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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  IN TANGIER  LIPOPROTEIN  DISEASE  by ROCKFORD WILLIAM B.Sc,  SAMBORSKI  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 REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE  STUDIES  (Department o f P a t h o l o g y )  We a c c e p t t h i s  thesis  to the required  as conforming standard  THE UNIVERSITY OF B R I T I S H COLUMBIA J a n u a r y 21 1987  ( c ) R o c k f o r d W.  Samborski,  1987  THE  In  presenting  degree  this  at the  thesis  in  partial fulfilment  of  University of  British Columbia,  I agree  freely available for reference copying  of  department publication  this or of  thesis for by  this  his  or  and study.  Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  DE-6(3/81)  that the  representatives.  may be It  thesis for financial gain shall not  permission.  requirements  I further agree  scholarly purposes her  the  for  an  advanced  Library shall make it  that permission  for extensive  granted  head  is  by the  understood be  that  allowed without  of  my  copying  or  my written  ABSTRACT This  s t u d y was  underlying Tangier  the  (TD).  i n Tangier  n o r m a l human HDL concentration  in  by  up  was  Initially, plasma  added t o TD  i n the  TD  observations  acytransferase triglyceride  not  be  completely  o f HDL  The  was  plasma  not  seen  l o s s of  accounted  f o r by  a c c o m p a n i e d by  f o r by  TD  a  HDLthe  2.3-fold  These  lecithin:  p l a s m a . However, activity  cholesterol  preliminary  of c h o l e s t e r y l ester  plasma i s r e s p o n s i b l e  resulting triglyceride-rich,  However, t h i s  HDL  f o r the  changes  i n TD  o f HDL  c h o l e s t e r y l - p o o r HDL  f o r the  f i n d i n g does not  t h a t may  contribute  in  was  human s k i n f i b r o b l a s t exclude other  t o the  HDL  pathways  rapid turnover  of  plasma.  m e t a b o l i s m o f n o r m a l HDL  i n v i t r o was defect  TD  During  composition.  catabolism  modified  i n the  of HDL-triglyceride.  shown t o have a n o r m a l a f f i n i t y receptor.  Sufficient  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. ~  i n the  t r a n s f e r p r o t e i n i n TD  The  examined.  t o w i t h i n normal l e v e l s .  accounted  evidence suggested t h a t the  HDL-lipid  was  HDL  plasma to r a i s e the  e s t e r and  activity,  level  metabolism of normal  i n a s i m i l a r manner. The  concentration  could  lipoprotein in  of HDL-cholesterol  c o u l d be  l o s s of HDL-cholesteryl i n the  density  mechanism(s)  i n a t i m e d e p e n d e n t manner. T h i s was  c o n t r o l samples t r e a t e d  increase  the  in vitro  concentration  t o 50%  cholesterol  of high  of HDL-cholesterol  incubation the fell  i n v e s t i g a t e the  hypercatabolism  disease  incubated  designed to  also studied metabolism  i n an i n TD.  by  TD  f i b r o b l a s t s and  attempt to  identify  However, b o t h TD  a  monocytes  cellular  f i b r o b l a s t s and  m o n o c y t e s were n o r m a l w i t h r e s p e c t bind/internalize It  to t h e i r  and d e g r a d e normal HDL  i s concluded  that  in vitro.  and p r o t e i n c o m p o s i t i o n  t o r e m o v a l f r o m t h e p l a s m a component. Thus,  not  i n the c e l l s  the defect  of these  to  t h e h y p e r c a t a b o l i s m o f n o r m a l HDL  involves alterations of HDL-lipid  the hypothesis that  ability  i n TD r e s i d e s  patients.  these studies i n the plasma  i n TD  prior support and  TABLE OF CONTENTS ABSTRACT  i  TABLE OF CONTENTS  i i i  ABBREVIATIONS  xii  L I S T OF TABLES  viii  L I S T OF FIGURES  ix  ACKNOWLEDGEMENTS  xi -  DEDICATION 1  x  INTRODUCTION  1.1  Introduction  1  1.2  Lipoproteins  4  1.2.1  Introduction  4  1.2.2  Apolipoproteins  6  1.2.3  Lipoprotein structure  9  1.3  L i p o p r o t e i n metabolism  9  1.3.1  Introduction  9  1.3.2  Lipoprotein biogenesis  11  1.3.3  Lipoprotein catabolism  ....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  Cell-lipoprotein  19  interaction  1.4  Reverse c h o l e s t e r o l t r a n s p o r t  26  1.5  Tangier  28  disease  1.5.1  Introduction  1.5.2  History  1.5.3  Clinical  1.5.4  Biochemical  1.6  28 ..28  features  29  features  Biochemical defect  i n Tangier iii  31 disease  33  i  v  1.7  Rationale  1.8  S p e c i f i c aims....  2  for this  study  36 36  MATERIALS AND METHODS  2.1.1  Control  subjects  2.1.2  Tangier patient  37 37  2.2  Material  40  2.3  Lipoproteins  41  2.3.1  Isolation of lipoproteins  41  2.3.1.1  Preparative ultracentrifugation.  2.3.1.2  Heparin-Magnesium  2.3.2  Iodination  of lipoproteins  2.3.2.1  Preparation of  2.3.2.2  Characterization 1 2 5  2.3.3 2.3.3.1  precipitation  1 2 5  I-lipoproteins  43  [ H] l a b e l i n g o f plasma and l i p o p r o t e i n s 3  tri[ H]oleate 3  2.3.3.3  3  labeling  2.3.4 2.3.4.1  3  ester  44 labeling  p l a s m a a n d HDL  Characterization  of Lipoproteins..  ...45 45  Determination of l i p o p r o t e i n particle  2.3.4.2  labeling of  lipoproteins  [ H] c h o l e s t e r y l of  44  44  [ H] c h o l e s t e r o l plasma  42  of  o f VLDL 2.3.3.2  ....42 42  I-lipoproteins  Glyceryl  41  size  Lipoprotein  analysis  iv  45 46  2.3.5  Enzyme A s s a y s  2.3.5.1  46  N o n - s p e c i f i c plasma h y d r o l y s i s cholesteryl  2.3.5.2 2.4  of  esters  46  Bovine m i l k l i p o p r o t e i n  lipase  C e l l culture  2.4.1  47  Isolation  and c u l t u r e  of  human f i b r o b l a s t s 2.4.2  47  Preparation of c e l l lipoprotein  2.4.3  lines prior to  binding experiments  C u l t u r e o f human p e r i p h e r a l  ..49  blood  monocytes 2.5  49  Metabolism of l i p o p r o t e i n s cultured  47  by  cells i n vitro  51  2.5.1  1 2 5  I - L D L m e t a b o l i s m 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-HDL  52  3 3.1  metabolism by monocytes..  RESULTS C a t a b o l i s m o f n o r m a l HDL b y T a n g i e r In  3.1.1  3  vitro Effect  53 o f i n c u b a t i o n a t 37°C o n  electrophoretic 3.1.2  plasma  Effect  mobility  lipoprotein  i n vitro  o f i n c u b a t i o n a t 37 ° C o n  HDL-cholesterol composition i n v i t r o 3.1.3  Effect  Effect  56  o f i n c u b a t i o n a t 37°C o n  lipoprotein 3.1.4  53  lipid  composition i n v i t r o  59  o f i n c u b a t i o n a t 37°C on HDL  apoprotein composition i n v i t r o  v  62  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 modification incubation  o f n o r m a l HDL  the  during 70  i n TD p l a s m a  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  R o l e 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  M e t a b o l i s m o f HDLprj by c u l t u r e d fibroblast  skin  in vitro  76  3.3.1  Introduction  76  3.3.2  M e t a b o l i s m 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 fibroblasts  3.5  by c u l t u r e d  D  in vitro  ....84  lipase  in vitro  C a t a b o l i s m o f n o r m a l HDL fibroblasts Tangier  3.6.2  T a n g i e r monocytes  Modification  4.2  Possible  97 97 ..100  o f n o r m a l HDL  b y TD  Plasma  The m e t a b o l i s m o f TD  108 and TD  The m e t a b o l i s m o f fibroblasts  modified  fibroblasts HDLrr.  D  by  in vitro  in vitro  vi  112  normal  in vitro  The m e t a b o l i s m o f LDLpp by fibroblasts  103  i n the  o f HDL  l i p o p r o t e i n s by n o r m a l  4.3.2  Tangier  fibroblasts  factors involved  modification  4.3.1  by  DISCUSSION  4.1  4.3  90  and m o n o c y t e s i n c u l t u r e  3.6.1  4  skin  C a t a b o l i s m o f VLDLpp by B o v i n e m i l k lipoprotein  3.6  LDLrr.  112 normal 113  The  lipolysis  A cellular  o f VLDLpp by  defect  o f HDL  vii  BmLpL i n  catabolism  vitro in  TD  L I S T OF TABLES Table  I  Classification  Table  II  Major plasma a p o l i p o p r o t e i n s  Table  III  Clinical  Table  IV  Lipid  Effect  ;  composition  TD p l a s m a and TD  containing Table V  7  p r e s e n t a t i o n o f TD. .  and p r o t e i n  control,  5  o f plasma l i p o p r o t e i n s  30  of  plasma  e x o g e n o u s HDL  o f 37°C i n c u b a t i o n on  53 HDL  compositon f o l l o w i n g t h e a d d t i o n of normal HDL t o TD p l a s m a Table VI  Effect  60  o f 37°C i n c u b a t i o n on  apolipoproteins  HDL  following the  a d d i t i o n o f n o r m a l HDL t o TD p l a s m a Table VII  Role  67  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 VIII  Role  73  Table  Chemical  IX  o f LCAT i n m o d i f i c a t i o n p r o c e s s composition  o f LDLpQ  ...85  Table X  LDL k i n e t i c b i n d i n g p a r a m e t e r s  88  T a b l e XI  Chemical  93  composition  of V L D L ^  viii  L I S T OF  FIGURES  Figure  1.  