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A biochemical investigation into the mechanism of hypercatabolism of high density lipoprotein in Tangier… Samborski, Rockford William 1987

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A BIOCHEMICAL INVESTIGATION INTO THE MECHANISM OF HYPERCATABOLISM OF HIGH DENSITY LIPOPROTEIN IN TANGIER DISEASE by ROCKFORD WILLIAM SAMBORSKI B . S c , Simon F r a s e r U n i v e r s i t y , 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA January 21 1987 (c)Rockford W. Samborski, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia 1956 Main Mall Vancouver, Canada Department V6T 1Y3 DE-6(3/81) ABSTRACT T h i s study was designed t o i n v e s t i g a t e the mechanism(s) u n d e r l y i n g the hypercatabolism of h i g h d e n s i t y l i p o p r o t e i n i n T a n g i e r d i s e a s e (TD). I n i t i a l l y , the metabolism of normal HDL incubated i n Tangier plasma i n v i t r o was examined. S u f f i c i e n t normal human HDL was added t o TD plasma to r a i s e the c o n c e n t r a t i o n of H D L - c h o l e s t e r o l t o w i t h i n normal l e v e l s . During i n c u b a t i o n the c o n c e n t r a t i o n of H D L - c h o l e s t e r o l i n the TD plasma f e l l by up t o 50% i n a time dependent manner. T h i s was not seen i n c o n t r o l samples t r e a t e d i n a s i m i l a r manner. The l o s s of HDL-c h o l e s t e r o l i n the TD c o u l d be completely accounted f o r by the l o s s of H D L - c h o l e s t e r y l e s t e r and was accompanied by a 2 . 3 - f o l d i n c r e a s e i n the c o n c e n t r a t i o n o f H D L - t r i g l y c e r i d e . These o b s e r v a t i o n s c o u l d not be accounted f o r by l e c i t h i n : c h o l e s t e r o l a c y t r a n s f e r a s e a c t i v i t y , c h o l e s t e r y l e s t e r h y d r o l y s i s , or the. ~ t r i g l y c e r i d e l e v e l i n the TD plasma. However, p r e l i m i n a r y evidence suggested t h a t the a c t i v i t y o f c h o l e s t e r y l e s t e r t r a n s f e r p r o t e i n i n TD plasma i s r e s p o n s i b l e f o r the changes i n H D L - l i p i d composition. The r e s u l t i n g t r i g l y c e r i d e - r i c h , c h o l e s t e r y l - p o o r HDL was shown to have a normal a f f i n i t y f o r the human s k i n f i b r o b l a s t HDL r e c e p t o r . However, t h i s f i n d i n g does not exclude o t h e r pathways of HDL c a t a b o l i s m t h a t may c o n t r i b u t e t o the r a p i d t u r n o v e r o f m o d i f i e d HDL i n TD plasma. The metabolism of normal HDL by TD f i b r o b l a s t s and monocytes i n v i t r o was a l s o s t u d i e d i n an attempt t o i d e n t i f y a c e l l u l a r d e f e c t of HDL metabolism i n TD. However, both TD f i b r o b l a s t s and monocytes were normal with r e s p e c t to t h e i r a b i l i t y t o b i n d / i n t e r n a l i z e and degrade normal HDL i n v i t r o . I t i s concluded t h a t the hypercatabolism o f normal HDL i n TD i n v o l v e s a l t e r a t i o n s of H D L - l i p i d and p r o t e i n c o m p o s i t i o n p r i o r t o removal from the plasma component. Thus, t h e s e s t u d i e s support the hypothesis t h a t the d e f e c t i n TD r e s i d e s i n the plasma and not i n the c e l l s o f these p a t i e n t s . TABLE OF CONTENTS ABSTRACT i TABLE OF CONTENTS i i i ABBREVIATIONS x i i LIST OF TABLES v i i i LIST OF FIGURES ix ACKNOWLEDGEMENTS xi -DEDICATION x i v 1 INTRODUCTION 1.1 I n t r o d u c t i o n 1 1.2 L i p o p r o t e i n s 4 1.2.1 I n t r o d u c t i o n 4 1.2.2 A p o l i p o p r o t e i n s 6 1.2.3 L i p o p r o t e i n s t r u c t u r e 9 1.3 L i p o p r o t e i n metabolism 9 1.3.1 I n t r o d u c t i o n 9 1.3.2 L i p o p r o t e i n b i o g e n e s i s 11 1.3.3 L i p o p r o t e i n c a t a b o l i s m ....16 1.3.3.1 Exchange and t r a n s f e r r e a c t i o n s 16 1.3.3.2 Enzymatic m o d i f i c a t i o n 17 1.3.3.3 C e l l - l i p o p r o t e i n i n t e r a c t i o n 19 1.4 Reverse c h o l e s t e r o l t r a n s p o r t 2 6 1.5 Tang i e r d i s e a s e 28 1.5.1 I n t r o d u c t i o n 2 8 1.5.2 H i s t o r y ..28 1.5.3 C l i n i c a l f e a t u r e s 29 1.5.4 Biochemical f e a t u r e s 31 1.6 Biochemical d e f e c t i n T a n g i e r d i s e a s e 33 i i i 1.7 R a t i o n a l e f o r t h i s study 36 1.8 S p e c i f i c aims.... 36 2 MATERIALS AND METHODS 2.1.1 C o n t r o l s u b j e c t s 37 2.1.2 T a n g i e r p a t i e n t 37 2.2 M a t e r i a l 40 2.3 L i p o p r o t e i n s 41 2.3.1 I s o l a t i o n of l i p o p r o t e i n s 41 2.3.1.1 P r e p a r a t i v e u l t r a c e n t r i f u g a t i o n . 41 2.3.1.2 Heparin-Magnesium p r e c i p i t a t i o n ....42 2.3.2 I o d i n a t i o n of l i p o p r o t e i n s 42 2.3.2.1 P r e p a r a t i o n of 1 2 5 I - l i p o p r o t e i n s 42 2.3.2.2 C h a r a c t e r i z a t i o n o f 1 2 5 I - l i p o p r o t e i n s 43 2.3.3 [ 3H] l a b e l i n g o f plasma and l i p o p r o t e i n s 44 2.3.3.1 G l y c e r y l t r i [ 3 H ] o l e a t e l a b e l i n g o f VLDL 44 2.3.3.2 [ 3H] c h o l e s t e r o l l a b e l i n g o f plasma l i p o p r o t e i n s 44 2.3.3.3 [ 3H] c h o l e s t e r y l e s t e r l a b e l i n g o f plasma and HDL ...45 2.3.4 C h a r a c t e r i z a t i o n of L i p o p r o t e i n s . . 45 2.3.4.1 Determination of l i p o p r o t e i n p a r t i c l e s i z e 45 2.3.4.2 L i p o p r o t e i n a n a l y s i s 46 i v 2.3.5 Enzyme Assays 4 6 2.3.5.1 N o n - s p e c i f i c plasma h y d r o l y s i s of c h o l e s t e r y l e s t e r s 4 6 2.3.5.2 Bovine m i l k l i p o p r o t e i n l i p a s e 47 2.4 C e l l c u l t u r e 47 2.4.1 I s o l a t i o n and c u l t u r e o f human f i b r o b l a s t s 47 2.4.2 P r e p a r a t i o n of c e l l l i n e s p r i o r t o l i p o p r o t e i n b i n d i n g experiments ..49 2.4.3 C u l t u r e of human p e r i p h e r a l b l o o d monocytes 49 2.5 Metabolism of l i p o p r o t e i n s by c u l t u r e d c e l l s i n v i t r o 51 2.5.1 1 2 5 I - L D L metabolism by f i b r o b l a s t s 51 2.5.2 1 2 5 I - H D L metabolism by f i b r o b l a s t s 52 2.5.3 1 2 5 I - H D L 3 metabolism by monocytes.. 52 3 RESULTS 3.1 Catabolism o f normal HDL by T a n g i e r plasma In v i t r o 53 3.1.1 E f f e c t of i n c u b a t i o n a t 37°C on l i p o p r o t e i n e l e c t r o p h o r e t i c m o b i l i t y i n v i t r o 53 3.1.2 E f f e c t o f i n c u b a t i o n a t 37 °C on HD L - c h o l e s t e r o l composition i n v i t r o 56 3.1.3 E f f e c t o f i n c u b a t i o n a t 37°C on l i p o p r o t e i n l i p i d c o mposition i n v i t r o 59 3.1.4 E f f e c t o f i n c u b a t i o n a t 37°C on HDL a p o p r o t e i n composition i n v i t r o 62 v 3.2 I n v e s t i g a t i o n of mechanisms u n d e r l y i n g the m o d i f i c a t i o n of normal HDL d u r i n g i n c u b a t i o n i n TD plasma 70 3.2.1 Role of c h o l e s t e r y l e s t e r h y d r o l y s i s 70 3.2.2 Role o f LCAT 72 3.2.3 Role o f h y p e r t r i g l y c e r i d e m i a 74 3.3 Metabolism o f HDLprj by c u l t u r e d s k i n f i b r o b l a s t i n v i t r o 76 3.3.1 I n t r o d u c t i o n 76 3.3.2 Metabolism o f HDLpp by s k i n f i b r o b l a s t s 76 3.4 Metabolism o f L D L r r . D by c u l t u r e d s k i n f i b r o b l a s t s i n v i t r o ....84 3.5 Catabolism o f VLDLpp by Bovine m i l k l i p o p r o t e i n l i p a s e i n v i t r o 90 3.6 Catabolism o f normal HDL by T a n g i e r f i b r o b l a s t s and monocytes i n c u l t u r e 97 3.6.1 T a n g i e r f i b r o b l a s t s 97 3.6.2 T a n g i e r monocytes ..100 4 DISCUSSION 4.1 M o d i f i c a t i o n of normal HDL by TD Plasma 103 4.2 P o s s i b l e f a c t o r s i n v o l v e d i n the m o d i f i c a t i o n of HDL 108 4.3 The metabolism of TD and TD m o d i f i e d l i p o p r o t e i n s by normal f i b r o b l a s t s i n v i t r o 112 4.3.1 The metabolism of H D L r r . D by normal f i b r o b l a s t s i n v i t r o 112 4.3.2 The metabolism o f LDLpp by normal f i b r o b l a s t s i n v i t r o 113 v i The l i p o l y s i s of VLDLpp by BmLpL i n v i t r o A c e l l u l a r d e f e c t of HDL c a t a b o l i s m i n TD v i i LIST OF TABLES Table I C l a s s i f i c a t i o n of plasma l i p o p r o t e i n s 5 Table I I Major plasma a p o l i p o p r o t e i n s 7 Table I I I C l i n i c a l p r e s e n t a t i o n of TD. . ; 30 Table IV L i p i d and p r o t e i n composition o f c o n t r o l , TD plasma and TD plasma c o n t a i n i n g exogenous HDL 53 Table V E f f e c t o f 37°C i n c u b a t i o n on HDL compositon f o l l o w i n g the a d d t i o n of normal HDL t o TD plasma 60 Table VI E f f e c t of 37°C i n c u b a t i o n on HDL a p o l i p o p r o t e i n s f o l l o w i n g the a d d i t i o n of normal HDL t o TD plasma 67 Table VII Role o f CE h y d r o l y s i s i n m o d i f i c a t i o n process 71 Table V I I I Role o f LCAT i n m o d i f i c a t i o n process 73 Table IX Chemical composition o f LDLpQ ...85 Table X LDL k i n e t i c b i n d i n g parameters 88 Table XI Chemical composition of V L D L ^ 93 v i i i LIST OF FIGURES Fi g u r e 1. S t r u c t u r e of l i p o p r o t e i n 10 Figu r e 2. Overview of c h o l e s t e r o l metabolism 12 F i g u r e 3. Route of the LDL r e c e p t o r i n mammalian c e l l s 23 F i g u r e 4. TD p a t i e n t (JKW) f a m i l y t r e e 39 F i g u r e 5. E f f e c t of 37° i n c u b a t i o n on l i p o p r o t e i n e l e c t r o p h o r e s i s p a t t e r n f o l l o w i n g the a d d i t i o n of normal HDL t o TD plasma 55 F i g u r e 6. The r e l a t i v e t o t a l c h o l e s t e r o l c o n t e n t o f normal HDL incubated i n c o n t r o l or TD plasma 58 F i g u r e 7. Gel f i l t r a t i o n chromatography o f 1 2 5 I - H D L - a p o p r o t e i n s f o l l o w i n g i n c u b a t i o n a t 4°C or 37°C 63 F i g u r e 8. Gel f i l t r a t i o n o f [ 3H] c h o l e s t e r o l l a b e l l e d TD plasma c o n t a i n i n g HDL f o l l o w i n g i n c u b a t i o n a t 4° and 37° 68 F i g u r e 9. Gel f i l t r a t i o n chromatography o f 1 2 5 j _ H D L f o l l o w i n g i n c u b a t i o n i n TD plasma a t 4° and 37° 69 F i g u r e 10. Role of h y p e r t r i g l y c e r i d e m i a i n the m o d i f i c a t i o n p r o c e s s 75 F i g u r e 11. 10% SDS - P A G E of 1 2 5 I - H D L T D and 1 2 5 I - H D L 3 7 79 F i g u r e 12a. T o t a l b i n d i n g o f 1 2 5 I - H D L T D and 1 2 5 I - H D L 3 7 to normal f i b r o b l a s t s 80 F i g u r e 12b. S p e c i f i c b i n d i n g o f 1 2 5 I - H D L T D and 1 2 5 I - H D L 3 7 to normal f i b r o b l a s t s 81 i x F i g u r e 13. Scatchard a n a l y s i s of data from F i g u r e 12b...82 F i g u r e 14. Degradation of 1 2 5 I - H D L T D and 1 2 5 I - H D L 3 7 by normal f i b r o b l a s t s 83 F i g u r e 15. Bindi n g and I n t e r n a l i z a t i o n o f 1 2 5 I - l a b e l l e d LDI*^ and c o n t r o l LDL by normal f i b r o b l a s t s 8 6 F i g u r e 16. Degradation of 1 2 5 I - l a b e l l e d LDLpQ and c o n t r o l LDL by normal f i b r o b l a s t s 89 F i g u r e 17. Determination o f VLDL s i z e and d i s t r i b u t i o n by QLS 91 F i g u r e 18a. The h y d r o l y s i s o f VLDL by BmLpL 95 F i g u r e 18b. Eadie-Hofstee a n a l y s i s of data from F i g u r e 18a... 9 6 F i g u r e 19. Bi n d i n g o f 1 2 5 I - H D L 3 t o TD and c o n t r o l f i b r o b l a s t s .98 F i g u r e 20. Scatchard a n a l y s i s o f da t a from F i g u r e 19....99 F i g u r e 21. Bi n d i n g o f 1 2 5 I - H D L 3 t o TD and c o n t r o l monocytes du r i n g a 4 hour i n c u b a t i o n a t 37°C 101 F i g u r e 22. Bi n d i n g o f 1 2 5 I - H D L 3 t o TD and c o n t r o l monocytes d u r i n g a 20 hour i n c u b a t i o n a t 37°C......... 102 x ACKNOWLEDGEMENTS F i r s t and foremost I wish t o thank my s u p e r v i s o r Dr. Haydn P r i t c h a r d f o r h i s p a t i e n c e , guidance, enthusiasm and f r i e n d s h i p d u r i n g the course o f these s t u d i e s . I a l s o wish t o thank Dr. J . F r o h l i c h , Dr. D. Seccombe, Dr. N. J e t h a , Dr. W. L. Dunn, Dr. N. Urquhart, Mr. R.Mcleod, Mr. C.Yeung, and Mr. D. Sparks f o r t h e i r i n t e r e s t and sugg e s t i o n s c o n c e r n i n g the work pre s e n t e d i n t h i s t h e s i s . I am inde b t e d t o the e n t i r e s t a f f of the C l i n i c a l Chemistry Department, Hematology Department and M i c r o b i o l o g y Department of Shaughnessy H o s p i t a l without whose h e l p t h i s t h e s i s would not be p o s s i b l e . I am a l s o indebted t o the B r i t i s h Columbian Heart Foundation f o r f i n a n c i a l support i n the form o f a student t r a i n e e s h i p . x i ABBREVIATIONS apo A p o l i p o p r o t e i n BmLpL Bovine m i l k l i p o p r o t e i n l i p a s e B m a x Maximum b i n d i n g determined by Scatchard a n a l y s i s BSA Bovine serum albumin CE C h o l e s t e r y l e s t e r ( s ) CEH CE h y d r o l y s i s CETP C E - t r a n s f e r p r o t e i n CHD. Coronary h e a r t d i s e a s e FC Free c h o l e s t e r o l FED... Fish-eye d i s e a s e FH F a m i l i a l h y p e r c h o l e s t e r o l e m i a FP F i c o 11 - Paque HDL High d e n s i t y l i p o p r o t e i n HDL-C. ....HDL-cholesterol HDLrppj. . T a n g i e r m o d i f i e d HDL HDL37 Normal HDL i s o l a t e d from plasma a f t e r b e i n g incubated a t 37°C f o r 24 h. HTGL. Hepatic t r i g l y c e r i d e l i p a s e Hypoalpha. F a m i l i a l h y p o a l p h a l i p o p r o t e i n e m i a IDL. Intermediate d e n s i t y l i p o p r o t e i n s . E q u i l i b r i u m d i s s o c i a t i o n constant K^j Michaelis-menton c o n s t a n t LBTI Lima bean t r y p s i n i n h i b i t o r LCAT L e c i t h i n : c h o l e s t e r o l a c y l t r a n s f e r a s e LDL. Low d e n s i t y l i p o p r o t e i n LDL-C L D L - c h o l e s t e r o l x i i L D L T D T a n g i e r LDL (1.006 t o 1.063 g/ml) LPDP L i p o p r o t e i n d e f i c i e n t human plasma LPDS L i p o p r o t e i n d e f i c i e n t fe'tal c a l f serum LpL L i p o p r o t e i n l i p a s e LTP L i p i d t r a n s f e r p r o t e i n MEM..... M o d i f i e d E a gles medium PAGE . . . i P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s PBS Phosphate b u f f e r e d s a l i n e P l Phosphol i p i d QLS.. . . . . Q u a s i e l a s t i c l a s e r l i g h t s c a t t e r i n g RCT .Reverse c h o l e s t e r o l t r a n s p o r t SDS Sodium d o d e c y l s u l p h a t e TCA T r i c h l o r o a c e t i c a c i d TD T a n g i e r d i s e a s e TG T r i g l y c e r i d e TLC T h i n l a y e r chromatography VLDL... Very low d e n s i t y l i p o p r o t e i n V L D L T D TD VLDL V m a x Maximum v e l o c i t y o f r e a c t i o n WBC White b l o o d c e l l s x i i i To Mr. Ken W i l l i a m s DEDICATION x i v 1 INTRODUCTION 1.1 I n t r o d u c t i o n T h i s year, as i n o t h e r s b e f o r e i t , more Canadians w i l l d i e from coronary h e a r t d i s e a s e (CHD) than from any o t h e r s i n g l e d i s e a s e (1). I t i s f o r t h i s reason t h a t so much time, e f f o r t , and money have been d e d i c a t e d t o the study of the mechanisms which u n d e r l y CHD. The m u l t i f a c t o r i a l nature of t h i s d i s o r d e r i s w e l l known and has r e c e n t l y been reviewed (2). Though f a c t o r s such as smoking, h y p e r t e n s i o n and o b e s i t y c o n t r i b u t e t o i t s p r o g r e s s i o n , i t i s the l e v e l of c h o l e s t e r o l i n plasma t h a t i s c o n s i d e r e d t o be the major a t h e r o g e n i c f a c t o r (3). S t u d i e s have shown t h a t the i n c i d e n c e of CHD i s p o s i t i v e l y c o r r e l a t e d w i t h the plasma l e v e l s of low d e n s i t y l i p o p r o t e i n (LDL) c h o l e s t e r o l (4,5). While i n c r e a s e d l e v e l s of LDL c h o l e s t e r o l p r e d i s p o s e s i n d i v i d u a l s t o CHD, plasma h i g h d e n s i t y l i p o p r o t e i n (HDL) c h o l e s t e r o l has been shown t o be i n v e r s e l y r e l a t e d t o CHD i n c i d e n c e (4,5). The r e c e n t -r e p o r t of the L i p i d Research C l i n i c Program has confirmed the p r o t e c t i v e a c t i o n of HDL a g a i n s t CHD and f u r t h e r e s t a b l i s h e d the importance o f H D L - c h o l e s t e r o l metabolism i n CHD (6). Thus, the c e n t r a l q u e s t i o n i n l i p o p r o t e i n r e s e a r c h i s : "How does LDL p r e d i s p o s e , and HDL p r o t e c t , an i n d i v i d u a l from a t h e r o s c l e r o s i s ? " The mechanisms which r e g u l a t e LDL metabolism and c e l l u l a r c h o l e s t e r o l homeostasis have been w e l l s t u d i e d . A major c o n t r i b u t i o n t o t h i s f i e l d was made d u r i n g the mid 70's by Brown and G o l d s t e i n (7). T h e i r work, f o r which they j o i n t l y r e c e i v e d the 1985 Nobel p r i z e i n P h y s i o l o g y or Medicine, was c e n t e r e d on understanding the b i o c h e m i c a l d e f e c t i n f a m i l i a l h y p e r c h o l e s t e r o l e m i a (FH) (7,8,9). B r i e f l y , Brown and G o l d s t e i n 1 showed t h a t i n normal i n d i v i d u a l s LDL i s c l e a r e d from plasma by c e l l s v i a a s p e c i f i c plasma membrane r e c e p t o r , and t h a t t h i s r e c e p t o r i s d e f e c t i v e i n p a t i e n t s with FH. LDL accumulates i n the plasma of these p a t i e n t s and p r e d i s p o s e s them t o CHD a t an e a r l y age. The r e s u l t s of t h i s work has ..greatly i n c r e a s e d our knowledge of the r o l e of t h i s l i p o p r o t e i n i n the a t h e r o s c l e r o t i c p r o c e s s . The mechanism by which HDL p r o t e c t s a g a i n s t CHD i s not as w e l l understood. However, i t i s known t h a t HDL p l a y s a c e n t r a l r o l e i n the m o b i l i z a t i o n of c h o l e s t e r o l from c e l l s and i t s t r a n s f e r t o the l a r g e r l i p o p r o t e i n s . T h i s p r o c e s s i s r e f e r r e d to as " r e v e r s e c h o l e s t e r o l t r a n s p o r t " and i s d i s c u s s e d i n more d e t a i l i n S e c t i o n 1.4. The c u r r e n t knowledge of HDL metabolism has been advanced by s t u d i e s i n a group of p a t i e n t s who, i n the absence o f pronounced h y p e r t r i g l y c e r i d e m i a , p r e s e n t with a f a m i l i a l form o f HDL d e f i c i e n c y . These p a t i e n t s are c l a s s i f i e d i n t o one o f the f o l l o w i n g c a t e g o r i e s (10): 1) F a m i l i a l apo A - I / C - I I I d e f i c i e n c y . 2) HDL d e f i c i e n c y w i t h p l a n a r xanthomas. 3) Apo A - I M i l a n o . 4) F i s h - e y e d i s e a s e (FED). 5) F a m i l i a l h y p o a l p h a l i p o p r o t e i n e m i a (Hypoalpha). 6) T a n g i e r d i s e a s e (TD). While a l l of these p a t i e n t s p r e s e n t w i t h d e f i c i e n c y o f HDL, not a l l of them are a t r i s k f o r a t h e r o s c l e r o s i s . While the i n d i v i d u a l s w i t h apo A - I / C - I I I d e f i c i e n c y and hypoalpha develop e a r l y a t h e r o s c l e r o s i s those w i t h FED or Apo A - I M ^ l a n o do not 2 s u f f e r from premature v a s c u l a r d i s e a s e . Thus, w h i l e some p a t i e n t s with HDL d e f i c i e n c y are g r e a t l y p r e d i s p o s e d t o h e a r t d i s e a s e o t h e r s f u n c t i o n q u i t e w e l l without any a p p r e c i a b l e q u a n t i t i e s of HDL. T h e r e f o r e , i t i s of g r e a t importance t h a t we understand: 1) The primary d e f e c t ( s ) l e a d i n g t o H D L - d e f i c i e n c y i n these p a t i e n t s , and 2) The compensatory (adaptive) mechanisms which prevent development of a t h e r o s c l e r o s i s i n some of these d i s o r d e r s . While r e c e n t work has shown t h a t the HDL d e f i c i e n c y i n F a m i l i a l apo A - I / C - I I I d e f i c i e n c y (11), and apo A-I M j L x a n o (12) are a s s o c i a t e d w i t h a b n o r m a l i t i e s of t h e apo A-I gene, the p r e c i s e m o l e c u l a r d e f e c t s i n most o f these d i s o r d e r s remain unknown. I f we want t o understand the r e l a t i o n s h i p between HDL metabolism and a t h e r o s c l e r o s i s i t i s e s s e n t i a l t h a t the primary b i o c h e m i c a l d e f e c t s u n d e r l y i n g f a m i l i a l H D L - d e f i c i e n c y d i s o r d e r s be e l u c i d a t e d . For the p a s t 3 years, the Shaughnessy H o s p i t a l L i p i d Research Group has been s t u d y i n g the o n l y p a t i e n t i n Canada wi t h TD and i t i s t h i s p a t i e n t on whom the work i n t h i s t h e s i s i s c e n t e r e d upon. TD i s a r a r e d i s o r d e r o f plasma l i p i d t r a n s p o r t t h a t i s reviewed i n S e c t i o n 1.5. Though t h i s d i s o r d e r has o n l y been d e s c r i b e d i n t h i r t y t h r e e p a t i e n t s world wide i t s s i g n i f i c a n c e i n understanding CHD i s o b v i o u s l y tremendous. I t i s the purpose o f t h i s r e s e a r c h t o i n v e s t i g a t e the b i o c h e m i c a l d e f e c t i n TD i n the hope t h a t the r e s u l t s w i l l c o n t r i b u t e t o our knowledge o f HDL metabolism i n TD p a t i e n t s and l e a d t o g r e a t e r u nderstanding o f HDL metabolism i n normal i n d i v i d u a l s . In the f o l l o w i n g pages I s h a l l b r i e f l y review the c u r r e n t 3 knowledge of l i p o p r o t e i n s t r u c t u r e , f u n c t i o n and metabolism i n the normal i n d i v i d u a l and then the c u r r e n t l i t e r a t u r e on t o TD and i t s l i p o p r o t e i n a b n o r m a l i t i e s . In c l o s i n g I w i l l d i s c u s s the p o s s i b l e d e f e c t ( s ) i n T a n g i e r d i s e a s e and the o b j e c t i v e s of t h i s r e s e a r c h . 1.2 L i p o p r o t e i n s 1.2.1 I n t r o d u c t i o n Due t o the hydrophobic nature of l i p i d s , t h e i r s o l u b i l i t y i n water i s v e r y low. As a consequence, t h e i r t r a n s p o r t i n b l o o d i s d i f f i c u l t . However, a system f o r i n c r e a s i n g the s o l u b i l i t y of l i p i d s i n b l o o d has been developed by v e r t e b r a t e s and i t i s known c o l l e c t i v e l y as plasma l i p o p r o t e i n s . L i p o p r o t e i n s accomplish t h i s f u n c t i o n by s u r r o u n d i n g m i c e l l e s of n e u t r a l l i p i d w i t h a h y d r o p h i l i c s h e l l c o n s i s t i n g mainly of c h o l e s t e r o l , p h o s p h o l i p i d and p r o t e i n molecules. The p r o t e i n c o n s t i t u e n t s c a l l e d a p o l i p o p r o t e i n s (apo), range i n m o l e c u l a r weight from 7 t o 350 kDa and are l o c a l i z e d on the s u r f a c e of the l i p o p r o t e i n p a r t i c l e (13). The l i p i d components of the l i p o p r o t e i n s c o n s i s t of v a r i o u s amounts of p h o s p h o l i p i d (PL), t r i g l y c e r i d e (TG), f r e e c h o l e s t e r o l (FC) and c h o l e s t e r y l e s t e r (CE). L i p o p r o t e i n s can be c l a s s i f i e d on the b a s i s o f t h e i r h y d r a t e d d e n s i t y , f l o a t a t i o n r a t e , e l e c t r o p h o r e t i c m o b i l i t y , p a r t i c l e s i z e , m e t a b o l i c f u n c t i o n and p r o t e i n c o n s t i t u e n t s . C u r r e n t l y , l i p o p r o t e i n s are most commonly c l a s s i f i e d i n t o f o u r major groups on the b a s i s of t h e i r hydrated d e n s i t y (Table I ) . In t h i s c l a s s i f i c a t i o n system t h e r e i s an o v e r l a p of p h y s i c a l p r o p e r t i e s and m e t a b o l i c f u n c t i o n ; Chylomicrons c a r r y d i e t a r y 4 TABLE 1 CLASSIFICATION OF LIPOPROTEINS IN THEIR HYDRATED STATE (reproduced from r e f 13) L i p o p r o t e i n c l a s s D e n s i t y (gm/ml) Diameter (nm) Mean mol e c u l a r weight E l e c t r o -p h o r e t i c m o b i l i t y Chylomicrons <0.94 1 0 2 - 1 0 3 5 . 0 X 1 0 9 o r i g a n VLDL 0.94 -1.006 30-70 7.5X10 6 pre-beta LDL 1.006-1.063 15-25 2.5X10 6 beta HDL 2 1.063 -1.125 6-14 3 . 9 X 1 0 5 alpha HDL 3 1.125-1 .21 6 - 1 0 1 . 9 X 1 0 5 alpha TG's from the e n t e r o c y t e s t o e x t r a h e p a t i c t i s s u e f o r u t i l i z a t i o n or storage; very low d e n s i t y l i p o p r o t e i n s (VLDL) c o n t a i n TG's s y n t h e s i z e d i n the l i v e r ; low d e n s i t y l i p o p r o t e i n s (LDL) d e r i v e d from VLDL c a t a b o l i s m and the h i g h d e n s i t y l i p o p r o t e i n s (HDL) are the major c a r r i e r s of plasma c h o l e s t e r o l . The c l a s s i f i c a t i o n of plasma l i p o p r o t e i n s on the b a s i s of t h e i r f l o a t a t i o n d e n s i t y may be i n a p p r o p r i a t e as t h e r e appears t o be c o n s i d e r a b l e h e t e r o g e n e i t y between p a r t i c l e s separated by t h i s method and those separated by o t h e r methods (14,15,16). In a d d i t i o n , work by Scanu e t a l have c l e a r l y shown t h a t t h e r e are important d i f f e r e n c e s i n the d e n s i t y ranges among s u b j e c t s and t h a t r i g o r o u s d e n s i t y l i m i t s are not u n i v e r s a l l y a p p l i c a b l e t o d e f i n e l i p o p r o t e i n s of i n d i v i d u a l s e r a (17). I t i s hoped t h a t w i t h the development of new methodologies f o r i s o l a t i n g , s e p a r a t i n g , and c h a r a c t e r i z i n g l i p o p r o t e i n s , the c o n f u s i o n surrounding l i p o p r o t e i n n o m e n c l a t u r e - w i l l be r e s o l v e d . However at pre s e n t t h i s system i s s t i l l the accepted standard i n l i p o p r o t e i n r e s e a r c h . 