o f L D L g . 145 . R e d u c t i o n i n s u r f a c e e n e r g y i s an i m p o r t a n t p r e -r e q u i s i t e t o e m u l s i o n f o r m a t i o n and s t a b i l i t y . Low i n t e r f a c i a l t e n s i o n s p r o m o t e t h e f o r m a t i o n o f s m a l l d r o p l e t s i z e s ( H a r k i n s and Z o l l m a n , 1 3 2 6 ) . I t i s known t h a t y o l k and f r a c t i o n s o f y o l k p o s s e s s s u r f a c t a n t p r o p e r t i e s ( J o h n s o n and S a u n d e r s , 1 3 7 1 ; Powrie., 1955 ; V i n c e n t e t a l . , 1966) b u t r..o i n f o r m a t i o n e x i s t s f o r t h e MF and LDLg c o m p o n e n t s o f y o l k . S u r f a c e t e n s i o n d i l u t i o n c u r v e s o f n a t i v e a n d l y o p h i l i z e d MF and LDLg s o l u t i o n s ( i s o l a t e d - f r o m e g g s o f t h e s e c o n d f l o c k o f h e n s 41 wk o l d ) a r e shown i n F i g u r e 1 5. B o t h c o m p o n e n t s o f y o l k n o t i c a b l y r e d u c e d s u r f a c e t e n s i o n s . The d i f f e r e n c e b e t w e e n n a t i v e and l y o p h i l i z e d s t a t e s was m i n i m a l . The s u r f a c e t e n s i o n s o f LDLg s o l u t i o n s w e re a l w a y s s u b s t a n t i a l l y l o w e r t h a n t h o s e o f c o m p a r a b l e MF s o l u t i o n s . C o n s i d e r i n g t h e p r o p o s e d s t r u c t u r e s f c r t h e s e p a r t i c l e s ( C h a p t e r I ) s u c h a n e f f e c t i s n o t u n e x p e c t e d . The p h o s p h o l i p i d i n t h e u l t r a -p a r t i c l e s o f t h e LDLg p a r t i c l e m i g h t a c t as a r e s e r v o i r of. s u r f a c t a n t m o l e c u l e s . P h o s p h o l i p i d s l o w e r s u r f a c e t e n s i o n s m a r k e d l y ( P o w r i e , 1955 ; V i n c e n t e t a l . , 1 9 6 6 ) . I n d i l u t e -s o l u t i o n , p h o s p h o l i p i d s m i g h t e x i s t a s m o l e c u l a r l y d i s p e r s e d m o l e c u l e s i n e q u i l i b r i u m w i t h b ound LDLg p h o s p h o l i p i d s . Bangham (196 3 ) ha.s shown t h i s o c c u r r e d w i t h p u r e l e c i t h i n s o l s . S i m i l a r l y m i g r a t i o n and t r a n s f e r o f p h o s p h o l i p i d s h a s b e e n shown t o o c c u r w i t h i n o t h e r l i p o p r o t e i n s y s t e m s s u c h a s p l a s m a l i p o p r o t e i n s , m i l k l i p o p r o t e i n s a nd b l o o d c h y l o m i c r o n s Ti W CD — 1 M ! — i C n •00 • a c *—* O P H] J -o CD CL * r + cn H -O < 3 CD O H> * P J > 3 1— 1 P J f - 1 r+ < H I 1—1 T l cn H* t r 01 • o I - 1 h-1 •< O <T> 'H CL ^ Hi h-> H. H* O N 3 0) Cu cn cn CO o » - n O - f r 1— 1 £ o re i i o i — H-M IS1 (D a n e s A o o -T" m ' 9nT ( r e v i e w e d by M a r g o l i s , 1 9 6 9 ) . F r e e z e d r y i n g o f LDLg p r o b a b l y r e s u l t s i n t h e l i b e r a t i o n o f o t h e r c o m p o n e n t s w h i c h w o u l d c o m p e t e w i t h p h o s p h o l i p i d s a t t h e ' i n t e r f a c e ; a h i g h e r s u r f a c e t e