Structure of lipoprotein  10  Figure  2.  Overview o f c h o l e s t e r o l metabolism  12  Figure  3.  Route o f t h e LDL r e c e p t o r i n mammalian  cells  Figure  4.  TD p a t i e n t  Figure  5.  Effect  23  (JKW)  family tree  o f 3 7 ° i n c u b a t i o n on  electrophoresis pattern addition Figure  6.  n o r m a l HDL  Figure  7.  total  following the 55  c h o l e s t e r o l content of  incubated  c o n t r o l o r TD p l a s m a  Gel f i l t r a t i o n 1 2 5  lipoprotein  o f n o r m a l HDL t o TD p l a s m a  The r e l a t i v e  in  39  58  chromatography  I-HDL-apoproteins  of  following  i n c u b a t i o n a t 4°C o r 37°C Figure  8.  Gel f i l t r a t i o n labelled  63  o f [ H] c h o l e s t e r o l 3  TD p l a s m a c o n t a i n i n g HDL  following  i n c u b a t i o n a t 4 ° and 3 7 ° Figure  9.  Gel f i l t r a t i o n 125j_  H D L  68  chromatography o f  following incubation i n  TD p l a s m a a t 4 ° a n d 3 7 ° Figure  10.  69  Role o f 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 process  Figure  11.  10% S D S - P A G E o f  Figure  12a.  Total binding 1 2 5  Figure  12b.  I-HDL  Specific 1 2 5  I-HDL  3 7  1 2 5  of  1 2 5  of  T D  1 2 5  I-HDL  I-HDL  T D  79  80  and  fibroblasts ix  3 7  and  fibroblasts  1 2 5  t o normal  and  T D  I-HDL  t o normal  binding 3 7  I-HDL  75  81  Figure  13.  Scatchard  Figure  14.  Degradation of I-HDL  1 2 5  Figure  15.  Binding 1 2 5  Figure  17.  T D  fibroblasts  1 2 5  86  I-labelled  c o n t r o l LDL by n o r m a l o f VLDL  LDLpQ  fibroblasts  d i s t r i b u t i o n b y QLS  91  The h y d r o l y s i s o f VLDL b y BmLpL  Figure  18b.  Eadie-Hofstee  from F i g u r e  18a...  Binding  of  t o TD  and 20.  Scatchard  Figure  21.  Binding and  22.  Binding and  3  analysis of data  of  1 2 5  I-HDL  3  .98 from F i g u r e  19....99  t o TD  c o n t r o l monocytes  during Figure  I-HDL  96  control fibroblasts  Figure  95  analysis  of data  1 2 5  89  size  18a.  19.  83  L D I * ^ a n d c o n t r o l LDL  Figure  Figure  12b...82  and  normal f i b r o b l a s t s  Determination and  I-HDL  from F i g u r e  and I n t e r n a l i z a t i o n o f  Degradation o f and  1 2 5  by n o r m a l  3 7  I-labelled  by F i g u r e 16.  analysis of data  a 4 hour i n c u b a t i o n a t 37°C of  1 2 5  I-HDL  3  101  t o TD  c o n t r o l monocytes  during  a 20 h o u r i n c u b a t i o n a t 3 7 ° C . . . . . . . . . 102  x  ACKNOWLEDGEMENTS First Pritchard  and foremost  I wish  f o r h i s patience, guidance,  during t h e course o f these I a l s o wish t o thank Dr.  t o t h a n k my s u p e r v i s o r D r . Haydn enthusiasm  studies. Dr. J . F r o h l i c h ,  D r . D. Seccombe,  N. J e t h a , D r . W. L. Dunn, D r . N. U r q u h a r t ,  C.Yeung, a n d Mr. D. S p a r k s  and f r i e n d s h i p  for their  interest  c o n c e r n i n g t h e work p r e s e n t e d i n t h i s I am i n d e b t e d t o t h e e n t i r e  staff  Mr. R . M c l e o d , Mr. and s u g g e s t i o n s  thesis. 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 o f S h a u g h n e s s y H o s p i t a l w i t h o u t whose h e l p t h i s  t h e s i s w o u l d n o t be  possible. I am a l s o for  financial  indebted t o the B r i t i s h s u p p o r t i n t h e form  Columbian  o f a student  xi  Heart  Foundation  traineeship.  ABBREVIATIONS apo  Apolipoprotein  BmLpL  Bovine milk  B  Maximum b i n d i n g  m  a  x  by  lipoprotein  lipase  determined  Scatchard a n a l y s i s  BSA  B o v i n e serum  CE  Cholesteryl ester(s)  CEH  CE h y d r o l y s i s  CETP  CE-transfer protein  CHD.  Coronary h e a r t  FC  Free  FED...  Fish-eye  disease  FH  Familial  hypercholesterolemia  FP  F i c o 11 - Paque  HDL  High d e n s i t y  HDL-C.  albumin  disease  cholesterol  lipoprotein  ....HDL-cholesterol  HDLrppj. .  Tangier modified  HDL37  N o r m a l HDL incubated  HDL  isolated  from plasma a f t e r  a t 3 7 ° C f o r 24  HTGL.  Hepatic  triglyceride  Hypoalpha.  Familial  IDL.  Intermediate  h.  lipase  hypoalphalipoproteinemia density  lipoproteins  .Equilibrium dissociation K^j  Michaelis-menton  LBTI  Lima b e a n t r y p s i n  LCAT  Lecithin:  LDL.  Low  LDL-C  LDL-cholesterol  constant  constant inhibitor  cholesterol acyltransferase  density  lipoprotein  xii  being  LDL  T a n g i e r LDL (1.006 t o 1.063 g/ml)  T D  LPDP  Lipoprotein  d e f i c i e n t human  LPDS  Lipoprotein  d e f i c i e n t fe'tal c a l f  LpL  Lipoprotein  lipase  LTP  Lipid  MEM.....  Modified . . . i  PAGE  transfer  E a g l e s medium  Polyacrylamide g e l electrophoresis Phosphate b u f f e r e d  Pl  Phosphol i p i d  RCT  ....Quasielastic  laser light  SDS  Sodium  TCA  Trichloroacetic  TD  Tangier  TG  Triglyceride  TLC  Thin  VLDL...  V e r y low d e n s i t y  VLDL  TD VLDL  V  m  a  WBC  saline  .Reverse c h o l e s t e r o l  x  T D  serum  protein  PBS  QLS..  plasma  scattering  transport  dodecylsulphate acid  disease  layer  chromatography lipoprotein  Maximum v e l o c i t y o f r e a c t i o n White b l o o d  cells  xiii  DEDICATION To Mr. Ken  Williams  xiv  1  INTRODUCTION  1.1  Introduction This year,  from c o r o n a r y disease  as  i n others before  heart disease  (CHD)  (1). I t i s f o r t h i s  underly  CHD.  known and  The  has  multifactorial  i s the  level  o f low  CHD,  factor  study nature  levels  o f LDL  report of the  Lipid  Research C l i n i c a g a i n s t CHD  importance of HDL-cholesterol question  predispose, The  and  ( 2 ) . Though f a c t o r s  (HDL)  the plasma  (4,5).  p r o t e c t , an  and  Goldstein  the  1985  cholesterol  the biochemical  i n CHD  individual  ( 6 ) . Thus,  hypercholesterolemia  (FH)  does  (7,8,9).  1  to been  recent the  the  LDL  from a t h e r o s c l e r o s i s ? "  m e t a b o l i s m and  70's  jointly  or Medicine,  cellular  major  made d u r i n g t h e mid  defect i n  levels  f u r t h e r e s t a b l i s h e d the  r e s e a r c h i s : "How  i n Physiology  has  confirmed  ( 7 ) . T h e i r work, f o r w h i c h t h e y  Nobel p r i z e  understanding  was  be  the  individuals  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 field  to  While  Program has and  as  i t s progression,  ( 4 , 5 ) . The  mechanisms w h i c h r e g u l a t e LDL  contribution to this  such  incidence  metabolism  in lipoprotein HDL  disorder i s well  cholesterol predisposes  p r o t e c t i v e a c t i o n o f HDL  central  of t h i s  cholesterol  t o CHD  and  effort,  o f t h e mechanisms w h i c h  correlated with  (LDL)  inversely related  single  ( 3 ) . S t u d i e s h a v e shown t h a t  plasma h i g h d e n s i t y l i p o p r o t e i n  shown t o be  die  i n plasma t h a t i s c o n s i d e r e d  is positively  density lipoprotein  increased  other  obesity contribute to  of c h o l e s t e r o l  the major a t h e r o g e n i c i n c i d e n c e o f CHD  and  f r o m any  t h a t s o much t i m e ,  r e c e n t l y been reviewed  smoking, h y p e r t e n s i o n it  than  reason  money have b e e n d e d i c a t e d t o t h e  i t , more C a n a d i a n s w i l l  was  by  Brown  received  centered  on  familial  Briefly,  Brown and  Goldstein  showed t h a t cells  i n normal  via a specific  receptor  i n d i v i d u a l s LDL  p l a s m a membrane r e c e p t o r ,  i s defective  in patients  plasma of these p a t i e n t s age.  The  of the  r e s u l t s of  r o l e of  The  i s cleared  and  w i t h FH.  l i p o p r o t e i n i n the  mechanism by  w h i c h HDL  protects  against  role  i n the  mobilization  in Section  studies  of  cholesterol  current  transport"  k n o w l e d g e o f HDL  hypertriglyceridemia,  following  categories  as  central its to  i n more  are  classified  2)  HDL  A-I/C-III  3)  Apo  4)  Fish-eye disease  5)  Familial hypoalphalipoproteinemia  6)  Tangier disease  deficiency i  l  a  them a r e  been advanced  pronounced  form o f  i n t o one  of  by  HDL the  (10):  n  o  deficiency.  with planar  atherosclerosis  xanthomas.  .  at  i n d i v i d u a l s w i t h apo early  a  absence of  familial  F a m i l i a l apo  a l l of  process.  and  i s discussed  1)  M  knowledge  process i s referred  i n the  present with a  These p a t i e n t s  A-I  the  early  i s not  plays  metabolism has  who,  (FED). (Hypoalpha).  (TD).  While a l l of these p a t i e n t s not  and  HDL  an  in  1.4.  i n a group of p a t i e n t s  deficiency.  