1.2.2 A p o l i p o p r o t e i n s The d i s t r i b u t i o n of the a p o l i p o p r o t e i n s i n human plasma i s w e l l known and has r e c e n t l y been reviewed by M o r r i s e t e t a l (18) and Smith e t a l (19). Table I I p r e s e n t s the d i s t r i b u t i o n and molecular weights of the major a p o l i p o p r o t e i n s . The o u t s t a n d i n g f e a t u r e of a l l a p o l i p o p r o t e i n s i s t h e i r amphipathic nature; the presence of both h y d r o p h i l i c and hydrophobic r e g i o n s i n the t e r t i a r y s t r u c t u r e of the molecules a l l o w s them t o i n t e r a c t w i t h both aqueous and l i p i d environments s i m u l t a n e o u s l y . A number of 6 TABLE I I APOLIPOPROTEINS OF THE HUMAN PLASMA LIPOPROTEINS (Reproduced from r e f . 9) A p o l i p o r o t e i n s M o l e c u l a r weight L i p o p r o t e i n (dalton) d i s t r i b u t i o n Apo A-I 28,331 HDL Apo A-II 17,380 HDL Apo B-48 200,000 Chylomicrons Apo B-100 350,000 VLDL, LDL Apo C-I 7,000 HDL, VLDL Apo C-II 8,837 Chylomicrons, VLDL, HDL Apo C-III 8,751 Chylomicrons, VLDL, HDL Apo D 32,500 HDL Apo E 34,145 Chylomicrons, VLDL, HDL 7 s p e c i f i c f u n c t i o n s f o r the a p o l i p o p r o t e i n s have been i d e n t i f i e d . These i n c l u d e : 1) C o f a c t o r f o r enzymes which m e t a b o l i z e l i p o p r o t e i n s , (apo C-II, l i p o p r o t e i n l i p a s e ; apo A-I, l e c i t h i n c h o l e s t e r o l a c y l t r a n s f e r a s e ) . 2) Ligand f o r i n t e r a c t i o n w i t h c e l l u l a r r e c e p t o r s , (apo B-100, LDL r e c e p t o r ; apo E, remnant r e c e p t o r ; apo A-I, HDL r e c e p t o r ) . 3) S t r u c t u r a l a p o l i p o p r o t e i n f o r l i p o p r o t e i n p a r t i c l e , (apo B-48 and apo B-100, chylomicrons and VLDL; apo A-I, HDL) . 4) L i p i d , t r a n s f e r / e x c h a n g e a c t i v i t y , (apo D). The primary amino a c i d sequences have been determined f o r most of these p r o t e i n s (20). Three a p o l i p o p r o t e i n s , apo A-I, apo .„.,,, A - I I , and apo C-II are s y n t h e s i z e d as p r e p r o a p o l i p o p r o t e i n s . The p r e p e p t i d e i s c o - t r a n s l a t i o n a l l y c l e a v e d , w h i l e the p r o p e p t i d e undergoes p o s t - t r a n s l a t i o n a l c l e a v a g e . In the case o f apo A-II c leavage occurs p r i m a r i l y i n t r a c e l l u l a r l y (21), w h ile f o r apo A-I (22) and apo C-II (23) cleavage o c c u r s p r i m a r i l y i n the plasma. In a d d i t i o n t o p r o t e o l y t i c cleavage, a p o l i p o p r o t e i n s may a l s o be m o d i f i e d p o s t - t r a n s l a t i o n a l l y by the a d d i t i o n of carbohydrates (20) or a c y l groups (24). The genomic s t r u c t u r e s of a number o f these a p o l i p o p r o t e i n s have been r e p o r t e d i n c l u d i n g those of apo A-I (25,26), apo A - I I (27), apo E (28), apo C-II (29), apo C - I I I (30) and most r e c e n t l y apo B-100 (31). I t appears t h a t a number of the a p o l i p o p r o t e i n genes have remarkably s i m i l a r s t r u c t u r e s and the observed 8 s i m i l a r i t y i n the s t r u c t u r a l o r g a n i z a t i o n of i n t r o n s and exons i n these genes have prompted the s p e c u l a t i o n t h a t they are e v o l u t i o n a r y r e l a t e d (27). 1.2.3 L i p o p r o t e i n S t r u c t u r e I n v e s t i g a t i o n of the composition, s t r u c t u r e , and p r o p e r t i e s of i n t a c t l i p o p r o t e i n s has been accomplished by u s i n g a v a r i e t y o f methods i n c l u d i n g n u c l e a r magnetic resonance s p e c t r o s c o p y , e l e c t r o n s p i n resonance spectroscopy, e l e c t r o n microscopy, immunochemistry and c r o s s - l i n k i n g experiments t o mention a few. None o f the i n f o r m a t i o n gathered from these s t u d i e s p e r m i t s a d e t a i l e d d e s c r i p t i o n of the plasma l i p o p r o t e i n s a t a m o l e c u l a r l e v e l due t o the dynamic nature of these p a r t i c l e . However, from t h i s i n f o r m a t i o n i t has been p o s s i b l e t o c o n s t r u c t models which do p r o v i d e i n s i g h t i n t o the m o l e c u l a r o r g a n i z a t i o n o f the main components of the plasma l i p o p r o t e i n s (17,19). A g e n e r a l f e a t u r e o f a l l models i s the l o c a l i z a t i o n of TG and CE i n the hydrophobic core of the p a r t i c l e which i s than surrounded by a monolayer of p r o t e i n , PL and FC (Figure 1.). For a more comprehensive review o f l i p o p r o t e i n s t r u c t u r e the r e a d e r i s r e f e r r e d t o Smith e t a l (19). 1.3 L i p o p r o t e i n Metabolism 1.3.1 I n t r o d u c t i o n C h o l e s t e r o l i s s y n t h e s i z e d from 2 carbon (acetate) u n i t s through a pathway i n v o l v i n g i n excess of 30 enzymes (32). D a i l y , t h e r e i s a f l u x of approximately 2500 mg of c h o l e s t e r o l i n t o the body. D i e t a r y sources account f o r about 20% w h i l e the remainder i s d e r i v e d from endogenous s y n t h e s i s (33). C h o l e s t e r o l i s 9 F i g u r e 1. S t r u c t u r e o f L i p o p r o t e i n s Model of human LDL showing the d i s t r i b u t i o n o f PL, FC, and p r o t e i n on the s u r f a c e of the p a r t i c l e and TG and CE i n the c o r e of the p a r t i c l e , [ r e p r i n t e d from r e f (179)]. 10 u t i l i z e d f o r a number of d i v e r s e purposes: 1) As a s t r u c t u r a l component of the plasma membranes, where i t modulates f l u i d i t y and maintains the b a r r i e r between the c e l l and i t s environment. 2) P r e c u r s o r f o r b i l e a c i d s . 3) P r e c u r s o r f o r s t e r o i d hormones. 4) P r e c u r s o r f o r v i t a m i n D. C h o l e s t e r o l i s not broken down i n the body but r a t h e r i s e x c r e t e d i n the f e c e s as b i l e s a l t s . Thus the i n f l u x o f c h o l e s t e r o l i n t o the body i s balanced by a p r o p o r t i o n a t e e f f l u x . C u r r ent understanding o f l i p o p r o t e i n metabolism i s o u t l i n e d i n F i g u r e 2. Sancta s i m p l i c i t a s two sepa r a t e pathways of c h o l e s t e r o l metabolism can be i d e n t i f i e d . The f i r s t d e a l s w i t h the metabolism of c h o l e s t e r o l d e r i v e d from the d i e t (exogenous pathway) and the second d e a l s w i t h the metabolism of c h o l e s t e r o l s y n t h e s i z e d i n the body (endogenous, pathway). There i s a g r e a t d e a l o f i n t e r a c t i o n between theses two pathways. The b i o g e n e s i s and c a t a b o l i s m o f i n d i v i d u a l l i p o p r o t e i n s w i l l be examined independently and F i g u r e 2 i s i n c l u d e d t o a i d i n the d i s c u s s i o n t h a t f o l l o w s . 1.3.2 L i p o p r o t e i n B i o g e n e s i s Chylomicrons: A f t e r b e i n g e m u l s i f i e d by b i l e s a l t s i n the l a r g e i n t e s t i n e , d i e t a r y f a t i s adsorbed, broken down and subsequently r e s y n t h e s i z e d by the i n t e s t i n a l e p i t h e l i u m . The e n t e r o c y t e s then s e c r e t e the TG i n t o the c i r c u l a t i o n as chylomicrons, which are than c a t a b o l i z e d t o remnant p a r t i c l e s and f i n a l l y c l e a r e d by the l i v e r as o u t l i n e d i n F i g u r e 2. 11 Exogenous Pathway Endogenous Pathway . Dietary Cholesterol frrrnrrnr Lipoprotein Lipase LDL Receptor ~ ' Extrahepatic Tissues Lipoprotein Lipase LCAT F i g u r e 2. Seperate pathways f o r recep t o r - m e d i a t e d metabolism o f l i p o p r o t e i n c a r r y i n g endogenous and exogenous c h o l e s t e r o l [ r e p r i n t e d from r e f ( 7 ) ] . 12 E l e c t r o n m i c r o s c o p i c , and b i o c h e m i c a l s t u d i e s have e l u c i d a t e d the pathway of chylomicron assembly and s e c r e t i o n i n the e n t e r o c y t e (34). In the lumen of the smooth endoplasmic r e t i c u l u m nascent chylomicrons are formed when the newly s y n t h e s i z e d TG are coated with PL and a p o l i p o p r o t e i n s (35). Apo B i s thought t o be e s s e n t i a l f o r the assembly of nascent T G - r i c h l i p o p r o t e i n t h a t can be s e c r e t e d by the c e l l (36,37). V e s i c l e s , c o n t a i n i n g the l i p i d d r o p l e t s , t r a v e l t o the G o l g i apparatus where the a p o l i p o p r o t e i n s are g l y c o s y l a t e d ( 9 ) . The nascent chylomicrons are then r e l e a s e d from the G o l g i apparatus i n s e c r e t o r y v e s i c l e s t h a t t r a v e l t o the l a t e r a l s u r f a c e of the c e l l where they, f u s e with the plasmalemma and r e l e a s e the chylomicrons i n t o the i n t e r c e l l u l a r space. The chylomicrons t r a v e l i n t o t h e c e n t r a l l a c t e a l s of the m i c r o v i l l i , flow through the lymphatics and v i a the t h o r a c i c duct e n t e r the c i r c u l a t i o n a t the l e v e l o f the j u g u l a r v e i n (9). Chylomicrons c o n t a i n apo A's; apo C s , apo E and the s m a l l e r form o f apo B (apo B-48). However, i t i s b e l i e v e d t h a t o n l y apo B-48 i s a s s o c i a t e d with the p a r t i c l e p r i o r t o i t s s e c r e t i o n i n lymph (9). O v e r a l l , the s e c r e t i o n of chylomicrons i s a d i r e c t f u n c t i o n of the r a t e of f a t a b s o r p t i o n (38) . Very Low D e n s i t y L i p o p r o t e i n s (VLDL): The l i v e r i s the major s i t e f o r s e c r e t i o n of l i p o p r o t e i n s c o n t a i n i n g endogenously d e r i v e d l i p i d s . TG i s s e c r e t e d by the l i v e r i n t o the c i r c u l a t i o n as VLDL p a r t i c l e s , which are formed i n s i d e the c e l l i n a s i m i l a r manner to chylomicrons. VLDL c o n t a i n apo B 1 Q 0 which d i f f e r s from B 4 8 a s s o c i a t e d with chylomicrons. As w i t h chylomicrons, VLDL's a l s o c o n t a i n a s m a l l amount of non-apo B a p o l i p o p r o t e i n s . Of t h e s e p r o t e i n , apo E and apo C s h a v e been shown, a l o n g w i t h B-100, t o be a s s o c i a t e d w i t h t h e n a s c e n t VLDL p a r t i c l e s a t t h e l e v e l o f G o l g i body ( 3 9 ) . V a r i a t i o n i n VLDL s y n t h e s i s i s m a i n l y t h e r e s u l t o f t h e t h e r a t e o f u p t a k e f a t t y a c i d s o r g l u c o s e f r o m the. b l o o d and t h e e x t e n t t o w h i c h t h e s e p r e c u r s o r s a r e u t i l i z e d f o r t h e h e p a t o c y t e s own n e e d s ( 4 0 ) . Low d e n s i t y L i p o p r o t e i n s ( L D L ) : P l a s m a LDL a r e p r i m a r i l y p r o d u c e d by t h e a c t i o n o f l i p o p r o t e i n l i p a s e and h e p a t i c t r i g l y c e r i d e l i p a s e on VLDL p a r t i c l e s i n v i v o (9 ). The f o r m a t i o n o f LDL f r o m VLDL i s a c c o m p l i s h e d by l o s s o f TG and P L and, p e r h a p s most c r i t i c a l l y , b y t h e l o s s o f apo E ( 4 1 ) . I n a d d i t i o n , r e c e n t e v i d e n c e t h a t s u g g e s t s t h a t some LDL may be d i r e c t l y s e c r e t e d by t h e l i v e r ( 4 2 ) . H i g h D e n s i t y L i p o p r o t e i n s (HDL): The p l a s m a HDL a r e a h e t e r o g e n o u s g r o u p o f p a r t i c l e s ( 4 3 ) . The g e n e s i s o f m a t u r e HDL i n v o l v e s t h e p r o d u c t i o n o f HDL p r e c u r s o r s , d e f i n e d a s b e i n g c o m p l e x e s o f a p o p r o t e i n / P L / F C . The p r o d u c t i o n o f t h e s e HDL p r e c u r s o r s i n v o l v e s t h r e e s e p a r a t e p r o c e s s e s : 1) d i r e c t s e c r e t i o n o f d i s c o i d a l h i g h d e n s i t y s t r u c t u r e s f r o m h e p a t i c and i n t e s t i n a l c e l l s ( " n a s c e n t " HDL p a r t i c l e s ) . 2) l i p i d and p r o t e i n c o n s t i t u e n t s r e l e a s e d f r o m l i p o l y z e d T G - r i c h l i p o p r o t e i n s ( " s u r f a c e r e m n a n t s " ) . 3) D i r e c t a s s o c i a t i o n o f PL w i t h f r e e a p o l i p o p r o t e i n A - I . The " n a s c e n t " HDL s t r u c t u r e s i d e n t i f i e d i n l i v e r p e r f u s a t e s and i n t e s t i n a l lymph> were i n i t i a l l y t h o u g h t t o be t h e m a j o r HDL p r e c u r s o r s (43) . However, t h e e x p e r i m e n t s upon w h i c h t h i s 14 h y p o t h e s i s i s based have come under sharp c r i t i c i s m (43) . The i n a b i l i t y t o i d e n t i f y such s t r u c t u r e s along s e c r e t o r y pathways and the f a c t t h a t LpL i s h i g h l y a c t i v e under the c o n d i t i o n s of the p e r f u s i o n experiments has l e a d t o support f o r the h y p o t h e s i s t h a t the "nascent" HDL p a r t i c l e s are the l i p o l y t i c products o f VLDL or chylomicrons. A n a l y s i s of the d i s c o i d a l p a r t i c l e s from l i v e r o r i n t e s t i n e show t h a t the a p o l i p o p r o t e i n composition o f these p a r t i c l e s i s s u p p o r t i v e of them b e i n g d e r i v e d from l i p o l y s i s o f the T G - r i c h l i p o p r o t e i n i n s i t u (43). Thus i t i s c u r r e n t l y b e l i e v e d t h a t d i s c o i d a l p r e c u r s o r s o r i g i n a t i n g from the s u r f a c e coat o f l i p o l y z e d T G - r i c h l i p o p r o t e i n s are the major, i f not the only, source o f HDL p r e c u r s o r s (43,44). M a t u r a t i o n of HDL p r e c u r s o r s t o s p h e r i c a l HDL i s dependent on the a c t i o n o f the plasma enzyme; l e c i t h i n : c h o l e s t e r o l a c y l t r a n s f e r a s e (LCAT). Hamilton has shown t h a t HDL p r e c u r s o r s formed in v i v o can be transformed t o s p h e r i c a l p a r t i c l e s by the LCAT r e a c t i o n i n v i t r o (45). In p a t i e n t s w i t h LCAT d e f i c i e n c y , where the mat u r a t i o n of p r e c u r s o r HDL i s absent, l a r g e amounts of d i s c o i d a l s t r u c t u r e s accumulate i n the p a t i e n t s plasma (46). Mature HDL e x i s t s i n plasma as t h r e e d i s t i n c t p o p u l a t i o n s ; HDL^, HDL 2, and HDL 3. HDL 2 and HDL 3 are the major HDL p o p u l a t i o n s p r e s e n t i n the plasma o f man. The main f e a t u r e s o f the two are summarized as f o l l o w s : 1) The core diameter o f HDL 2 i s approximately 50% l a r g e r than t h a t o f HDL 3 and thus c o n t a i n s 3 - 4 - f o l d more CE and TG than HDL 3. 2) The s u r f a c e area of HDL 2 i s 2 - f o l d g r e a t e r than HDL 3 and the d i f f e r e n c e i n p r o t e i n c o n t e n t i s about 50%. 15 HDL-^  i s l a r g e r than HDL 2 and i s r i c h i n apo E. HDL-^  i s p r e s e n t i n o n l y s m a l l amounts i n the plasma of man but i s p r e s e n t i n l a r g e q u a n t i t i e s i n r a t s (43) . A p o s s i b l e analogue of HDL-^  has been d e s c r i b e d i n the plasma of animals f e d d i e t s r i c h i n c h o l e s t e r o l and i s r e f e r r e d t o as HDL C- Recent evidence i n d i c a t e s t h a t c o n v e r s i o n between subspecies occurs (47). The mechanisms r e s p o n s i b l e f o r these c o n v e r s i o n s w i l l be d i s c u s s e d i n the f o l l o w i n g s e c t i o n . 1.3.3 L i p o p r o t e i n C a t a b o l i s m 1.3.3.1 Exchange and T r a n s f e r R e a c t i o n s The composition of the plasma l i p o p r o t e i n s i s c o n t i n u a l l y b e i n g m o d i f i e d i n v i v o as a r e s u l t of a dynamic exchange and net t r a n s f e r of l i p i d s between p a r t i c l e s and c e l l s . I n t e r e s t i n t h i s p r ocess has stemmed from the i n i t i a l o b s e r v a t i o n t h a t CE formed by the a c t i o n of LCAT i n the HDL's the major p o r t i o n of CE p r e s e n t i n the plasma i s a s s o c i a t e d w i t h the LDL's (48). T h i s o b s e r v a t i o n suggested t h a t a s p e c i f i c mechanism f o r i n t e r l i p o p r o t e i n t r a n s f e r of CE might e x i s t i n plasma. Evidence f o r such a p r o c e s s came i n 1975 when Z i l v e r s m i t e t a l r e p o r t e d the presence o f a s p e c i f i c l i p i d t r a n s f e r p r o t e i n (LTP) i n the plasma of h y p e r c h o l e s t e r o l e m i a animals (49). S i n c e then a number of r e p o r t s have been p u b l i s h e d (50) which have shown t h a t the t r a n s f e r of CE i s c a t a l y z e d by such p r o t e i n s as: 1) CE-TG t r a n s f e r p r o t e i n (CETP/LTP 1). 2) CE-PL t r a n s f e r p r o t e i n (LTP 2 ) . 3) Apo D. The exact mechanism of t r a n s f e r i s u n c l e a r but i t appears 16 t h a t TG and CE compete with each ot h e r f o r t r a n s f e r c a t a l y z e d by the LTP•s i n a c o n c e n t r a t i o n dependent f a s h i o n (51). The r e a c t i o n i s b i - d i r e c t i o n a l and seems t o be based on the exchange of CE f o r TG (51,52,53) or PL (53,54,55) between l i p o p r o t e i n p a r t i c l e s . The l i p i d t r a n s f e r p r o t e i n s appear t o be r e s p o n s i b l e f o r the con v e r s i o n o f HDL 2 t o HDL-^  (47) . 1 . 3 . 3 . 2 Enzymatic M o d i f i c a t i o n Enzymes, which modify plasma l i p i d s p l a y a key r o l e i n the i n t e r c o n v e r s i o n o f c i r c u l a t i n g l i p o p r o t e i n s . Three o f the most important o f these enzymes are: 1) L e c i t h i n : c h o l e s t e r o l a c y l t r a n s f e r a s e (LCAT; EC 2.3.1.43). 2) L i p o p r o t e i n l i p a s e (LpL; EC 3.1.1.34). 3) H e p a t i c t r i g l y c e r i d e l i p a s e (HTGL). .. LCAT i s an e x t r a c e l l u l a r enzyme of h e p a t i c o r i g i n t h a t c i r c u l a t e s i n the plasma and c a t a l y z e s the t r a n s f e r o f the C-2 1 f a t t y a c i d from p h o s p h a t i d y l c h o l i n e t o the 3•-hydroxy1 group of c h o l e s t e r o l (56). Human LCAT was f i r s t p u r i f i e d t o homogeneity by A l b e r s e t a l (57) and has been shown t o have an apparent M r of 65 kDa by sodium d o d e c y l s u l p h a t e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (SDS-PAGE: 57) or 59 kDa by sedime n t a t i o n e q u i l i b r i u m u l t r a c e n t r i f u g a t i o n (58). A g r e a t d e a l on the b i o c h e m i s t r y and pathophysiology o f the LCAT r e a c t i o n i s now a v a i l a b l e which c l e a r l y i n d i c a t e s the c e n t r a l r o l e t h a t LCAT p l a y s i n the pro c e s s of c h o l e s t e r o l m o b i l i z a t i o n from t i s s u e (56,59). I t i s g e n e r a l l y accepted t h a t LCAT, i n a s s o c i a t i o n w i t h HDL, form the c h o l e s t e r y l e s t e r i f i c a t i o n complex i n plasma (60). Though apo A-I i s the most common c o f a c t o r f o r the LCAT r e a c t i o n 17 (61) other l i p o p r o t e i n s such as apo C-I (62) and apo E (63) may a l s o a c t as c o f a c t o r s . The nature of the LCAT/HDL i n t e r a c t i o n i s not f u l l y understood and thus i s the s u b j e c t of numerous i n v e s t i g a t i o n s (64). In normal plasma, the number of LCAT molecules i s approximately 1 5 0 - f o l d l e s s than the amount of c i r c u l a t i n g HDL p a r t i c l e s (65). T h i s has l e a d t o the s p e c u l a t i o n t h a t a s m a l l subpopulation of HDL p a r t i c l e s e x i s t t h a t c o n t a i n the LCAT molecules (52) and t h a t these p a r t i c l e s are the major ac c e p t o r of c h o l e s t e r o l from p e r i p h e r a l c e l l membranes (9). However, the exact nature of these p a r t i c l e s has y e t t o be e l u c i d a t e d and i s c u r r e n t l y under i n v e s t i g a t i o n i n our l a b o r a t o r y . Recent evidence i n d i c a t e s t h a t the LCAT r e a c t i o n i s r e s p o n s i b l e f o r the c o n v e r s i o n of HDL 3 t o HDL 2 (47). . LpL i s an enzyme t h a t i s l o c a t e d on the l u m i n a l s u r f a c e of the e n d o t h e l i a l c e l l (66) and c a t a l y s e s the h y d r o l y s i s of d i - and t r i a c y l g l y c e r i d e s a s s o c i a t e d w i t h chylomicrons and VLDL. LpL i s -s p e c i f i c f o r primary e s t e r s but has no a b s o l u t e s t e r e o s p e c i f i c i t y . T h i s enzyme r e q u i r e s apo C-II as a c o f a c t o r . However, the exact mechanism by which apo C-II promotes t h i s r e a c t i o n i s unknown. LpL i s anchored t o the e n d o t h e l i a l membrane by a s p e c i f i c r e c e p t o r (66) and upon i n t r a v e n o u s i n j e c t i o n of h e p a r i n i s r e l e a s e d i n t o the plasma. LpL has been i s o l a t e d from a number of sources i n c l u d i n g bovine m i l k , r a t h e a r t and r a t adipose t i s s u e . LpL i s a g l y c o p r o t e i n w i t h s i m i l a r carbohydrate content but t h e r e are few o t h e r s i m i l a r i t i e s and many d i f f e r e n c e s e x i s t between the enzymes from d i f f e r e n t t i s s u e s . HTGL i s another e x t r a c e l l u l a r l i p o l y t i c enzyme t h a t i s s e c r e t e d by hepatocytes and i s r a p i d l y r e l e a s e d i n t o the l i v e r 18 p e r f u s a t e upon the a d d i t i o n of h e p a r i n i n t o the p e r f u s i o n medium (66). T h i s enzyme does not r e q u i r e any a p o l i p o p r o t e i n f o r l i p o l y t i c a c t i v i t y a g a i n s t T G - r i c h l i p o p r o t e i n s and i s immunologically d i s t i n c t from LpL. R e c e n t l y , Brown (67) has suggested t h a t HTGL i s e s s e n t i a l i n m e t a b o l i z i n g the remnants produced by LpL a c t i o n on VLDL, c o n v e r t i n g them t o LDL. In a d d i t i o n t o HTGL's r o l e i n the c a t a b o l i s m of T G - r i c h p a r t i c l e s i t has been shown t h a t HTGL p l a y s a c e n t r a l r o l e i n HDL metabolism; c o n v e r t i n g HDL 2 t o HDL 3 by h y d r o l y z i n g l i p o p r o t e i n PL (47). Thus through t h e i r c o n c e r t e d a c t i o n , LCAT, LpL and HTGL p l a y a key r o l e s i n the d e g r a d a t i o n , formation and remodeling of l i p o p r o t e i n p a r t i c l e s . 1 . 3 . 3 . 3 C e l l - L i p o p r o t e i n i n t e r a c t i o n s L i p o p r o t e i n s i n t e r a c t w i t h c e l l s e i t h e r through s p e c i f i c r e c e p t o r s or v i a a non-receptor mediated p r o c e s s e s (9). The non^ r e c e p t o r p r o c e s s e s of c e l l / l i p o p r o t e i n metabolism have not been w e l l c h a r a c t e r i z e d but i t would appear t h a t l i p o p r o t e i n s e n t e r the c e l l by b u l k phase e n d o c y t o s i s (68). C u r r e n t l y , the exact c o n t r i b u t i o n o f t h i s pathway t o the c l e a r a n c e o f most l i p o p r o t e i n s i s unknown. S t u d i e s on LDL c a t a b o l i s m suggest t h a t up t o t w o - t h i r d s of LDL c l e a r a n c e i s mediated through the LDL r e c e p t o r (69). The f i n d i n g s of D i e t s c h y e t a l support t h i s o b s e r v a t i o n , and f u r t h e r suggest t h a t the non-receptor mediated process c o n t r i b u t e s t o l e s s than 33% of LDL c l e a r a n c e (70). 19 In the f o l l o w i n g s e c t i o n , r e c e p t o r mediated c e l l - l i p o p r o t e i n i n t e r a c t i o n w i l l be d i s c u s s e d . The c r i t e r i a used f o r e s t a b l i s h i n g t h a t a l i p o p r o t e i n i s i n t e r a c t i n g with a c e l l r e c e p t o r i s as f o l l o w s (71): 1) The r e c e p t o r should demonstrate s p e c i f i c i t y . 2) The l i g a n d should b i n d t o a f i n i t e number of s p e c i f i c b i n d i n g s i t e s . 3) The l i g a n d should have a v e r y h i g h a f f i n i t y f o r t h e i r r e c e p t o r (Values f o r the e q u i l i b r i u m d i s s o c i a t i o n constant (K^) of 10" 9 t o 1 0 ~ 1 0 M are common). 4) The b i n d i n g o f the l i g a n d t o the r e c e p t o r s h o u l d mediate a p h y s i o l o g i c event, and the number of r e c e p t o r s occupied by the l i g a n d should be r e l a t e d t o the magnitude of the b i o l o g i c a l e f f e c t . 