n s i o n i s i n d i c a t i v e o f t h i s ( F i g u r e 1 5 ) . S i n c e t h e o u t e r l a m e l l a e o f t h e m y e l i n f i g u r e a r e s u s p e c t e d t o be p r o t e i n , p h o s p h o l i p i d and c h o l e s t e r o l , d i s p e r -s i o n o f m o l e c u l e s i n d i l u t e s o l u t i o n s w o u l d n o t be e x p e c t e d t o be. s i g n i f i c a n t a nd l a m e l l a e w o u l d r e m a i n i n t a c t . As shown i n F i g u r e 1 5 , f r e e z e d r i e d MF s o l u t i o n s w e r e somewhat l o w e r i n s u r f a c e t e n s i o n t h a n t h e n a t i v e s a m p l e s . D i s r u p t i o n o f MF by f r e e z e d r y i n g m i g h t e x p o s e more, s u r f a c t a n t m o l e c u l e s t o t h e a q u e o u s e n v i r o n m e n t a n d a s a c o n s e q u e n c e , more m o l e c u l e s w o u l d go i n t o s o l u t i o n w i t h t h e r e s u l t o f a d e c r e a s e i n s u r f a c e t e n s i o n . The s h a p e s o f t h e c u r v e s shown i n F i g u r e s 15 and 16 s u p p o r t t h e a b o v e h y p o t h e s i s . A c c o r d i n g t o M c B a i n e t a l . ( 1 9 3 7 ) t h r e e d i f f e r e n t t y p e s o f s u r f a c e a c t i v i t y c u r v e s c a n be con.-'-s t r u c t e d . The t y p e I I I c u r v e i s r e p r e s e n t a t i v e . o f t h o s e compounds i n s o l u t i o n w h i c h r e s u l t i n a g r e a t d e c r e a s e i n s u r f a c e t e n s i o n w i t h o n l y a s l i g h t i n c r e a s e i n s u r f a c t a n t c o n c e n t r a t i o n . Compounds s u c h a s p h o s p h o l i p i d s , a r e s u r f a c t a n t s s i n c e t h e i r m o l e c u l e s p o s s e s s b o t h l i p o p h i l i c and h y d r o p h i l i c g r o u p s and a r e a b l e t o a d s o r b a t b o t h p h a s e s o f a n e m u l s i o n . The s u r f a c e -a c t i v i t y c u r v e s o f LDLg ( F i g u r e s 15 a n d 16) a r e a l m o s t i d e n t i c a l t o t y p e I I I c u r v e s . P r e s u m a b l y t h e u l t r a p a r t i c l e s o f t h e LDLg fr 4 0 1 — I — • . 1 I — ' — — f 0 0.05 0.1 0.25 0.5 C O N C E N T R A T I O N , X FIGURE 16. S u r f a c e t e n s i o n o f l y o p h i l i z e d MF ( a ) , F I I ( t ) , and LDLg ( c ) i s o l a t e d f r o m eggs o f hens a b o u t . 1 . 5 y e a r s o l d . 00 149 . a r e c omposed o f p h o s p h o l i p i d s w h i c h a r e i n e q u i l i b r i u m w i t h p h o s p h o l i p i d m o l e c u l e s i n s o l u t i o n . V i c e n t e t a l . ( 1 9 6 5 ) h a v e r e p o r t e d a s u r f a c e t e n s i o n o f 42 d y n e s / c m ( o f 0 . 