at  CHD  by  this  our  from c e l l s  larger lipoproteins. This  "reverse cholesterol  The  CHD  atherosclerotic  u n d e r s t o o d . However, i t i s known t h a t  detail  that  accumulates  p r e d i s p o s e s them t o  well  as  and  t h i s work has ..greatly i n c r e a s e d  this  t r a n s f e r to the  LDL  from plasma  risk  present with deficiency  for atherosclerosis.  A-I/C-III deficiency t h o s e w i t h FED  2  and  o r Apo  While  M  l  a  n  o  HDL,  the  hypoalpha  A-I ^  of  do  develop not  suffer  from premature v a s c u l a r  w i t h HDL others HDL.  d e f i c i e n c y are  disease.  greatly predisposed  f u n c t i o n q u i t e w e l l w i t h o u t any  Therefore, 1)  The  i t i s of great  primary defect(s)  patients, 2)  The  Thus, w h i l e to heart  appreciable  i m p o r t a n c e t h a t we leading  some  patients  disease  quantities understand:  to HDL-deficiency  i n these  and  compensatory  (adaptive)  mechanisms w h i c h  prevent  d e v e l o p m e n t o f a t h e r o s c l e r o s i s i n some o f t h e s e While recent  work has  Familial  apo  (12)  associated  are  A-I/C-III  shown t h a t  deficiency  the  (11),  with abnormalities  precise molecular defects  HDL  and  apo  of the  apo  A-I  i n most o f t h e s e d i s o r d e r s  m e t a b o l i s m and  atherosclerosis i t i s essential that  be  the  past  3 years,  R e s e a r c h G r o u p has and  the  remain  r e l a t i o n s h i p between  familial  that  upon. TD  i s reviewed  been d e s c r i b e d significance  the  HDL-deficiency  i n TD  the  i s a rare disorder i n Section  in thirty  i n the  1.5.  three  research  HDL  primary disorders  u n d e r s t a n d i n g o f HDL  patient  Lipid  i n Canada  work i n t h i s  thesis i s  of plasma l i p i d  Though t h i s  with  transport  disorder  has  only  p a t i e n t s w o r l d wide i t s  to  hope t h a t  metabolism  only  whom t h e  i n u n d e r s t a n d i n g CHD  k n o w l e d g e o f HDL  In the  Shaughnessy H o s p i t a l  been s t u d y i n g  purpose of t h i s  defect  the  i t i s t h i s p a t i e n t on  centered  the  gene,  elucidated. For  TD  underlying  in  MjLxano  A-I  want t o u n d e r s t a n d t h e  defects  disorders.  deficiency  unknown. I f we  biochemical  of  i n v e s t i g a t e the  the  i n TD  metabolism  i s obviously  tremendous. I t i s biochemical  results will  contribute  patients  lead to  and  to  our  greater  i n normal i n d i v i d u a l s .  f o l l o w i n g pages I s h a l l 3  briefly  review the  current  knowledge o f  lipoprotein structure,  the  normal  i n d i v i d u a l and  and  i t s lipoprotein abnormalities.  possible defect(s)  then the  i n Tangier  f u n c t i o n and current  metabolism  literature  In c l o s i n g I w i l l  disease  and  the  on  in TD  to  discuss  o b j e c t i v e s of  the this  research. 1.2  Lipoproteins  1.2.1  Introduction Due  to the  water i s very difficult. lipids  hydrophobic nature of  low.  As  i n blood  has  b e e n d e v e l o p e d by  surrounding  hydrophilic  shell  and  (13).  are  The  (apo),  localized  lipid  and  range i n m o l e c u l a r on  hydrated  density,  particle  size,  surface  (PL),  with  can  be  phospholipid  called  the  classification  p r o p e r t i e s and  classified  on  the  lipoprotein particle  (TG),  various  free cholesterol  basis of  their  rate, electrophoretic mobility,  f u n c t i o n and  protein  b a s i s of t h e i r hydrated system t h e r e  metabolic  350  (CE).  floatation  metabolic  this  a  l i p o p r o t e i n s c o n s i s t of  triglyceride  is  i t i s known  weight from 7 t o  of the  in  of  accomplish  constituents.  l i p o p r o t e i n s a r e most commonly c l a s s i f i e d  m a j o r g r o u p s on this  the  cholesteryl ester  Currently,  protein constituents  components o f t h e  Lipoproteins  and  c o n s i s t i n g mainly of c h o l e s t e r o l ,  amounts o f p h o s p h o l i p i d (FC)  vertebrates  m i c e l l e s of neutral l i p i d  p r o t e i n m o l e c u l e s . The  i n blood  solubility  as p l a s m a l i p o p r o t e i n s . L i p o p r o t e i n s  apolipoproteins  solubility  a consequence, t h e i r t r a n s p o r t  f u n c t i o n by  kDa  their  However, a s y s t e m f o r i n c r e a s i n g t h e  collectively  and  lipids,  i s an  density  overlap  of  into (Table  I ) . In  physical  function; Chylomicrons carry  4  four  dietary  TABLE 1  Lipoprotein class  Chylomicrons  CLASSIFICATION OF LIPOPROTEINS IN THEIR HYDRATED ( r e p r o d u c e d f r o m r e f 13)  Density (gm/ml)  <0.94  STATE  Diameter (nm)  Mean molecular weight  Electrophoretic mobility  10 -10  5.0X10  origan  2  3  9  VLDL  0.94-1.006  30-70  7.5X10  6  LDL  1.006-1.063  15-25  2.5X10  6  beta  pre-beta  HDL  1.063-1.125  6-14  3.9X10  5  2  alpha  HDL  1.125-1.21  6-10  1.9X10  5  3  alpha  TG's  from t h e  enterocytes  or storage;  very  low  synthesized  i n the  major c a r r i e r s The  their be  density  liver;  f r o m VLDL c a t a b o l i s m the  to extrahepatic  the  high  be  heterogeneity  those separated  a d d i t i o n , work by  l i p o p r o t e i n s (LDL)  density  derived  l i p o p r o t e i n s (HDL)  o f p l a s m a l i p o p r o t e i n s on  f l o a t a t i o n d e n s i t y may  method and  density  TG's  inappropriate  other  methods  Scanu e t a l have c l e a r l y  basis  as t h e r e  between p a r t i c l e s  by  the  separated  (14,15,16).  by  that rigorous  are  universally applicable  with  l i p o p r o t e i n s of  the  not  i n d i v i d u a l sera  d e v e l o p m e n t o f new  (17).  and  surrounding  l i p o p r o t e i n nomenclature-will  at present  1.2.2  is still  the  accepted  and to  that  confusion  resolved. standard  However in  research.  Apolipoproteins The  distribution  w e l l known and and  system  be  are  isolating,  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  this  lipoprotein  I t i s hoped  methodologies f o r  separating,  this  shown t h a t t h e r e  r a n g e s among s u b j e c t s  limits  to  In  density  density  of  appears  important d i f f e r e n c e s i n the  define  are  of plasma c h o l e s t e r o l .  classification  considerable  utilization  l i p o p r o t e i n s (VLDL) c o n t a i n  low  and  tissue for  has  Smith e t a l  molecular feature  of the  apolipoproteins  r e c e n t l y b e e n r e v i e w e d by  (19).  Table  weights of the  II presents  of a l l a p o l i p o p r o t e i n s  s t r u c t u r e of the  b o t h aqueous and  lipid  is  Morriset  (18)  is their  The  and  outstanding  amphipathic nature;  hydrophobic regions  molecules allows  them t o  environments simultaneously.  6  et a l  distribution  major a p o l i p o p r o t e i n s .  p r e s e n c e o f b o t h h y d r o p h i l i c and tertiary  the  i n human p l a s m a  in  the  the  i n t e r a c t with A number  of  TABLE I I  APOLIPOPROTEINS OF THE HUMAN PLASMA ( R e p r o d u c e d from r e f . 9)  Apoliporoteins  M o l e c u l a r weight (dalton)  LIPOPROTEINS  Lipoprotein distribution  Apo A - I  28,331  HDL  Apo A - I I  17,380  HDL  Apo B-48  200,000  Chylomicrons  Apo B-100  350,000  VLDL,  LDL  Apo C-I  7,000  HDL,  Apo C - I I  8,837  Chylomicrons,  VLDL,  HDL  Apo C - I I I  8,751  Chylomicrons,  VLDL,  HDL  VLDL,  HDL  VLDL  Apo D  32,500  HDL  Apo E  34,145  Chylomicrons,  7  specific These  functions  f o r the  apolipoproteins  have been  identified.  include: 1)  Cofactor (apo  f o r enzymes w h i c h m e t a b o l i z e  C-II,  l i p o p r o t e i n l i p a s e ; apo  cholesterol 2)  Ligand (apo apo  3)  HDL  Structural B-48  lecithin  acyltransferase).  B-100, LDL  (apo  A-I,  for interaction with c e l l u l a r  A-I,  lipoproteins,  receptor;  apo  E,  receptors,  remnant  receptor;  receptor).  apolipoprotein and  apo  for lipoprotein  B-100, c h y l o m i c r o n s and  particle, VLDL; apo  A-I,  HDL) . 4)  Lipid,transfer/exchange (apo  The  D).  p r i m a r y amino a c i d s e q u e n c e s h a v e b e e n d e t e r m i n e d  most o f t h e s e p r o t e i n s .„.,,, A - I I ,  activity,  and  apo  C-II  are  (20).  Three a p o l i p o p r o t e i n s ,  synthesized  as  undergoes p o s t - t r a n s l a t i o n a l cleavage.  (22) In  and  apo  addition  C-II  (23)  or  a c y l groups  The  (27), apo  apo  B-100  E  (28),  (31).  (21),  while  addition  of  apo  A-II  f o r apo  i n the  apolipoproteins  The  A-I  plasma.  may  also  be  carbohydrates  (24).  