5) The number of r e c e p t o r s i s not con s t a n t and can be r e g u l a t e d by the absence or presence o f the l i g a n d . Chylomicrons: Catabolism o f chylomicrons by LpL r e s u l t s i n the p r o d u c t i o n o f remnant p a r t i c l e s t h a t may then be f u r t h e r c a t a b o l i z e d by the a c t i o n o f HTGL (9). The r e s u l t i n g c h y l o m i c r o n remnants c o n t a i n apo B-48 and apo E, but have l o s t t he major p o r t i o n of t h e i r apo C p r o t e i n s . The c l e a r a n c e o f remnant p a r t i c l e s by the l i v e r i s mediated by a s p e c i f i c , h i g h a f f i n i t y r e c e p t o r (71). Windier e t al have r e p o r t e d t h a t the apo E o f the chylomicron remnant i n t e r a c t s w i t h a s p e c i f i c h e p a t i c r e c e p t o r (41). T h i s p o s s i b i l i t y i s f u r t h e r supported by the o b s e r v a t i o n s t h a t : 1) apo B-48 does not i n t e r a c t w i t h t h i s r e c e p t o r and 20 2) t h a t p a t i e n t s with mutant phenotypes of apo E have a d e f e c t i v e a b i l i t y t o c l e a r , chylomicrons from t h e i r plasma (71). The e x p r e s s i o n of t h i s r e c e p t o r by the l i v e r does not appear t o be r e g u l a t e d and thus importance of t h i s r e c e p t o r t o l i v e r c h o l e s t e r o l homeostasis i s obscure (9). VLDL: VLDL are heterogenous i n s i z e , c o m p o s i t i o n and m e t a b o l i c f a t e (72). Large VLDL p a r t i c l e s l o s e the m a j o r i t y o f t h e i r apo C s and p i c k up apo E d u r i n g l i p o l y s i s . The r e s u l t i n g VLDL remnant p a r t i c l e are c l e a r e d by the l i v e r through i n t e r a c t i o n w i t h a s p e c i f i c , h i g h a f f i n i t y r e c e p t o r t h a t i s d i s t i n c t from the chylomicron remnant r e c e p t o r (72) . T h i s r e c e p t o r , which was i n i t i a l l y r e f e r r e d t o as the h e p a t i c apo B,E r e c e p t o r but i s known as the LDL r e c e p t o r , i s the major c a t a b o l i c pathway f o r VLDL e n d o c y t o s i s (72) . While the LDL r e c e p t o r can r e c o g n i z e e i t h e r apo E or apo B of,VLDL remnant p a r t i c l e s , the apo B of the l a r g e r VLDL p a r t i c l e s i s not r e c o g n i z e d by the LDL r e c e p t o r (72) . I t appears t h a t the apo E t r a n s f e r s from the HDL p o o l t o l a r g e r VLDL p a r t i c l e s o n l y a f t e r s i g n i f i c a n t l i p o l y s i s has o c c u r r e d (72) . T h i s mechanism prevents the l i v e r from q u i c k l y removing newly s y n t h e s i z e d VLDL's from the c i r c u l a t i o n . S t u d i e s i n p a t i e n t s who l a c k a f u n c t i o n a l apo B,E r e c e p t o r have shown t h a t t h e r e are o t h e r mechanisms f o r c l e a r i n g VLDL remnant p a r t i c l e s from c i r c u l a t i o n (68). There i s some evidence t h a t the chylomicron remnant r e c e p t o r may be i n v o l v e d i n the c l e a r a n c e of VLDL remnants but a t p r e s e n t the s i g n i f i c a n c e of t h i s pathway i n VLDL removal i s unknown. Large VLDL i s o l a t e d from a p a t i e n t w i t h h y p e r t r i g l y c e r i d e m i a have been shown t o i n t e r a c t 21 w i t h a s p e c i f i c B-VLDL r e c e p t o r p r e s e n t on macrophages (70). The s i g n i f i c a n c e o f t h i s r e c e p t o r i n normolipemic i n d i v i d u a l s has not yet been f u l l y e s t a b l i s h e d as l a r g e VLDL from normal i n d i v i d u a l s do not i n t e r a c t with the r e c e p t o r . LDL: The LDL r e c e p t o r i s a c e l l s u r f a c e g l y c o p r o t e i n of approximately 120 kDa which c y c l e s between the c e l l - s u r f a c e and the e n d o c y t i c v e s i c l e s (7). The LDL r e c e p t o r i s s y n t h e s i z e d i n the rough endoplasmic r e t i c u l u m and appears on the s u r f a c e of c e l l approximately 45 min l a t e r (7). The r e c e p t o r then migrates t o , and gathers i n , s p e c i f i c domains of the c e l l membrane c a l l e d coated p i t s (7). A n a l y s i s of the coated p i t s has shown t h a t a p r o t e i n ( c l a t h e r i n ) i s c l o s e l y a s s o c i a t e d w i t h the membrane and se r v e s as an anchor f o r the LDL r e c e p t o r s (7). LDL b i n d s t o the r e c e p t o r and t r i g g e r s a response t h a t l e a d s t o i n v a g i n a t i o n of the coated p i t and i n t e r n a l i z a t i o n o f the bound LDL. T h i s e n d o c y t i c v e s i c l e than fuses w i t h o t h e r v e s i c l e s g i v i n g r i s e t o an endosome. As the pH of the endosome begins t o f a l l below 6.5 the LDL d i s s o c i a t e s from the r e c e p t o r s and the LDL r e c e p t o r s c l u s t e r i n a segment of the endosome. The LDL r e c e p t o r c y c l e i s completed when t h i s segment o f membrane pinches o f f from the endosome and r e t u r n s the r e c e p t o r s t o the c e l l - s u r f a c e (7). The remaining endosome than fuses w i t h a primary lysosome and the LDL c o n s t i t u e n t s are h y d r o l y z e d . 22 F i g u r e 3. Route o f LDL r e c e p t o r i n mammalian c e l l s The LDL r e c e p t o r begins i t ' s c y c l e i n t h e ER from which i t t r a v e l s t o the G o l g i complex, c e l l s u r f a c e , coated p i t , endosome, and back t o the s u r f a c e . HMG CoA reductase denotes 3-hydroxy-3-m e t h y l g l u t a r y l CoA re d u c t a s e ; ACAT denotes acyl-CoA: c h o l e s t e r o l a c y l t r a n s f e r a s e . V e r t i c a l arrows i n d i c a t e the d i r e c t i o n of r e g u l a t o r y a f f e c t s . [ r e p r i n t e d from (7)] The LDL-derived c h o l e s t e r o l c o o r d i n a t e l y r e g u l a t e s the c e l l ' s c h o l e s t e r o l content by: 1) I n h i b i t i n g c e l l u l a r c h o l e s t e r o l s y n t h e s i s a t s e v e r a l l e v e l s : i ) Suppression of t r a n s c r i p t i o n o f HMG CoA r e d u c t a s e gene. i i ) A c c e l e r a t i o n o f the d e g r a d a t i o n of the above enzyme. 2) A c t i v a t i o n o f a c h o l e s t e r o l e s t e r i f y i n g enzyme, a c y l CoA: c h o l e s t e r o l a c y l t r a n s f e r a s e (ACAT). 3) Suppression o f the s y n t h e s i s o f the LDL r e c e p t o r by low e r i n g c e l l u l a r messenger RNA l e v e l s . The r o u t e of the LDL r e c e p t o r i n mammalian c e l l s and the e f f e c t o f LDL-derived c h o l e s t e r o l on i n t r a c e l l u l a r c h o l e s t e r o l homeostasis i s summarized i n F i g u r e 3. HDL: The complexity of l i p o p r o t e i n metabolism i s e x e m p l i f i e d by the i n v e s t i g a t i o n o f i n v i v o c e l l - H D L ' i n t e r a c t i o n . U n l i k e LDL, which i s c a t a b o l i z e d as a u n i t , HDL c o n s t i t u e n t s appear t o be c a t a b o l i z e d independently as i n d i c a t e d by the f o l l o w i n g o b s e r v a t i o n s i n r a t (43): 1) HDL-FC i s c l e a r e d more r a p i d l y from c i r c u l a t i o n than i s HDL-CE. 2) The kidney i s the major s i t e o f apo A-I removal w h i l e the l i v e r i s the major s i t e o f HDL-CE removal. 3) HDL a p o l i p o p r o t e i n s are not removed from plasma a t the same r a t e . 4) The r a t i o o f uptake of HDL-CE/HDL-protein v a r i e s g r e a t l y i n the body. The study of i n v i v o c e l l - H D L i n t e r a c t i o n s i s f u r t h e r c o m p l icated 24 by the f a c t t h a t both the p r o t e i n and l i p i d components of HDL's can be a c q u i r e d from or t r a n s f e r r e d t o o t h e r l i p o p r o t e i n c l a s s e s . S t u d i e s on the i n t e r a c t i o n of HDL wit h c u l t u r e d c e l l s i n v i t r o have been e a s i e r t o i n t e r p r e t . HDL has been shown t o remove c h o l e s t e r o l from c e l l s i n v i t r o . I t has been suggested t h a t the removal of c h o l e s t e r o l from c e l l may be mediated by the i n t e r a c t i o n o f HDL w i t h a s p e c i f i c c l a s s o f h i g h a f f i n i t y b i n d i n g s i t e s on the c e l l s u r f a c e (73). T h i s s u g g e s t i o n i s based on the o b s e r v a t i o n t h a t c h o l e s t e r o l enrichment of c u l t u r e d c e l l s i n c r e a s e s t h e i r a b i l i t y t o b i n d HDL (74) . However, i t i s important t o emphasize t h a t w h i l e LDL i s i n t e r n a l i z e d by c e l l s a f t e r b i n d i n g t o the LDL r e c e p t o r HDL i s not i n t e r n a l i z e d by most c e l l s examined (74,75,76) wi t h the e x c e p t i o n o f p e r i p h e r a l monocytes (77). C u r r e n t l y , a s p e c i f i c HDL b i n d i n g s i t e has been d e s c r i b e d i n the f o l l o w i n g c e l l t y p es: 1) A r t e r i a l smooth muscle c e l l s (75), 2) Human, r a b b i t and p i g hepatocytes (76,78,79), 3) Rat i n t e s t i n a l mucosal and a d r e n a l c o r t i c a l c e l l s (80), and most r e c e n t l y , 4) Human p e r i p h e r a l b l o o d monocytes (77). Recently, Graham and Oram have used l i g a n d b l o t t i n g techniques t o show t h a t 1 2 5 I - H D L binds t o a 110 kDa p r o t e i n , which they b e l i e v e i s the HDL r e c e p t o r (81). The p o s s i b l e i n t e r a c t i o n o f HDL w i t h a c e l l - s u r f a c e r e c e p t o r on c u l t u r e d c e l l s i m p l i e s t h a t HDL possesses s p e c i f i c r e c o g n i t i o n s i t e s f o r t h i s r e c e p t o r w i t h i n one or more of i t s components. The r e c o g n i t i o n s i t e s may l i e w i t h i n the HDL ap o p r o t e i n s , p a r t i c u l a r l y apo A-I 25 but p o s s i b l y apo A - I I and others (82,83,84). These f i n d i n g s were f u r t h e r supported by the r e s u l t s of a r e c e n t study by Oram e t a l who showed t h a t m o d i f i c a t i o n of HDL t y r o s i n e r e s i d u e s and/or c r o s s l i n k i n g o f HDL a p o p r o t e i n s a l t e r s the l i g a n d s i t e r e c o g n i t i o n (85). However, t h i s i s s u e remains u n s e t t l e d as a r e c e n t r e p o r t from Tabas and T a l l c o n f l i c t s w i t h the above view. These i n v e s t i g a t o r s concluded t h a t s u r f a c e l i p i d s , not p r o t e i n s , are the major components t h a t i n t e r a c t w i t h c u l t u r e d c e l l s (86). C u r r e n t l y , the p h y s i c a l c h a r a c t e r i s t i c s of the p u t a t i v e HDL r e c e p t o r and the mechanism by which i t may f a c i l i t a t e c h o l e s t e r o l t r a n s p o r t from c e l l s are unknown (73). 1.4 Reverse c h o l e s t e r o l t r a n s p o r t The m o b i l i z a t i o n of c h o l e s t e r o l from c e l l s and i t s subsequent t r a n s p o r t t o the l i v e r f o r e x c r e t i o n as b i l e s a l t s i s r e f e r r e d t o as r e v e r s e c h o l e s t e r o l t r a n s p o r t (RCT). T h i s p r o c e s s i n v o l v e s the f o l l o w i n g events: 1) T r a n s f e r of FC from c e l l membranes onto HDL p a r t i c l e s . 2) E s t e r i f i c a t i o n of FC v i a the LCAT r e a c t i o n . 3) T r a n s f e r of CE t o LDL and VLDL. 4) Uptake of C E - r i c h p a r t i c l e s by the l i v e r . The i n d i v i d u a l components of t h i s pathway have been e x t e n s i v e l y s t u d i e d and r e c e n t l y reviewed by F i e l d i n g (9). The i n i t i a l t r a n s f e r of FC from the c e l l membrane t o the l i p o p r o t e i n a c c e p t o r occurs by the d e s o r p t i o n of FC from the c e l l membrane. T h i s i s f o l l o w e d by d i f f u s i o n down a c o n c e n t r a t i o n g r a d i e n t , a process d r i v e n by the LCAT r e a c t i o n (56), t o the HDL p a r t i c l e . Besides c e l l membrane c h o l e s t e r o l the LCAT r e a c t i o n can a l s o use plasma l i p o p r o t e i n c h o l e s t e r o l . However, even though the m a j o r i t y of FC s u b s t r a t e o r i g i n a t e s i n LDL and VLDL (87,88), when equal masses of c e l l and plasma l i p o p r o t e i n c h o l e s t e r o l are p r esent, up t o 80% of the FC e s t e r i f i e d by the LCAT r e a c t i o n may be d e r i v e d from c e l l membranes (9,89). The a b i l i t y of membranes t o donate FC v a r i e s from c e l l type t o c e l l type (9) and may r e f l e c t , among ot h e r t h i n g s , the nature of the HDL/ c e l l i n t e r a c t i o n . C h o l e s t e r o l l o a d i n g of c e l l s has been shown t o i n c r e a s e the number of HDL b i n d i n g s i t e s on the c e l l - s u r f a c e s u g g e s t i n g t h a t the f l u x of c h o l e s t e r o l from c e l l may be r e g u l a t e d by d i f f e r e n t i a l e x p r e s s i o n of the HDL r e c e p t o r (73). The mechanism which promotes d i s s o c i a t i o n of the HDL from the c e l l - s u r f a c e i s unknown. However, once HDL e n t e r s the plasma i t i n t e r a c t s w i t h s p e c i f i c LTP's which promote the t r a n s f e r o f CE from HDL t o e i t h e r LDL or VLDL f o r c l e a r a n c e by the l i v e r . In a d d i t i o n t o t h i s mechanism, HDL can d i r e c t l y d e l i v e r the CE t o the l i v e r by i n t e r a c t i n g w i t h a h e p a t i c HDL b i n d i n g s i t e (76). In summary, RCT i s a complex p r o c e s s i n v o l v i n g l i p o p r o t e i n s , LTP's, LCAT, l i p o l y t i c enzymes, and l i p o p r o t e i n r e c e p t o r s . T h i s p r o c e s s prevents the accumulation o f c h o l e s t e r o l i n s i t e s not i n v o l v e d i n c h o l e s t e r o l c l e a r a n c e . Any d e f e c t i n t h i s pathway w i l l a f f e c t the body's a b i l i t y t o m o b i l i z e c h o l e s t e r o l and l e a d t o c h o l e s t e r o l accumulation i n the body. TD i s such a p e r t u r b a t i o n and t h e r e f o r e the f o l l o w i n g d i s c u s s i o n w i l l review the c u r r e n t understanding of TD. 27 1.5 T a n g i e r Disease 1.5.1 I n t r o d u c t i o n TD i s a r a r e autosomal r e c e s s i v e d i s o r d e r of plasma l i p i d t r a n s p o r t . The t y p i c a l f i n d i n g s i n p a t i e n t s w i t h t h i s d i s o r d e r i n c l u d e : 1) An absence of normal HDL. 2) Moderate h y p e r t r i g l y c e r i d e m i a . 3) The wide spread t i s s u e accumulation of c h o l e s t e r y l e s t e r s , e s p e c i a l l y prominent i n the r e t i c u l o e n d o t h e l i a l c e l l s , i n lymph nodes, thymus, bone marrow and r e c t a l mucosa. The presence of HDL d e f i c i e n c y and e n l a r g e d yellow-orange t o n s i l s or t o n s i l l a r remnants i s c o n s i d e r e d pathognomonic of the d i s e a s e (90). 1.5.2 H i s t o r y Unusual t o n s i l s were removed from a 5 y e a r o l d boy from T a n g i e r I s l a n d , V i r g i n i a i n 19 60. M i c r o s c o p i c examination o f the t o n s i l s r e v e a l e d the presence of numerous foam c e l l s . The p a t i e n t was r e f e r r e d t o the N a t i o n a l I n s t i t u t e of H e a l t h w i t h the t e n t a t i v e d i a g n o s i s of e i t h e r h i s t i o c y t o s i s o r l i p i d - s t o r a g e d i s e a s e . However, wi t h the d i s c o v e r y of h y p o a l p h a l i p o p r o t e i n e m i a i t became c l e a r t h a t t h i s was a new d i s o r d e r , l a t e r c a l l e d T a n g i e r d i s e a s e because of the p a t i e n t ' s home on the T a n g i e r I s l a n d i n Cheasepeake Bay (91). The o l d e r s i s t e r of the o r i g i n a l p a t i e n t had the same d i s o r d e r and f i v e y e ars l a t e r Hoffmann and F r e d r i c k s o n d e s c r i b e d a second p a i r of s i b l i n g s from an u n r e l a t e d k i n d r e d (92). P r e s e n t l y , twenty f i v e years s i n c e the f i r s t p a t i e n t was 28 d i s c o v e r e d , t h i r t y t h r e e p a t i e n t s with t h i s d i s e a s e have been r e p o r t e d (91-97). The p a t i e n t d e s c r i b e d i n t h i s study i s the f i r s t and only known TD p a t i e n t i n Canada. 1.5.3 C l i n i c a l F e a t u r e s The c l i n i c a l f e a t u r e s i n homozygous and heterozygous TD p a t i e n t s have r e c e n t l y been reviewed by Sch a e f e r e t a l (96). Tab l e I I I shows the frequency of c l i n i c a l a b n o r m a l i t i e s found on p h y s i c a l examination of the homozygotes. None of the s e a b n o r m a l i t i e s have been observed i n h e t e r o z y g o t e s (96). The Framingham study (98) has shown t h a t decreased plasma H D L - c h o l e s t e r o l (HDL-C) l e v e l s are a s s o c i a t e d w i t h premature a t h e r o s c l e r o s i s . However, i n TD heterozygotes or homozygotes be l l o w 35 years of age, no CHD or c e r e b r o v a s c u l a r d i s e a s e has been noted. In s u b j e c t s betv/een 35 and 65 y e a r s of age 26% of het e r o z y g o t e s and 45% of homozygotes had some evidence o f CHD, as compared t o 4% or l e s s i n a normal p o p u l a t i o n (96). The i n c r e a s e d p r e v a l e n c e o f CHD i n TD i s c o n s i s t e n t w i t h t h e concept t h a t p a t i e n t s w i t h f a m i l i a l HDL d e f i c i e n c y may be a t an i n c r e a s e d r i s k f o r premature v a s c u l a r d i s e a s e . However, the s t r i k i n g l y a c c e l e r a t e d a t h e r o s c l e r o s i s such as t h a t observed i n p a t i e n t s w i t h FH i s r a r e i n TD p a t i e n t s p o s s i b l y because of t h e i r reduced plasma l e v e l s o f LDL (96) . 29 TABLE I I I CLINICAL PRESENTATION OF 33 PATIENTS WITH TANGIER DISEASE (Courtesy Dr. J F r o h l i c h ) Symptom Mode of P r e s e n t a t i o n F e a t u r e s P h y s i c a l on Examinatii T o n s i l l a r a b n o r m a l i t i e s 8/33 (24%) 26/33 (79%) Splenomegaly 2/33 (6%) 16/3 3 (48%) Hepatomegaly - - 14/33 (42%) Lymphadenopathy - - 6/33 (18%) Neuropathy 11/33 (33%) 18/33 (55%) Corneal i n f i l t r a t i o n - - 14/29 (48%) Abnormal r e c t a l mucosa - - 10/10 (100%) Bone marrow foam c e l l s - - 9/13 (69%) Abnormal s k i n f i n d i n g s 1/28 (3%) - -Hypocholesterolemia 3/28 (10%) 31/33 (94%) Chest p a i n 1/33 (3%) — -30 1.5.4 Biochemical F e a t u r e s Biochemical a b n o r m a l i t i e s noted i n TD homozygotes i n c l u d e plasma c h o l e s t e r o l , L D L - c h o l e s t e r o l (LDL-C), and HDL-C v a l u e s which are approximately 37%, 34%, and 4% of normal, r e s p e c t i v e l y . P a t i e n t s have u s u a l l y moderately e l e v a t e d f a s t i n g plasma TG l e v e l s . I n d i v i d u a l v a r i a t i o n i n the plasma TG l e v e l s i s c o n s i d e r a b l e and i s h i g h l y dependent on d i e t (90). Plasma PL c o n c e n t r a t i o n s i n TD plasma range from 30 t o 50% of c o n t r o l v a l u e s . T h i s l i k e l y r e f l e c t s the HDL d e f i c i e n c y (90). Clearance of chylomicrons i n plasma i s delayed w i t h consequent accumulation of chylomicron remnants i n plasma (90). V L D L r p p (d<1.006 g/ml) migrate more s l o w l y than normal VLDL on paper e l e c t r o p h o r e s i s , y e t t h e i r morphology, gr o s s chemical composition and q u a l i t a t i v e a p o l i p o p r o t e i n content are s i m i l a r (99,100). T h i s d i f f e r e n c e i n m o b i l i t y may be e x p l a i n e d by the observed q u a n t i t a t i v e abnormality i n C - a p o l i p o p r o t e i n s (93)... Intermediate d e n s i t y l i p o p r o t e i n s (IDL:d=l.006-1.019 g/ml) do not accumulate i n the plasma of TD p a t i e n t s (90) and the l e v e l s of LDL are reduced t o approximately 33% of normal (101). L D L T D are d e f i c i e n t i n CE and e n r i c h e d i n TG (99,100). S e v e r a l i n v e s t i g a t o r s have r e p o r t e d t h a t L D L T D migrates f a s t e r than c o n t r o l LDL on paper e l e c t r o p h o r e s i s (95,100). Others have found L D L T D w i t h normal e l e c t r o p h o r e t i c m o b i l i t y (94) . The reason f o r these d i f f e r e n c e s i s u n c l e a r and may r e f l e c t the v a r i e d composition o f LDL i s o l a t e d from d i f f e r e n t p a t i e n t s w i t h TD. In TD plasma the c o n c e n t r a t i o n of p r o t e i n i n the HDL r e g i o n was o n l y 0.4 t o 4.5% o f normal. The l i p i d content of TD HDL v a r i e s not o n l y among p a t i e n t s but a l s o i n the same p a t i e n t s 31 (90). Agarose g e l e l e c t r o p h o r e s i s and e l e c t r o n microscopy have shown a s t r i k i n g h e t e r o g e n e i t y of the TD HDL. E l e c t r o n m i c r o s c o p i c s t u d i e s by F o r t e e t a l (102) have r e v e a l e d the presence of t h r e e m o r p h o l o g i c a l l y d i s t i n c t p a r t i c l e s i n the HDL d e n s i t y r e g i o n : 1) Large t r a n s l u c e n t forms, over 100 nm i n diameter, which appear t o r e p r e s e n t chylomicron remnants. 2) Round p a r t i c l e s , 20-25 nm i n diameter. 3) Small p a r t i c l e s , 5.5-7.5 nm i n diameter. While normal HDL have not been r e c o v e r e d from TD plasma, both the major a p o p r o t e i n s o f HDL, apo A-I and apo A - I I , are p r e s e n t . The apo A-I c o n c e n t r a t i o n o f TD plasma i s t y p i c a l l y l e s s than 1% of normal, and apo A - I I l e v e l s a r e about 5-7% o f normal (90). Although s t r u c t u r a l s t u d i e s of the apo A-I and apo A - I I have not r e v e a l e d major a b n o r m a l i t i e s they do not exclude the p o s s i b i l i t y of minor s t r u c t u r a l a l t e r a t i o n (90). Apo B, a l l of the C*s, D and E have been i d e n t i f i e d i n TD plasma (101). A study by A l a u p o v i c e t a l (93) has shown t h a t i n TD homozygotes t h e r e i s normal l e v e l s of apo B, a v a r i a b l e but g e n e r a l i z e d decrease i n apo C and apo E l e v e l s and a d e f i c i e n c y of apo D. Apo B i n TD plasma has normal amino a c i d c o m p o s i t i o n and immunochemical p r o p e r t i e s (90). Apo E i s a l s o normal as judged by i t s amino a c i d a n a l y s i s and p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (90). C u r r e n t l y , data on the o t h e r a p o l i p o p r o t e i n s are not a v a i l a b l e . The decreased plasma c o n c e n t r a t i o n of apo C's i s c o n s i s t e n t w i t h the p o s t u l a t e t h a t HDL a c t s as a r e s e r v o i r f o r C 32 a p o l i p o p r o t e i n s . C o n s e r v a t i o n of the C a p o l i p o p r o t e i n s i n normal plasma i s accomplished by the t r a n s f e r of apo C's from the r a p i d l y c a t a b o l i z e d l i p o p r o t e i n p o o l (Chylomicrons and VLDL) t o the slower t u r n i n g over HDL pool.. In TD t h e r e i s i n s u f f i c i e n t HDL p r e s e n t t o p r o v i d e the necessary r e s e r v o i r f o r the apo C's hence t h e i r f a s t e r removal. Examination of p o s t h e p a r i n LpL a c t i v i t y i n TD plasma has shown v a r i a b l e r e s u l t s . LpL a c t i v i t y i s decreased i n some p a t i e n t s (103,104) but not i n o t h e r s (105). No c l e a r c u t d e f i c i e n c y i n the a c t i v i t y of LpL or HTGL has y e t been demonstrated (90). While low LCAT a c t i v i t y has been r e p o r t e d i n some p a t i e n t s w i t h TD (95,103,104) normal o r e l e v a t e d l e v e l s of LCAT have a l s o been d e s c r i b e d (105,106). 1.6 B i o c h e m i c a l D e f e c t i n T a n g i e r Disease S e v e r a l hypotheses have been formulated t o e x p l a i n the m o l e c u l a r d e f e c t i n TD. These i n c l u d e : 1) A c c e l e r a t e d c a t a b o l i s m of HDL d e s p i t e normal r a t e s o f apo A-I s y n t h e s i s (107-114). 2) Abnormal s t r u c t u r e of apo A-I (115). 3) Impaired p o s t t r a n s l a t i o n a l m o d i f i c a t i o n of proapo A-I (116-119). 4) A f a i l u r e o f apo A-I t o a s s o c i a t e w i t h HDL (120,121). However, no unequivocal evidence of a s t r u c t u r a l a p o l i p o p r o t e i n d e f e c t c a u s i n g HDL d e f i c i e n c y has been e s t a b l i s h e d . Recent s t u d i e s of the apo A-I gene from our TD p a t i e n t have f a i l e d t o demonstrate a c o d i n g a b n o r m a l i t y (120). Furthermore, t h e r e does not appear t o be a d e f e c t i n 33 t r a n s c r i p t i o n , t r a n s l a t i o n o r s e c r e t i o n o f t h e apo A - I . S c h a e f e r e t a l have shown t h a t p a t i e n t s w i t h TD h a v e o n l y a s l i g h t d e c r e a s e i n apo A - I s y n t h e s i s and s e c r e t i o n r a t e s ( 1 1 0 ) . I n a d d i t i o n , i t has b e e n d e m o n s t r a t e d t h a t p r o a p o l i p o p r o t e i n A - I c a n be c o n v e r t e d t o m a t u r e a p o l i p o p r o t e i n A - I b y a c o n v e r t a s e i n n o r m a l p l a s m a and t h a t t h i s c o n v e r t a s e a c t i v i t y i s n o r m a l i n TD p l a s m a (118,119). The t e r t i a r y s t r u c t u r e o f TD apo A - I was e x a m i n e d by Weech e t a l u s i n g f o u r m o n o c l o n a l a n t i b o d i e s t o n o r m a l A - I ( 1 2 3 ) . T h ey showed t h a t t h e m o l e c u l a r w e i g h t , p i and C N B r - c l e a v a g e f r a g m e n t s o f TD and n o r m a l apo A - I were n o t d i f f e r e n t . I n a d d i t i o n , t h e y a l s o e x c l u d e d t h e p o s s i b i l i t y t h a t d i m e r s , a g g r e g a t e s o r f r a g m e n t s o f apo A - I c o u l d be r e s p o n s i b l e f o r t h e r a p i d c a t a b o l i s m i n t h i s d i s e a s e . R e c e n t l y , s t u d i e s b y P r i t c h a r d e t a l h a v e d e m o n s t r a t e d t h a t apo A - I p u r i f i e d f r o m a p a t i e n t w i t h TD was f u n c t i o n a l l y n o r m a l i n a c t i v a t i n g b o t h p u r i f i e d a n d p l a s m a LCAT ( 1 2 4 ) . Thus i t w o u l d a p p e a r t h e p r o d u c t i o n o f apo A - I i n TD i s n o r m a l and t h e apo A - I i s o l a t e d f r o m TD p a t i e n t s i s a b i o l o g i c a l l y a c t i v e p r o t e i n . A s t h e m a j o r i t y o f HDL p r e c u r s o r s a r e d e r i v e d d u r i n g l i p o l y s i s o f T G - r i c h l i p o p r o t e i n s a d e f e c t i n t h e l i p o l y s i s o f T G - r i c h l i p o p r o t e i n s w o u l d i m p a i r t h e p r o d u c t i o n o f HDL and may r e s u l t i n HDL d e f i c i e n c y . However, t h i s e x p l a n a t i o n f o r t h e HDL d e f i c i e n c y i n TD h a s y e t t o be s u b s t a n t i a t e d . I n v i v o e x p e r i m e n t s on t h e m e t a b o l i s m o f HDL i n TD p a t i e n t s h a v e g e n e r a t e d a g r e a t d e a l o f i n t e r e s t i n g i n f o r m a t i o n . S c h a e f e r and c o w o r k e r s h ave shown t h a t n o r m a l HDL i n j e c t e d i n t o TD p a t i e n t s h a d a g r e a t l y r e d u c e d p l a s m a r e s i d e n c e t i m e ( 1 1 4 ) . The 34 observed h y p e r c a t a b o l i s m of HDL i n TD was f u r t h e r c o r r o b o r a t e d by s t u d i e s i n which l a r g e r amounts of HDL were i n f u s e d ( i e p a r t i a l plasma exchange) (125). C u r r e n t l y , a l l the cumulative evidence i n the l i t e r a t u r e suggests t h a t the d e f e c t i n TD l i e s i n enhanced c a t a b o l i s m of HDL. However, t o date the exact b i o c h e m i c a l nature of t h i s d e f e c t i s unknown. 35 1.7 R a t i o n a l e f o r t h i s study Hypercatabolism of HDL i n TD plasma may be the r e s u l t of e i t h e r i n c r e a s e d c e l l u l a r c a t a b o l i s m or i n c r e a s e d plasma c a t a b o l i s m without c e l l u l a r involvement. Work by S c h a e f e r e t a l has e s t a b l i s h e d t h a t the d e f e c t i n TD r e s u l t s i n the h y p e r c a t a b o l i s m of HDL from plasma. However, t h e i r s t u d i e s c o u l d not d i f f e r e n t i a t e between m o d i f i c a t i o n of HDL composition by plasma f a c t o r s p r i o r t o c a t a b o l i s m or the d i r e c t uptake and d e g r a d a t i o n o f i n d i v i d u a l HDL components. Thus, t h i s r e s e a r c h w i l l examine the r o l e s i n which TD plasma f a c t o r ( s ) and/or TD c e l l s p l a y i n the "hypercatabolism" of HDL i n TD p a t i e n t s . In our approach t o s t u d y i n g the mechanisms u n d e r l y i n g HDL c a t a b o l i s m we have focused on the f o l l o w i n g s t u d i e s : 1) Examination o f the c a t a b o l i s m of normal HDL i n TD plasma d u r i n g i n c u b a t i o n i n v i t r o . 2) Examination of the i n t e r a c t i o n of normal HDL w i t h TD c e l l s i n v i t r o . 1 . 