2 5 ' t o 1 5 % s o l u t i o n s ) f o r L D L p , a v a l u e c l o s e t o t h a t r e p o r t e d h e r e f o r L D L g , ( a b o u t 41 d y n e s / c m ) . T y p e I c u r v e s show a g r a d u a l d e c r e a s e i n s u r f a c e t e n s i o n w i t h i n c r e a s i n g s o l u t e c o n c e n t r a t i o n . P r o t e i n s a n d f a t t y a c i d s a r e t y p i c a l compounds w h i c h p o s s e s s t h i s p r o p e r t y . The s u r f a c e t e n s i o n c u r v e s o f MF ( F i g u r e s 15 and 16) a r e s i m i l a r t o t y p e I c u r v e s , i n d i c a t i n g t h a t p r o t e i n o f MF may be r e l e a s e d f r o m t h e o u t e r s u r f a c e . On l y o p h i l i z a t i o n , some o h o s p h o l i p i d s may become more i n t i m a t e l y c o n n e c t e d w i t h p r o t e i n s and t h u s t h e s u r f a c e c h a r a c t e r may n e t be a p p r e c i a b l y d i f f e r e n t ; i n f a c t , v e r y l i t t l e d i f f e r e n c e was f o u n d . F i g u r e 16 shows t h e s u r f a c e a c t i v i t y o f l y o p h i l i z e d MF, LDLg and F I I f r a c t i o n s i s o l a t e d f r o m e g g s o f t h e f i r s t f l o c k o f h e n s ( a b o u t 1 1/2 y e a r s o f a g e ) . T h e s e c u r v e s a r e s i m i l a r i n s h a p e t o t h o s e i n F i g u r e 15 w h e r e 41 week o l d b i r d s o f a d i f f e r e n t f l o c k w e r e u s e d . C o m p o s i t i o n a l l y t h e two f l o c k s a r e a l s o s i m i l a r ( C h a p t e r I I ) . C u r v e b o f F i g u r e 16 r e p r e s e n t s a F I I d i l u t i o n s e r i e s . S i n c e t h e F I I was composed o f b o t h MF ( c u r v e a ) and LDLg ( c u r v e c ) p a r t i c l e s , s u r f a c e t e n s i o n v a l u e s w e r e e x p e c t e d t o l i e b e t w e e n c u r v e s o f t h e d i s p e r s i o n s w i t h t h e i n d i v i d u a l p a r t i c l e s . T h i s was f o u n d t r u e o n l y f o r d i l u t i o n s b e l o w a b o u t 0.6%. 150 DISCUSSION Chang e t a l . (197 2) h a v e p u b l i s h e d a n e l e c t r o n m i c r o s c o p y s t u d y o f m a y o n n a i s e e m u l s i o n s w h i c h , f c r t h e f i r s t t i m e , r e v e a l e d t h e c o m p l e x i t y o f y o l k - s t a b i l i z e d e m u l s i o n s . A t l e a s t f o u r d i s t i n c t z o n e s w i t h i n and a b o u t an o i l d r o p l e t ' s s u r f a c e w e r e f o u n d . The r e s u l t s o f t h i s c u r r e n t s t u d y i n d i c a t e t h a t MF a n d LDLg c o m p o n e n t s o f e g g y o l k a r e e f f i c i e n t e m u l s i f i e r s . T h e s e p a r t i c l e s w e r e s i m i l a r t o w h o l e egg y o l k i n t h e i r a b i l i t y t o l o w e r s u r f a c e t e n s i o n and f o r m s t a b l e e m u l s i o n s . C o m p l e x z o n e s w e r e f o u n d a b o u t o i l d r o p l e t s i n a l l y o l k o r y o l k c o m p o n e n t s t a b i l i z e d e m u l s i o n s . H o w e v e r , i n c o n t r a s t t o t h e r e s u l t s o f Chang e t a l . ( 1 9 7 2 ) , no i n t e r n a l z o n e was ever-o b s e r v e d . Chang e t a l . (197 2) f o u n d p a r t i c l e s r e s i d i n g w e l l b e n e a t h t h e o i l d r o p l e t ' s s u r f a c e (up t o l u m ) . E v e n a f t e r l y o p h i l i z a t i o n , w h i c h c a u s e d MF a n d LDLg d i s r u p t i o n , t h e r e was no e v i d e n c e t h a t MF a n d LDLg o r