genomic s t r u c t u r e s  have been r e p o r t e d  the  apo  propeptide  case of  cleavage occurs p r i m a r i l y  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 (20)  In the  intracellularly  to p r o t e o l y t i c cleavage,  A-I,  preproapolipoproteins.  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  cleavage occurs p r i m a r i l y  apo  for  of  including apo  C-II  a number o f  those of  (29),  I t appears t h a t  apo  apo  these A-I  C-III  a number o f  genes have r e m a r k a b l y s i m i l a r s t r u c t u r e s 8  and  apolipoproteins  ( 2 5 , 2 6 ) , apo  (30) the the  and  most  A-II recently  apolipoprotein observed  similarity  i n the  structural organization  t h e s e genes have prompted t h e evolutionary 1.2.3  related  of  spin  None o f  the  detailed level this  the  information  provide  of  protein, of  the PL  electron  properties  using  a  variety  spectroscopy, microscopy,  of the  from t h e s e s t u d i e s  plasma l i p o p r o t e i n s a t  few.  permits a  a  molecular  i t has  models which  been p o s s i b l e  to construct  molecular organization  plasma l i p o p r o t e i n s localization  FC  (Figure  lipoprotein structure  1.).  o f TG  the  For  of  the  (17,19). A g e n e r a l and  CE  i n the  p a r t i c l e w h i c h i s t h a n s u r r o u n d e d by and  and  However,  the  in  are  dynamic n a t u r e o f t h e s e p a r t i c l e .  a l l models i s the  core of  gathered  i n s i g h t i n t o the  components o f  structure,  nuclear magnetic resonance  information  to  exons  c r o s s - l i n k i n g experiments to mention a  description  due  they  b e e n a c c o m p l i s h e d by  resonance spectroscopy,  i m m u n o c h e m i s t r y and  do  composition,  i n t a c t l i p o p r o t e i n s has  electron  that  and  Structure  of the  o f methods i n c l u d i n g  speculation  introns  (27).  Lipoprotein Investigation  of  from  main feature  hydrophobic  a monolayer  a more c o m p r e h e n s i v e  reader i s referred to  of  review  Smith et  al  (19). 1.3  Lipoprotein  1.3.1  Metabolism  Introduction Cholesterol  i s synthesized  t h r o u g h a pathway i n v o l v i n g there i s a  derived  i n excess of  f l u x of approximately  body. D i e t a r y is  from 2 c a r b o n  sources account  2500 mg  9  units  30  enzymes  of  cholesterol  f o r a b o u t 20%  f r o m endogenous s y n t h e s i s  (acetate)  (33).  (32).  w h i l e the Cholesterol  Daily,  into  the  remainder is  Figure  1.  Structure of Lipoproteins  M o d e l o f human LDL s h o w i n g  the d i s t r i b u t i o n  p r o t e i n on t h e s u r f a c e o f t h e p a r t i c l e of the p a r t i c l e ,  [reprinted  from r e f  10  and TG  (179)].  o f PL,  and CE  FC,  i n the  and core  utilized  f o r a number o f d i v e r s e  1) As a s t r u c t u r a l  component o f t h e p l a s m a membranes, where i t  modulates f l u i d i t y cell  and m a i n t a i n s  2)  Precursor  forbile  3)  Precursor  f o rsteroid  hormones.  4)  Precursor  f o rvitamin  D.  excreted  the barrier  acids.  i s n o t b r o k e n down i n t h e b o d y b u t r a t h e r i s  i n t h e f e c e s as b i l e  cholesterol  into  Current  understanding  cholesterol  salts.  t h e body i s b a l a n c e d  F i g u r e 2. S a n c t a  Thus t h e i n f l u x o f by a p r o p o r t i o n a t e  o f l i p o p r o t e i n metabolism  simplicitas  two s e p a r a t e  m e t a b o l i s m c a n be i d e n t i f i e d .  pathway) a n d t h e s e c o n d d e a l s w i t h synthesized  i n t h e body  independently that  The f i r s t  deals  with  (exogenous  the metabolism o f c h o l e s t e r o l  of individual  two p a t h w a y s . T h e b i o g e n e s i s  lipoproteins will  be examined  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 t h e d i s c u s s i o n  follows.  1.3.2  Lipoprotein Chylomicrons:  large  i s outlined  (endogenous, p a t h w a y ) . T h e r e 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 t h e s e s catabolism  efflux.  pathways o f  t h e m e t a b o l i s m o f c h o l e s t e r o l d e r i v e d from t h e d i e t  and  between t h e  and i t s e n v i r o n m e n t .  Cholesterol  in  purposes:  intestine,  subsequently enterocytes  A f t e r being  e m u l s i f i e d by b i l e  then  salts  i nthe  d i e t a r y f a t i s a d s o r b e d , b r o k e n down a n d  r e s y n t h e s i z e d by t h e i n t e s t i n a l  chylomicrons, finally  Biogenesis  e p i t h e l i u m . The  s e c r e t e t h e TG i n t o t h e c i r c u l a t i o n a s  which a r e than  c l e a r e d by t h e l i v e r  c a t a b o l i z e d t o remnant p a r t i c l e s and a s o u t l i n e d i n F i g u r e 2.  11  Exogenous Pathway  Endogenous Pathway  . Dietary Cholesterol  LDL Receptor  ~  frrrnrrnr  '  Extrahepatic Tissues  Lipoprotein Lipase  Lipoprotein Lipase  F i g u r e 2.  LCAT  S e p e r a t e pathways f o r r e c e p t o r - m e d i a t e d m e t a b o l i s m o f lipoprotein  carrying  cholesterol  [reprinted  12  endogenous and exogenous from r e f ( 7 ) ] .  E l e c t r o n m i c r o s c o p i c , and elucidated  t h e pathway o f c h y l o m i c r o n  the enterocyte reticulum  nascent  thought  containing  chylomicrons  that  essential c a n be  the l i p i d  smooth  and  droplets,  the c e l l  travel  a r e then r e l e a s e d from  secretory vesicles  that travel  travel  into the  the G o l g i  the lymphatics the l e v e l apo it  Cs,  of the jugular vein apo  E and  Vesicles,  nascent  apparatus  in  surface of the  cell  r e l e a s e the  space.  The  chylomicrons  of the m i c r o v i l l i ,  that  (9). Chylomicrons  t h e s m a l l e r form o n l y apo  to i t s secretion  chylomicrons  TG-rich  flow  through  and v i a t h e t h o r a c i c d u c t e n t e r t h e c i r c u l a t i o n  i s believed  prior  lacteals  B-48  i n lymph  i s a direct  B  apparatus  ( 9 ) . The  to the l a t e r a l  intercellular  into the central  (36,37).  to the G o l g i  where they, f u s e w i t h t h e plasmalemma and chylomicrons  newly  f o r the assembly o f nascent  s e c r e t e d by  in  a p o l i p o p r o t e i n s ( 3 5 ) . Apo  where t h e a p o l i p o p r o t e i n s a r e g l y c o s y l a t e d chylomicrons  secretion  endoplasmic  a r e f o r m e d when t h e  a r e c o a t e d w i t h PL  t o be  lipoprotein  a s s e m b l y and  ( 3 4 ) . I n t h e lumen o f t h e  s y n t h e s i z e d TG is  b i o c h e m i c a l s t u d i e s have  o f apo  B  c o n t a i n apo  at  A's;  (apo B - 4 8 ) . However,  i s a s s o c i a t e d with the (9). O v e r a l l ,  particle  the secretion  of  function of the rate of f a t absorption  (38) . Very site  Low  D e n s i t y L i p o p r o t e i n s (VLDL):  for secretion  derived  lipids.  TG  a s VLDL p a r t i c l e s ,  of lipoproteins i s s e c r e t e d by  containing the l i v e r  which a r e formed  inside  manner t o c h y l o m i c r o n s . VLDL c o n t a i n apo B  4 8  The  B  liver  major  endogenously  i n t o the the c e l l  1 Q 0  i s the  circulation in a  similar  which d i f f e r s  a s s o c i a t e d w i t h c h y l o m i c r o n s . As w i t h c h y l o m i c r o n s ,  from  VLDL's  a l s o c o n t a i n a s m a l l amount o f non-apo B a p o l i p o p r o t e i n s . Of  these 100,  protein, to  level the  be  of  result  the  the  and  the  extent  the  triglyceride LDL  action  secreted  by  High  the  that liver  production  1)  direct from  (LDL):  by  precursors  Plasma  LDL  at  the  is  mainly  glucose are  are  from  utilized  the  loss  loss  of  that  of apo  some L D L  The  TG  and  E  (41).  may  primarily  hepatic  (9).  in vivo  by  formation  PL  and,  In  be  (HDL):  The  plasma  (43).  The  genesis  particles of  three  HDL  precursors,  and  The  addition,  directly  HDL  of  are  of  defined  production  separate  secretion of  hepatic  or  l i p o p r o t e i n l i p a s e and  apoprotein/PL/FC.  involves  acids  B-  (40).  Lipoproteins  involves  of  these  synthesis  with  (42).  group  precursors  needs  along  VLDL p a r t i c l e s  fatty  VLDL p a r t i c l e s  heterogenous  complexes  which  shown,  i n VLDL  uptake  suggests  of  been  nascent  i s accomplished  Density  the  of  to  critically,  evidence  have  Variation  of  l i p a s e on  most  the  rate  own  C s  Lipoproteins  from VLDL  perhaps recent  (39).  the  by  apo  with  of  density  produced  and  body  hepatocytes  Low  of  E  associated  Golgi  the. b l o o d for  apo  a  mature  as  HDL  being  these  HDL  processes:  d i s c o i d a l high  intestinal  cells  density  structures  ("nascent"  HDL  particles). 