8 S p e c i f i c aims 1) To study the l i p i d and p r o t e i n changes i n normal HDL d u r i n g i n c u b a t i o n w i t h the plasma from a p a t i e n t w i t h TD. 2) To study the i n t e r a c t i o n of the TD m o d i f i e d HDL w i t h normal f i b r o b l a s t c u l t u r e s . 3) To c u l t u r e s k i n f i b r o b l a s t s from c o n t r o l s and a TD p a t i e n t and compare t h e i r a b i l i t y t o b i n d and degrade normal HDL. 4) To c u l t u r e p e r i p h e r a l b l o o d monocytes from c o n t r o l s and a TD p a t i e n t and compare t h e i r a b i l i t y t o b i n d and degrade normal HDL. 36 2 MATERIALS AND METHODS 2.1 S u b j e c t s 2.1.1 C o n t r o l subj e c t s Healthy normolipidemic c o n t r o l s u b j e c t s were r e c r u i t e d amongst l a b o r a t o r y s t a f f . H y p e r t r i g l y c e r i d e m i c s u b j e c t s and p a t i e n t s w i t h homozygous form of TD o r LCAT d e f i c i e n c y were r e c r u i t e d from the L i p i d C l i n i c a t Shaughnessy h o s p i t a l . 2.1.2 T a n g i e r p a t i e n t Case H i s t o r y : J.K.W., a 56 year o l d male, presented i n March 1982 w i t h a p r o g r e s s i v e weakness and numbness and p a r e s t h e s i a e of both hands. These symptoms were f i r s t noted i n 1981 along w i t h drooping of h i s r i g h t lower e y e l i d . H i s p a s t h i s t o r y i n c l u d e d : t o n s i l l e c t o m y i n c h i l d h o o d , b i l a t e r a l sympathectomy f o r "bad c i r c u l a t i o n i n the f e e t " i n 1947 and splenectomy f o r "hypersplenism" i n 1964. H i s t o l o g i c a l examination of the s p l e e n showed numerous f a t - l a d e n c e l l s . In the l a t e 1960's he-noted enlargement o f h i s nose and ear l o b e s . P l a s t i c surgery of h i s nose f o r rhinophyma was c a r r i e d out i n 19 69 w i t h numerous f o l l o w - u p procedures due t o poor wound h e a l i n g . In 1980 he s u f f e r e d an a t t a c k of c h o l e c y s t i t i s and was t r e a t e d by cholecystectomy. H i s t o l o g i c a l a n a l y s i s of a wedge b i o p s y of the l i v e r done a t t h a t time showed numerous foam c e l l s . In November 1985 the p a t i e n t s t a r t e d l o s i n g weight and i n February 1986 t h e d i a g n o s i s of Insulin-dependent d i a b e t e s m e l l i t u s was made. P h y s i c a l Examination: He i s a t a l l man, (184 cm), w i t h l a r g e e a r l o b e s . A few y e l l o w s t r e a k s are e v i d e n t on h i s pharynx. N e u r o l o g i c a l examination r e v e a l e d marked wasting and weakness of the hands and f e e t . A r i g h t lower e c t r o p i o n w i t h healed c o r n e a l 37 u l c e r s was pr e s e n t . The f a c i a l muscles were a t r o p h i c and weak. There was a l o s s o f s e n s i t i v i t y t o touch and v i b r a t i o n over h i s hands and f e e t i n a glove and s t o c k i n g d i s t r i b u t i o n . The b i c e p s and t r i c e p s r e f l e x e s were depressed, the an k l e j e r k s absent and p l a n t a r responses were downgoing. H i s t o l o g y : S t u d i e s were done on b i o p s i e s o b t a i n e d from the p a t i e n t s s k i n , s u r a l nerve, r e c t a l mucosa and l i v e r by F r o h l i c h e t a l (personal communication). A n a l y s i s by l i g h t and e l e c t r o n microscopy r e v e a l e d the presence of a l a r g e number of foam c e l l s i n a l l o f these t i s s u e s . These f i n d i n g s were not d i f f e r e n t from those d e s c r i b e d by F r e d r i c k s o n e t a l (91). Family h i s t o r y : The p a t i e n t ' s p arents were f i r s t c o u s i n s . H i s f a t h e r d i e d a t age 50 of a h e a r t a t t a c k . H i s mother d i e d i n her e a r l y f o r t i e s d u r i n g surgery. H i s o n l y s i s t e r , aged 55, i s a l i v e and w e l l . The f a m i l y emigrated t o Canada from B r i s t o l , England i n 1919. The p a t i e n t has no progeny. The f a m i l y t r e e i s shown i n F i g u r e 4. 3 8 W family £ 9 * M <X o, Q3 Tv 6 5 rj6 Q7 j& d 1 0 aTd75 x 21 13 6, 6 , 0 , 6 , 6 5 9 g 7 g, F i g u r e 4. Family t r e e o f p a t i e n t JKW (•) (Courtesy o f Dr. M. Hayden) 39 2.2 M a t e r i a l s The f o l l o w i n g companies s u p p l i e d chemicals or equipment as i n d i c a t e d : Amersham Canada L t d . , O n t a r i o , Canada: 1 2 5 I - N a I . Amicon Canada L t d . , O n t a r i o , Canada: 0.45uM f i l t e r s , m i c r o c o n c e n t r a t i o n system. A n a l t e c h Inc, Newark, Del., USA: S i l i c a g e l G (250 urn). Bio-Rad L a b o r a t o r i e s (Canada) L t d . , O n t a r i o , Canada: B i o g e l AO.5m and A5.0m, Bio-Rad PAGE system. Boehringer Mannheim, West Germany: Reagent k i t s f o r the FC, TG, and CE ass a y s . Corning M e d i c a l , Ca, USA: Agarose g e l e l e c t r o p h o r e s i s system. Gibco Canada, O n t a r i o , Canada: MEM, RPMI-1640, FCS, t r y p s i n and a n t i m i c r o b i a l s . NEN Research Products, Quebec, Canada: [ 3H]-CE, [ 3 H ] - c h o l e s t e r o l , and [ 3 H ] - g l y c e r y l t r i o l e a t e . Pharmacia (Canada) Inc., Quebec, Canada: Sephadex G-50, FP. Tago L t d . , Ca., USA: Apo A-I r a d i a l immunodiffusion p l a t e s . Western S c i e n t i f i c , B.C., Canada: U l t r a c e n t r i f u g a t i o n tubes, t i s s u e c u l t u r e d i s h e s . N.B. A l l ot h e r m a t e r i a l were standard l a b o r a t o r y s t o c k . 40 2.3 L i p o p r o t e i n s 2.3.1 I s o l a t i o n o f l i p o p r o t e i n s 2.3.1.1 P r e p a r a t i v e u l t r a c e n t r i f u g a t i o n VLDL (d< 1.006 g/ml) was i s o l a t e d from the f a s t i n g plasma of the p a t i e n t s w i t h TD and LCAT d e f i c i e n c y and from poo l e d plasma from normal i n d i v i d u a l s by p r e p a r a t i v e u l t r a c e n t r i f u g a t i o n a t 15°C f o r 18 h a t 42,000 rpm (1.14 x 10 5 g ) . The p a r t i c l e s were f u r t h e r p u r i f i e d by a second c e n t r i f u g a t i o n a t the same d e n s i t y . LDL and HDL and HDL 3 were i s o l a t e d i n t h e d e n s i t y range 1.006-1.063 g/ml; 1.063-1.21 g/ml and 1.12-1.21 g/ml, r e s p e c t i v e l y by s e r i a l u l t r a c e n t r i f u g a t i o n a t 15°C f o r 24 h a t 42,000 rpm (1.13 x 1 0 5 g) and washed by r e c e n t r i f u g a t i o n a t the upper d e n s i t y l i m i t . T o t a l plasma l i p o p r o t e i n s were i s o l a t e d from plasma a c c o r d i n g t o Rudel e t a l (127). B r i e f l y , the d e n s i t y o f the plasma was a d j u s t e d t o d = 1.225 g/ml w i t h s o l i d NaBr and then s u b j e c t e d t o u l t r a c e n t r i f u g a t i o n a t 15°C f o r 40 h a t 40,000 rpm. The top f r a c t i o n was removed by tube s l i c i n g and the bottom f r a c t i o n was recovered i n the o r i g i n a l plasma volume t h e r e a f t e r d e s i g n a t e d as l i p o p r o t e i n d e f i c i e n t human plasma (LPDP). L i p o p r o t e i n d e f i c i e n t f e t a l c a l f serum (LPDS) was prepared by the methods of Brown and G o l d s t e i n (128) . The d e n s i t y o f the f e t a l c a l f serum was a d j u s t e d t o d = 1.225 g/ml and s u b j e c t e d t o u l t r a c e n t r i f u g a t i o n a t 15°C f o r 40 h a t 40,000 rpm. The top f r a c t i o n , c o n t a i n i n g t o t a l l i p o p r o t e i n s , was removed by tube s l i c i n g and the bottom f r a c t i o n (LPDS) was saved. A l l f r a c t i o n s were d i a l y z e d a g a i n s t f o u r changes of 0.15 M NaCl c o n t a i n i n g 1 mM EDTA and 0.03% NaN 3 ( b u f f e r A), s t e r i l i z e d by 0.45 uM f i l t r a t i o n and s t o r e d a t -20°C u n t i l used. 2.3.1.2 P r e c i p i t a t i o n o f VLDL and LDL from plasma HDL c o u l d be i s o l a t e d from plasma by p r e c i p i t a t i o n of VLDL and LDL w i t h heparin-manganese as d e s c r i b e d by Warnick e t a l (129). In a t y p i c a l p r e p a r a t i o n , 500 u l of plasma was mixed wi t h 50 u l of 1 M MnCl 2 c o n t a i n i n g 0.15 M NaCl and 2500 u n i t s / m l h e p a r i n . The sample was then v o r t e x e d and p l a c e d a t 4°C f o r 3 0 min, then c e n t r i f u g e d a t 6000 x g f o r 10 min a t 15°C. The supernatant c o n t a i n e d o n l y a l p h a - l i p o p r o t e i n s as determined by agarose g e l e l e c t r o p h o r e s i s . 2.3.2 I o d i n a t i o n o f l i p o p r o t e i n s 2.3.2.1 P r e p a r a t i o n o f 1 2 5 I - l a b e l e d l i p o p r o t e i n s 1 2 5 I - l i p o p r o t e i n s were prepared by the i o d i n e monocholoride method of MacFarlane (130) as m o d i f i e d by B i l h e i m e r e t a l (131). B r i e f l y , 1-3 mg of HDL p r o t e i n was e q u i l i b r a t e d w i t h 0.4 M g l y c i n e b u f f e r , pH 10 ( b u f f e r B) by passage through a Sephadex G-50 column (1.0 x 20.0 cm). The f o l l o w i n g were then added t o a p l a s t i c screw top v i a l ; 10 u l of s t o c k IC1 (0.33 M) d i l u t e d 10-f o l d , 10 u l 1 2 5 I - N a I (1 mCi), 6 drops of b u f f e r B and 1-3 mg HDL. The 1 2 5 I - H D L was separated from the f r e e 1 2 5 i by passage down through a Sephadex G-50 columns (1.0 x 20.0 cm) and e l u t e d w i t h 0.15 M NaCl, 1 M EDTA, pH 7.4 ( b u f f e r C). The f i n a l s p e c i f i c a c t i v i t y v a r i e d between 100 and 3 00 cpm/ng p r o t e i n . A l l the i o d i n a t e d l i p o p r o t e i n p r e p a r a t i o n s were s t e r i l i z e d by passage through a 0.4 5 uM m i l l i p o r e f i l t e r and s t o r e d a t 4°C f o r a maximum of 3 weeks. 42 2.3.2.2 C h a r a c t e r i z a t i o n o f I - L i p o p r o t e i n s The amount of r a d i o a c t i v i t y a s s o c i a t e d w i t h apo B was determined u s i n g a m o d i f i e d Holmquist procedure (132). B r i e f l y , 100 u l of 10 0 - f o l d d i l u t e d 1 2 5 I - L D L was mixed w i t h 200 u l of u n l a b e l e d LDL (1 mg/ml) and 300 u l of i s o p r o p a n o l was added t o the mixture which was then v o r t e x e d and s t o r e d a t room temperature f o r 10 min p r i o r t o c e n t r i f u g a t i o n a t 13,000 x g f o r 10 min a t 4°C. The p e l l e t , c o n t a i n i n g the p r e c i p i t a t e d apo B, and the supernatant were separated and counted f o r 60 seconds i n a 1271 RiaGamma c o u n t e r (LKB).The r a d i o a c t i v i t y a s s o c i a t e d w i t h each f r a c t i o n was counted (cpm). The r e s u l t s were than expressed as percentage o f r a d i o a c t i v i t y a s s o c i a t e d w i t h the apo B. The amount of r a d i o a c t i v i t y a s s o c i a t e d w i t h the l i p i d component was assayed by l i p i d e x t r a c t i o n o f 10 u l o f 1 2 5 I -l i p o p r o t e i n by the method of F o l c h e t a l (133) and measuring the r a d i o a c t i v i t y i n the c h l o r o f o r m phase. 10 u l o f c a r r i e r LDL (1 mg/ml) was i n c l u d e d i n a l l samples. The r e s u l t s are expressed as percentage r a d i o a c t i v i t y a s s o c i a t e d w i t h the l i p i d . The r a d i o a c t i v i t y a s s o c i a t e d w i t h the p r o t e i n component was determined by p r e c i p i t a t i o n o f 10 u l of 1 2 5 I - l i p o p r o t e i n by the a d d i t i o n of 200 u l of 50% t r i c h l o r o a c e t i c a c i d (TCA). 200 u l of bovine serum albumin (BSA:5 mg/ml) was added t o a l l samples t o ac t as a c a r r i e r p r o t e i n and t o b i n d any f r e e l i p i d . A f t e r c e n t r i f u g a t i o n f o r 10 min a t 17,000 x g the amount of r a d i o a c t i v i t y a s s o c i a t e d w i t h the p e l l e t and supernatant were determined. The r e s u l t s are expressed as percentage r a d i o a c t i v i t y a s s o c i a t e d w i t h p r o t e i n . I o d i n a t e d l i p o p r o t e i n s were f u r t h e r c h a r a c t e r i z e d by agarose 43 g e l e l e c t r o p h o r e s i s u s i n g the Corning E l e c t r o p h o r e s i s System as mo d i f i e d f o r l i p o p r o t e i n s (134). I o d i n a t e d a p o l i p o p r o t e i n s were c h a r a c t e r i z e d by sodium d o d e c y l s u l p h a t e p o l y a c r y l a m i d e g e l e l e c t o p h o r e s i s (SDS-PAGE) under r e d u c i n g c o n d i t i o n s on e i t h e r 10 or 12.5% acrylamide g e l s a c c o r d i n g t o the method o f Laemmli (135). Agarose g e l s and SDS-polyacrylamide g e l s c o n t a i n i n g r a d i o l a b e l e d l i p o p r o t e i n s were f u r t h e r a n a l y z e d i n d i r e c t l y by autoradiography o f g e l s f o r 1-7 days o r d i r e c t l y by s l i c i n g g e l s i n t o 0.5 cm s e c t i o n s and coun t i n g the gamma r a d i a t i o n . 2.3.3 [ 3H] l a b e l i n g o f plasma and l i p o p r o t e i n s 2.3.3.1 L a b e l i n g o f VLDL w i t h g l y c e r y l t r i [ 3 H ] o l e a t e VLDL was l a b e l l e d with g l y c e r y l t r i [ 3 H ] o l e a t e a c c o r d i n g t o the method of F i e l d i n g (136). B r i e f l y , 100 u C i g l y c e r y l t r i [ 3 H ] o l e a t e (1.0 Ci/mM) was s o l u b i l i z e d i n 1 ml o f d i m e t h y l s u l f o x i d e , d i l u t e d t o 4 ml w i t h b u f f e r C. To t h i s s o l u t i o n was added 5 mg of VLDL-TG w i t h c o n s t a n t s t i r r i n g . A f t e r i n c u b a t i o n f o r 3 h a t 37°C, the r e a c t i o n mixture was d i a l y z e d a g a i n s t b u f f e r C a t 4°C ov e r n i g h t . The l a b e l l e d VLDL was r e i s o l a t e d a t d = 1.006 gm/ml by u l t r a c e n t r i f u g a t i o n f o r 18 h a t 42,000 rpm and and d i a l y z e d e x t e n s i v e l y a g a i n s t b u f f e r C. 2.3.3.2 L a b e l i n g o f plasma l i p o p r o t e i n s w i t h [ 3H] c h o l e s t e r o l A l i q u o t s o f plasma (2 ml) were i n c u b a t e d f o r 16 h a t 4°C i n the presence of 10 u C i [7- 3H] c h o l e s t e r o l (23.7 Ci/mM) as p r e v i o u s l y d e s c r i b e d by P r i t c h a r d e t a l (137) . In t h i s l a b e l i n g procedure more than 90% of the l a b e l was a s s o c i a t e d w i t h the 44 d i f f e r e n t l i p o p r o t e i n f r a c t i o n (d<1.21 g/ml). Moreover, as s u f f i c i e n t time was p e r m i t t e d f o r the complete e q u i l i b r a t i o n of the r a d i o l a b e l w i t h the l i p o p r o t e i n s the s p e c i f i c a c t i v i t y o f FC was the same i n a l l l i p o p r o t e i n f r a c t i o n s . 2.3-3.3 L a b e l i n g o f plasma and HDL w i t h [ 3H] CE Plasma or HDL were l a b e l l e d w i t h [ 3H] CE by a procedure s i m i l a r t o t h a t of Sparks e t a l (138). B r i e f l y , 3-7 u C i of [ c h o l e s t e r y l - 1 , 2 , 6 , 7 - 3 H ( N ) ] - c h o l e s t e r y l l i n o l e a t e (100 Ci/mM) was d r i e d down i n a g l a s s v i a l under n i t r o g e n . One ml a l i q u o t s o f plasma o r HDL (1-10 mg/ml) were i n t r o d u c e d and the mixture was i ncubated f o r 14 h a t 4°C. The sample was then t r a n s f e r r e d i n t o a second v i a l and used the same day. 2.3.4 C h a r a c t e r i z a t i o n o f L i p o p r o t e i n s 2.3.4.1 Determination o f l i p o p r o t e i n p a r t i c l e s i z e Plasma was s u b f r a c t i o n a t e d i n t o VLDL, LDL and HDL s i z e d p a r t i c l e s by g e l f i l t r a t i o n chromatography. In a t y p i c a l p r e p a r a t i o n , a 2 ml a l i q u o t of l a b e l l e d plasma was a p p l i e d t o a B i o g e l A5.0m chromatographic column (90 x 1.5 cm) and e l u t e d a t 4.3 ml/h w i t h 0.15 M NaCl c o n t a i n i n g 10 mM T r i s / H C l , pH 7.4, 0.01% EDTA, and 0.3% NaN 3 ( b u f f e r D). E l u e n t from the column was c o l l e c t e d i n 2 ml f r a c t i o n s and the amount of l a b e l p r e s e n t i n each f r a c t i o n was determined. The peak co r r e s p o n d i n g t o HDL was f u r t h e r analyzed by passage through a B i o g e l AO.5m chromatographic column (90 x 1.5 cm) u s i n g b u f f e r D and a flow r a t e of 21 ml/h. Both columns were p r e c a l i b r a t e d by the e l u t i o n of p u r i f i e d , 1 2 5 I - V L D L , 1 2 5 I - L D L and 1 2 5 I - H D L . A more a c c u r a t e d e t e r m i n a t i o n of VLDL and LDL p a r t i c l e s i z e was o b t a i n e d by q u a s i e l a s t i c l i g h t s c a t t e r i n g (QLS) u s i n g a 45 NiComp 270 submicron p a r t i c l e s i z e r a c c o r d i n g t o the method of Mayer e t a l (139), c o u r t e s y of Dr. P. C u l l i s , Dept of Biochemistry, U n i v e r s i t y of B r i t i s h Columbia. 2.3.4.2 L i p o p r o t e i n l i p i d a n a l y s i s C h o l e s t e r o l , CE and TG were determined e n z y m a t i c a l l y by standard procedures (140,141). PL were q u a n t i t a t e d by d e t e r m i n a t i o n of l i p i d phosphorus a c c o r d i n g t o the method of Anderson e t a l (142). VLDL, LDL, HDL and HDL 3 p r o t e i n were measured by the method of Lowry e t a l (143) as m o d i f i e d f o r l i p o p r o t e i n s (144) u s i n g BSA as a standard. Apo C-II l e v e l s were determined by Dr. W. McConnathy, Oklahoma M e d i c a l Research Foundation, by p r e v i o u s l y d e s c r i b e d immunochemical methods (145). Apo A-I l e v e l s were determined by r a d i a l - i m m u n o d i f f u s i o n u s i n g preprepared a n t i - a p o A-I p l a t e s . 2.3.5 Enzyme Assays 2.3.5.1 H y d r o l y s i s o f CE i n plasma The r a t e of CE h y d r o l y s i s (CEH) i n plasma was determined by i n c u b a t i n g plasma p r e l a b e l e d w i t h [ 3H] CE ( S e c t i o n 2.3.3.3) or plasma c o n t a i n i n g HDL p r e l a b e l e d w i t h [ 3H] CE, f o r 24 h a t 4°C and 37°C. The r e a c t i o n was stopped by the a d d i t i o n o f 4 ml chloroform:methanol (2:1). The l i p i d s were e x t r a c t e d by the method o f F o l c h (133) and the CE's sep a r a t e d from the FC by t h i n l a y e r chromatography (TLC) on s i l i c a g e l G p l a s t i c p l a t e s i n a petroleum e t h e r : e t h e r : a c e t i c a c i d (70:10:1) s o l v e n t system. The l i p i d was v i s u a l i z e d by exposure t o i o d i n e vapor and the area of the s i l i c a g e l co r r e s p o n d i n g t o c h o l e s t e r o l o r CE was then c u t out and p l a c e d i n a 7 ml g l a s s s c i n t i l l a t i o n v i a l . F i v e ml of 46 Omnifluor i n toluene (4 g/1) were added t o each v i a l and the sample was assayed f o r 3H s c i n t i l l a t i o n . The r a t e o f CEH was determined and the r e s u l t s expressed as percentage h y d r o l y s i s of t o t a l CE per 24 hour. 2.3.5.2 Bovine m i l k l i p o p r o t e i n l i p a s e Bovine m i l k l i p o p r o t e i n l i p a s e (BmLpL), p u r i f i e d from bovine skimmed m i l k by a f f i n i t y chromatography on heparin-Sepharose 4B as d e s c r i b e d p r e v i o u s l y (146), was a g i f t from R.McLeod. The enzyme was homogeneous as determined by SDS-PAGE. The p u r i f i e d enzyme had a s p e c i f i c a c t i v i t y o f 8 umoles f r e e f a t t y a c i d r e l e a s e d per hour p e r ml. VLDL was l a b e l l e d w i t h g l y c e r o l t r i [ 3 H ] o l e a t e as p r e v i o u s l y d e s c r i b e d ( S e c t i o n 2.3.3.1) and the r a t e o f h y d r o l y s i s o f the l a b e l l e d VLDL's by BmLpL were determined e s s e n t i a l l y as d e s c r i b e d by F e i l d i n g e t a l (136). B r i e f l y , the assay mixture c o n t a i n e d g l y c e r o l t r i [ 3 H ] o l e a t e l a b e l l e d VLDL, a t the i n d i c a t e d TG c o n c e n t r a t i o n s , p u r i f i e d BmLpL (0.7 ug/ml), 0.16 M NaCl, 125 mg/ml bovine serum albumin, 0.35 M T r i s - H C l , pH 8.2 ( f i n a l volume, 1.3 m l ) . The mixtures were i n c u b a t e d f o r 45 min a t 37°C i n a shaking water bath and l i p o l y s i s was stopped by the a d d i t i o n of i s o p r o p a n o l : s u l p h u r i c a c i d (40:1). The amount o f [ 3 H ] o l e i c a c i d r e l e a s e d was determined a c c o r d i n g t o Schotz e t a l (147). The r e s u l t s are expressed as umoles o f o l e i c a c i d r e l e a s e d per mg BmLpL per min. 2.4 C e l l C u l t u r e 2.4.1 I s o l a t i o n and c u l t u r e o f human f i b r o b l a s t s S k i n was b i o p s i e d from the medial p a r t o f the forearm of the TD p a t i e n t and c u l t u r e d i n 35 mm d i s h e s and m o d i f i e d Eagles 47 medium (MEM) c o n t a i n i n g 20% f e t a l c a l f serum (FCS), 4.0 IU/ml p e n i c i l l i n (base),4.0 ug/ml str e p t o m y c i n (base) and 10.0 ng/ml fungizone and maintained i n a h u m i d i f i e d C0 2 (5%) i n c u b a t o r a t 37°C. The ex p l a n t was not d i s t u r b e d d u r i n g the f i r s t f i v e days of c u l t u r e . On the s i x t h day the medium was r e p l a c e d w i t h f r e s h medium. A f t e r 14 days i n c u l t u r e , the medium was changed t o MEM c o n t a i n i n g 5% FCS, 1.0 IU/ml p e n i c i l l i n , 1.0 ug/ml s t r e p t o m y c i n and 2.5 ng/ml fungizone. When the c e l l monolayer reached c o n f l u e n c y the c e l l s were d i s s o c i a t e d w i t h a 0.05% T r y p s i n / 0 . 2 % EDTA s o l u t i o n , d i l u t e d 3 times w i t h medium and r e p l a t e d . Age matched c o n t r o l f i b r o b l a s t l i n e s were o b t a i n e d from t h e NIGMS Human g e n e t i c mutant c e l l r e p o s i t o r y and a l l the c e l l l i n e s were used between 5th and 15th passage. T a n g i e r - d e r i v e d and c o n t r o l - d e r i v e d f i b r o b l a s t s had s i m i l a r r a t e s o f growth as determined by v i s u a l i n s p e c t i o n (cytometry) and p r o t e i n d e t e r m i n a t i o n . 48 2.4.2 P r e p a r a t i o n o f c e l l l i n e s p r i o r t o l i p o p r o t e i n b i n d i n g experiments P r e p a r a t i o n o f human s k i n f i b r o b l a s t s f o r LDL b i n d i n g and degradation assays was performed a c c o r d i n g t o the method of Brown et a l (128). On day 0 of the experiment, c o n f l u e n t monolayers of c o n t r o l and T a n g i e r c e l l s from s t o c k f l a s k s were d i s s o c i a t e d with 0.05% t r y p s i n / 0 . 0 2 % EDTA s o l u t i o n and were seeded a t a c o n c e n t r a t i o n o f 5.0 x l O 4 c e l l s per d i s h i n t o 35 x 15 mm p e t r i d i s h e s i n 2 ml of MEM c o n t a i n i n g 10% FCS. On day 3, the medium was r e p l a c e d w i t h 2 ml of f r e s h growth medium c o n t a i n i n g 10% FCS. On day 5, each monolayer was washed w i t h 2 ml s t e r i l e phosphate b u f f e r e d s a l i n e (PBS). Afterwards 2 ml of f r e s h growth medium c o n t a i n i n g 5% l i p o p r o t e i n - d e f i c i e n t f e t a l c a l f serum (LPDS) was added t o the c u l t u r e s . Experiments were performed on day 7 a f t e r the c e l l s had been incubated f o r 48 h i n the presence o f LPDS. The d i s h e s were approximately 60-75 % c o n f l u e n t with c e l l s and contained 40-100 ug of c e l l p r o t e i n . F i b r o b l a s t s f o r HDL b i n d i n g experiments were seeded a t the same c o n c e n t r a t i o n as p r e v i o u s l y d e s c r i b e d f o r LDL experiments. The media were changed every 3 days u n t i l t he c e l l s had reached confluency ( u s u a l l y 10-12 d a y s ) . On the day o f the experiment the media were removed and the c e l l s were in c u b a t e d i n MEM c o n t a i n i n g 2% BSA f o r 2 h p r i o r t o the s t a r t o f the experiment t o d i s s o c i a t e c e l l a s s o c i a t e d HDL. 2.4.3 C u l t u r e s o f Human P e r i p h e r a l Blood Monocytes Human p e r i p h e r a l b l o o d monocytes were i s o l a t e d from f r e s h l y drawn blood u s i n g a method m o d i f i e d from t h a t o f Cohn e t a l (148). In a t y p i c a l p r e p a r a t i o n , venous b l o o d (69 ml) was drawn 49 i n t o v a c u t a i n e r s c o n t a i n i n g 0.1 % EDTA. A one ml sample of b l o o d was analyzed f o r leukocyte counts u s i n g a C o u l t e r Counter (Model S ) . White b l o o d c e l l s (WBC) d i f f e r e n t i a l v a l u e s f o r i n d i v i d u a l c e l l types were obtained by c o u n t i n g 200 c e l l on smears made of the b l o o d sample s t a i n e d w i t h Wrights s t a i n and examined under o i l w i t h a compound microscope. These 2 procedures were done i n the D i v i s i o n o f Hematology, Shaughnessy H o s p i t a l . The remaining blood was then c e n t r i f u g e d a t approximately 321 x g f o r 10 min a t room temperature. The c e l l p e l l e t was resuspended i n an equal volume PBS c o n t a i n i n g 0.1% EDTA a t 37°C. The f i n a l volume was a d j u s t e d t o 7 0 ml w i t h PBS and then 35 ml was g e n t l y l a y e r e d over 15 ml of F i c o l l - P a q u e (FP) i n a 50 ml c o n i c a l c e n t r i f u g a t i o n tube. The sample was then c e n t r i f u g e d a t room temperature f o r 30 min a t approximately 427 x g. A f t e r c e n t r i f u g a t i o n of the sample f o r 30 min, the top l a y e r was a s p i r a t e d and the mononuclear c e l l l a y e r was t r a n s f e r r e d t o a 15 ml c o n i c a l c e n t r i f u g e tube. The c e l l s were washed twice w i t h 15 ml of warm PBS (37°C). A f t e r the l a s t wash, the c e l l p e l l e t was suspended i n 10 ml o f RPMI-1640 c o n t a i n i n g 20% d e l i p i d a t e d FCS, 1.0 IU/ml p e n i c i l l i n , 1.0 ug/ml st r e p t o m y c i n and 2.5 ng/ml fungizone. The l e u k o c y t e c o n c e n t r a t i o n was determined by c o u n t i n g an a l i q u o t of the c e l l suspension u s i n g a haemocytometer. The y i e l d of l e u c o c y t e s was approximately 85% of the i n i t i a l number. Assuming a s i m i l a r y i e l d f o r monocytes the c e l l suspension was a d j u s t e d t o 1.0 x 1 0 6 monocytes per ml and one ml a l i q u o t s were then p i p e t t e d i n t o 1.5 x 17 mm t i s s u e c u l t u r e d i s h e s . The d i s h e s were incubated i n a C0 0 c o n t r o l l e d i n c u b a t o r 50 a t 37°C f o r 1 h. The non-adherent c e l l s were then removed and the monocytes washed f o u r times with 1 ml a l i q u o t s o f PBS. The c e l l s were used immediately f o r b i n d i n g s t u d i e s . Monocyte p r e p a r a t i o n s were g r e a t e r than 95% pure as determined by immunoperoxidase s t a i n i n g . Average monocyte y i e l d from 69 ml o f plasma was 1-2 x 10 7 c e l l s . 