c o m p o n e n t s o f t h e s e p a r t i c l e s p e n e t r a t e d t h e o i l d r o p l e t ' s s u r f a c e t o a n y a p p r e c i a b l e e x t e n t . Some c o m p o n e n t s o f l y o p h i l i z e d MF w e r e f o u n d b e n e a t h t h e o i l d r o p l e t ' s s u r f a c e b u t t h e y d i d n o t p e n e t r a t e f u r t h e r . I t c a n be s u g g e s t e d t h a t MF a n d LDLg a n d / o r t h e i r c o m p o n e n t s , a s w e l l a s p l a s m a LDL ( s i n c e t h e y a r e t h o u g h t t o be s t r u c t u r a l l y and c o m p o s i t i o n a l l y s i m i l a r t o g r a n u l a r LDL, C h a p t e r I I ) may n e v e r r e s i d e a n y w h e r e b u t a t o r a b o v e a n o i l 1 5 1 . d r o p l e t ' s s u r f a c e , a t l e a s t i n s a l t - f r e e e m u l s i o n s . T h i s c o n s i d e r a b l y l i m i t s t h e t y p e o f y o l k c omponent w h i c h m i g h t r e s i d e b e n e a t h t h e . s u r f a . e e . H o w e v e r , i t must t e b o r n e i n m i n d t h a t t h e e m u l s i o n s s t u d i e d by Chang e t a l . ( 1 9 7 2 ) w ere c o m p l e x e m u l s i o n s w h i c h c o n t a i n e d i n g r e d i e n t s ( s a l t , s u g a r , v i n e g a r ) n o t u s e d i n t h i s s t u d y . T h e s e i n g r e d i e n t s may h a v e c a u s e d d i f f e r e n t d e g r a d a t i v e o r a g g r e g a t i n g e f f e c t s and t h u s t h e r e i s a p o s s i b i l i t y t h a t MF and LDLg ( a l s o L D L P ) may c o n t r i b u t e t o t h i s i n t e r n a l z o n e o f s t a b i l i z a t i o n . H o w e v e r , c i r c u m s t a n t i a l e v i d e n c e s u g g e s t s t h a t MF, LDLg a n d LDLp do r e s i d e a t o r a b o v e - t h e o i l d r o p l e t ' s s u r f a c e . Chang e t a l . (197 2) p r o p o s e d t h a t t h e i n t e r n a l o r " s p e c k l e d " z o n e was c o mposed o f a l l o r some o f t h e f o l l o w i n g s t r u c t u r e s : 1. c o a l e s c e d LDL m i c e l l e s f r o m t h e p l a s m a o r g r a n u l a r , p h a s e s o f e g g y o l k ; 2. one o r more ( a g g r e g a t e d ) p h o s p h o l i p i d u l t r a -p a r t i c l e s d i s l o d g e d f r o m LDL m i c e l l e s d u r i n g e m u l s i f i c a t i o n ; and 3. v a r i o u s c o m b i n a t i o n s o f h i g h - d e n s i t y l i p o p r o t e i n s (HDL, a - and 0 - l i p o v i t e l l i n ) . A l l t h e p a r t i c l e s o f t h e " s p e c k l e d " z o n e were o f t h e o r d e r o f 50 8 i n d i a m e t e r and a g g r e g a t e s o f t h e s e b a s i c u n i t s w e r e o b s e r v e d t o be up t o a b o u t 2 50 R i n d i a m e t e r . T h i s s i z e r e s t r i c t i o n t e n d s t o e l i m i n a t e t h e p o s s i b i l i t y o f i n t a c t , c o a l e s c e d o r a g g r e g a t e d LDL m i c e l l e s r e s i d i n g i n t h i s z o n e . LDL m i c e l l e s a r e t h e m s e l v e s a t l e a s t 25 0 8 i n d i a m e t e r a n d a number o f them e i t h e r c o a l e s