2)  3) The and  lipid  and  protein  constituents  TG-rich  lipoproteins  Direct  association of  "nascent"  intestinal  precursors  HDL  lymph>  ("surface PL  structures were  (43) . H o w e v e r ,  with  free  lipolyzed  apolipoprotein  thought  experiments  14  from  remnants").  identified  initially the  released  A-I.  in  liver  perfusates  to  be  major  upon  the  which  this  HDL  hypothesis  i s b a s e d h a v e come u n d e r s h a r p c r i t i c i s m  inability  to identify  and  the fact  the  perfusion  that  that  such s t r u c t u r e s  LpL i s h i g h l y  experiments has l e a d t o support  o r i n t e s t i n e show t h a t  lipolysis  of the TG-rich  currently believed surface  coat  that  o f them b e i n g  LCAT r e a c t i o n  (43,44).  t o s p h e r i c a l HDL i s d e p e n d e n t  discoidal  in vitro  (45).  I n p a t i e n t s w i t h LCAT d e f i c i e n c y ,  accumulate  HDL i s a b s e n t , l a r g e amounts o f  i n the patients  M a t u r e HDL e x i s t s i n p l a s m a a s t h r e e HDL^, H D L ,  and  populations  present  HDL . HDL 3  The c o r e than t h a t and  2)  and H D L  o f HDL  TG t h a n  3  distinct  populations;  a r e t h e m a j o r HDL  as f o l l o w s :  d i a m e t e r o f HDL  The s u r f a c e the  2  plasma ( 4 6 ) .  i n t h e p l a s m a o f man. T h e main f e a t u r e s o f  two a r e s u m m a r i z e d 1)  precursors  c a n be t r a n s f o r m e d t o s p h e r i c a l p a r t i c l e s by t h e  structures  2  cholesterol  ( L C A T ) . H a m i l t o n h a s shown t h a t HDL  where t h e m a t u r a t i o n o f p r e c u r s o r  the  o r i g i n a t i n g from t h e  t h e a c t i o n o f t h e p l a s m a enzyme; l e c i t h i n :  in v i v o  Thus i t i s  l i p o p r o t e i n s a r e t h e major, i f  M a t u r a t i o n o f HDL p r e c u r s o r s  formed  from  (43).  d i s c o i d a l precursors  s o u r c e o f HDL p r e c u r s o r s  acyltransferase  composition of  derived  lipoprotein i n situ  of l i p o l y z e d TG-rich  the only,  products of  o f t h e d i s c o i d a l p a r t i c l e s from  the apolipoprotein  these p a r t i c l e s i s supportive  pathways  f o rthe hypothesis  t h e " n a s c e n t " HDL p a r t i c l e s a r e t h e l i p o l y t i c  liver  on  secretory  a c t i v e under t h e c o n d i t i o n s o f  VLDL o r c h y l o m i c r o n s . A n a l y s i s  not  along  (43) . The  3  2  i s approximately  and t h u s c o n t a i n s  50% l a r g e r  3-4-fold  more CE  HDL . 3  area  o f HDL  2  i s 2-fold greater  difference i n protein content 15  t h a n HDL  i s a b o u t 50%.  3  and  HDL-^  i s l a r g e r than HDL  only  small  and  and  amounts i n t h e  quantities described  2  in rats i n the  (43) . A p o s s i b l e  as  i n apo  p l a s m a o f man  plasma of  i s referred to  is rich  HDL -  Recent evidence  C  section.  1.3.3  Lipoprotein  1.3.3.1  E x c h a n g e and  The  transfer  of  p r o c e s s has by  the  in vivo  Transfer  as  a result  LCAT i n t h e  plasma  of  initial  i s associated  suggested that transfer  of  CE  presence of  that  in  a specific  might e x i s t  the  that  LDL's  of  (50)  1)  CE-TG t r a n s f e r p r o t e i n  (CETP/LTP  2)  CE-PL t r a n s f e r p r o t e i n  (LTP  3)  Apo  this  CE This  for  i n plasma.  Evidence reported in  Since then a  w h i c h h a v e shown t h a t  such p r o t e i n s  net  formed  (48).  (LTP)  (49).  in  CE  transfer protein  of  by  Interest  al  animals  i s catalyzed  continually  when Z i l v e r s m i t e t  lipid  have been p u b l i s h e d  is  major p o r t i o n  w i t h the  plasma of h y p e r c h o l e s t e r o l e m i a  The  cells.  a s p e c i f i c mechanism  the  CE  cholesterol  a d y n a m i c e x c h a n g e and  HDL's t h e  s u c h a p r o c e s s came i n 1975  of  been  Reactions  plasma l i p o p r o t e i n s  for  transfer  discussed  observation  of  in  large  mechanisms  stemmed f r o m t h e  interlipoprotein  reports  The  and  i n the  observation  be  has  indicates  l i p i d s between p a r t i c l e s  action  present  (47).  rich  in  in  Catabolism  composition of the  being modified  i s present  i s present  animals fed d i e t s  f o r these conversions w i l l  following  but  HDL-^  a n a l o g u e o f HDL-^  c o n v e r s i o n between s u b s p e c i e s o c c u r s responsible  E.  the number the  as:  1).  2).  D.  e x a c t mechanism o f  transfer  16  i s u n c l e a r but  i t appears  t h a t TG and CE compete w i t h e a c h o t 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  fashion  LTP•s i n a c o n c e n t r a t i o n  dependent  (51).  The r e a c t i o n  is  b i - d i r e c t i o n a l and seems t o be b a s e d on t h e e x c h a n g e o f CE f o r  TG  (51,52,53) o r PL (53,54,55) b e t w e e n l i p o p r o t e i n p a r t i c l e s . The  lipid  transfer proteins  conversion  o f HDL  appear t o be r e s p o n s i b l e  t o HDL-^ (47) .  2  Enzymatic  1 . 3 . 3 . 2  Modification  Enzymes, w h i c h m o d i f y p l a s m a l i p i d s interconversion  for the  of circulating  play  a key r o l e i n t h e  l i p o p r o t e i n s . T h r e e o f t h e most  i m p o r t a n t o f t h e s e enzymes a r e : 1)  Lecithin:cholesterol acyltransferase  2)  Lipoprotein  3)  Hepatic t r i g l y c e r i d e  l i p a s e (LpL;  (LCAT; EC 2 . 3 . 1 . 4 3 ) .  EC 3 . 1 . 1 . 3 4 ) .  lipase  (HTGL).  .. LCAT i s an e x t r a c e l l u l a r enzyme o f h e p a t i c circulates fatty  a c i d from p h o s p h a t i d y l c h o l i n e  cholesterol Albers kDa  i n t h e plasma and c a t a l y z e s  (56).  origin  that  t h e t r a n s f e r o f t h e C-2  t o t h e 3•-hydroxy1 group o f  Human LCAT was f i r s t  purified  t o h o m o g e n e i t y by  e t a l (57) a n d h a s b e e n shown t o h a v e a n a p p a r e n t M  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  (SDS-PAGE: 57) o r 59 kDa b y s e d i m e n t a t i o n ultracentrifugation  (58). A g r e a t  1  deal  o f 65  r  g e l electrophoresis equilibrium  on t h e b i o c h e m i s t r y  and  p a t h o p h y s i o l o g y o f t h e LCAT r e a c t i o n i s now a v a i l a b l e w h i c h clearly  indicates the central role that  of c h o l e s t e r o l m o b i l i z a t i o n It  i s generally  from t i s s u e  accepted that  form t h e 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  LCAT p l a y s (56,59).  LCAT, i n a s s o c i a t i o n w i t h HDL, complex  Though apo A - I i s t h e most common c o f a c t o r  17  i n the process  i n plasma ( 6 0 ) . f o r t h e LCAT r e a c t i o n  (61)  other  l i p o p r o t e i n s s u c h as  a l s o a c t as not  fully  c o f a c t o r s . The  understood  investigations  and  (64).  that the  HDL  a small  thus  acceptor  i s the  150-fold  subpopulation (52)  (62)  and  that  exact  e l u c i d a t e d and  the  has  cell  f o r the  enzyme t h a t  endothelial c e l l  triacylglycerides specific  (66)  and  associated  However, t h e  mechanism by  a specific  heparin  receptor  i s released  number o f s o u r c e s  there  e x i s t between t h e  (66)  by  and  i n t o the  are  apo  w h i c h apo  few  plasma. LpL  other  (9).  to  be  our  LCAT r e a c t i o n  is  (47).  2  luminal  surface  hydrolysis  C-II  C-II  as  of  of d i -  VLDL. L p L  a  and is -  cofactor.  promotes  this  e n d o t h e l i a l membrane  upon i n t r a v e n o u s  i s a glycoprotein  major  absolute  i s anchored to the  has  i n j e c t i o n of  been i s o l a t e d from  rat heart  and  with s i m i l a r carbohydrate  similarities  h e p a t o c y t e s and  cellular  and  lipolytic  many d i f f e r e n c e s  enzyme t h a t  i s rapidly released 18  a  rat  enzymes f r o m d i f f e r e n t t i s s u e s .  HTGL i s a n o t h e r e x t r a secreted  no  including bovine milk,  a d i p o s e t i s s u e . LpL content but  has  enzyme r e q u i r e s  i s unknown. LpL  the the  contain  the  yet  w i t h c h y l o m i c r o n s and  This  by  on  catalyses  f o r primary e s t e r s but  exact  the  of  speculation  membranes  t o HDL  3  i s located  stereospecificity.  reaction  amount  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  o f HDL  may  LCAT  these p a r t i c l e s are  responsible  the  number o f  particles exist that  Recent evidence i n d i c a t e s t h a t  i s an  (63)  numerous  lead to the  laboratory.  . LpL  of  n a t u r e o f t h e s e p a r t i c l e s has  conversion  E  LCAT/HDL i n t e r a c t i o n i s  o f c h o l e s t e r o l from p e r i p h e r a l  However, t h e  apo  l e s s than the  This  o f HDL  and  subject  In normal plasma,  p a r t i c l e s (65).  LCAT m o l e c u l e s  C-I  nature of the  molecules i s approximately circulating  apo  i n t o the  is liver  perfusate  upon t h e  a d d i t i o n of heparin  ( 6 6 ) . T h i s enzyme d o e s n o t lipolytic  activity  immunologically  r e q u i r e any  against TG-rich  distinct  i n t o the  apolipoprotein for  l i p o p r o t e i n s and  f r o m LpL.  Recently,  addition has  LpL  a c t i o n on VLDL, c o n v e r t i n g  t o HTGL's r o l e  i n the  b e e n shown t h a t HTGL p l a y s  c o n v e r t i n g HDL through t h e i r roles  i n the  2  t o HDL  3  concerted  by  catabolism  degradation,  formation  the  of TG-rich i n HDL  lipoprotein  a c t i o n , LCAT, L p L and  (67)  has  remnants  them t o LDL.  a central role  hydrolyzing  is  Brown  s u g g e s t e d 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 p r o d u c e d by  p e r f u s i o n medium  and  In  particles i t metabolism;  PL  (47).  HTGL p l a y  remodeling of  a  Thus  key  lipoprotein  particles. Cell-Lipoprotein interactions  1.3.3.3  Lipoproteins receptors  interact with  or v i a a non-receptor  receptor processes  cell  by  bulk  e i t h e r through  mediated processes  i t would appear t h a t  phase e n d o c y t o s i s  (68). C u r r e n t l y , the  o f t h i s pathway t o t h e  clearance  lipoproteins  i s unknown. S t u d i e s  LDL  to two-thirds  receptor  observation, process  o f LDL  ( 6 9 ) . The and  ( 9 ) . The  non^  clearance  on  o f most  catabolism  suggest  et a l support  f u r t h e r suggest t h a t the  c o n t r i b u t e s t o l e s s t h a n 33%  19  exact  i s mediated through the  f i n d i n g s of Dietschy  non-receptor  o f LDL  been  lipoproteins enter  contribution  up  specific  o f c e l l / l i p o p r o t e i n metabolism have not  w e l l c h a r a c t e r i z e d but the  cells  clearance  that LDL  this mediated (70).  In the f o l l o w i n g s e c t i o n , interaction will that  r e c e p t o r mediated  be d i s c u s s e d . The c r i t e r i a  a lipoprotein  cell-lipoprotein  used  i s i n t e r a c t i n g with a c e l l  for establishing  r e c e p t o r i s as  follows (71): 1) The r e c e p t o r s h o u l d d e m o n s t r a t e 2) The l i g a n d binding  specificity.  should bind t o a f i n i t e  number o f s p e c i f i c  sites.  3) The l i g a n d  s h o u l d have a v e r y h i g h a f f i n i t y  receptor  (Values f o r t h e e q u i l i b r i u m  constant  (K^) o f 1 0 "  9  to 10~  1 0  for their  dissociation  M a r e common).  4) The b i n d i n g o f t h e l i g a n d t o t h e r e c e p t o r s h o u l d a p h y s i o l o g i c event, by t h e l i g a n d biological  a n d t h e number o f r e c e p t o r s  s h o u l d be r e l a t e d  mediate occupied  t o t h e magnitude o f t h e  effect.  5) The number o f r e c e p t o r s i s n o t c o n s t a n t  a n d c a n be  r e g u l a t e d by t h e a b s e n c e o r p r e s e n c e o f t h e l i g a n d . Chylomicrons:  Catabolism  o f chylomicrons  t h e p r o d u c t i o n o f remnant p a r t i c l e s c a t a b o l i z e d by t h e a c t i o n  o f HTGL  by LpL r e s u l t s i n  t h a t may t h e n  ( 9 ) . The r e s u l t i n g  r e m n a n t s c o n t a i n apo B-48 a n d apo E, b u t h a v e l o s t portion  of t h e i r  be  further chylomicron  t h e major  apo C p r o t e i n s . T h e c l e a r a n c e o f r e m n a n t  p a r t i c l e s by t h e l i v e r  i s mediated by a s p e c i f i c ,  receptor  e t al h a v e r e p o r t e d t h a t t h e apo E  (71).Windier  chylomicron  remnant i n t e r a c t s w i t h  (41). T h i s p o s s i b i l i t y  a specific  i s f u r t h e r supported  high  affinity  hepatic receptor  by t h e o b s e r v a t i o n s  that: 1) apo B-48 d o e s n o t i n t e r a c t w i t h t h i s  20  of the  r e c e p t o r and  2)  that p a t i e n t s with defective ability  m u t a n t p h e n o t y p e s o f apo to clear, chylomicrons  E have  a  from t h e i r  plasma  (71). The be  expression regulated  cholesterol  of t h i s  and  r e c e p t o r by  the  liver  thus importance of t h i s  homeostasis i s obscure  receptor  fate  their  Cs  apo  (72). and  VLDL remnant p a r t i c l e i n t e r a c t i o n with distinct  from the  apo  i s known a s t h e  receptor pool has  E o r apo  only  i s not  remnant p a r t i c l e s  are  apo  other  t h i s pathway i n VLDL r e m o v a l a patient with  resulting  receptor  that i s  (72) . T h i s  the hepatic  apo  B,E  i s the major c a t a b o l i c LDL  receptor  can  LDL  E t r a n s f e r s from the  HDL  after  by  the  the  significant the  liver  lipolysis from q u i c k l y  circulation. B,E  receptor  mechanisms f o r c l e a r i n g VLDL (68). There  remnant r e c e p t o r may  o f VLDL r e m n a n t s b u t  of  through  l a c k a f u n c t i o n a l apo  from c i r c u l a t i o n  chylomicron  The  recognized  VLDL's f r o m t h e  i n p a t i e n t s who  and  B of,VLDL remnant p a r t i c l e s ,  (72) . T h i s mechanism p r e v e n t s  have shown t h a t t h e r e  clearance  receptor,  l a r g e r VLDL p a r t i c l e s  removing newly s y n t h e s i z e d  that the  liver  (72) . W h i l e t h e  t o l a r g e r VLDL p a r t i c l e s  Studies  the  affinity  (72) . I t a p p e a r s t h a t t h e  occurred  lipolysis.  r e f e r r e d t o as LDL  to  liver  l o s e the m a j o r i t y  remnant r e c e p t o r  initially  e i t h e r apo  B of the  high  chylomicron  pathway f o r VLDL e n d o c y t o s i s recognize  E during  a r e c l e a r e d by  a specific,  r e c e p t o r , w h i c h was r e c e p t o r but  apo  to  composition  L a r g e VLDL p a r t i c l e s  p i c k up  appear  (9).  VLDL: VLDL a r e h e t e r o g e n o u s i n s i z e , metabolic  does not  at present  be the  i s some  evidence  involved in  the  s i g n i f i c a n c e of  i s unknown. L a r g e VLDL i s o l a t e d  h y p e r t r i g l y c e r i d e m i a h a v e b e e n shown t o 21  from  interact  with  a specific  significance yet  B-VLDL r e c e p t o r p r e s e n t  of this  been f u l l y  receptor  i n normolipemic  ( 7 0 ) . The  i n d i v i d u a l s has not  e s t a b l i s h e d a s l a r g e VLDL f r o m n o r m a l  do n o t i n t e r a c t w i t h  individuals  the receptor.  LDL: The LDL r e c e p t o r approximately  on m a c r o p h a g e s  is a cell  surface glycoprotein of  120 kDa w h i c h c y c l e s b e t w e e n t h e c e l l - s u r f a c e  the  endocytic vesicles  the  r o u g h e n d o p l a s m i c r e t i c u l u m a n d a p p e a r s on t h e s u r f a c e o f  cell to,  approximately and g a t h e r s  coated  pits  protein serves  45 min l a t e r  in, specific  the  pits  membrane  t h e membrane and  ( 7 ) . LDL b i n d s  than  fuses with  o f t h e b o u n d LDL.  A s t h e pH o f t h e endosome b e g i n s  i n a segment o f t h e endosome.  c o m p l e t e d when t h i s  This  to f a l l  constituents are  fuses with  hydrolyzed.  22  6.5  The LDL r e c e p t o r c y c l e i s  segment o f membrane p i n c h e s  endosome t h a n  below  receptors  o f f from t h e  endosome and r e t u r n s t h e r e c e p t o r s t o t h e c e l l - s u r f a c e remaining  to the  other v e s i c l e s g i v i n g r i s e to  LDL d i s s o c i a t e s f r o m t h e r e c e p t o r s and t h e LDL  cluster  called  a response t h a t leads t o i n v a g i n a t i o n of  p i t and i n t e r n a l i z a t i o n  endosome.  migrates  h a s shown t h a t a  associated with  a s an a n c h o r f o r t h e LDL r e c e p t o r s  endocytic v e s i c l e an  domains o f t h e c e l l  (clatherin) i s closely  coated  i s synthesized i n  ( 7 ) . The r e c e p t o r t h e n  (7). A n a l y s i s of the coated  r e c e p t o r and t r i g g e r s the  ( 7 ) . The LDL r e c e p t o r  and  a primary  ( 7 ) . The  l y s o s o m e and t h e LDL  Figure  3.  Route o f LDL r e c e p t o r  The LDL r e c e p t o r travels to the Golgi  begins i t ' s cycle complex,  and b a c k t o t h e s u r f a c e . methylglutaryl  HMG  cell  cells  i n t h e ER f r o m w h i c h i t  surface,  c o a t e d p i t , endosome,  CoA r e d u c t a s e d e n o t e s  3-hydroxy-3-  CoA r e d u c t a s e ; ACAT d e n o t e s a c y l - C o A : c h o l e s t e r o l  acyltransferase. regulatory  i n mammalian  V e r t i c a l arrows i n d i c a t e t h e d i r e c t i o n o f  affects. [reprinted  from ( 7 ) ]  The cell's  LDL-derived  cholesterol  cholesterol  coordinately  regulates  the  c o n t e n t by:  1) I n h i b i t i n g c e l l u l a r  cholesterol  synthesis  at  several  levels: i)  S u p p r e s s i o n o f t r a n s c r i p t i o n o f HMG  CoA  reductase  gene. ii)  Acceleration  2) A c t i v a t i o n  o f t h e d e g r a d a t i o n o f t h e a b o v e enzyme.  