2.5 Metabolism o f l i p o p r o t e i n s by c u l t u r e d c e l l s i n v i t r o 2.5.1 1 2 5 I - L D L Metabolism by F i b r o b l a s t s On the day of the experiment, the LPDS medium was removed and the monolayer washed wi t h PBS. 1 2 5 I - L D L (0-100 mg/ml) i n 2 ml of medium c o n t a i n i n g BSA (2.0 mg/ml) and l i m a bean t r y p s i n i n h i b i t o r (LBTI) (0.6 mg/ml) was added t o t h e d i s h e s . S t u d i e s on the time dependency o f b i n d i n g i n d i c a t e d t h a t b i n d i n g reached s a t u r a t i o n a f t e r f o u r hours o f i n c u b a t i o n w i t h LDL (a dose o f 50 ug/ml) and remain c o n s t a n t f o r the next f o u r hours. Thus, an i n c u b a t i o n time o f s i x hours was used i n thes e s t u d i e s . At the end o f the i n c u b a t i o n p e r i o d , 2 ml of the medium was removed i n t o tubes c o n t a i n i n g 0.5 ml of 50% TCA. The TCA-soluble, non-iodide r a d i o a c t i v i t y was determined a c c o r d i n g t o the method of G o l d s t e i n and Brown (128). C e l l monolayers were washed w i t h 3 x 2 ml a l i q u o t s o f i c e - c o l d PBS c o n t a i n i n g 2 mg/ml BSA f o l l o w e d by 3 x 2 ml a l i q u o t s o f i c e - c o l d PBS alone. The c e l l monolayers were then l y s e d i n 1.0 ml 0.1M NaOH f o r 30 min a t 37°C. 1 2 5 I - r a d i o a c t i v i t y and c e l l p r o t e i n was determined and the r e s u l t s f o r LDL b i n d i n g and d e g r a d a t i o n were expressed as ng LDL p r o t e i n p e r mg of c e l l p r o t e i n . The s p e c i f i c component o f b i n d i n g was determined as the d i f f e r e n c e between the t o t a l b i n d i n g component and the non-51 s p e c i f i c b i n d i n g component (128)• The n o n - s p e c i f i c component was determined by conducting the above experiment i n the presence of excess u n l a b e l l e d l i g a n d (128) .. 2.5.2 1 2 5 I - H D L Metabolism by F i b r o b l a s t s On the day of the experiment, f i b r o b l a s t s were washed wi t h 2 x 1 ml a l i q u o t s of MEM and p r e i n c u b a t e d w i t h 2 ml MEM f o r 2 h. The c e l l s were then incubated w i t h 2 ml MEM c o n t a i n i n g BSA (2 mg/ml), LBTI (6 mg/ml) and e i t h e r 1 2 5 I - H D L (0-100 mg/ml) or 1 2 5 l -HDL 3 (0-100 mg/ml) f o r 2 h a t 37°C. At the end of the i n c u b a t i o n p e r i o d , HDL c e l l a s s o c i a t i o n and d e g r a d a t i o n assays were performed a c c o r d i n g t o the methods of Oram e t a l (75). These d e t e r m i n a t i o n s are almost i d e n t i c a l t o those d e s c r i b e d above f o r the LDL a n a l y s e s . 2.5.3 1 2 5 I - H D L 3 Metabolism by Monocytes . ...To, determine 1 2 5 I - H D L 3 a s s o c i a t e d w i t h and degraded by C o n t r o l and T a n g i e r monocytes, the f o l l o w i n g assay was used. F r e s h l y i s o l a t e d monocytes were incubated w i t h RPMI-1640 c o n t a i n i n g 20% LPDS and 1 2 5 I - H D L (0-200 ug/ml) f o r 4 o r 20 h a t 37°C. C e l l v i a b i l i t y was examined a t the end o f the experiment by t r y p a n b l u e e x c l u s i o n . V i s u a l i n s p e c t i o n i n d i c a t e d > 90% v i a b i l i t y . Monocyte c e l l a s s o c i a t e d 1 2 5 I - l a b e l was determined i n a s i m i l a r manner as p r e v i o u s l y d e s c r i b e d f o r f i b r o b l a s t s ( S e c t i o n 2.5.1) 52 3 R e s u l t s 3.1 Catabolism of normal HDL by T a n g i e r plasma i n v i t r o T a b l e IV shows the p r o t e i n and l i p i d content of c o n t r o l and of TD plasma be f o r e and a f t e r the a d d i t i o n of exogenous normal HDL. The h y p e r t r i g l y c e r i d e m i a , h y p o c h o l e s t e r o l e m i a and h y p o a l p h a l i p o p r o t e i n e m i a of the TD plasma was p r e v i o u s l y d e s c r i b e d i n ot h e r p a t i e n t s w i t h t h i s d i s o r d e r (90). The a d d i t i o n of normal HDL t o TD plasma, r e s u l t e d i n an i n c r e a s e i n the FC and CE content i n both the t o t a l plasma and HDL f r a c t i o n . Apo A-I l e v e l s of the TD plasma were a l s o r a i s e d t o w i t h i n t h e normal range by the a d d i t i o n of normal HDL. The t o t a l amount o f CE and TG i n the Tan g i e r plasma remained s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l samples due t o the low CE and h i g h TG content o f the TD LDL and VLDL f r a c t i o n . 3.1.1 E f f e c t o f i n v i t r o i n c u b a t i o n on l i p o p r o t e i n e l e c t r o p h o r e t i c m o b i l i t y Normal HDL was added t o TD plasma i n s u f f i c i e n t q u a n t i t y t o r a i s e the HDL-C l e v e l s t o approximately 40 mg/dl. One ml a l i q u o t s of t h i s mixture were t r a n s f e r r e d t o g l a s s t e s t tubes (13 x 100 mm), s e a l e d and incubated a t 37°C f o r 0,4,12 and 24 h. The samples were examined by agarose g e l e l e c t r o p h o r e s i s as d e s c r i b e d i n S e c t i o n 2.3.2.2 (F i g u r e 5 ). In c o n t r o l plasma the m o b i l i t y of LDL was 0.7 cm p r i o r t o i n c u b a t i o n . During the i n c u b a t i o n a t 37°C the m o b i l i t y of the normal LDL i n c r e a s e d l i n e a r l y from 0.7 cm t o 1.2 cm i n samples incubated f o r 24 h. T h i s f i n d i n g i s c o n s i s t e n t with the o b s e r v a t i o n s of C a r l s o n e t a l (149). The m o b i l i t y of the HDL i n c o n t r o l plasma was 2.7 cm p r i o r t o i n c u b a t i o n and remained 53 TABLE IV. LIPID AND PROTEIN CONTENT OF PLASMA AND LIPOPROTEINS IN TD PLASMA IN THE PRESENCE AND ABSENCE OF EXOGENOUS HIGH DENSITY LIPOPROTEIN. FRACTION SAMPLE COMPOSITION (mg/dl) (n=3) CE FC TG APO A-I PLASMA CONTROL TANGIER TANGIER + HDL a 115+18 * * 45±3 71+10* 49+9 98+78 27+2 43+13 ** 297+15 *** 146+7 2+0.5 *** 276+48*** 129+6 HDL VLDL/LDL CONTROL TANGIER TANGIER + HDL CONTROL TANGIER TANGIER + HDL 38±9 2+1 33+5 76±16 63±4* 38+12' *** 9+1 18+3 1+0.5*** 3+1** 11±4 40+10 * * 23+3 33+15 28+6' 79+28 N.D. N.D. N.D. N.D. 286+16*** N.D. 248+49*** N.D. S u f f i c i e n t normal HDL was added to an a l i q u o t o f TD plasma t o r a i s e the HDL-C l e v e l t o a p p r o x i m a t e l y 45 mg/dL p r i o r to the de t e r m i n a t i o n o f l i p i d and a p o p r o t e i n A-I c o n c e n t r a t i o n s . k HDL and VLDL/LDL f r a c t i o n s r e f e r t o heparin/manganese n o n p r e c i p i t a b l e and p r e c i p i t a b l e l i p o p r o t e i n s , r e s p e c t i v e l y . c N.D.= not determined. Apoprotein A-I l e v e l s were not determined i n the heparin/manganese p r e c i p i t a t e d plasma. S i g n i f i c a n c e o f d i f f e r e n c e from c o n t r o l : * p<0.05; ** p<0.01; p<0.001. FC= f r e e c h o l e s t e r o l ; CE= e s t e r i f i e d c h o l e s t e r o l ; T G = t r i g l y c e r i d e 54 T A N G I E R C O N T R O L + -jpf ' I p l a s m a § 1 3 Oh 4h 1 i ; $ B 'E £ S p l a s m a < ' I I i i i 1 1 1 0 1 2 3 4 4 3 2 1 O M o b i l i t y (cm) F i g u r e 5. E f f e c t o f i n c u b a t i o n on l i p o p r o t e i n e l e c t r o p h o r e s i s p a t t e r n f o l l o w i n g t h e a d d i t i o n o f normal HDL t o TD plasma. 1% agarose g e l e l e c t r o p h o r e s i s p a t t e r n s o f TD plasma c o n t a i n i n g normal HDL (A) and c o n t r o l plasma (B) incubated a t 37° f o r the i n d i c a t e d times. D i r e c t i o n o f e l e c t r o p h o r e s i s i s from cathode t o anode ( l e f t t o r i g h t and r i g h t t o l e f t f o r TD and c o n t r o l r e s p e c t i v e l y ) . The m o b i l i t y o f l i p o p r o t e i n s i n the c o n t r o l plasma were : LDL; 0.7 cm; VLDL; 1.3 cm; HDL; 2.7 cm. 55 unchanged d u r i n g the i n c u b a t i o n p e r i o d . T h i s i s a l s o c o n s i s t e n t with p r e v i o u s r e p o r t s (149). C a r l s o n suggest t h a t the i n c r e a s e d m o b i l i t y i s due to a change i n charge of the LDL p a r t i c l e when i t becomes o x i d i z e d and/or p i c k s up f r e e f a t t y a c i d s (149). The m o b i l i t y of the LDLpj-, was 1.1 cm p r i o r t o i n c u b a t i o n and i n c r e a s e d l i n e a r l y d u r i n g the 24 h i n c u b a t i o n t o a v a l u e of 1.4 cm. The i n c r e a s e d m o b i l i t y of LDL,pD compared t o c o n t r o l LDL i s c o n s i s t e n t with p r e v i o u s r e p o r t s (95,100). The HDL i n TD plasma had a m o b i l i t y o f 2.7 cm p r i o r t o the i n c u b a t i o n and the m o b i l i t y remained r e l a t i v e l y unchanged d u r i n g the 24 h i n c u b a t i o n p e r i o d . The r e l a t i v e i n t e n s i t y o f the HDL band d i m i n i s h e d d r a m a t i c a l l y d u r i n g the i n c u b a t i o n p e r i o d i n TD plasma compared t o c o n t r o l s i n d i c a t i n g t h a t c a t a b o l i s m o f the HDL p a r t i c l e was o c c u r i n g i n v i t r o . 3 . 1 . 2 E f f e c t o f i n c u b a t i o n on H D L - c h o l e s t e r o l P r e v i o u s work by P r i t c h a r d and F r o h l i c h (12 4) examined the i n v i v o r a t e o f HDL c a t a b o l i s m i n the TD p a t i e n t s t u d i e d i n t h i s t h e s i s . They showed t h a t the l e v e l s o f HDL-C and t o t a l plasma apo A-I decreased r a p i d l y i n TD plasma f o l l o w i n g the i n f u s i o n o f 3 1 of normal plasma. The peak v a l u e o f HDL-C c o n c e n t r a t i o n was 20 mg/dl but over the 66 h a f t e r plasmapheresis i t r e t u r n e d t o the o r i g i n a l l e v e l o f 2 mg/dl. T h i s r e s u l t was almost i d e n t i c a l t o t h a t d e s c r i b e d f o r " p a t i e n t A" by Schaefer e t a l (12 5). These data i n d i c a t e t h a t both of these TD p a t i e n t s had s i m i l a r HDL-C turnov e r . P r i t c h a r d and F r o h l i c h a l s o noted r a p i d l o s s o f apo A-I from the plasma of the TD p a t i e n t (124). The disappearance o f apo A-I fo l l o w e d a b i e x p o n e n t i a l curve with an i n i t i a l f a s t component and 56 a subsequent slow component. The f a i l u r e o f apo A-I t o reach normal l e v e l s and i t s r a p i d c l e a r a n c e from the plasma compartment was s i m i l a r t o t h a t r e p o r t e d by Schaefer e t a l (125). S i n c e HDL-C i n TD plasma r a p i d l y decreases i n v i v o a f t e r i n f u s i o n o f l a r g e amounts of HDL (107-114, 125), the f o l l o w i n g experiment examined the changes i n HDL-C of c o n t r o l HDL d u r i n g i n c u b a t i o n i n TD plasma i n v i t r o . Exogenous normal HDL and an equal volume of b u f f e r C was added t o c o n t r o l plasma t o r a i s e HDL-C l e v e l s t o 40 mg/dl. One ml samples o f c o n t r o l and HDL-en r i c h e d TD plasma were incubated a t 37°C f o r 0,4,8,12 and 24 h. HDL-C l e v e l s were determined as d e s c r i b e d i n S e c t i o n 2.2.5.2. The r e s u l t s o f t h i s experiment are pres e n t e d i n F i g u r e 6. T h i s experiment demonstrates t h a t the p r e v i o u s l y observed l o s s o f c h o l e s t e r o l from normal HDL i n f u s e d i n t o TD plasma a l s o occurs i n v i t r o . A 35% decrease i n HDL-C o c c u r r e d over the f i r s t 4 h of the i n c u b a t i o n with a f u r t h e r decrease t o 50% of i n i t i a l v a l u e s a f t e r 24 h. In c o n t r o l plasma, HDL-C l e v e l s i n c r e a s e d s l i g h t l y d u r i n g the 24 h i n c u b a t i o n ( F i g u r e 6 ) . Thus t h i s decrease i n HDL-C i n the TD plasma i n v i t r o suggests t h a t the "hypercatabolism" o f HDL i n f u s e d i n t o TD plasma i s dependent on plasma f a c t o r s and does not r e q u i r e c e l l u l a r involvement. In the f o l l o w i n g experiment, the changes i n plasma, HDL, and LDL/VLDL l i p i d were f o l l o w e d over a 12 h p e r i o d . 57 0 . 4 8 12 16 20 24 Time (h) F i g u r e 6i The r e l a t i v e t o t a l c h o l e s t e r o l c o n t e n t o f normal HDL inc u b a t e d i n c o n t r o l o r TD plasma. Normal HDL was added t o samples of TD plasma as d e s c r i b e d i n Table IV and incubated f o r the i n d i c a t e d times a t 37°C ... The i n t i t a l HDL c o n c e n t r a t i o n of the c o n t r o l and TD plasma i s i n d i c a t e d i n Table IV. The f i g u r e shows the amount of c h o l e s t e r o l a s s o c i a t e d with HDL r e l a t i v e t o t h e i n i t i a l v a l u e from c o n t r o l (A) or Tan g i e r plasma (H)• The d a t a r e p r e s e n t mean + S.D. f o r t h r e e independent experiments. 58 3.1.3 E f f e c t o f i n v i t r o i n c u b a t i o n on l i p o p r o t e i n l i p i d c o mposition The r e l a t i v e changes i n the amount of FC, CE and TG i n the plasma, HDL and VLDL/LDL f r a c t i o n s f o l l o w i n g the i n v i t r o i n c u b a t i o n o f normal HDL wit h TD plasma are shown i n Table V. In c o n t r o l plasma, CE l e v e l s i n c r e a s e d by 17 + 6% d u r i n g the i n c u b a t i o n p e r i o d . However, t h i s i n c r e a s e c o u l d be i n h i b i t e d by the a d d i t i o n o f 5 mM d i i s o p r o p y l f l u o r p h o s p h a t e (DiFP) which completely i n h i b i t s LCAT a c t i v i t y (150). The o b s e r v a t i o n t h a t the CE content o f the TD plasma remained unchanged d u r i n g the i n c u b a t i o n p e r i o d r e f l e c t s the low LCAT a c t i v i t y i n the plasma of t h i s p a t i e n t [68% of normal (124)]. C o n t r o l plasma l e v e l s o f FC decreased by 24 +8% but remained unchanged i n the TD plasma which was c o n s i s t e n t w i t h d i f f e r e n c e s i n the r a t e o f CE p r o d u c t i o n i n both samples. Plasma l e v e l s o f TG remained unchanged i n both c o n t r o l and TD plasma. C o n t r o l HDL-FC l e v e l s decreased by 17 +9% over a 12 h i n c u b a t i o n i n d i c a t i n g the u t i l i z a t i o n of t h i s f r a c t i o n as a s u b s t r a t e f o r the LCAT r e a c t i o n . However, l i t t l e change i n HDL-FC was seen i n TD plasma d u r i n g the f i r s t 8 h of i n c u b a t i o n but the l e v e l s decreased by 34 +17% d u r i n g the l a s t 4 h. These r e s u l t s i n d i c a t e t h a t the i n i t i a l l o s s o f c h o l e s t e r o l from normal HDL incubated i n T a n g i e r plasma i s i n the form o f CE and not FC. In c o n t r o l plasma, HDL-CE l e v e l s i n c r e a s e d l i n e a r l y d u r i n g the f i r s t 8 h of i n c u b a t i o n a f t e r which no f u r t h e r s i g n i f i c a n t i n c r e a s e s were seen. By c o n t r a s t , HDL-CE l e v e l s i n TD plasma decreased by 43 +4% i n the f i r s t 4 h of the i n c u b a t i o n but d i d 59 TABLE V . CHANGES IN THE RELATIVE LIPID COMPOSITION OF TD LIPOPROTEINS FOLLOWING INCUBATION IN THE PRESENCE OF EXOGENOUS NORMAL HIGH DENSITY LIPOPROTEINS. Samples of c o n t r o l , TD and TD + HDL plasma (Table IV) were incubated f o r the i n d i c a t e d time at 37 C. The re a c t i o n s were stopped by r a p i d c o o l i n g to 0°C. The amount of l i p i d was determined before and a f t e r p r e c i p i t a t i o n of VLDL and LDL by heparin/manganese. The r e s u l t s are expressed as means+1 SD of the amount recovered r e l a t i v e to time= Oh. The i n i t i a l values for^each sample are i n d i c a t e d i n Table IV. S i g n i f i c a n c e of d i f f e r e n c e of TD from c o n t r o l : * p<0.05; * p<0.01; p<0.001 FRACTION PERCENTAGE OF INITIAL VALUE JBATI0M CHOLESTERYL ESTER FREE CHOLESTEROL TRIACYLGLYCEROL IE (h) CONTROL TANGIER CONTROL TANGIER CONTROL TANGIER n=4 n=4 n=4 n=4 n=3 n=3 0 100 100 100 100 100 100 4 108+4 98+3** 84+5 101+8* 99+1 100 + 1 8 118 + 8 109+2 79+8 97+8 105+9 102 + 4 12 117 + 6 103+14 76+8 103+17 101+3 101 + 1 0 100 100 100 100 100 100 4 107+7 57+4*** 96+7 108+20 119+17 194±6*** 8 116+14 60+6*** 92 + 5 102+7 132±14 224+4*** 12 120+12 se±i4*** 6 3+9 60+17 121+14 193+31** 0 100 100 100 100 100 100 4 109+7 151+32* a i+6 101+14 9 5+4 9 2 + 2 8 119+7 188+29* 76±10 95+11 99 + 14 91 + 5 12 116+9 . 174+29* •'. .. 75+10 116+25 97+7 90+2 O PLASMA HDL VLDL/LDL not decrease f u r t h e r d u r i n g the remaining 8 h. The d i f f e r e n c e between TD and c o n t r o l , f o r both the a b s o l u t e change and the r a t e of change, are h i g h l y s i g n i f i c a n t (P<0.001) (Table V ) . In c o n t r o l plasma, HDL-TG l e v e l s rose by 32 +14% d u r i n g the f i r s t 8 h of i n c u b a t i o n w i t h no s i g n i f i c a n t changes o c c u r i n g d u r i n g the remaining 4 h. By c o n t r a s t , the r e l a t i v e TG content of the normal HDL i n TD plasma r o s e t o 224 ±4% o f the i n i t i a l amount over the f i r s t 8 h. T h i s o b s e r v a t i o n suggests t h a t t h e l o s s of CE from the core of the HDL may be a s s o c i a t e d w i t h the a c q u i s i t i o n of TG from the VLDL/LDL f r a c t i o n . I t was noted t h a t the l o s s of HDL-CE and the i n c r e a s e i n HDL-TG were, on a molar b a s i s , e s s e n t i a l l y the same f o r both c o n t r o l and TD plasma. Thus, these r e s u l t s suggest t h a t t h e r e m a y be an exchange of 1 molecule o f TG f o r every molecule of C E i n the core o f the HDL p a r t i c l e . In c o n t r o l plasma, VLDL/LDL l e v e l s of CE i n c r e a s e d by 19 +7% d u r i n g the f i r s t 8 h of i n c u b a t i o n w i t h no f u r t h e r i n c r e a s e s observed d u r i n g the l a s t 4 h. T h i s i n c r e a s e probably r e f l e c t s the t r a n s f e r of CE from HDL. The r a t e of t h i s t r a n s f e r was s i g n i f i c a n t l y (P<0.001) g r e a t e r i n TD plasma. During the f i r s t 8 h t h e r e was an 88 +29% i n c r e a s e i n the CE con t e n t o f TD VLDL/LDL with no s i g n i f i c a n t changes d u r i n g the l a s t 4 h o f i n c u b a t i o n . VLDL/LDL FC c h o l e s t e r o l l e v e l s decreased i n c o n t r o l plasma by 19 +6% i n the f i r s t 4 h f o l l o w e d by a f u r t h e r decreases d u r i n g the subsequent 8 h of i n c u b a t i o n . However, l i t t l e change i n VLDL/LDL FC was observed i n TD plasma d u r i n g the f i r s t 8 h. VLDL/LDL TG l e v e l s remained unchanged i n c o n t r o l plasma d u r i n g the 12 h i n c u b a t i o n p e r i o d . However, i n TD VLDL/LDL t h e r e was an 61 approximate 10% decrease d u r i n g the f i r s t 4 h of i n c u b a t i o n f o l l o w e d by a small decrease d u r i n g the remaining 8 h. 3.1.4 E f f e c t o f i n c u b a t i o n on HDL a p o p r o t e i n composition The r e l a t i v e amount of apo A-I remaining i n the c o n t r o l and TD plasma a f t e r a 24 h i n c u b a t i o n was 108 +5 and 105 +6 mg/dl r e s p e c t i v e l y , which was approximately 95% o f the i n i t i a l v a l u e s . T h i s demonstrates t h a t no protease, s p e c i f i c f o r normal apo A-I, e x i s t s i n the plasma of the p a t i e n t w i t h TD d i s e a s e . Thus, these r e s u l t s suggest t h a t the observed l o s s o f apo A-I from TD plasma i n v i v o p r o b a b l y r e q u i r e s c e l l u l a r i n t e r a c t i o n . I t has p r e v i o u s l y been r e p o r t e d t h a t apo A-I and A - I I from normal HDL r e d i s t r i b u t e among TD l i p o p r o t e i n s i n v i v o (125). T h e r e f o r e , i n the f o l l o w i n g i n v e s t i g a t i o n , t h e i n v i t r o t r a n s f e r of t hese a p o p r o t e i n s between the l i p o p r o t e i n c l a s s e s was s t u d i e d . The HDL-C l e v e l of 700 u l of TD plasma was a d j u s t e d t o approximately 45 mg/dl by the a d d i t i o n o f 170 u l o f 1 2 5 I - H D L (100 cpm/ng). H a l f o f the sample was incubated a t 4°C and the o t h e r h a l f was incubated a t 37°C. A f t e r i n c u b a t i n g f o r 24 h the plasma l i p o p r o t e i n s were separated by g e l f i l t r a t i o n on B i o g e l A5m and the amount of l a b e l r e covered i n each l i p o p r o t e i n c l a s s determined. More than 95% of the ^ - 2 5 i r a d i o a c t i v i t y a p p l i e d t o the column was recovered with the l i p o p r o t e i n f r a c t i o n s . The • r e s u l t s o f t h i s experiment are presented i n F i g u r e 7. At 4°C, apo A-I and A-II (represented by t o t a l 1 2 5 I ) r e d i s t r i b u t e d among the l i p o p r o t e i n s w i t h 91% o f a c t i v i t y remaining w i t h the HDL, 7% wit h LDL and l e s s than 1% wit h VLDL a f t e r 24 hours. I n c u b a t i o n a t 37°C g r e a t l y i n c r e a s e d the 62 40 50 60 Fraction number 70 F i g u r e 7. G e l f i l t r a t i o n chromatography o f 125 I-HDL-apoproteins f o l l o w i n g i n c u b a t i o n i n TD plasma a t 4°G o r 37°C. 125 I-HDL (100 cpm/ng) was added t o TD plasma to g i v e a f i n a l HDL-C c o n c e n t r a t i o n of 45 mg/dl. A f t e r i n c u b a t i o n a t 4°C ( £ ) and 37°C ( H ) f o r 24 h, the l i p o p r o t e i n s i n 2 mL a l i q u o t s o f plasma were r e s o l v e d on a B i o g e l A5m column. The amount of 1 2 5 i r e c o v ered i n the c o l l e c t e d f r a c t i o n s i s shown. The f r a c t i o n s i n which normal VLDL, LDL and HDL e l u t e d were determined by p r e c a l i b r a t i o n of the column w i t h 1 2 5 I - l i p o p r o t i e n s . 63 r e d i s t r i b u t i o n of r a d i o a c t i v i t y from HDL t o LDL and VLDL. 18% of the r a d i o a c t i v i t y was recovered i n the LDL f r a c t i o n and 2% w i t h the VLDL. The remaining 79% of the X £ d I - a p o p r o t e m was recovered from the HDL f r a c t i o n . These r e s u l t s i n d i c a t e t h a t HDL a p o p r o t e i n s d i s s o c i a t e from normal HDL and r e a s s o c i a t e w i t h V L D L T D L D L T D d u r i n g i n v i t r o i n c u b a t i o n . S i n c e r e d i s t r i b u t i o n of HDL a p o p r o t e i n i s g r e a t e r at 37°C than a t 4°C t h i s o b s e r v a t i o n suggests t h a t r a t e of movement of these a p o p r o t e i n s are temperature dependent and may be a s s o c i a t e d w i t h the temperature dependent changes i n the l i p i d composition of the HDL t h a t o c c u r r e d d u r i n g the i n c u b a t i o n (Table V). However, 8% of the r a d i o a c t i v i t y r e d i s t r i b u t e d d u r i n g the 4°C i n c u b a t i o n even though t h e r e were no s i g n i f i c a n t changes i n the HDL l i p i d c o mposition. Thus, i t appears t h a t some r e d i s t r i b u t i o n o f HDL a p o p r o t e i n occurs independently of the l i p i d exchange p r o c e s s . In a d d i t i o n t o the t r a n s f e r of 1 2 5 I - a p o p r o t e i n t o V L D L r p D and l i t , L D L r p D , -L^-'I was a l s o recovered i n a minor peak which e l u t e d a f t e r HDL ( f r a c t i o n 68 i n F i g u r e 7). T h i s peak was more apparent a f t e r i n c u b a t i o n a t 37°C. While we have been unable t o c h a r a c t e r i z e the nature of t h i s f r a c t i o n we do know t h i s does not r e p r e s e n t f r e e l a b e l ( f r e e 1 2 5 i e l u t e s much l a t e r ; g r e a t e r than f r a c t i o n 100). Schaefer e t a l (110,12 5) have p r e v i o u s l y shown t h a t apo A-I and apo A-II d i s t r i b u t e unevenly among TD plasma l i p o p r o t e i n f o l l o w i n g i n t r a v e n o u s a d m i n i s t r a t i o n . The r e d i s t r i b u t i o n o f these a p o p r o t e i n s d u r i n g i n c u b a t i o n of normal " I - H D L i n TD plasma was t h e r e f o r e a l s o s t u d i e d i n our i n v i t r o experiments. The B i o g e l A 5.0m column f r a c t i o n s c o n t a i n i n g VLDL, LDL and 64 HDL ( F i g u r e 7) were concentrated and the i n d i v i d u a l a p o p r o t e i n s were r e s o l v e d by 12.5% SDS-PAGE. The r e s u l t s of t h i s experiment are shown i n Table VI. A f t e r i n c u b a t i o n a t 4°C, o n l y 7% and 8% of the apo A-I and apo A-II were recovered i n the VLDL/LDL f r a c t i o n s r e s p e c t i v e l y . T h i s suggests t h a t the t r a n s f e r of the two a p o p r o t e i n s a t 4°C occurred a t the same r a t e and, p o s s i b l y , c o n c u r r e n t l y . However, at 37°C, the r e l a t i v e t r a n s f e r r a t e s of apo A-I and apo A-II were s i g n i f i c a n t l y d i f f e r e n t . A t o t a l of 26.7% o f the apo A^-II was t r a n s f e r r e d t o VLDL/LDL i n 24 h whereas only 14.9% of the apo A-I was r e c o v e r e d i n t h i s f r a c t i o n . These r e s u l t s are s i m i l a r t o the i n v i v o o b s e r v a t i o n s of S c h a e f e r e t a l (110,125) i n t h a t a g r e a t e r percentage of the apo A - I I became a s s o c i a t e d w i t h the lower d e n s i t y l i p o p r o t e i n s than d i d apo A-I. Furthermore, these r e s u l t s suggest t h a t the t r a n s f e r of a p o p r o t e i n s occurs i n v i t r o and does not r e q u i r e the c o n c u r r e n t c e l l u l a r metabolism of the plasma l i p o p r o t e i n s . The above experiment i n d i c a t e s t h a t the i n c u b a t i o n has no major e f f e c t s on the s i z e d i s t r i b u t i o n of the v a r i o u s l i p o p r o t e i n s . In an e a r l i e r experiment TD l i p o p r o t e i n s were l a b e l l e d w i t h [ H] c h o l e s t e r o l a f t e r i n c u b a t i o n with normal HDL a t 4°C and 37°C and then separate on a B i o g e l A 5.0m column (Fi g u r e 8 ) . T h i s experiment demonstrates t h a t t h e r e were no major changes i n l i p o p r o t e i n p a r t i c l e s i z e d u r i n g i n c u b a t i o n a t 4°C or 37°C. The e f f e c t of i n c u b a t i o n on the s i z e o f the HDL p a r t i c l e was f u r t h e r examined i n the f o l l o w i n g study. The B i o g e l A 5.0m column HDL peak ( F r a c t i o n s 51-72) was pooled, c o n c e n t r a t e d and r e -65 a p p l i e d t o an B i o g e l A 0.5m column. The r e s u l t s of t h i s experiment are presented i n F i g u r e 9. These r e s u l t s i n d i c a t e t h a t the s i z e the HDL p a r t i c l e was not s i g n i f i c a n t l y changed d u r i n g the experiment by comparison w i t h 1 2 5 I - H D L . While the AO.5m column can separate HDL 2 and HDL 3, the column p r o f i l e o f both c o n t r o l and TD m o d i f i e d 1 2 5 I - H D L showed o n l y one peak. The presence of a s i n g l e subspecies o f HDL i s not s u r p r i s i n g as the i n v i t r o c o n v e r s i o n of HDL 3 t o HDL, i s w e l l documented (47). 