c e d o r a g g r e g a t e d w o u l d r e s u l t i n a much l a r g e r s t r u c t u r e . E v e n i f 152 . s u r f a c e u l t r a p a r t i c l e s w e r e l o s t , b i n d i n g o f t h e r e m a i n i n g p r o -f i l e c o r e s (15C A5) d u r i n g e m u l s i f i c a t i o n w o u l d s t i l l r e s u l t i n a l a r g e c o m p l e x . M o r e o v e r , t h e p o l a r s u r f a c e o f i n t a c t , a g g r e g a t e d o r c o a l e s c e d LDL ( p r o p o s e d s t r u c t u r e . C h a p t e r I ) , w o u l d , by s o l u b i l i t y p r e f e r e n c e , m o s t l i k e l y r e s i d e i n a n a q u e o u s e n v i r o n m e n t r a t h e r t h a n a l i p o p h i l i c one. S i m i l a r l y u l t r a p a r t i c l e s , t h o u g h t t c be m i c e l l e s o f p h o s p h o l i p i d ( C h a p t e r I ) , w o u l d a l s o p r e f e r a p o l a r e n v i r o n m e n t . S c h n e i d e r and T a t t r i e ( 1 9 6 8 ) showed t h a t p h o s p h o l i p i d s w e r e o n l y s l i g h t l y s o l u b l e i n a n h y d r o u s t r i g l y c e r i d e s a nd w e r e c o m p l e t e l y i n s o l u b l e when w a t e r was p r e s e n t . M o l e c u l a r l y - d i s p e r s e d p h o s p h o l i p i d s l i b e r a t e d d u r i n g e m u l s i f i c a t i o n w o u l d most l i k e l y r e s i d e a t t h e o i l / w a t e r i n t e r f a c e b e c a u s e o f t h e i r p o l a r a n d n o n - p o l a r p r o p e r t i e s , b u t m i c e l l e s o f p h o s p h o l i p i d ( e x t e r n a l p o l a r g r o u p s ) w o u l d u n d o u b t e d l y p r e f e r an a q u e o u s e n v i r o n m e n t . O n l y i n a h i g h l i p i d c o n c e n t r a t i o n s i t u a t i o n , w i t h l i t t l e 'water a v a i l a b l e , w o u l d t h e r e v e r s e p h o s p h o l i p i d s t r u c t u r e ( p o l a r end o r i e n t e d t o w a r d the: c e n t e r ) o c c u r . ( L u z z o t i , 1 9 6 8 ) . I n ' L D L g e m u l s i o n s ( F i g u r e s 9, 10 and 1 1 ) , i t a p p e a r e d a s i f t h e LDL m i c e l l e was d i s r u p t e d . No " s p e c k l e d " z o n e s w e r e p r e s e n t . E l e c t r o n - d e n s e p a r t i c l e s o f up t o 200 % m i g h t be s i n g l e o r a g g r e g a t e d u l t r a p a r t i c l e s a n d w h i c h m i g h t be c o n -s i d e r e d u n u s e d r e s e r v o i r s o f p h o s p h o l i p i d . T h e s e p a r t i c l e s c o u l d be i n p a r t p r o t e i n a g g r e g a t e s o r p h o s p h o l i p i d - p r o t e i n a g g r e g a t e s f o r m e d d u r i n g t h e e m u l s i f i c a t i o n p r o c e s s . L i b e r a t e d 1 5 3 . n e u t r a l l i p i d c o r e s m i g h t h a v e r e m a i n e d e n t r a p p e d w i t h i n t h e e m u l s i f i e r c o m p l e x o r may h a v e merged w i t h t h e e m u l s i o n ' s o i l p h a s e . P r o t e i n m o l e c u l e s a p p e a r e d t o h a v e b e e n r e l e a s e d and have, m i g r a t e d t o t h e I n t e r f a c e o f t h