of a cholesterol  cholesterol  e s t e r i f y i n g enzyme, a c y l  acyltransferase  (ACAT).  3) S u p p r e s s i o n o f t h e s y n t h e s i s  o f t h e LDL r e c e p t o r b y  l o w e r i n g c e l l u l a r m e s s e n g e r RNA The of  r o u t e o f t h e LDL r e c e p t o r LDL-derived  cholesterol  h o m e o s t a s i s i s summarized HDL:  levels.  i n mammalian c e l l s  on i n t r a c e l l u l a r i n Figure  and t h e e f f e c t  cholesterol  3.  The c o m p l e x i t y o f l i p o p r o t e i n m e t a b o l i s m i s  exemplified  by t h e i n v e s t i g a t i o n  of i n vivo  U n l i k e LDL, w h i c h i s c a t a b o l i z e d  as a u n i t ,  a p p e a r t o be c a t a b o l i z e d following  CoA:  observations  cell-HDL'interaction. HDL  constituents  i n d e p e n d e n t l y as i n d i c a t e d  in rat  1) HDL-FC i s c l e a r e d  by t h e  (43):  more r a p i d l y  from c i r c u l a t i o n t h a n i s  HDL-CE. 2) The k i d n e y the  liver  i s the major s i t e i s the major s i t e  3) HDL a p o l i p o p r o t e i n s same  o f apo A - I r e m o v a l o f HDL-CE  while  removal.  a r e n o t removed f r o m p l a s m a a t t h e  rate.  4) The r a t i o o f u p t a k e o f H D L - C E / H D L - p r o t e i n v a r i e s  greatly  i n t h e body. The  study of i n vivo  cell-HDL interactions 24  i s further  complicated  by t h e f a c t can  that  be a c q u i r e d Studies  b o t h t h e p r o t e i n and l i p i d  components  from o r t r a n s f e r r e d t o o t h e r  on t h e i n t e r a c t i o n o f HDL w i t h  o f HDL's  lipoprotein classes. cultured  cells in  v i t r o h a v e b e e n e a s i e r t o i n t e r p r e t . HDL h a s b e e n shown t o remove cholesterol  from c e l l s  invitro.  I t has been s u g g e s t e d t h a t t h e  removal o f c h o l e s t e r o l from c e l l interaction sites  o f HDL w i t h a s p e c i f i c  on t h e c e l l  observation increases  may b e m e d i a t e d b y t h e  that  their  surface  class of high  (73). This  suggestion  affinity  i s b a s e d on t h e  c h o l e s t e r o l enrichment o f c u l t u r e d ability  to bind  binding  HDL (74) . However,  cells i t i s  i m p o r t a n t t o e m p h a s i z e t h a t w h i l e LDL i s i n t e r n a l i z e d b y c e l l s after binding cells  t o t h e LDL r e c e p t o r  examined  HDL i s n o t i n t e r n a l i z e d b y most  (74,75,76) w i t h t h e e x c e p t i o n  monocytes  (77). Currently,  described  i n the following c e l l  1) A r t e r i a l  of peripheral  a s p e c i f i c HDL b i n d i n g  s i t e has been  types:  smooth m u s c l e c e l l s ( 7 5 ) ,  2) Human, r a b b i t a n d p i g h e p a t o c y t e s ( 7 6 , 7 8 , 7 9 ) , 3) R a t i n t e s t i n a l and  most  mucosal and a d r e n a l  which they b e l i e v e  implies receptor  monocytes ( 7 7 ) .  Graham a n d Oram h a v e u s e d l i g a n d b l o t t i n g  t e c h n i q u e s t o show t h a t  interaction  1 2 5  I - H D L b i n d s t o a 110 kDa p r o t e i n ,  i s t h e HDL r e c e p t o r  ( 8 1 ) . The p o s s i b l e  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  t h a t HDL p o s s e s s e s s p e c i f i c within  c e l l s (80),  recently,  4) Human p e r i p h e r a l b l o o d Recently,  cortical  recognition  one o r more o f i t s c o m p o n e n t s .  s i t e s may l i e w i t h i n  t h e HDL a p o p r o t e i n s ,  25  on c u l t u r e d sites  cells  for this  The r e c o g n i t i o n  particularly  apo A - I  but  p o s s i b l y apo  further who  A-II  s u p p o r t e d by  and the  others  r e s u l t s of a recent  showed t h a t m o d i f i c a t i o n  crosslinking recognition recent  o f HDL (85).  report  ( 8 2 , 8 3 , 8 4 ) . T h e s e f i n d i n g s were  o f HDL  apoproteins a l t e r s the  However, t h i s  f r o m T a b a s and  issue  Tall  the  m a j o r components t h a t  Currently, receptor  and  transport 1.4  the  the  from c e l l s  ligand  surface  w h i c h i t may  a r e unknown  lipids,  i n v o l v e s the  of  a  above view.  not  proteins,  putative  facilitate  (86).  HDL  cholesterol  (73).  liver  for excretion  cholesterol transport  following  as  transport  to the  reverse  al  site  remains u n s e t t l e d  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  r e f e r r e d t o as  and as  its bile  (RCT). T h i s  salts  is  process  events:  1)  Transfer  2)  Esterification  o f FC  3)  Transfer  t o LDL  4)  Uptake of  FC  from c e l l  o f CE  membranes o n t o HDL  v i a the and  particles.  LCAT r e a c t i o n .  VLDL.  C E - r i c h p a r t i c l e s by  the  liver.  i n d i v i d u a l components o f t h i s p a t h w a y h a v e b e e n  extensively  studied  initial  t r a n s f e r of  acceptor  o c c u r s by  This  and/or  i n t e r a c t with cultured c e l l s  mechanism by  subsequent t r a n s p o r t  The  residues  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  Reverse c h o l e s t e r o l The  Oram e t  c o n f l i c t s w i t h the  These i n v e s t i g a t o r s concluded t h a t are  tyrosine  s t u d y by  i s followed  process driven Besides c e l l  by  and FC the  by  r e c e n t l y r e v i e w e d by from t h e  cell  desorption  F i e l d i n g (9).  membrane t o t h e  of  FC  from the  cell  d i f f u s i o n down a c o n c e n t r a t i o n  the  LCAT r e a c t i o n  (56),  membrane c h o l e s t e r o l t h e  to the  lipoprotein membrane.  gradient,  HDL  The  a  particle.  LCAT r e a c t i o n c a n  p l a s m a l i p o p r o t e i n c h o l e s t e r o l . However, e v e n t h o u g h t h e  also  use  majority  of  FC  substrate  masses o f to  80%  of the  from c e l l varies  cell  f l u x of  differential The  it  esterified  from c e l l  by  ability  nature of the  loading  of  binding  s i t e s on  cholesterol  (9)  HDL/  c e l l s has  ( 8 7 , 8 8 ) , when  from c e l l  expression of the  are may  and  cell  may  may  be  regulated  receptor  l i v e r by In  the  involved will  a f f e c t the  cholesterol  perturbation current  enzymes, and  clearance.  body's a b i l i t y accumulation  and  the  binding  therefore  the  understanding of  cholesterol  Any  defect  to mobilize  i n the  b o d y . TD  following TD.  27  from plasma  transfer liver.  site  of  to  (76).  lipoproteins,  in sites  This not  i n t h i s pathway  cholesterol i s such  discussion  CE  In  CE  lipoprotein receptors.  accumulation of  in cholesterol  HDL  enters the  i s a complex p r o c e s s i n v o l v i n g  L T P ' s , LCAT, l i p o l y t i c process prevents the  HDL  that  by  d i r e c t l y d e l i v e r the  interacting with a hepatic  summary, RCT  the  (73).  o r VLDL f o r c l e a r a n c e by can  FC  among  increase  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 t h e  the  donate  interaction.  c e l l - s u r f a c e i s unknown. However, o n c e HDL  t o t h i s mechanism, HDL  up  derived  c e l l - s u r f a c e suggesting  HDL  addition  be  reflect,  mechanism w h i c h p r o m o t e s d i s s o c i a t i o n o f  e i t h e r LDL  equal  present,  o f membranes t o  b e e n shown t o  the  to  the  VLDL  LCAT r e a c t i o n  type  f r o m HDL  to  and  the  ( 9 , 8 9 ) . The  type to c e l l  the  number o f HDL  i n LDL  plasma l i p o p r o t e i n c h o l e s t e r o l  membranes  Cholesterol  the  and FC  other things,  the  originates  and  lead  a will  review  1.5  Tangier  1.5.1  Disease  Introduction TD  i s a r a r e autosomal r e c e s s i v e  transport.  The  typical  disorder  of plasma  findings i n patients with t h i s  lipid  disorder  include: 1)  An  absence of normal  2)  Moderate  3)  The  HDL.  hypertriglyceridemia.  wide spread t i s s u e accumulation  esters, cells,  e s p e c i a l l y prominent  of c h o l e s t e r y l  i n the r e t i c u l o e n d o t h e l i a l  i n lymph n o d e s , thymus, bone marrow and  rectal  mucosa. The tonsils  p r e s e n c e o f HDL or t o n s i l l a r  disease  d e f i c i e n c y and  enlarged  remnants i s c o n s i d e r e d  yellow-orange  pathognomonic of  (90).  