66 TABLE VI. DISTRIBUTION OF 1 2 5 I - A P O A-I AND A - I I IN TANGIER LIPOPROTEINS AFTER INCUBATION WITH 1 2 5 - I HDL. A l i q u o t s of the column f r a c t i o n s o b t a i n e d from the experiment d e s c r i b e d i n f i g u r e 4 were pool e d as f o l l o w s : VLDL (28-32), LDL (33-50) and HDL (51-72). The r e s u l t i n g samples were concentrated by amicon f i l t r a t i o n and a l i q u o t s were r e s o l v e d i n t o i n d i v i d u a l a p o p r o t e i n s by e l e c t r o p h o r e s i s i n 12.5% p o l y a c l y a m i d e c o n t a i n i n g 1%SDS and 1% 2-mercaptoethanol. The bands were v i s u a l i z e d by s t a i n i n g i n commassie b l u e and areas c o r r e s p o n d i n g t o apo A-I and apo A - I I were c u t from the g e l and t h e amount of. • 3 I a s s o c i a t e d w i t h each o f these p r o t e i n s was determined. The r e s u l t s i n d i c a t e d the r e l a t i v e amount o f l a b e l t h a t was a s s o c c i a t e d w i t h each a p o p r o t e i n i n each l i p o p r o t e i n c l a s s . Data i s presented as mean+1 SD f o r 3 experiments. *** i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e (P<0.001) between the samples i n c u b a t e d a t 4°C and 37°C. PERCENTAGE DISTRIBUTION OF 1 2 5 I 4°C 37°C APO A-I VLDL 0.7+0.1 2.4+0,6*** LDL 6.0+0.5 12.5+1.5*** HDL 93.3+0.6 * * * 85.1+1.7 APO A-II VLDL 0.9+0.1 2.5±0.6*** LDL 7.8±0.4 24.2+0.8*** HDL 91.3+0.5 73.0+1.0*** 67 (f) c D O a " a O 20 30 40 50 60 70 80 Fraction number F i g u r e 8. Gel f i l t r a t i o n o f [ 3H] c h o l e s t e r o l l a b e l e d TD plasma c o n t a i n i n g HDL f o l l o w i n g i n c u b a t i o n a t 4° and 37°. HDL was added t o TD plasma t o g i v e a f i n a l HDL-C c o n c e n t r a t i o n of 45 mg/dl. A f t e r i n c u b a t i o n f o r 24 hours a t 4°C (©) and 37°C (Bj) , 2 ml a l i q u o t s of plasma were l a b e l l e d with [ 3H] c h o l e s t e r o l as d e s c r i b e d i n 'Methods' and the plasma l i p o p r o t e i n s : were r e s o l v e d on a B i o g e l A5.0m column. The f r a c t i o n s i n which normal VLDL, LDL and HDL e l u t e d were determined by p r e c a l i b r a t i o n o f the column with 1 2 5 I - l i p o p r o t i e n s . 68 T 1 1 j 1 1 1 j r F i g u r e 9. G e l f i l t r a t i o n chromatography o f I-HDL f o l l o w i n g i n c u b a t i o n i n TD plasma a t 4° and 37°. Both 4° (©) and 37° <-(H) HDL peaks ( F i g u r e 7, f r a c t i o n s 51-72) were concentrated and a volume of sample c o r r e s p o n d i n g t o 1 x 10 6 cpms was then r e s o l v e d on a B i o g e l A 0.5m column. The f r a c t i o n s i n which normal HDL e l u t e d was determined by p r e c a l i b r a t i o n of the column w i t h 1 2 5 I - H D L . 3.2 I n v e s t i g a t i o n o f mechanisms u n d e r l y i n g t h e m o d i f i c a t i o n o f normal HDL d u r i n g i n c u b a t i o n i n TD plasma 3.2.1 Role o f c h o l e s t e r y l e s t e r h y d r o l y s i s The p o s s i b i l i t y t h a t the l o s s o f CE from HDL inc u b a t e d w i t h TD plasma i n v i t r o may r e s u l t from CE h y d r o l y s i s (CEH) was examined by l a b e l i n g c o n t r o l and TD plasma ( i n some TD samples exogenous HDL was included) w i t h [ 3H] CE f o r 4 h a t 4°C. A f t e r i n c u b a t i n g the samples a t 37°C or 4°C f o r 24 h the r a t e o f CEH was determined. The r e s u l t s o f t h i s experiment are shown i n Tab l e VII (the r a t e o f CEH i s expressed as percentage h y d r o l y s i s o f t o t a l CE o c c u r i n g d u r i n g a 24 h ) . The r e s u l t s c l e a r l y show the r a t e of CEH i n TD plasma i s i n d i s t i g u i s h a b l e from t h a t o f two d i f f e r e n t c o n t r o l s (Table V I I , rows 1-3). In a subsequent experiment, normal HDL was added t o the TD plasma p r i o r t o [ 3H] CE l a b e l i n g and subsequent i n c u b a t i o n . T h i s r e s u l t e d i n a s l i g h t , but i n s i g n i f i c a n t , decrease i n the r a t e o f CEH i n TD plasma (Table V I I, row 4 ) . F i n a l l y , even when HDL was p r e l a b e l e d , b e f o r e a d d i t i o n t o t h e TD plasma, the r a t e o f CEH was not s i g n i f i c a n t l y i n c r e a s e d over c o n t r o l v a l u e s (Table V I I , row 5 ) . These r e s u l t s do not support the h y p o t h e s i s t h a t the l o s s o f CE from normal HDL i n TD plasma d u r i n g i n v i t r o i n c u b a t i o n i s due t o CEH. 70 TABLE V I I . CE HYDROLYSIS IN NORMAL HDL IN TD PLASMA. CE h y d r o l y s i s (CEH) was determined i n c o n t r o l s (2) and TD plasma by i n c u b a t i n g plasma p r e l a b e l l e d w i t h [ 3H] CE f o r 24 h a t 4°C and 37°C. In a d d i t i o n the CEH a c t i v i t y was a l s o examined by i n c u b a t i o n o f TD plasma wi t h HDL p r e l a b e l l e d w i t h [ 3H] CE. The amount of r a d i o l a b e l l e d CE h y d r o l y z e d d u r i n g t h e i n c u b a t i o n was determined (Methods). The r e s u l t s a re expressed as the percentage of CE t h a t was h y d r o l y z e d d u r i n g the i n c u b a t i o n p e r i o d . Percentage H y d r o l y s i s (%) C o n t r o l 1 4.8 C o n t r o l 2 6.1 TD 6.1 Tan g i e r + *HDL 3.0 * HDL p r e l a b e l l e d w i t h [ 3H] CE The r e s u l t s a re the mean of d u p l i c a t e i n c u b a t i o n s from a s i n g l e experiment. 71 3.2.2 Role o f L e c i t h i n : c h o l e s t e r o l a c y l t r a n s f e r a s e The p r e c e d i n g s t u d i e s on the m o d i f i c a t i o n o f HDL t h a t occurs d u r i n g i n c u b a t i o n w i t h TD plasma ( S e c t i o n 3.1) demonstrate the d i f f e r e n c e s i n the r a t e of s y n t h e s i s and i n t e r l i p o p r o t e i n t r a n s f e r of CE between TD and c o n t r o l plasma. As LCAT p l a y s a c e n t r a l r o l e i n t h i s process i n c o n t r o l plasma, the r o l e o f LCAT i n the m o d i f i c a t i o n o f HDL by TD plasma was examined. Table V I I I shows t h a t complete i n h i b i t i o n o f LCAT a c t i v i t y by 5 mM DiFP had no e f f e c t on the e f f l u x of c h o l e s t e r o l from normal HDL i n TD plasma. Furthermore, when normal HDL was added t o the plasma of a p a t i e n t w i t h homozygous LCAT d e f i c i e n c y / t h e l o s s of HDL-C was s i g n i f i c a n t l y lower than the e q u i v a l e n t l o s s i n TD plasma (Table V I I I ) . The sm a l l but s i g n i f i c a n t l o s s of HDL-C from.normal HDL i n the LCAT d e f i c i e n t plasma i s probably r e l a t e d t o the i n i t i a l r a t e s o f CE t r a n s f e r which a r e not i n f l u e n c e d by chemical i n h i b i t i o n o f LCAT (151). A s i m i l a r l o s s of HDL-C was seen i n c o n t r o l plasma c o n t a i n i n g 5 mM DiFP. These r e s u l t s i n d i c a t e t h a t LCAT i s not d i r e c t l y r e s p o n s i b l e f o r the m o d i f i c a t i o n o f normal HDL i n TD plasma d u r i n g i n v i t r o i n c u b a t i o n s . 72 TABLE V I I I . EFFECT OF LCAT ACTIVITY AND TG LEVEL ON THE LOSS OF CHOLESTEROL FROM NORMAL HDL INCUBATED IN TD PLASMA'-' Plasma from TD and LCAT d e f i c i e n t p a t i e n t s were r e c o n s t i t u t e d w i t h HDL t o a f i n a l HDL-C c o n c e n t r a t i o n of 45 mg/dL. A d d i t i o n a l samples of c o n t r o l and TD plasma were supplemented w i t h 5 mM DiFP t o i n h i b i t LCAT a c t i v i t y . After-i n c u b a t i o n a t 37°C f o r 24 h, HDL-C l e v e l s were determined. Plasma HDL-C TG (% of i n t i t a l value) (mg/dl) - DiFP + DiFP C o n t r o l 124 105.0 85.0 LCAT D e f i c i e n t 823 94.0 N.D* TD + HDL 284 57.4 56.3 N.D = not determined. The r e s u l t s are the means of d u p l i c a t e i n c u b a t i o n s from a s i n g l e experiment. 73 3.2.3 Role o f h y p e r t r i g l y c e r i d e m i a The r o l e o f moderate h y p e r t r i g l y c e r i d e m i a i n the TD plasma i n the m o d i f i c a t i o n o f HDL was examined i n the f o l l o w i n g s t u d i e s . In these s t u d i e s the a b i l i t y of moderately h y p e r t r i g l y c e r i d e m i a plasma t o modify HDL was determined. In h y p e r t r i g l y c e r i d e m i a ' s u b j e c t s , the plasma TG l e v e l s ranged from 200 t o 341 mg/dl w h i l e the HDL-C l e v e l s ranged from 17 to 54 mg/dl. A f t e r i n c u b a t i o n at 37°C f o r 12 h the l o s s of HDL-C was determined as d e s c r i b e d above and the r e s u l t s are presented i n F i g u r e 10. In none of these samples d i d the l o s s o f the HDL-C exceed 20%. Furthermore, when the HDL-C l e v e l s o f two samples were : supplemented w i t h s u f f i c i e n t exogenous HDL t o r a i s e the HDL-C l e v e l s t o above 50 mg/dl, the l o s s of HDL-C was s t i l l o n l y 15% and 17% of i n i t i a l v a l u e s r e s p e c t i v e l y . These data show t h a t the l o s s of c h o l e s t e r o l from HDL incubated i n TD plasma i s f a r g r e a t e r than can be e x p l a i n e d by a simple e q u i l i b r a t i o n o f l i p i d (9). C o n s i s t e n t w i t h these r e s u l t s i s the p r e v i o u s o b s e r v a t i o n t h a t when LCAT d e f i c i e n t plasma, supplemented w i t h exogenous HDL, was incubated f o r 24 h a t 37°C, the HDL-C l e v e l s decreased t o 94% of the i n i t i a l v a l u e d e s p i t e the presence o f h y p e r t r i g l y c e r i d e m i a [TG = 323 mg/dl (Table V I I I ) . 74 Q a) o o 0) CD CO c D L. I— 70-50-30-10--10-1 I 1 I . i 1 Tangier | c H # ® • HyperTG Normal o o o 1 » i i i « 1 1 100 200 300 Plasma TG (mg/d l ) 400 F i g u r e 10. Loss o f HDL-C d u r i n g i n c u b a t i o n o f plasma from h y p e r t r i g l y c e r i d e m i c s u b j e c t s . Plasma from h y p e r t r i g l y c e r i d e m i c s u b j e c t s (TG range 200 t o 341 mg/dl) was incubated f o r 12 h a t 37°C o r 4°C and the HDL-C val u e s were determined b e f o r e and a f t e r t h i s i n c u b a t i o n . 75 3.3 In v i t r o c a t a b o l i s m o f H D L incubated i n T D plasma 3.3. 1 I n t r o d u c t i o n I n f u s i o n of normal H D L i n TD plasma r e s u l t s i n the r a p i d , b i e x p o n e n t i a l decay of H D L - C and H D L - p r o t e i n from plasma. In s e c t i o n 3.1 we d e s c r i b e d the r a p i d l o s s of c h o l e s t e r o l from HDL incubated w i t h TD plasma i n v i t r o . We a l s o showed t h a t s i g n i f i c a n t amounts of H D L - p r o t e i n were t r a n s f e r r e d t o VLDLpjj L D L T D p a r t i c l e s d u r i n g the i n c u b a t i o n . F u r t h e r , we demonstrated t h a t the r e s u l t i n g H D L p a r t i c l e ( H D L r p D ) i s c h e m i c a l l y d i s t i n c t from normal H D L . The r a p i d l o s s of H D L - p r o t e i n from TD plasma i n v i v o may be due t o i n c r e a s e d c e l l u l a r c a t a b o l i s m o f , \ H D L r j D and thus t h i s p r o p o s a l was examined i n the f o l l o w i n g study. H D L , i s o l a t e d from normal plasma incubated f o r t h e same time as TD plasma ( c o n t a i n i n g normal H D L ) was used as a c o n t r o l i n t h i s experiment. The changes t h a t occur i n H D L d u r i n g i n v i t r o i n c u b a t i o n o f normal plasma are w e l l documented (149, 151-161) These changes i n c l u d e a g e n e r a l i n c r e a s e i n H D L - T G and an a s s o c i a t e d i n c r e a s e i n p a r t i c l e s i z e . While the mechanism of t h i s c o n v e r s i o n i s g e n e r a l l y understood l i t t l e i s known about the a b i l i t y o f these p a r t i c l e s t o i n t e r a c t w i t h t h e f i b r o b l a s t H D L r e c e p t o r . 3.3.2 Examination o f the i n v i t r o metabolism o f HDLTJTQ and H D L 3 7 by c u l t u r e d s k i n f i b r o b l a s t Twenty-five ml, samples of plasma was o b t a i n e d from a p o o l of normolipemic donors and from a p a t i e n t w i t h TD. The H D L - C l e v e l o f TD plasma was i n c r e a s e d t o a p proximately 4 5 mg/dl by the a d d i t i o n of normal H D L . An equal volume of b u f f e r C was added to the c o n t r o l plasma and both samples were i n c u b a t e d f o r 24 h a t 76 37°C. A f t e r i n c u b a t i o n the H D L was i s o l a t e d from the samples as d e s c r i b e d i n s e c t i o n 2.3.1.1. The H D L 1 s were then i o d i n a t e d , analyzed by S D S - P A G E ( F i g u r e 11) and used f o r H D L b i n d i n g and deg r a d a t i o n assays as o u t l i n e d i n s e c t i o n 2.7.2. As i n d i c a t e d i n F i g u r e 12a, n e i t h e r H D L r p Q nor H D L 3 7 reached s a t u r a t i o n i n the c o n c e n t r a t i o n range used (0 t o 50 ug/ml). The i n a b i l i t y t o d e t e c t b i n d i n g s a t u r a t i o n i s e x p l a i n e d by the r e l a t i v e l y l a r g e n o n - s p e c i f i c b i n d i n g component which obscures the h i g h a f f i n i t y b i n d i n g component. The s p e c i f i c component of H D L b i n d i n g was determined as d e s c r i b e d i n s e c t i o n 2.7.1 and the r e s u l t s are p r e s e n t e d i n F i g u r e 12b. Scatchard a n a l y s i s o f the s p e c i f i c b i n d i n g component f o r H D L T D and H D L 3 7 were done ( F i g u r e 13). These a n a l y s i s i n d i c a t e d K d's of 14.93 and_.12.3 5 ug/ml were o b t a i n e d f o r H D L r j , D and H D L 3 7 , r e s p e c t i v e l y . In a d d i t i o n , the maximum c a p a c i t y o f l i g a n d b i n d i n g ( B m a x ) was determined t o be 189 and 183 ug/mg c e l l p r o t e i n / 2 h f o r H D L T D and H D L 3 7 , r e s p e c t i v e l y . These s t e a d y - s t a t e k i n e t i c parameters i n d i c a t e t h a t H D L r p p has e s s e n t i a l l y the same a f f i n i t y f o r the H D L r e c e p t o r as does H D L 3 7 . The r e s u l t s o f t h i s study i n d i c a t e t h a t i n v i t r o i n c u b a t i o n of normal H D L i n TD plasma does not i n c r e a s e i t s a b i l i t y t o i n t e r a c t with the f i b r o b l a s t H D L r e c e p t o r . As the p r o t e i n composition f o r both H D L , p D and H D L 3 7 were n e a r l y i d e n t i c a l ( F i g u r e 14) these r e s u l t s suggest t h a t changes i n core l i p i d composition do not e f f e c t the a b i l i t y o f t h i s p a r t i c l e t o i n t e r a c t w i t h the H D L r e c e p t o r . A n a l y s i s o f the i n c u b a t i o n medium f o r d e g r a d a t i o n products 77 showed t h a t d e g r a d a t i o n products of HDL T D were d e t e c t a b l e a t the hi g h e r l i g a n d c o n c e n t r a t i o n s w h i l e no d e g r a d a t i o n p r o d u c t s o f HDL37 were d e t e c t e d (Figure 17). These p r e l i m i n a r y r e s u l t s suggest t h a t the l i p i d / p r o t e i n changes observed t o occur i n HDL incubated i n TD plasma do not d r a s t i c a l l y a l t e r the a b i l i t y o f the p a r t i c l e t o i n t e r a c t w i t h the HDL r e c e p t o r on c u l t u r e d f i b r o b l a s t s i n v i t r o . While the HDL T D p a r t i c l e was degraded a t a f a s t e r r a t e than HDL 3 7, the exact s i g n i f i c a n c e o f t h i s o b s e r v a t i o n remains u n c l e a r . 78 60 j i i i | i i i | i i i [ I I i [ T ' i ' i ' i i i i j i Distance from top of gel (cm) F i g u r e 11. SDS-PAGE on 10.0% g e l s o f 1 2 5 I - l a b e l l e d H D L ^ Q (©) and H D L 3 7 ( • ) . 25 ug of p r o t e i n was a p p l i e d t o each g e l i n the presence o f 1% 2-mercaptoethanol. A f t e r e l e c t r o p h o r e s i s f o r 2 . 5 hours a t 300V the p r o t e i n s were v i s u a l i s e d by s t a i n i n g the g e l s w i t h bromophenol b l u e . The g e l s were subsequently c u t i n t o 0 . 5 cm s e c t i o n s and the 1 2 5 I r a d i o a c t i v i t y determined. The r e s u l t s presented f o r HDLpQ (0) and HDL 3 7 (•) are expressed as percentage o f t o t a l r a d i o a c t i v i t y counted and are the r e s u l t s o f a s i n g l e d e t e r m i n a t i o n . 79 350—1—i—j—i—j—i—j—i—j—i—f 0 10 20 30 40 50 HDL (ug/ml) F i g u r e 12a. T o t a l b i n d i n g o f 1 2 5 I - H D L T D and 1 2 5 I - H D L 3 7 t o normal f i b r o b l a s t s Normal f i b r o b l a s t s were incubated f o r 2 hours a t 37°C i n the presence o f i n c r e a s i n g c o n c e n t r a t i o n s of e i t h e r HDL,pD (•) or HDL 3 7 (®) i n the presence or absence of excess u n l a b e l l e d HDL. The r e s u l t s are the average + range of d u p l i c a t e d e t e r m i n a t i o n s . 80 200-T 1 1 r j 1 j 1 1 1 f 0 10 20 30 40 50 HDL (ug/ml) F i g u r e 12b. S p e c i f i c b i n d i n g o f 1 2 5 I - H D L T D and 1 2 5 I - H D L 3 7 t o normal f i b r o b l a s t s Normal f i b r o b l a s t s were incubated f o r 2 hours a t 37°C i n the presence o f i n c r e a s i n g c o n c e n t r a t i o n s o f e i t h e r HDLpQ (B) or HDL 3 7 (Q) i n the presence o r absence of excess u n l a b e l l e d l i g a n d . The r e s u l t s are the average + range of d u p l i c a t e d e t e r m i n a t i o n s . 8 1 20-1 , , • i , , , r OH T j 1 J 1 J - r 1 0 50 100 150 200 Bound (ug/m!) F i g u r e 13. S c a t c h a r d a n a l y s i s o f the d a t a p r e s e n t e d i n F i g u r e 12b 82 50-r CN -o.E CD Q) Q q) _1° x £ cn c -j j 1 j 1 j 1 j r——p 10 20 30 40 50 HDL (ug/mi) F i g u r e 14, Degradation o f 1 2 5 I - H D L r r , D and 1 2 5 I - H D L 3 7 by normal f i b r o b l a s t s . Normal f i b r o b l a s t s were incubated f o r 2 hours a t 37°C i n the presence o f i n c r e a s i n g c o n c e n t r a t i o n s of e i t h e r H D L T D (H) or HDL 3 7 (0) i n the presence o r absence of excess u n l a b e l l e d l i g a n d . The t o t a l HDL d e g r a d a t i o n was determined as d e s c r i b e d i n the methods. The r e s u l t s are the average + range of d u p l i c a t e d e t e r m i n a t i o n s . 83 3.4 Metabolism o f L D L ^ Q by s k i n f i b r o b l a s t s LDL from TD and c o n t r o l plasma were i s o l a t e d by u l t r a c e n t r i f u g a t i o n and analyzed f o r l i p i d and p r o t e i n composition. T a b l e IX,presents the chemical composition of the normal LDL and.LDL T D. As has been p r e v i o u s l y r e p o r t e d (90), L D L T D i s e n r i c h e d i n PL, TG and p r o t e i n and d e p l e t e d i n i t s content of CE, and t o a l e s s e r extent FC, r e l a t i v e t o normal LDL. The mean diameter (nm) of the TD LDL was not d i f f e r e n t from normal LDL as determined by QLS (TD: 19.2nm; normal 18.7nm). A n a l y s i s o f TD plasma by agarose g e l e l e c t r o p h o r e s i s showed i n c r e a s e d e l e c t r o p h o r e t i c m o b i l i t y o f the B band ( F i g u r e 5; u s u a l p o s i t i o n o f normal LDL). While L D L T D i s a c h e m i c a l l y d i s t i n c t p a r t i c l e from c o n t r o l LDL, l i t t l e i s known about i t s metabolism. As TD p a t i e n t s u s u a l l y have o n l y 30% normal l e v e l s o f LDL i n t h e i r plasma (90), the p o s s i b i l i t y t h a t LDLpj-j i s more r a p i d l y c l e a r e d by c e l l s was examined. The c a t a b o l i s m o f LDLrr, D by c u l t u r e d s k i n f i b r o b l a s t s was s t u d i e d as d e s c r i b e d i n s e c t i o n 2.5.1. Samples of plasma were o b t a i n e d from a p o o l o f normolipemic donors and from a p a t i e n t w i t h TD. LDL was i s o l a t e d as d e s c r i b e d i n s e c t i o n 2.3.1.1. The LDL 1s were then i o d i n a t e d , and used f o r LDL b i n d i n g and degradation assays as o u t l i n e d i n s e c t i o n 2.7.1. The s p e c i f i c b i n d i n g o f TD and c o n t r o l 1 2 5 L D L t o c u l t u r e d f i b r o b l a s t s was determined ( F i g u r e 15). B i n d i n g o f c o n t r o l 1 2 5 I - L D L was s a t u r a t e d a t approximately 3 0 ug/ml which i s s i m i l a r the va l u e o f 25 ug/ml r e p o r t e d by Brown and G o l d s t e i n (128). However, the b i n d i n g of 1 2 5 I - L D L T D d i d not appear t o be s a t u r a t e d i n the l i g a n d range examined. 84 Table IX. Chemical composition of LDL (1.006 - 1.063-g/ml), Percent weight (mg.%) C o n t r o l TD Free c h o l e s t e r o l (FC) 9.2 3.4 E s t e r i f i e d c h o l e s t e r o l (EC) 29 .5 3.4 T r i a c y l g l y c e r o l (TG) 7.5 26.5 P h o s p h o l i p i d s (PL) 20.9 29.7 P r o t e i n 32.5 37.0 85 LDL (ug/ml) F i g u r e 15. B i n d i n g and i n t e r n a l i z a t i o n o f TD and c o n t r o l 1 2 5 I - L D L by normal f i b r o b l a s t s . Normal f i b r o b l a s t s were incubated f o r 6 hours a t 37°C i n i n c r e a s i n g c o n c e n t r a t i o n s of e i t h e r TD (0) o r c o n t r o l (#) LDL i n the presence or absence of excess u n l a b e l l e d l i g a n d . The s p e c i f i c component of a s s o c i a t i o n was determined as d e s c r i b e d i n the methods. The r e s u l t s are the average + range of d u p l i c a t e d e t e r m i n a t i o n s . 86 The s p e c i f i c component of b i n d i n g of t h i s experiment and t h a t o f a second experiment were examined by double r e c i p r o c a l p l o t a n a l y s i s (Table X). In both experiments, the f o r L D L T D was 3-5 f o l d lower than t h a t f o r c o n t r o l LDL. The V m a x f o r L D L T D was c o n s i s t e n t l y 3-10 f o l d lower than f o r c o n t r o l LDL. These r e s u l t s i n d i c a t e t h a t major d i f f e r e n c e s e x i s t between L D L T D and c o n t r o l LDL w i t h r e s p e c t t o t h e i r a b i l i t y t o i n t e r a c t w i t h human s k i n f i b r o b l a s t s LDL r e c e p t o r . A n a l y s i s o f the medium f o r deg r a d a t i o n products showed t h a t the r a t e o f d e g r a d a t i o n of L D L T D was approximately 50% the r a t e of c o n t r o l LDL ( F i g u r e 16). The r e s u l t s obtained f o r c o n t r o l LDL i n these experiments are s i m i l a r , t o those p r e v i o u s l y r e p o r t e d f o r c o n t r o l LDL (128) and thus these r e s u l t s i n d i c a t e t h a t L D L T D i s both c h e m i c a l l y and m e t a b o l i c a l l y d i s t i n c t from normal LDL. 87 TABLE X. KINETIC ANALYSIS OF LDL CELL BINDING DATA vmax ug/ml ng/mg c e l l p r o t e i n / 6 h Expt. 1 TD 13.4 1723 c o n t r o l 43.3 4411 Expt.2 TD 10.6 892 c o n t r o l 52.3 7590 88 50-r i i i | i i i | i i i | i i i | i i i | i i i | i LDL (ug/ml) F i g u r e 16. Degradation of TD and c o n t r o l I-LDL by normal f i b r o b l a s t s . Normal f i b r o b l a s t s were incubated f o r 6 hours a t 37°C i n i n c r e a s i n g c o n c e n t r a t i o n s of e i t h e r TD (fl) or c o n t r o l (Q) LDL i n the presence or absence of excess u n l a b e l l e d l i g a n d . The t o t a l LDL d e g r a d a t i o n was determined as d e s c r i b e d i n the methods. The r e s u l t s are the average + range of d u p l i c a t e d e t e r m i n a t i o n s . 89 3.5 Catabolism o f VLDLj, D by bovine m i l k l i p o p r o t e i n l i p a s e A common f e a t u r e of both TD and LCAT d e f i c i e n c y i s the presence of h y p e r t r i g l y c e r i d e m i a , however, the b i o c h e m i c a l mechanism(s) u n d e r l y i n g the h y p e r t r i g l y c e r i d e m i a remain u n c l e a r . While both the TD and LCAT d e f i c i e n t p a t i e n t s have L p l l e v e l s i n the low-normal range a c l e a r L p l abnormality has not been demonstrated (90). Thus, the p o s s i b i l i t y t h a t the T G - r i c h p a r t i c l e s accumulate i n TD and LCAT d e f i c i e n c y as a r e s u l t o f a d e f e c t i v e i n t e r a c t i o n between LpL and i t s l i p o p r o t e i n s u b s t r a t e s was examined i n t h i s study. The composition and s i z e o f the VLDL accumulating i n the plasma of the TD p a t i e n t and a LCAT d e f i c i e n t p a t i e n t ( V L D L L C A T ) was f i r s t s t u d i e d t o determine i f any a b n o r m a l i t i e s i n e i t h e r the l i p i d o r the p r o t e i n moiety o f the l i p o p r o t e i n s e x i s t e d . F i g u r e 17 p r e s e n t s the r e s u l t s o f s i z e e s t i m a t i o n o f VLDL by QLS. P a r t i c l e s from normal i n d i v i d u a l s were composed o f a major p o p u l a t i o n o f mean diameter 34.3 nm wit h a range o f 31 t o 3 6 nm. T h i s p a r t i c l e s i z e agrees w e l l w i t h t h a t p r e v i o u s l y r e p o r t e d f o r the VLDL p o o l as determined by e l e c t r o n microscopy (100). A minor p o p u l a t i o n was a l s o p r e s e n t i n normal VLDL (100 to 170 nm). T h i s most l i k e l y r e p r e s e n t s contaminating chylomicron p a r t i c l e s . The major p o p u l a t i o n i n VLDLpp had a mean diameter o f 44.3 nm w i t h a 1 broader range than c o n t r o l s (41 t o 52 nm). T h i s s l i g h t l y l a r g e r mean diameter and broader d i s t r i b u t i o n has a l s o been r e p o r t e d f o r VLDL i s o l a t e d from another p a t i e n t w i t h TD and analysed by EM (100). A minor p o p u l a t i o n (200 t o 400 nm) probably corresponds t o u n c a t a b o l i z e d chylomicrons as r e p o r t e d i n o t h e r s t u d i e s (99) . The R E L A T I V E V O L U M E "O > TJ O r~ m co N m O > o 0) (O CO O o 3 ' u : O " o o-o o o TJ g CO TJ •3 C H O > > < CO CO F i g u r e 17. Determination o f VLDL s i z e and d i s t r i b u t i o n by QLS VLDL were i s o l a t e d by u l t r a c e n t r i f u g a t i o n a t 1.006 g/ml from the plasma of TD LCAT d e f i c i e n t and normal i n d i v i d u a l s . Approximately 500 u l of each f r a c t i o n (3 mg TG/ml) was used f o r de t e r m i n a t i o n . T h i s f i g u r e shows a r e p r e s e n t a t i v e p a r t i c u l a r s i z e / d i s t r i b u t i o n p r o f i l e from a NiComp submicron p a r t i c l e s i z e r . P r o f i l e obtained with e q u i v a l e n t samples a t o t h e r time frames gave e s s e n t i a l l y i d e n t i c a l r e s u l t s . The peak h e i g h t s r e f e r t o the r e l a t i v e d i s t r i b u t i o n o f p a r t i c l e s o f w i t h i n the t o t a l p o p u l a t i o n assayed. The a n a l y s i s assumes a non ga u s s i a n d i s t r i b u t i o n and s o l i d p a r t i c l e s . 