e e m u l s i o n : a t h i n moder-a t e l y o s m i o p h i l i c z o n e c a n be s e e n . T h e s e o b s e r v a t i o n s s u g g e s t t h a t y o l k p a r t i c l e s o t h e r t h a n t h e MF, LDLg o r LDLp o r c o m p o n e n t s o f t h e s e p a r t i c l e s r e s i d e w i t h i n the: Cha^ng e t a l . ( 1 9 7 2 ) " s p e c k l e d " a r e a . I t i s more, l i k e l y t h a t HDL r a t h e r t h a n LDL e x i s t b e n e a t h t h e o i l d r o p l e t ' s s u r f a c e . S i n c e t h e l i p o v i t e l l i n s a r e i n s o l u b l e i n w a t e r ( B u r l e y , 1971) and s i n c e a b o u t 37% o f t h e HDL l i p i d c o m p o n e n t i s t h o u g h t t c be a t o r n e a r t h e HDL's s u r f a c e ( E v a n s e t a l . , 1 9 5 8 ) , c o m p a t a b i l i t y w i t h a l i p i d e n v i r o n m e n t m i g h t be e x p e c t e d . . K r a t o h v i l e t a l . (19 62 ) f o u n d t h a t HDL o f a b o u t 90 R i n d i a m e t e r d i s s o c i a t e i n a c i d s o l u t i o n i n t c s u b u n i t s o f a b o u t 4 5 t o 5 0 8 i n d i a m e t e r . The v i n e g a r i n c o r p o r a t e d i n t o t h e Chang e t a l . ( 1 9 7 2 ) e m u l s i o n w o u l d h a v e been c o n d u c i v e t o HDL d i s s o c i a t i o n and t h e p a r t i c l e s w i t h i n t h e " s p e c k l e d " a r e a m i g h t , i n p a r t , be HDL s u b u n i t s , a s w e l l a s v a r i o u s a g g r e g a t i o n s , o f w h o l e HDL and HDL s u b u n i t s . F i g u r e s 12 and 13 a r e t y p i c a l m i c r o g r a p h s o f n a t i v e a n d l y o p h i l i z e d y o l k p r e p a r e d o/w e m u l s i o n s ; no o t h e r i n g r e d i e n t s w e r e i n c l u d e d . . No " s p e c k l e d " a r e a s w e r e o b s e r v e d a n d , s i n c e s a l t was n o t a d d e d , egg y o l k g r a n u l e s r e m a i n e d i n t a c t . HDL a r e f o u n d o n l y w i t h i n t h e g r a n u l a r p h a s e o f egg 154 . y o l k . I t t h e r e f o r e a p p e a r s as i f no " s p e c k l e d " zone was f o r m e d b e c a u s e no HDL m i c r o - p a r t i c l e s w e r e r e l e a s e d . The HDL r e m a i n e d ' e n t r a p p e d w i t h i n t h e g r a n u l e m a t r i x . M y e l i n f i g u r e s a l s o f o r m e d v a r i o u s z o n e s a t o r a b o v e t h e s u r f a c e o f an o i l d r o p l e t b u t d i d n o t f o r m " s p e c k l e d " a r e a s b e n e a t h t h e o i l d r o p l e t ' s s u r f a c e . The m i c r o g r a p h s o f Chang e t a l . ( 1 9 7 2 ) o c c a s i o n a l l y showed an i n t a c t m y e l i n f i g u r e a b o v e t h e s u r f a c e a r e a . F i g u r e 5 shows t h a t n o t a l l MF w e r e b r o k e n down d u r i n g t h e e m u l s i f i c a t i o n p r o c e s s ; some r e m a i n e d i n t a c t . T h i s may be d i r e c t l y c o r r e l a t e d t o t h e s t r u c t u r e o f MF, a s . l i g h t a n d h e a v y MF h a v e b e e n o b s e r v e d ( C h a p t e r I ) . D i s r u p t e d MF g e