91 nature and s i g n i f i c a n c e o f the peaks a t 36 and 63 nm are as y e t unknown. V L D L L C A T o c c u r r e d i n a s i n g l e p o p u l a t i o n of 70 to 100 nm c o n f i r m i n g the r e s u l t s o f ot h e r i n v e s t i g a t o r s who have d e s c r i b e d l a r g e VLDL i n LCAT d e f i c i e n t plasma (162). The l i p i d and p r o t e i n composition of the p a r t i c l e s i s pres e n t e d i n T a b l e XI. The r e l a t i v e p r o p o r t i o n s of TG, FC, CE, P i and p r o t e i n i n the normal p a r t i c l e s were s i m i l a r t o the valu e s r e p o r t e d i n o t h e r s t u d i e s (162). D e s p i t e an i n c r e a s e i n p a r t i c l e s i z e ( F i g u r e 17) , V L D L r p D had e s s e n t i a l l y normal l i p i d and p r o t e i n c omposition. The composition of V L D L L C A T r e f l e c t e d the absence of c h o l e s t e r o l e s t e r i f i c a t i o n . The most marked d i f f e r e n c e between c o n t r o l VLDL and V L D L L C A T was i n the CE content which was 5.4% and 0.7%, r e s p e c t i v e l y . There was a l s o a t w o - f o l d i n c r e a s e i n the FC content of the VLDL, a f i n d i n g s i m i l a r t o t h a t r e p o r t e d p r e v i o u s l y (162). In a d d i t i o n , the r e l a t i v e amount o f apo C-II a s s o c i a t e d w i t h the VLDLpp was the same as t h a t observed i n c o n t r o l VLDL but i t was 1 0 - f o l d l e s s i n V L D L L C A T (Table X). C l e a r l y , the r e s u l t s o f t h i s study show t h a t V L D L r p D i s c h e m i c a l l y s i m i l a r t o VLDL i s o l a t e d from c o n t r o l s u b j e c t s except t h a t i t i s a l a r g e r p a r t i c l e ; w h i l e V L D L L C A T ^ s e n r i c n e d with FC and i t i s an even l a r g e r p a r t i c l e . I t i s p o s s i b l e t h a t the i n c r e a s e d s i z e o f the VLDL p a r t i c l e s may a l t e r t h e i r a b i l i t y t o i n t e r a c t w i t h LpL. In a d d i t i o n , F i e l d i n g and coworkers have shown t h a t normal VLDL supplemented w i t h excess FC i n v i t r o was a poor s u b s t r a t e f o r LpL (163). Thus, i n the f o l l o w i n g study the a b i l i t y o f these p a r t i c l e s t o a c t as a s u b s t r a t e f o r BmLpL was examined. 92 T a b l e XI. Chemical composition of VLDL (d<1.006 g/ml), Percent weight (mg.%) C o n t r o l TD LCAT d e f i c i e n c y Free c h o l e s t e r o l (FC) 4.5 5.6 7.6 E s t e r i f i e d c h o l e s t e r o l (EC) 5.4 3.6 0.7 T r i a c y l g l y c e r o l (TG) 64.0 58.5 72.0 P h o s p h o l i p i d s (PL) 16.8 19.6 15.3 P r o t e i n 9.4 13 .5 4.1 Apo C-II 0.7 0.7 0. 07 TG/FC 14.2 10.3 9.5 FC/PL 0.26 0.29 0.49 FC/EC 0. 83 1.26 10.9 93 BmLpL resembles p e r i p h e r a l t i s s u e l i p a s e ( i . e . adipose) both w i t h r e s p e c t t o i t s chromatography on heparin-Sepharose and s t i m u l a t i o n by apo C-II (164). I t can be p u r i f i e d t o near homogeneity (14 6) and i t i s s t a b l e . For these reasons i t was used t o study the h y d r o l y s i s of c o n t r o l VLDL, VLDLpp and V L D L L C A T i n v i t r o . F i g u r e 18a shows the h y d r o l y s i s o f the VLDL's by Bmlpl. E a d i e - H o f s t e e a n a l y s i s (165) o f the data from F i g u r e 18a i s pr e s e n t e d i n F i g u r e 18b and i n d i c a t e s t h a t the 1^ f o r the normal VLDL, V L D L T D , and V L D L L C A T w e r e 1-13, 2.25, and 2.42 mg/ml TG, ! r e s p e c t i v e l y . S i n c e the r e c i p r o c a l o f 1^ i s a measure o f the a f f i n i t y o f the s u b s t r a t e f o r i t s b i n d i n g s i t e these r e s u l t s suggest t h a t both TD and LCAT d e f i c i e n t VLDL's do not b i n d as a v i d l y t o the enzyme as the c o n t r o l VLDL. However, a t h i g h e r c o n c e n t r a t i o n s , they may be more r a p i d l y h y d r o l y z e d as i n d i c a t e d by the 3-4 f o l d i n c r e a s e i n the maximal v e l o c i t y ( V m a x ) ( C o n t r o l , 8.47; TD, 25.4; LCAT d e f i c i e n t ; 31.3 umoles/min/mg LpL) (Figure 18b). Thus the VLDL from both the TD and the LCAT d e f i c i e n t p a t i e n t s appear t o be good s u b s t r a t e s f o r the enzyme Bmlpl. 94 E \ — i & _J a E TJ e « a 9 < UL u. o . E 3 14 12 10 L C A T De f . T a n g i e r C o n t r o l 0.5 1.0 1.5 T r i g l y c e r i d e ( m g / m l ) F i g u r e 18a. The h y d r o l y s i s o f VLDL by BmLpL VLDL were i s o l a t e d from LCAT d e f i c i e n t , TD and normal i n d i v i d u a l s and l a b e l l e d with [ 3H] t r i o l e i n as d e s c r i b e d i n the methods. The i n d i c a t e d amount of VLDL-TG was incubated w i t h p u r i f i e d BmLpL f o r 45 min a t 37°C and the amount of [ 3 H ] o l e i c a c i d r e l e a s e d was determined. Each p o i n t r e p r e s e n t s the mean of d u p l i c a t e i n c u b a t i o n s , 95 40 c i 0> E « » © 30 « 20 < u. u. o E >. 10 ** u o > L E G E N D • Control • LCAT Def m Tangier \ v \ v \ \ \ \ \ \ \ x 0 5 10 15 v e l o c i t y / s u b s t r a t o F i g u r e 18b. E a d i e - H o f s t e e a n a l y s i s o f data from F i g u r e 18a The KJJ, f o r the h y d r o l y s i s o f VLDL by BmLpL was c a l c u l a t e d from the data i n A by an Eadie-Hofstee p l o t (negative s l o p e o f the curve) and V m a x ( i n t e r c e p t on the Y - a x i s ) . A n a l y s i s o f the data by a Hanes p l o t (165) gave v a l u e s of 1^ and V j n a x t h a t were w i t h i n 5% of those r e p o r t e d i n the f i g u r e . 96 3.6 Role o f c e l l u l a r components i n t h e HDL d e f i c i e n c y o f TD Recent r e p o r t s have suggested t h a t t he r a p i d removal of HDL from plasma o f TD p a t i e n t s d u r i n g plasmapheresis i s due t o an a c c e l e r a t e d c e l l u l a r c a t a b o l i s m of HDL (110,125). We used two t i s s u e c u l t u r e systems t o examine the i n t e r a c t i o n s o f normal HDL with TD c e l l s i n v i t r o . 3.6.1 TD f i b r o b l a s t The data on i n t e r a c t i o n o f 1 2 5 I - l a b e l l e d HDL 3 with s k i n f i b r o b l a s t s from our TD p a t i e n t and an age matched c o n t r o l are pre s e n t e d i n F i g u r e 19. S p e c i f i c b i n d i n g o f 1 2 5 I - l a b e l l e d HDL 3 appeared t o reach s a t u r a t i o n a t c o n c e n t r a t i o n s lower than 25 mg/ml f o r both c o n t r o l and TD f i b r o b l a s t s . The r e s u l t s were f u r t h e r a n a lyzed by Sca t c h a r d a n a l y s i s ( F i g u r e 20). From t h i s a n a l y s i s the K d s o f HDL 3 b i n d i n g t o c o n t r o l and TD f i b r o b l a s t s were determined t o be 10.95 and 5.24 ug/ml r e s p e c t i v e l y . In a d d i t i o n , the maximum amount of HDL 3 bound by c o n t r o l and Ta n g i e r f i b r o b l a s t s were e s s e n t i a l l y i d e n t i c a l , 262 and 257 ng/mg c e l l p r o t e i n / 2 hours r e s p e c t i v e l y . These r e s u l t s a re s i m i l a r t o those r e p o r t e d by Oram e t a l f o r normal s k i n f i b r o b l a s t s (73). Hence TD f i b r o b l a s t s do not appear t o b i n d and/or i n t e r n a l i z e normal HDL abnormally. A n a l y s i s o f the medium f o r degra d a t i o n p r o d u c t s showed t h a t n e i t h e r c o n t r o l nor TD f i b r o b l a s t s degraded any d e t e c t a b l e amount of HDL 3. T h i s i s i n agreement w i t h p r e v i o u s o b s e r v a t i o n s t h a t s k i n f i b r o b l a s t s do not degrade HDL 3 (73,74,75). We concluded t h a t TD f i b r o b l a s t s behave l i k e normal f i b r o b l a s t s with r e s p e c t t o t h e i r a b i l i t y t o i n t e r a c t w i t h normal HDL. 97 400- i » i [ i t i I i i i I i i i i i i i i i i i i j i i i ; > i i K i i i ; i r i | i 20 40 60 80 100 HDL3 (ug/ml) F i g u r e 19. S p e c i f i c b i n d i n g o f normal 1 2 5 I - H D L 3 t o TD and c o n t r o l f i b r o b l a s t s . TD (B) and c o n t r o l (®) f i b r o b l a s t s were incubated f o r 2 hours at 37°C i n the presence of i n c r e a s i n g c o n c e n t r a t i o n s of c o n t r o l 125 I-HDL3 ( i n the presence or absence o f excess u n l a b e l e d l i g a n d ) . The s p e c i f i c component of a s s o c i a t i o n was determined as desc i n the methods. The r e s u l t s are the average + range of d u p l i c a t e d e t e r m i n a t i o n s from a r e p r e s e n t a t i v e experiment. 9 8 OH 6 0 1 E 50-40-30-£ a* S E 20-CP c 10-0-l I J i 1 i i I I i Tangier cells _ a 50 100 150 200 250 300 Bound (ug/ml) F i g u r e 20. S c a t c h a r d a n a l y s i s o f d a t a from F i g u r e 19 99 3.6.2 TD monocytes The above s t u d i e s of TD c e l l s were then c a r r i e d out u s i n g human p e r i p h e r a l monocytes. F i g u r e 21 shows the i n t e r a c t i o n of HDL3 w i t h f r e s h l y i s o l a t e d monocytes from TD and c o n t r o l c e l l s . The r e s u l t s show no d i f f e r e n c e s between the a b i l i t y o f TD and c o n t r o l monocytes t o i n t e r a c t w i t h 1 2 5 I - l a b e l l e d HDL 3. I t has been p r e v i o u s l y r e p o r t e d t h a t the K d f o r I - l a b e l l e d HDL b i n d i n g t o c u l t u r e d monocytes i s approximately 132 mg/ml (77). C o n s i s t e n t w i t h t h i s o b s e r v a t i o n i s the l a c k of s a t u r a t i o n o f c e l l a s s o c i a t e d HDL seen i n both c o n t r o l and TD monocytes i n the l i g a n d ranges we examined ( F i g u r e 21). Assman e t a l have suggested t h a t c e l l a s s o c i a t e d HDL i s r e l e a s e d by c o n t r o l monocytes but not r e l e a s e d by TD monocytes (166). However, our r e s u l t s i n d i c a t e no dramatic i n c r e a s e i n 1 2 5 I - l a b e l l e d HDL 3 r a d i o a c t i v i t y a s s o c i a t e d with TD monocytes d u r i n g a 4 hour i n c u b a t i o n p e r i o d . Even when the i n c u b a t i o n p e r i o d was i n c r e a s e d t o 20 hours no s i g n i f i c a n t d i f f e r e n c e s i n c e l l a s s o c i a t e d HDL was observed between TD and c o n t r o l monocytes ( F i g u r e 22). These r e s u l t s i n d i c a t e t h a t i n the absence of c h o l e s t e r o l TD monocytes do not have a d e f e c t i n c e l l / H D L i n t e r a c t i o n . 100 F i g u r e 21. B i n d i n g o f 1 2 5 I - H D L 3 t o TD and c o n t r o l monocytes d u r i n g a 4 h i n c u b a t i o n a t 37°. TD (H) and c o n t r o l (©) monocytes were in c u b a t e d f o r 4 h a t 37°C i n the presence of i n c r e a s i n g c o n c e n t r a t i o n s o f c o n t r o l 12 5 . • I-HDL 3 ( i n the presence or absence o f excess u n l a b e l l e d l i g a n d ) . The s p e c i f i c component of a s s o c i a t i o n was determined as d e s c r i b e d i n the methods. The r e s u l t s are the mean + SD of t h r e e s e p e r a t e d u p l i c a t e d e t e r m i n a t i o n s . 101 F i g u r e 22. A " l - H D L 3 b i n d i n g t o TD and c o n t r o l monocytes d u r i n g a 20 h i n c u b a t i o n a t 37°. TD (•) and c o n t r o l (©) monocytes were incubated f o r 20 h a t 37°C i n the presence of i n c r e a s i n g c o n c e n t r a t i o n s of c o n t r o l I-HDL 3 i n the presence or absence of excess u n l a b e l l e d l i g a n d . The s p e c i f i c component of a s s o c i a t i o n was determined as d e s c r i b e d i n the methods. The r e s u l t s are the mean + SD o f t h r e e seperate d u p l i c a t e d e t e r m i n a t i o n s . 102 4 D i s c u s s i o n 4.1 M o d i f i c a t i o n o f normal HDL by TD plasma P r e v i o u s l y , Assmann e t a l (109), S c h a e f e r e t a l (110,125) and P r i t c h a r d and F r o h l i c h (124) have demonstrated t h a t the i n f u s i o n of r e l a t i v e l y l a r g e amounts of normal HDL i n t o TD p a t i e n t s i s f o l l o w e d by the r a p i d removal of both HDL-C and HDL-p r o t e i n from the plasma compartment. Our p a t i e n t (124) had v i r t u a l l y i d e n t i c a l HDL t u r n o v e r k i n e t i c s as p a t i e n t A i n Scha e f e r ' s study (125). T h i s i n d i c a t e s the absence of any major h e t e r o g e n e i t y between these two cases w i t h r e s p e c t t o HDL tu r n o v e r i n v i v o . Thus, the r e s u l t s are c o n s i s t e n t w i t h the h y p o t h e s i s t h a t TD i s the r e s u l t o f a d e f e c t which r e s u l t s i n hy p e r c a t a b o l i s m of HDL. Although the s t u d i e s o f both Assmann (109) and Schaefer (110,125) reached s i m i l a r c o n c l u s i o n s , they d i d not address the r e s p e c t i v e r o l e s o f c e l l u l a r c a t a b o l i s m and plasma m o d i f i c a t i o n o f the HDL i n the observed h y p e r c a t a b o l i s m . From t h e i r s t u d i e s , i t i s not p o s s i b l e t o determine whether changes i n HDL composition o c c u r r e d p r i o r t o , o r as a consequence of, i n t e r a c t i o n w i t h TD c e l l s i n v i v o . T h i s study attempted t o d e s c r i b e the i n i t i a l changes t h a t occur i n normal HDL d u r i n g i t s exposure t o TD plasma. The a d d i t i o n of normal HDL t o TD plasma i n c r e a s e d the FC and CE content o f the HDL t o w i t h i n the normal range (Table I V ) . The s l i g h t i n c r e a s e i n HDL-TG (when compared t o c o n t r o l s ) was a s s o c i a t e d w i t h a s m a l l but s i g n i f i c a n t i n c r e a s e i n VLDL/LDL CE. These o b s e r v a t i o n s may be due t o the a c t i o n o f l i p i d t r a n s f e r p r o t e i n s t h a t appear t o remain p a r t i a l l y a c t i v e a t 4.°C (159,167). 103 A comparison of i n v i t r o metabolism of HDL i n c o n t r o l and TD plasma by agarose g e l e l e c t r o p h o r e s i s ( F i g u r e 5) i n d i c a t e d t h a t d u r i n g i n c u b a t i o n of plasma a t 37°C, t h e r e was a r a p i d , time dependent l o s s of HDL-CE i n TD plasma. T h i s i n i t i a l o b s e r v a t i o n was confirmed by f o l l o w i n g the changes i n HDL-C t h a t o c c u r r e d d u r i n g the i n c u b a t i o n p e r i o d ( F i g u r e 6). S i m i l a r t o the i n v i v o f i n d i n g s of Schaefer e t a l (110,125) and Assmann e t a l (109), the l o s s of c h o l e s t e r o l from HDL i n TD plasma I n v i t r o f o l l o w e d a monoexponetial decay curve. I t i s i n t e r e s t i n g t o note t h a t Blum e t a l have proposed a two-compartment model f o r i n v i v o HDL c a t a b o l i s m i n normal i n d i v i d u a l s w i t h one component b e i n g w i t h i n the plasma and exchanging w i t h a nonplasma compartment (168). Furthermore, Scha e f e r e t a l r e p o r t e d t h a t 1 2 5 I - H D L may be c a t a b o l i z e d o n l y v i a the plasma compartment i n TD and t h a t the f r a c t i o n a l c a t a b o l i c r a t e (FCR) of HDL i n homozygous and heterozygous TD p a t i e n t s are i n c r e a s e d (125). Thus, the r e s u l t s of i n v i t r o i n c u b a t i o n of normal HDL w i t h TD plasma are s u p p o r t i v e o f Schaefer's o b s e r v a t i o n t h a t the i n v i v o c a t a b o l i s m o f normal HDL i n TD plasma occurs o n l y i n the plasma compartment. Furthermore, the r e l a t i v e r a t e of l o s s of HDL-C from TD plasma d u r i n g i n v i t r o i n c u b a t i o n , d e s c r i b e d i n the p r e s e n t experiments, i s q u a n t i t a t i v e l y the same as the i n v i v o s t u d i e s d u r i n g the f i r s t 24 h. Thus, the data suggest t h a t the i n i t i a l decay of HDL-C seen i n v i v o may occur independently of any c e l l u l a r involvement. 104 F u r t h e r a n a l y s i s of the plasma and l i p o p r o t e i n f r a c t i o n s (Table V) i n d i c a t e t h a t the observed changes i n the s t r u c t u r e of HDL and VLDL/LDL i n c o n t r o l plasma d u r i n g i n c u b a t i o n are e s s e n t i a l l y i d e n t i c a l t o those p r e v i o u s l y r e p o r t e d (157). The i n c r e a s e i n HDL-CE and decrease i n HDL-FC most l i k e l y r e f l e c t the a c t i v i t y of LCAT (162). In a d d i t i o n , the i n c r e a s e i n VLDL/LDL CE and i n the HDL-TG content r e f l e c t s the a c t i v i t y of the LTP's (50). The data f o r TD plasma r e v e a l t h a t the m a j o r i t y of the c h o l e s t e r o l l o s t from HDL ( F i g u r e 6) was i n the form of CE (Table V ) . T h i s l o s s o f CE from the core of normal HDL was a s s o c i a t e d w i t h an i n c r e a s e d c o n c e n t r a t i o n i n VLDL/LDL CE (Table V ) . Furthermore, th e t r a n s f e r o f c h o l e s t e r o l from HDL t o VLDL/LDL was a s s o c i a t e d w i t h a marked i n c r e a s e i n the TG content of the core o f the HDL p a r t i c l e s . On a molar b a s i s , t h e r e was approximately a 1:1 exchange of CE e s t e r f o r TG c o n s i s t e n t w i t h the involvement o f a s p e c i f i c LTP (50). P r e v i o u s l y S c h a e f e r e t a l (12 5) have shown t h a t the HDL core c o n s t i t u e n t s are c a t a b o l i z e d a t a f a s t e r r a t e than s u r f a c e p r o t e i n components. T h i s o b s e r v a t i o n has been confirmed by the plasmapheresis o f our p a t i e n t w i t h TD (12 4) where 50% of CE was l o s t from the i n f u s e d HDL whereas only about 15% of apo A-I was l o s t from t h i s f r a c t i o n . These r e s u l t s suggest t h a t HDL i s not c a t a b o l i z e d as a whole p a r t i c l e i n TD plasma. While i t was p o s s i b l e t o reproduce the i n v i v o l o s s of HDL-C i n v i t r o , the l o s s of HDL p r o t e i n i n v i v o c o u l d not be mimicked i n v i t r o . The t o t a l amount of A-I i n TD plasma d i d not decrease d u r i n g the i n c u b a t i o n i n v i t r o . I t i s t h e r e f o r e u n l i k e l y t h a t an apo A-I s p e c i f i c p r o t e a s e e x i s t s i n TD plasma,. Thus, the l o s s of 105 A-I from TD plasma must be due t o i t s i n c r e a s e d r a t e o f removal. T h i s may i n v o l v e a number of mechanisms i n c l u d i n g : 1) In c r e a s e d c a t a b o l i s m o f the H D L r p D p a r t i c l e 2) T r a n s f e r o f apo A-I t o more r a p i d l y c a t a b o l i z e d p a r t i c l e s 3) In c r e a s e d c l e a r a n c e o f n o n - l i p o p r o t e i n a s s o c i a t e d apo A - I . With these p o s s i b i l i t i e s i n mind, we s e t out t o f o l l o w the movement of HDL ap o p r o t e i n s d u r i n g i n v i t r o i n c u b a t i o n o f 1 2 5 I -HDL w i t h TD plasma ( F i g u r e 7 and Tab l e V I ) . While t h e r e was some t r a n s f e r o f HDL ap o p r o t e i n s a t 4°C t h i s was s i g n i f i c a n t l y l e s s than t h a t observed a f t e r i n c u b a t i o n a t 37°C. The temperature dependence o f apopotein movement can be e x p l a i n e d by s e v e r a l mechanisms. At 37°C the f l u i d i t y the l i p o p r o t e i n i s probably g r e a t e r then a t 4°C and thus the a p o p r o t e i n s may be more f r e e t o d i s s o c i a t e from the p a r t i c l e . A second e x p l a n a t i o n i s t h a t h i g h e r temperatures may i n c r e a s e the a c t i v i t y o f plasma f a c t o r s t h a t promote a p o p r o t e i n d i s s o c i a t i o n . However, the most obvious e x p l a n a t i o n i t t h a t i n c r e a s e d t r a n s f e r i s d i r e c t l y r e l a t e d t o the i n c r e a s e d k i n e t i c energy of the HDl p a r t i c l e s and hence t h e r e are more c o l l i s i o n s o c u r r i n g a t h i g h e r temperatures. The o b s e r v a t i o n t h a t a s u b s t a n t i a l amount of p r o t e i n was t r a n s f e r r e d t o the LDL f r a c t i o n agreed w i t h p r e v i o u s f i n d i n g s t h a t i n TD plasma, a s i g n i f i c a n t amount of apo A-I and apo A - I I i s a s s o c i a t e d with apo B c o n t a i n i n g l i p o p r o t e i n s (125). F u r t h e r , the f i n d i n g t h a t apo A-I and A - I I move independently o f each o t h e r i s a l s o i n agreement w i t h the o b s e r v a t i o n t h a t d u r i n g i n v i v o i n c u b a t i o n experiments a g r e a t e r amount of apo A-II i s a s s o c i a t e d w i t h VLDL/LDL f r a c t i o n 106 than apo A-I i n plasma (109,110,125). In another study, Assmann r e p o r t e d a steady decay of apo A-I I s p e c i f i c r a d i o a c t i v i t y w i t h i n the HDL d e n s i t y range i n v i v o but no s i g n i f i c a n t change i n apo A-I s p e c i f i c r a d i o a c t i v i t y i n t h i s same d e n s i t y r e g i o n (109). An e x p l a n a t i o n f o r t h i s d i f f e r e n c e may be t h a t apo A-I c o n c e n t r a t i o n s i n the HDL d e n s i t y r e g i o n i n TD plasma were so low t o begin w i t h t h a t f o l l o w i n g i s o l a t i o n of l i p o p r o t e i n s no d i l u t i o n o f s p e c i f i c a c t i v i t y was d e t e c t a b l e . Our r e s u l t s agree w i t h Schaefer's f i n d i n g s t h a t both apo: A-I, and apo A - I I , are l o s t from the HDL f r a c t i o n d u r i n g i n v i v o or i n v i t r o i n c u b a t i o n . The data p r e s e n t e d i n t h i s study suggest t h a t i n i t i a l events i n the metabolism of HDL i n TD plasma i n v o l v e core l i p i d exchanges t h a t a r e accompanied by a t r a n s f e r of-apo A-I and apo A - I I t o B l i p o p r o t e i n s . The remaining HDL i s a CE poor, T G - r i c h p a r t i c l e w i t h an a p p a r e n t l y normal diameter. The data i n t h i s s t u d y are c o n s i s t e n t w i t h the n o t i o n t h a t i t i s t h i s i n i t i a l m o d i f i c a t i o n o f HDL t h a t a c c e l e r a t e s the removal o f HDL from TD plasma. T h i s h y p o t h e s i s i s supported by the r e s u l t s of o t h e r s (110,125) who have shown t h a t the T G - r i c h HDL f r a c t i o n i s r a p i d l y removed from plasma. 107 4.2 P o s s i b l e f a c t o r s i n v o l v e d i n the m o d i f i c a t i o n o f HDL In an attempt t o i d e n t i f y the f a c t o r ( s ) i n TD plasma r e s p o n s i b l e f o r the m o d i f i c a t i o n o f normal HDL, the r o l e of LCAT, CE h y d r o l y s i s , and h y p e r t r i g l y c e r i d e m i a was examined. N i c h o l s e t a l (151) have p r e v i o u s l y r e p o r t e d t h a t LCAT p l a y s a major r o l e i n the m o d i f i c a t i o n o f HDL d u r i n g plasma i n c u b a t i o n i n v i t r o . T h e r e f o r e , the r o l e o f t h i s enzyme i n the changes t h a t o c c u r i n the s t r u c t u r e o f normal HDL f o l l o w i n g i n c u b a t i o n i n TD plasma was examined. I n h i b i t i o n of LCAT i n TD plasma, by the a d d i t i o n o f 5 mM DiFP (150), d i d not a l t e r the r a t e of l o s s of c h o l e s t e r o l from the HDL po o l (Table VIII) s u g g e s t i n g t h a t LCAT was not i n v o l v e d i n the m o d i f i c a t i o n o f the HDL p a r t i c l e s . The l o s s o f CE from HDL d u r i n g the i n c u b a t i o n c o u l d be e x p l a i n e d by a simple i n c r e a s e i n the r a t e o f CE h y d r o l y s i s d u r i n g the i n c u b a t i o n . However, the r e s u l t s d e s c r i b e d i n s e c t i o n 3.2.1 (Table VII) do not support t h i s h y p o t h e s i s . I t i s p o s s i b l e t h a t the r a p i d m o d i f i c a t i o n o f the l i p i d c o r e o f HDL d e s c r i b e d i n these experiments i s due, i n p a r t , t o a d i s e q u i l i b r i u m i n the c o n c e n t r a t i o n of c h o l e s t e r o l amongst the . plasma l i p o p r o t e i n s r e s u l t i n g from the a d d i t i o n o f a s i g n i f i c a n t amount o f exogenous HDL. Furthermore, the a d d i t i o n o f normal HDL i n t o the moderately h y p e r t r i g l y c e r i d e m i c TD plasma may f a c i l i t a t e t h e movement o f TG t o the HDL f r a c t i o n . In an attempt t o account f o r t h e s e p o s s i b i l i t i e s , the metabolism of normal HDL i n the plasma of a p a t i e n t w i t h homozygous LCAT d e f i c i e n c y was examined. D e s p i t e a more severe h y p e r t r i g l y c e r i d e m i a (compared t o the TD plasma) and the a d d i t i o n of a s i g n i f i c a n t amount of exogenous normal HDL, o n l y v e r y small l o s s e s i n HDL-C were observed i n the 108 LCAT d e f i c i e n t plasma a f t e r i n c u b a t i o n f o r 24h a t 37°C (Table V I I I ) . In a d d i t i o n , we examined the metabolism o f normal HDL i n the plasma of f i v e h y p e r t r i g l y c e r i d e m i c p a t i e n t s (Figure 10). The r e s u l t s of t h i s study i n d i c a t e t h a t the m o d i f i c a t i o n of HDL by TD plasma may not be s o l e l y accounted f o r by a mass e q u i l i b r a t i o n of l i p o p r o t e i n l i p i d s . However the exact r o l e o f t h e h y p e r t r i g l y c e r i d e m i a i n TD plasma i n the HDL m o d i f i c a t i o n process remains t o be e x p l a i n e d . TG 1s are known t o accumulate i n the HDL d u r i n g i n v i t r o i n c u b a t i o n s of normal plasma as a consequence o f LCAT, which i n c r e a s e s the amount o f CE i n the l i p o p r o t e i n c o r e (63) and CETP which promotes an exchange of HDL-CE f o r TG from other l i p o p r o t e i n s (50). The movement of CE from normal HDL i n TD plasma i s e s s e n t i a l l y an i n d i r e c t assay of CE mass t r a n s f e r as d e s c r i b e d by F i e l d i n g (169). The r a t e of t r a n s f e r of CE t o VLDL and LDL i s determined by the d i f f e r e n c e between the t o t a l i n c r e a s e i n plasma CE and the i n c r e a s e i n HDL-CE. A n a l y s i s of the data f o r the f i r s t 4 h of the i n c u b a t i o n (Table V) by t h i s method i n d i c a t e a r a t e o f 38.4 and 84.72 mg CE/dl plasma/24h, f o r c o n t r o l and TD plasma r e s p e c t i v e l y . O b v i o u s l y , t h e r e i s a s i g n i f i c a n t d i f f e r e n c e i n the r a t e of CE t r a n s f e r i n TD plasma compared t o c o n t r o l plasma. F u r t h e r a n a l y s i s of the data i n T a b l e V i n d i c a t e d t h a t the l o s s of HDL-CE was a s s o c i a t e d w i t h an equimolar i n c r e a s e i n HDL-TG c o n t e n t . As CETP r e d i s t r i b u t e s LCAT-derived CE from t h e i r s i t e s o f s y n t h e s i s i n HDL t o the l e s s dense T G - r i c h l i p o p r o t e i n s (50), by m e d i a t i n g an equimolar exchange of CE and TG (50), i t 109 may be t h a t t h i s p rocess i s a c c e l e r a t e d i n TD. The s i m p l e s t and most obvious e x p l a n a t i o n f o r the observed i n c r e a s e i n CETP a c t i v i t y would be t h a t t h e r e i s an i n c r e a s e d amount o f CETP i n TD plasma. While we have y e t t o d i r e c t l y measure the mass of CETP, the a c t i v i t y of CETP i n the non-l i p o p r o t e i n f r a c t i o n of our TD's plasma has been shown t o be i n c r e a s e d by approximately 50% over c o n t r o l s (Mr. D. Sparks p e r s o n a l communication). A second p o s s i b l e e x p l a n a t i o n i s d e r i v e d from r e c e n t s t u d i e s which have shown t h a t the a c t i v i t y of LTP's are i n f l u e n c e d by the chemic a l composition of both the HDL p a r t i c l e and the T G - r i c h a c c e p t o r l i p o p r o t e i n . In both i n v i v o and i n v i t r o s t u d i e s i t has been shown t h a t the changes i n l i p i d composition were a s s o c i a t e d w i t h i n c r e a s e d b i n d i n g o f CETP t o both VLDL and HDL (50). Thus, i t i s p o s s i b l e t h a t the i n c r e a s e d t r a n s f e r a c t i v i t y seen i n TD plasma may not be due t o an i n c r e a s e d l e v e l o f CETP but i n s t e a d t o , t h e abnormal composition o f the T G - r i c h l i p o p r o t e i n s (LDL or VLDL) which may a c c e l e r a t e the l i p i d t r a n s f e r p r o c e s s . Support f o r t h i s h y p o t h e s i s comes from the r e c e n t o b s e r v a t i o n t h a t an i n c r e a s e d TG/CE r a t i o i n a c c e p t o r l i p o p r o t e i n s w i l l f a v o r a g r e a t e r net t r a n s f e r of CE i n t o T G - r i c h l i p o p r o t e i n s ( f o r review see r e f . 