n e r a l l y show t h r e e d i f f e r e n t s t a b i l i z i n g z o n e s , f o u r when t h e MF i s l y o p h i l i z e d . A l l c f t h e s e z o n e s w o u l d c o n t r i b u t e t o o v e r a l l e m u l s i o n s t a b i l i t y . The y o l k • p a r t i c l e s , MF and L D L g , a r e i m p l i c a t e d i n y o l k ' s a b i l i t y t o s t a b i l i z e e m u l s i o n s . I n m a y o n n a i s e e m u l s i o n s , f o r , i n s t a n c e , t h e p r e s e n c e o f s a l t w o u l d l i b e r a t e MF and LDLg f r o m t h e y o l k g r a n u l e p h a s e and t h e s e p a r t i c l e s w o u l d be f r e e t o c o n t r i b u t e t o y o l k ' s f u n c t i o n a l p r o p e r t i e s i.n f o r m i n g and s t a b i l i z i n g t h e e m u l s i o n . 1 5 5 . CONCLUSIONS The s u r f a c e t e n s i o n o f d i s p e r s i o n s o f MF a n d LDLg p a r t i c l e s a t c o n c e n t r a t i o n l e v e l s b e t w e e n 0.01 and 1% w e r e c o n s i d e r a b l y l o w e r t h a n t h e s u r f a c e t e n s i o n o f w a t e r . Thus i t i s n o t s u r p r i s i n g t h a t b o t h MF a n d LDLg h a v e b e e n f o u n d t o be a s e f f e c t i v e , a s w h o l e y o l k f o r e m u l s i f i c a t i o n o f v e g e t a b l e o i l . The h i g h s t a b i l i t y o f MF and LDLg e m u l s i o n s c a n be a t t r i b u t e d t o c o m p l e x p a r t i c u l a t e z o n e s f o r m e d on o i l d r o p l e t s u r f a c e s d u r i n g t h e e m u l s i f i c a t i o n p r o c e s s . I n e l e c t r o n m i c r o -g r a p h s , t h r e e d i s t i n c t z o n e s w ere o b s e r v e d i n e m u l s i o n w i t h n a t i v e MF, n a t i v e - LDLg a nd l y o p h i l i z e d LDLg w h i l e f o u r z o n e s w e r e n o t e d i n t h e l y o p h i l i z e d MF e m u l s i o n s . L y o p h i l i z a t i o n o f MF a n d y o l k i m p r o v e d e m u l s i o n s t a b i l i t y . F o r MF e m u l s i o n s , e n h a n c e d s t a b i l i t y c a n be a t t r i b u t e d t o e x t e n s i v e l a m e l l a e d i s r u p t i o n a n d o r i e n t a t i o n o f f r a g m e n t s o n d r o p l e t s u r f a c e s . I m p r o v e m e n t i n t h e s t a b i l i t y o f e m u l s i o n s w i t h l y o p h i l i z e d y o l k i s p r o b a b l y b r o u g h t a b o u t b y t h e i n c r e a s e d c o n c e n t r a t i o n o f l o w - d e n s i t y l i p o p r o t e i n s w h i c h w e r e r e l e a s e d f r o m t h e g r a n u l e s d u r i n g t h e f r e e z e d r y i n g p r o c e s s . 156. LITERATURE CITED B a l d w i n , R.E. 1 9 7 3 . F u n c t i o n a l p r o p e r t i e s i n f o o d s . I n Egg S c i e n c e and T e c h n o l o g y : E d . , W.J. S t a d e l m a n a n d O.J. C o t t e r i l l , P u b . , A v i P u b l i s h i n g Co. I n c . , W e s t p o r t , Conn. Bangham, A.D. 1 9 6 3 . P h y s i c a l s t r u c t u r e and b e h a v i o r o f l i p i d s a n d l i p i d e n z y m e s . I n A d v a n c e s i n L i p i d R e s e a r c h 1. 65. E d s . , R. P a o l e t t i and D. K r i t c h e v s k y . P u b . , A c a d e m i c P r e s s , New Y o r k , N.Y. B e c h e r , P. 1 9 6 6 . E m u l s i o n s : T h e o r y and P r a c t i c e . P u b . , R e i n h o l d Pub. C o r p . , New Y o r k . p . 1 5 0 - 2 0 9 . B u r l e y , R.W. 1 9 7 1 . L i p o p r o t e i n s . I n B i o c h e m i s t r y and M e t h o d o l o g y o f L i p i d s . E d s . , A.R. J o h n s o n a n d J . B . D a v e n p o r t . ' P u b . , W i l e y - I n t e r s c i e n c e , New Y o r k . B u r l e y , R.W. and W.H. Cook. 1 9 6 1 . I s o l a t i o n a n d c o m p o s i t i o n o f a v i a n egg y o l k g r a n u l e s and t h e i r c o n s t i t u e n t a a n d B - l i p o p r o t e i n s . C a n . J . B i o c h e m . P h y s i o l . 3 9 , 1 2 9 5 . C h a n g , C M . 1 9 6 9 . S t u d i e s o n egg y o l k . Ph.D. T h e s i s , U n i v . o f W i s . , M a d i s o n , W i s . C h a n g , C M . , W.D. P o w r i e and 0. Fennema. 197 2. E l e c t r o n m i c r o s c o p y o f m a y o n n a i s e . Can. I n s t . F o o d S c i . T e c h n o l . J . 5_, 134. C o r r a n , J.W. and W.C.N. L e w i s . 1924. L e c i t h i n a n d c h o l e s -t e r o l i n r e l a t i o n t o t h e p h y s i c a l n a t u r e o f c e l l m e m b r a n c e s . B i o c h e m i c a l J . 1_8 , 1 3 6 4 . C u n n i n g h a m , F.E. and P. V a r a d a r a j u l a . 1 9 7 3 . A r e v i e w o f t h e f a c t o r s a f f e c t i n g e m u l s i f i n g c h a r a c t e r i s t i c s of h e n s ' egg y o l k . W o r l d ' s P o u l t . S c i . J . 2 9 , 1 3 2 . E v a n s , R . J . , S.L. B a n d e m e r , K . H e i n l e i n and J.A. D a v i d s o n . 1 9 6 8 . B i n d i n g o f l i p i d t o p r o t e i n i n l i p o v i t e l l i n f r o m h e n ' s e g g . B i o c h e m . 3095 . H a r k i n s , W.D. a n d N. Z o l l m a n . 1 9 2 6 . I n t e r f a c i a l t e n s i o n and e m u l s i f i c a t i o n . I . The e f f e c t s o f b a s e s , s a l t s , and a c i d s u p o n t h e i n t e r f a c i a l t e n s i o n b e t w e e n a q u e o u s s o d i u m o l e a t e s o l u t i o n s a n d b e n z e n e . J . Am. Chem. S o c . 4 8 , 69. 157 . J o h n s o n , M.C.R. and L. S a u n d e r s . 1 9 7 1 . I n t e r f a c i a l s t u d i e s o f t r i g l y c e r i d e s o l u t i o n s o f e g g - p h o s p h a t i d y l c h o l i n e . Chem. P h y s . L i p i d s . 8_, 112. K r a t o h . v i l , J . P . , W.G. M a r t i n and W.K. Cook. L S 6 2 . M o l e c u l a r -w e i g h t and d i s s o c i a t i o n b e h a v i o r o f a- and B-l i p o v i t e l l i n i n a c i d s o l v e n t s . Can. J . B i o c h e m . P h y s i o l . 40_, 877 . L u z z o t i , V. 19 68. X - r a y d i f f r a c t i o n ' s t u d i e s o f l i p i d - w a t e r s y s t e m s . I n B i o l o g i c a l Membranes. " P h y s i c a l F a c t a n d F u n c t i o n . Ec.. , D. Chapman, P u b . , A c a d e m i c P r e s s . 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