50). Examination of the data presented i n Table IX i n d i c a t e s t h a t t h e TG/CE r a t i o i n c o n t r o l LDL and L D L r p D were 0.25 and 7.79, r e s p e c t i v e l y . In a d d i t i o n , a n a l y s i s o f the data presented i n T a b l e XI i n d i c a t e t h a t the TG/CE r a t i o i n c o n t r o l VLDL and V L D L T D were 11.85 and 16.25, r e s p e c t i v e l y . Thus, the TG/CE r a t i o i n both L D L T D and VLDLj^ are more f a v o r a b l e t o the CETP c a t a l y z e d 110 r e a c t i o n than are the r e s p e c t i v e c o n t r o l l i p o p r o t e i n s . Thus, these f i n d i n g s are s u p p o r t i v e o f the above h y p o t h e s i s . A t h i r d p o s s i b l e e x p l a n a t i o n of our data comes from the o b s e r v a t i o n o f Z i l v e r s m i t e t a l (170). They have d e s c r i b e d the presence o f CETP i n h i b i t o r p r o t e i n i n plasma. Thus, i t i s p o s s i b l e t h a t a d e f i c i e n c y of such a ' f a c t o r c o u l d e x p l a i n the e l e v a t e d l e v e l o f CETP a c t i v i t y seen i n TD plasma. In summary the r e s u l t s of our experiments are the f i r s t r e p o r t e d evidence of h y p e r c a t a b o l i s m of HDL i n TD plasma i n v i t r o r e s u l t i n g from an i n c r e a s e i n the CETP a c t i v i t y i n TD plasma. The exact mechanism u n d e r l y i n g t h i s i n c r e a s e remains u n c l e a r and needs t o be e l u c i d a t e d . I l l 4.3 The Metabolism o f TD and T D - m o d i f i e d l i p o p r o t e i n s by normal f i b r o b l a s t s 4.3.1 The i n v i t r o metabolism of H D L r p D by normal f i b r o b l a s t From the p r e v i o u s d i s c u s s i o n i t has become apparent t h a t the l o s s o f apo A-I from TD plasma must be a s s o c i a t e d w i t h an i n c r e a s e d r a t e o f removal. We have p o s t u l a t e d t h a t t h i s removal may be e x p l a i n e d by an i n c r e a s e d c e l l u l a r uptake of H D L r p D p a r t i c l e . T h e r e f o r e , the uptake of HDLJIQ was examined by i n c u b a t i n g i t w i t h normal s k i n f i b r o b l a s t s . In p r e l i m i n a r y experiments, we examined the c a t a b o l i s m o f H D L T D by f i b r o b l a s t s u s i n g HDL 3 7 as a c o n t r o l . Both t o t a l and s p e c i f i c b i n d i n g f o r both H D L r p D and HDL 3 7 were e s s e n t i a l l y the same. Comparison of the K d s from t h i s study t o t h a t d e s c r i b e d by Oram e t a l (K^: 18.2 ug/ml r e f . 74) i n d i c a t e t h a t t h e r e was l i t t l e d i f f e r e n c e between these l i g a n d s , with r e s p e c t t o r e c e p t o r a f f i n i t y , even though HDLj, D and HDL 3 7 had undergone i n c u b a t i o n i n v i t r o . An obvious e x p l a n a t i o n f o r these f i n d i n g s i s t h a t changes i n the core l i p i d o f an HDL p a r t i c l e do not e f f e c t i t s a b i l i t y t o i n t e r a c t w i t h the HDL r e c e p t o r . A second a l t e r n a t i v e i s t h a t i n c u b a t i o n does e f f e c t the p a r t i c l e s a b i l i t y t o i n t e r a c t with the HDL r e c e p t o r but t h a t these changes are masked by i n c r e a s e s or decreases i n a f f i n i t y f o r oth e r b i n d i n g s i t e s on the s u r f a c e o f the f i b r o b l a s t . For example, i f , d u r i n g the i n c u b a t i o n , the HDL T D p i c k e d up apo E i t s a b i l i t y t o i n t e r a c t with the the LDL r e c e p t o r would g r e a t l y be enhanced. The data p r e s e n t e d i n , F i g u r e 14 i n d i c a t e s t h a t more HDL T D than HDL 3 7 was degraded by f i b r o b l a s t s . However, the s i g n i f i c a n c e of t h e s e r e s u l t s i s not c l e a r as t h i s experiment was o n l y done once. Thus, more experiments are needed t o c o n f i r m these i n i t i a l f i n d i n g s . The human f i b r o b l a s t model may be i n a p p r o p r i a t e f o r s t u d y i n g the c a t a b o l i s m of HDI*^. T h e r e f o r e , f u r t h e r s t u d i e s u s i n g the p e r i p h e r a l b l o o d monocytes model should be examined i f the exact m e t a b o l i c f a t e of HDLpp i s t o be determined. U n f o r t u n a t e l y , t h e s e experiments c o u l d not be c a r r i e d out by myself due t o the d e a r t h of sample and time. In summary, the p r e c i s e m e t a b o l i c f a t e of t h i s p a r t i c l e i s u n c l e a r and w h i l e the r e s u l t s of t h i s study do not c o n v i n c i n g l y support the h y p o t h e s i s t h a t HDLpp i s h y p e r c a t a b o l i z e d by c u l t u r e d s k i n f i b r o b l a s t more work i s r e q u i r e d i f i t s r o l e i n h y p e r c a t a b o l i s m o f apo A-I and the pathogenesis of TD i s t o be f u l l y understood. 4.3.2 The metabolism o f LDI*j,D by normal f i b r o b l a s t i n v i t r o The i n c r e a s e d r a t e of removal of apo A-I may a l s o be e x p l a i n e d by the t r a n s f e r of apo A-I t o more r a p i d l y c a t a b o l i z e d p a r t i c l e s such as LDL. Therefore, the c a t a b o l i s m of LDLpp by c u l t u r e d f i b r o b l a s t s was examined. The p h y s i c a l , chemical and m e t a b o l i c nature of LDLpp was examined and the abnormal chemical composition o f LDI#pD r e p o r t e d by o t h e r s (90) was confirmed (Table I X ) . The experiments d e s c r i b e d i n F i g u r e s 15 and 16 are the f i r s t t o study the metabolism of L D L p p by normal f i b r o b l a s t s . The r e s u l t s demonstrated t h a t L D L r p D b i n d i n g and d e g r a d a t i o n was lower than 113 those of normal LDL. T h i s o b s e r v a t i o n may be e x p l a i n e d by the abnormal l i p i d and p r o t e i n composition i n the L D L r p D . The composition o f L D L r p D , a TG-r i c h p a r t i c l e which may c o n t a i n a d d i t i o n a l p r o t e i n s b e s i d e s apo B, i s s i m i l a r t o LDL i s o l a t e d from the plasma of h y p e r t r i g l y c e r i d e m i c p a t i e n t s (171) or f o r LDL m o d i f i e d by l i p i d t r a n s f e r p r o t e i n i n v i t r o (172). LDL p a r t i c l e s i s o l a t e d from these sources a l s o showed a decreased a b i l i t y t o i n t e r a c t with the LDL r e c e p t o r on the s u r f a c e of c u l t u r e d f i b r o b l a s t s (171,172). In summary, these experiments do not support a r o l e f o r LDLpjj i n the h y p e r c a t a b o l i s m of apo A-I i n TD plasma. I t i s p o s s i b l e t h a t t h e f i b r o b l a s t model i s the wrong c e l l t ype t o study and t h a t c e l l s which are a c t i v e i n the c l e a r a n c e of LDL from c i r c u l a t i o n (monocytes or hepatocytes) may be more a p p r o p r i a t e t o study. Thus; t h i s mechanism f o r the removal of apo A-I from plasma may s t i l l be important and t h e r e f o r e f u r t h e r work i s necessary b e f o r e e x c l u s i o n o f t h i s c l e a r a n c e mechanism. 114 4.4 The l i p o l y s i s o f VLDLrr. D by BmLpL The a s s o c i a t i o n between HDL l e v e l s and the r a t e o f l i p o l y s i s o f the VLDL has been known f o r s e v e r a l y ears (43). However, the r o l e o f HDL i n the metabolism o f plasma TG (and v i c e versa) i s l e s s w e l l understood i n p a t i e n t s w i t h severe d i s o r d e r s o f HDL metabolism. In the f o l l o w i n g study the p h y s i c a l , chemical and metabolic p r o p e r t i e s o f VLDL, i s o l a t e d from p a t i e n t s w i t h TD and LCAT d e f i c i e n c y , were s t u d i e d i n an attempt t o e l u c i d a t e the cause of the h y p e r t r i g l y c e r i d e m i a i n these d i s o r d e r s . In t h i s study, i t has been shown t h a t the VLDL p a r t i c l e s t h a t accumulate i n LCAT d e f i c i e n t and TD d i s e a s e plasma are s u i t a b l e s u b s t r a t e s f o r BmLpL i n v i t r o . The k i n e t i c parameters of the h y d r o l y s i s o f the VLDL p a r t i c l e s from the LCAT d e f i c i e n t and TD p a t i e n t i n d i c a t e t h a t the apparent V m a x v a l u e s are approximately f o u r and t h r e e times greater> r e s p e c t i v e l y , than t h a t f o r the c o n t r o l VLDL. The r e s u l t s o f t h i s a n a l y s i s a l s o i n d i c a t e d t h a t the apparent IC^ was i n c r e a s e d f o r both d i s o r d e r s . These r e s u l t s suggest t h a t the i n t e r a c t i o n between the V L D L r p D and V L D L L C A T with BmLpL i s weaker than t h a t observed with c o n t r o l s u b s t r a t e . The decreased a f f i n i t y o f the enzyme towards the TD and LCAT d e f i c i e n t s u b s t r a t e may r e f l e c t the i n c r e a s e d s i z e o f the s e abnormal l i p o p r o t e i n p a r t i c l e s and, p o s s i b l y , t h e i r d i f f e r e n t chemical composition. F i e l d i n g e t a l have shown t h a t the a d d i t i o n o f exogenous FC t o normal VLDL i n v i t r o decreased the r a t e o f h y d r o l y s i s by l i p o p r o t e i n l i p a s e (163). Thus, the i n c r e a s e d FC co n t e n t of V L D L L C A T and VLDLj, D may e x p l a i n the 115 abnormal KJJ/S observed (Table X I ) . However, i n TD the FC t o PL r a t i o i s comparable t o c o n t r o l thus s u g g e s t i n g t h a t o t h e r f a c t o r s , such as i n c r e a s e d p a r t i c l e s i z e , i s r e s p o n s i b l e f o r the decreased enzyme a f f i n i t y . In t h i s study, p o s t h e p a r i n LpL a c t i v i t y i s moderately decreased i n both d i s o r d e r s (J F r o h l i c h , u n p u b l i s h e d r e s u l t s ) which agrees w i t h p r e v i o u s data i n oth e r p a t i e n t s w i t h LCAT d e f i c i e n c y (173,174) o r TD (103,104). However, the data are somewhat d i f f i c u l t t o e v a l u a t e due t o d i f f e r e n c e s i n the amount of h e p a r i n i n j e c t e d , the time p r i o r t o sampling and the method of d e t e r m i n a t i o n o f the r a t e o f h y d r o l y s i s . In some experiments, f r e e f a t t y a c i d s would have been generated s i n c e i n v i t r o i n h i b i t o r s o f the l i p a s e (104) were not added t o the p o s t h e p a r i n plasma. I t appears u n l i k e l y t h a t a decrease i n the a b s o l u t e amount of p o s t h e p a r i n LpL a c t i v i t y i n i t s e l f i s d i r e c t l y a s s o c i a t e d w i t h the development of h y p e r t r i g l y c e r i d e m i a as low l i p a s e a c t i v i t y was a l s o observed i n a LCAT d e f i c i e n t s u b j e c t w i t h normal plasma l e v e l s o f t r i g l y c e r i d e (J F r o h l i c h , u n p u blished r e s u l t s ) . In a d d i t i o n , the c o n c e n t r a t i o n o f apo C-II, the major a c t i v a t o r o f l i p o p r o t e i n l i p a s e (163), i n the plasma of thes e s u b j e c t s does not appear t o be d i r e c t l y r e l a t e d t o the ex t e n t o f h y p e r t r i g l y c e r i d e m i a . T h i s may r e f l e c t the presence of s u f f i c i e n t apo C-II r e q u i r e d t o f u l l y a c t i v a t e the l i p a s e i n v i t r o (175). The p o s s i b i l i t y t h a t the h y p e r t r i g l y c e r i d e m i a p l a y s a r o l e i n the e t i o l o g y o f these two d i s o r d e r s i s i n t r i g u i n g . In LCAT d e f i c i e n c y , the few p a t i e n t s who do not develop h y p e r l i p i d e m i a 116 (176) do not have the r e n a l c o m p l i c a t i o n s t h a t a re o f t e n observed i n t h i s d i s o r d e r (46). T h i s suggests t h a t development of the r e n a l l e s i o n s may be secondary t o the presence o f abnormal p a r t i c l e s t h a t accumulate i n the m a j o r i t y of the LCAT d e f i c i e n t p a t i e n t s . On the other hand, n e i t h e r LCAT d e f i c i e n t p a t i e n t s ( e s p e c i a l l y those without r e n a l c o m p l i c a t i o n s ) nor TD p a t i e n t s are a t a markedly i n c r e a s e d r i s k o f h e a r t d i s e a s e (46,96) d e s p i t e t h e i r low l e v e l s o f HDL. The reasons f o r t h i s o b s e r v a t i o n are not c l e a r but i t i s probable t h a t decreases i n the o t h e r l i p o p r o t e i n r i s k f a c t o r s secondary t o the major d e f e c t may occur. The a b i l i t y o f chylomicrons and VLDL t o accept FC and/or CE from ot h e r l i p o p r o t e i n s i s w e l l e s t a b l i s h e d . F i e l d i n g and coworkers have r e p o r t e d t h a t p o s t p r a n d i a l l i p e m i a i s a s s o c i a t e d w i t h i n c r e a s e d net c h o l e s t e r o l t r a n s p o r t (177). Thus, i t i s p o s s i b l e t h a t t r a n s f e r o f CE t o these p a r t i c l e s may overcome some o f the d e f i c i e n c i e s i n c h o l e s t e r o l t r a n s p o r t t h a t are a s s o c i a t e d with decreased HDL l e v e l s . T h i s h y p o t h e s i s i s f u r t h e r supported by the p r e l i m i n a r y o b s e r v a t i o n t h a t t h e r e was an i n c r e a s e i n CE t r a n s f e r from normal HDL t o TD VLDL/LDL when normal HDL was incubated w i t h T a n g i e r l i p o p r o t e i n s (d<1.21 g/ml) and normal LPDP ( r e s u l t s not shown). The p o s s i b l e causes of h y p e r t r i g l y c e r i d e m i a i n LCAT d e f i c i e n c y and TD may i n c l u d e one or more of the f o l l o w i n g mechanisms: 1) Increased h e p a t i c p r o d u c t i o n o f T G - r i c h l i p o p r o t e i n s ; 2) Decreased c a t a b o l i s m o f t r i g l y c e r i d e l i p o p r o t e i n s due t o decreased l i p o p r o t e i n l i p a s e a c t i v i t y , which i n t u r n may be due t o a decrease i n l i p o p r o t e i n l i p a s e mass or an 117 imbalance between i t s a c t i v a t o r s and i n h i b i t o r s i n the p a t i e n t s plasma; 3) The i n a b i l i t y o f the l i p o p r o t e i n t o a c t as an e f f i c i e n t s u b s t r a t e ; and 4) decreased h e p a t i c c l e a r a n c e of T G - r i c h l i p o p r o t e i n s and/or t h e i r remnants. C u r r e n t l y , no data r e g a r d i n g TG p r o d u c t i o n i n e i t h e r LCAT d e f i c i e n c y or TD have been p u b l i s h e d . The r e s u l t s o f t h i s study i n d i c a t e t h a t the T G - r i c h l i p o p r o t e i n s t h a t accumulate i n v i v o are s u i t a b l e s u b s t r a t e s f o r l i p o p r o t e i n l i p a s e i n v i t r o . However, the i n v i t r o i n t e r a c t i o n of T G - r i c h l i p o p r o t e i n s w i t h the l i p a s e may not r e f l e c t the i n v i v o s i t u a t i o n . While t h e r e appears t o be s u f f i c i e n t amount of the LPL a c t i v a t o r (apo C-II) even i n the plasma o f the LCAT d e f i c i e n t p a t i e n t , no data a r e a v a i l a b l e r e g a r d i n g the presence of p o s s i b l e i n h i b i t o r s . The p o s t h e p a r i n LPL a c t i v i t y i s somewhat decreased i n both LCAT d e f i c i e n t and TD p a t i e n t s . However, the magnitude of the decrease suggests t h a t changes i n LPL a c t i v i t y alone may not p l a y a major r o l e i n the accumulation of the T G - r i c h l i p o p r o t e i n p a r t i c l e s i n these p a t i e n t s . In a d d i t i o n , i t has been demonstrated t h a t the VLDL from these p a t i e n t s are s u i t a b l e s u b s t r a t e s f o r LPL d e s p i t e d i f f e r e n c e s i n s i z e and composition. Thus, ot h e r mechanisms, such as the c a t a b o l i s m by h e p a t i c l i p a s e , i n t e r a c t i o n w i t h h e p a t i c r e c e p t o r s , o v e r p r o d u c t i o n , or r o l e of LCAT d e f i c i e n c y remain t o be e x p l o r e d . In summary, the p o s s i b i l i t y t h a t the d e f i c i e n c y of HDL i s due t o a decrease p r o d u c t i o n of HDL p r e c u r s o r s i s not supported 1 1 8 by the f i n d i n g t h a t VLDLpp i s a good s u b s t r a t e f o r BmLpL. However, f u r t h e r s t u d i e s are r e q u i r e d t o f u r t h e r e l u c i d a t e the s i g n i f i c a n c e o f t h i s h y p o t h e s i s e d d e f e c t . 119 4.5 A C e l l u l a r D e f e c t o f HDL Metabolism i n TD ? R e c e n t l y , Assmann e t a l have suggested t h a t the abnormality i n TD i s due t o a d e f e c t i n the i n t r a c e l l u l a r t r a f f i c k i n g of i n t e r n a l i z e d HDL. These i n v e s t i g a t o r s showed t h a t p e r i p h e r a l b l o o d monocytes, i s o l a t e d from TD p a t i e n t s , bound and i n t e r n a l i z e d normal HDL a t a r a t e comparable t o c o n t r o l monocytes but, u n l i k e c o n t r o l monocytes, f a i l e d t o r e l e a s e the HDL back i n t o the medium (166). F i g u r e 21 shows the amount o f 1 2 5 I - H D L 3 t h a t was a s s o c i a t e d w i t h c u l t u r e d monocytes from TD and c o n t r o l i n d i v i d u a l s a f t e r 4 hours of i n c u b a t i o n a t 37°. No major d i f f e r e n c e s were observed between c o n t r o l and TD monocytes thus i n d i c a t i n g t h a t s u r f a c e b i n d i n g of HDL t o these c e l l s was not abnormal i n TD d i s e a s e . Only n e g l i g i b l e c e l l - m e d i a t e d d e g r a d a t i o n of t h e 1 2 5 I - H D L 3 o c c u r r e d . T h i s was p r e v i o u s l y r e p o r t e d by o t h e r s (73,74,75) and was not s i g n i f i c a n t l y d i f f e r e n t i n TD c e l l s . These r e s u l t s are s i m i l a r t o those r e p o r t e d by Schmitz e t a l . These authors r e p o r t e d t h a t b i n d i n g o f HDL was o n l y modestly i n c r e a s e d i n f r e s h l y i s o l a t e d monocytes from TD p a t i e n t s (166). In a d d i t i o n , t h e s e authors have p r e v i o u s l y shown t h a t the e x p r e s s i o n of the HDL r e c e p t o r i n monocytes c o u l d be up r e g u l a t e d by i n c u b a t i n g the the monocytes i n medium c o n t a i n i n g a c e t y l a t e d LDL. These authors demonstrated t h a t c e l l - a s s o c i a t e d r a d i o a c t i v i t y c o u l d o n l y be i n c r e a s e d by 10-3 0% when c o n t r o l and TD monocytes were c u l t u r e d f o r 2 days i n 100 ug/ml a c e t y l - L D L p r i o r t o i n c u b a t i o n with 1 2 5 i -HDL 3. The most marked r e s u l t t h a t Schmitz e t a l r e p o r t e d was the apparent d e f e c t i n the r e l e a s e of HDL from these c e l l s . In an attempt t o demonstrate d e f e c t i v e c e l l u l a r t u r n o v e r of HDL i n TD 120 c e l l s t h a t had not been p r e i n c u b a t e d i n ac e t y l - L D L , the l e n g t h of the i n c u b a t i o n p e r i o d o f the experiment was extended. As approximately 80% of c e l l a s s o c i a t e d HDL i s r e l e a s e from c u l t u r e d monocytes w i t h i n 5 hours, i t was assumed t h a t any d e f e c t i n the r e l e a s e o f HDL 3 would manifest i t s e l f by the accumulation of l a b e l w i t h i n the c e l l s d u r i n g the extended i n c u b a t i o n . Moreover, any s i g n i f i c a n t d e g r a d a t i o n of the i n t e r n a l i z e d HDL 3 i n both the c o n t r o l and T a n g i e r c e l l s should be apparent a f t e r t h i s p e r i o d of i n c u b a t i o n . F i g u r e 22 shows t h a t the amount of 1 2 5 I - l a b e l l e d HDL 3 a s s o c i a t e d w i t h the c e l l s a f t e r 20 hours was not d i f f e r e n t between c o n t r o l o r the TD c e l l s . Thus, i t was concluded t h a t abnormal HDL metabolism cannot be demonstrated i n f r e s h l y i s o l a t e d monocytes. The reasons f o r our i n a b i l i t y t o demonstrate a d e f e c t i n f r e s h l y i s o l a t e d , monocytes are not c l e a r a t p r e s e n t . I t i s p o s s i b l e t h a t l i p i d l o a d i n g o f the monocytes by a c e t y l - L D L i s r e q u i r e d b e f o r e the d e f e c t can be demonstrated. However, i f t h i s i s a l s o the case i n v i v o , one should c o n s i d e r t h a t o r i g i n o f the CE which accumulate i n t i s s u e h i s t i o c y t e s . I t has been proposed t h a t r a p i d uptake of the HDL i n p a t i e n t s w i t h TD may supply the CE which accumulates and r e s u l t s i n foam c e l l development (166) although no experimental evidence i s a v a i l a b l e t o t e s t t h i s h y p o t h e s i s . I t i s a l s o p o s s i b l e t h a t LDL p r o v i d e s the c h o l e s t e r o l f o r the accumulating l i p i d . In f a c t , t h i s seems more f e a s i b l e s i n c e LDL from TD p a t i e n t s has abnormal l i p i d and p r o t e i n composition (90) and i n c r e a s e d e l e c t r o p h o r e t i c m o b i l i t y (95,100) 121 which may r e s u l t i n c e l l u l a r uptake v i a the scavenger pathway. Whether t h i s c o u l d r e s u l t i n the s i g n i f i c a n t decreases i n LDL-C observed i n TD (90) and e x p l a i n the k i n e t i c s o f apo A-I turnover i n TD p a t i e n t s remains t o be e v a l u a t e d . In an a d d i t i o n a l study, TD s k i n f i b r o b l a s t s were examined f o r t h e i r a b i l i t y t o b i n d / i n t e r n a l i z e and degrade HDL i n c u l t u r e . The r e s u l t s o f t h i s study showed t h a t TD f i b r o b l a s t s d i d not b i n d / i n t e r n a l i z e or degrade normal HDL i n an abnormal f a s h i o n . R e c e n t l y these r e s u l t s were confirmed i n our l a b by R. McLeod e t a l and by Dr. J.Oram at the U n i v e r s i t y of Washington, S e a t t l e Wa. Both o f these independent s t u d i e s showed t h a t the b i n d i n g , i n t e r n a l i z a t i o n , r e l e a s e and d e g r a d a t i o n of HDL by TD f i b r o b l a s t s was not d i f f e r e n t from c o n t r o l c e l l l i n e s . Furthermore, i n a subsequent s e r i e s of experiments the metabolism o f LDL, and the i n t r a c e l l u l a r a c t i v i t y of ACAT and HMGCoA reductase were a l s o shown t o be normal i n TD f i b r o b l a s t s (R. McLeod, unpublished r e s u l t s ) . In a r e c e n t paper, F r o h l i c h e t a l (178) have shown t h a t the i n t e r a c t i o n of HDL with a d i p o c y t e s i s o l a t e d from our TD p a t i e n t was s l i g h t l y l e s s than t h a t f o r c o n t r o l a d i p o c y t e s . F r o h l i c h concluded t h a t i t was u n l i k e l y t h a t TD a d i p o c y t e s p l a y a r o l e i n the e t i o l o g y of TD. I t i s s t i l l p o s s i b l e , however, t h a t the d e f e c t i n TD may i n v o l v e a c e l l u l a r component. Thus f u r t h e r s t u d i e s should examine the r o l e of TD l i v e r i n t h i s d i s o r d e r as apo A-I immunoreactive m a t e r i a l has been d e s c r i b e d i n h e p a t i c Kuppfer c e l l s i n TD ( J . F r o h l i c h , unpublished r e s u l t s ) . In a d d i t i o n , the experiments p r e s e n t e d here and those r e p o r t e d by Schmitz e t a l (166) 122 i n v e s t i g a t e d the i n t e r a c t i o n o f TD c e l l s w i t h normal HDL. However, i t i s p o s s i b l e t h a t the HDL p a r t i c l e t h a t i n t e r a c t s w i t h the TD c e l l s i n v i v o i s markedly d i f f e r e n t from t h a t used i n thes e experiments. In t h i s t h e s i s , i t has been demonstrated t h a t when normal H D L i s incubated w i t h TD plasma, the CE core i s r a p i d l y exchanged f o r TG from the lower d e n s i t y l i p o p r o t e i n s ( S e c t i o n 3.1). Thus, i t i s p o s s i b l e t h a t h y p e r c a t a b o l i s m o f H D L r j - , D p a r t i c l e may be more r e l e v a n t t o the a c t u a l i n v i v o s i t u a t i o n . D e s p i t e these o b s e r v a t i o n and those of Schmitz and coworkers, more i n v e s t i g a t i o n s i n H D L metabolism i n TD p a t i e n t s are r e q u i r e d i f the exact m o l e c u l a r d e f e c t i s t o be d e s c r i b e d . 123 REFERENCES 1. Ross R. N.Engl.J.Med. 1986, 314:488-500. 2. Levy RI. A t h e r o s c l e r o s i s . 1981, 1:312-325. 3. I n k e l e s S, E i s e n b e r g D. Medicine ( B a l t i m o r e ) . 1981, 60:110-123. 4. Gordon T, C a s t e l l i WP, H j o r t l a n d MC. e t a l . Am J Med. 1977, 251:351-374. 5. H e i s s G, e t a l . 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