1.5.2  History U n u s u a l t o n s i l s were removed f r o m a 5 y e a r o l d boy  Tangier  Island,  tonsils  revealed  was  Virginia the  r e f e r r e d to the  tentative diagnosis disease. it  the  National of  Island  disorder  older and  discovery a new  because of the  sister  of  The  with  the  patient  the  hypoalphalipoproteinemia  disorder,  later  p a t i e n t ' s home on  called  the  Tangier  (91).  of the  original  p a t i e n t had  f i v e y e a r s l a t e r H o f f m a n n and  a second p a i r of Presently,  I n s t i t u t e of Health  t h i s was  i n C h e a s e p e a k e Bay  The  examination of  e i t h e r h i s t i o c y t o s i s or l i p i d - s t o r a g e  However, w i t h t h e  disease  Microscopic  p r e s e n c e o f numerous foam c e l l s .  became c l e a r t h a t  Tangier  i n 19 60.  from  s i b l i n g s f r o m an  28  same  Fredrickson  unrelated  twenty f i v e y e a r s s i n c e the  the  first  kindred patient  described  (92). was  discovered, reported first  thirty  (91-97).  three patients with t h i s The p a t i e n t  described i n t h i s  and o n l y known TD p a t i e n t  1.5.3  Clinical The c l i n i c a l  f e a t u r e s i n homozygous and h e t e r o z y g o u s  examination  The Framingham  a b n o r m a l i t i e s found  study  (98) h a s shown t h a t  (HDL-C) l e v e l s However,  i n heterozygotes (96). decreased  i n TD h e t e r o z y g o t e s  o r homozygotes  o r c e r e b r o v a s c u l a r d i s e a s e has  I n s u b j e c t s betv/een  t o 4% o r l e s s  p r e v a l e n c e o f CHD patients with  35 and 65 y e a r s o f age 26% o f o f CHD,  HDL  d e f i c i e n c y may  the  accelerated  observed  a t h e r o s c l e r o s i s such  as t h a t  that  be a t an i n c r e a s e d r i s k  f o r p r e m a t u r e v a s c u l a r d i s e a s e . However,  strikingly i n patients  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 b e c a u s e o f t h e i r plasma l e v e l s  o f LDL  as  i n a n o r m a l p o p u l a t i o n ( 9 6 ) . The i n c r e a s e d  i n TD i s c o n s i s t e n t w i t h t h e c o n c e p t  familial  plasma  are a s s o c i a t e d with premature  h e t e r o z y g o t e s and 45% o f h o m o z y g o t e s h a d some e v i d e n c e compared  on  o f t h e h o m o z y g o t e s . None o f t h e s e  b e l l o w 35 y e a r s o f age, no CHD been noted.  TD  by S c h a e f e r e t a l ( 9 6 ) .  of c l i n i c a l  a b n o r m a l i t i e s have been o b s e r v e d  atherosclerosis.  i s the  Features  T a b l e I I I shows t h e f r e q u e n c y  HDL-cholesterol  study  been  i n Canada.  p a t i e n t s have r e c e n t l y been r e v i e w e d  physical  d i s e a s e have  (96) .  29  reduced  TABLE I I I  CLINICAL  PRESENTATION OF 33  WITH TANGIER (Courtesy  Dr. J  DISEASE Frohlich)  Mode o f Presentation  Symptom Tonsillar  abnormalities  PATIENTS  F e a t u r e s on P h y s i c a l Examinatii  8/33  (24%)  26/33  (79%)  Splenomegaly  2/33  (6%)  16/3 3  (48%)  Hepatomegaly  -  -  14/33  (42%)  Lymphadenopathy  -  -  6/33  (18%)  11/33  (33%)  18/33  (55%)  -  -  14/29  (48%)  -  -  10/10  (100%)  -  9/13  (69%)  Neuropathy Corneal  infiltration  Abnormal  rectal  Bone marrow foam  mucosa cells  1/28  (3%)  -  -  Hypocholesterolemia  3/28  (10%)  31/33  (94%)  Chest  1/33  (3%)  Abnormal  skin findings  pain  30  —  -  1.5.4  Biochemical Biochemical  Features  a b n o r m a l i t i e s noted  plasma c h o l e s t e r o l ,  37%,  34%,  P a t i e n t s have u s u a l l y m o d e r a t e l y Individual variation  c o n s i d e r a b l e and concentrations  i n TD  4%  of normal,  i n t h e p l a s m a TG  r e f l e c t s t h e HDL  of chylomicrons  (d<1.006 g/ml)  30  (99,100). observed  and  qualitative  This difference quantitative  t o 50%  of  Intermediate  deficient  i n CE  i n m o b i l i t y may  LDL  T D  LDL  n o r m a l VLDL  33%  and  of normal  (99,100). LDL  T D  o f LDL  isolated  from  may  o n l y 0.4  t o 4.5%  different  o f n o r m a l . The  v a r i e s n o t o n l y among p a t i e n t s b u t  31  lipid also  levels  (101).  LDL  faster  Others  (94) . The  reflect  do  not  of are  T D  Several  the  found  reason  for  varied  i n t h e HDL  content  i n the  than  have  patients with  I n TD p l a s m a t h e c o n c e n t r a t i o n o f p r o t e i n was  the  the  migrates  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  composition  similar  (IDL:d=l.006-1.019 g/ml)  e n r i c h e d i n TG  i s u n c l e a r and  on  chemical  e x p l a i n e d by  on p a p e r e l e c t r o p h o r e s i s ( 9 5 , 1 0 0 ) .  these differences  (90).  i n C - a p o l i p o p r o t e i n s (93)...  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 control  be  p a t i e n t s (90)  to approximately and  with  remnants i n plasma  more s l o w l y t h a n  density lipoproteins  are reduced  PL  (90).  a p o l i p o p r o t e i n content are  a c c u m u l a t e i n t h e p l a s m a o f TD LDL  is  control  i n plasma i s d e l a y e d  abnormality  TG  (90). Plasma  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, g r o s s composition  respectively.  levels  deficiency  of chylomicron  migrate  HDL-C v a l u e s  e l e v a t e d f a s t i n g plasma  plasma range from  consequent accumulation V L D L r p p  and  and  i s h i g h l y d e p e n d e n t on d i e t  values. This l i k e l y Clearance  homozygotes i n c l u d e  L D L - c h o l e s t e r o l (LDL-C),  which are approximately  levels.  i n TD  o f TD  TD. region HDL  same p a t i e n t s  ( 9 0 ) . A g a r o s e g e l e l e c t r o p h o r e s i s and shown a s t r i k i n g microscopic  heterogeneity  s t u d i e s by  o f t h e TD  Forte et a l  presence of three morphologically density  20-25 nm  3)  Small  5.5-7.5 nm  particles,  present. 1%  The  apo  A-I  have not  Apo plasma  B,  i n the  o f HDL,  apo  structural  a l l of the  A-II  HDL  A  study  homozygotes t h e r e  generalized decrease Apo  B i n TD  by  apo  A-I  and  A-II,  et a l  apo  A-I  do  of apo  exclude  apo  (93)  has  a n a l y s i s and  B,  A-II the  and  TD in  a deficiency composition  E i s a l s o normal  the  in  a v a r i a b l e but  polyacrylamide on  normal  shown t h a t  n o r m a l amino a c i d  ( 9 0 ) . Apo  less  (90).  E levels  p l a s m a has  and  not  i n apo  C and  are  plasma i s t y p i c a l l y  o f apo  a p o l i p o p r o t e i n s are not  apo  plasma,  E have been i d e n t i f i e d  (90). C u r r e n t l y , data  decreased  f r o m TD  i s normal l e v e l s  i t s amino a c i d  electrophoresis  The  diameter.  alteration  Alaupovic  immunochemical p r o p e r t i e s  j u d g e d by  in  l e v e l s a r e a b o u t 5-7%  D and  which  diameter.  s t u d i e s of the  C*s,  i n diameter,  been r e c o v e r e d  of minor s t r u c t u r a l  (101).  D.  in  nm  r e v e a l e d major a b n o r m a l i t i e s they  possibility  with  particles  the  remnants.  c o n c e n t r a t i o n o f TD  o f n o r m a l , and  (90). Although  and  have not  the major apoproteins  100  chylomicron  Round p a r t i c l e s ,  o f apo  Electron  have r e v e a l e d  distinct  2)  W h i l e n o r m a l HDL  TD  (102)  Large t r a n s l u c e n t forms, over appear t o r e p r e s e n t  than  HDL.  have  region:  1)  both  e l e c t r o n microscopy  as  gel  other  available.  p l a s m a c o n c e n t r a t i o n o f apo  t h e p o s t u l a t e t h a t HDL  C's  i s consistent  a c t s as a r e s e r v o i r f o r C  32  apolipoproteins. plasma  i s accomplished  rapidly the  present  t u r n i n g o v e r HDL  shown v a r i a b l e  results.  LpL not  i n the a c t i v i t y  demonstrated  (90). While  some p a t i e n t s w i t h TD  activity i n others o f LpL  low  S e v e r a l hypotheses  f o r t h e apo  ( 1 0 5 ) . No  o r HTGL h a s  C's  HDL  hence  has  i n some  clear yet  has  plasma  cut  been  been r e p o r t e d i n of  (105,106). Disease t o e x p l a i n the  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 o f HDL synthesis  i n TD  i s decreased  have been f o r m u l a t e d  d e f e c t i n TD.  A-I  VLDL) t o  is insufficient  activity  LCAT a c t i v i t y  Biochemical Defect i n Tangier  molecular  there  and  the  (95,103,104) n o r m a l o r e l e v a t e d l e v e l s  LCAT h a v e a l s o b e e n d e s c r i b e d 1.6  from  (Chylomicrons  reservoir  o f p o s t h e p a r i n LpL  (103,104) b u t  deficiency  C's  removal.  Examination  patients  o f apo  p o o l . . I n TD  to provide the necessary  faster  C a p o l i p o p r o t e i n s i n normal  by t h e t r a n s f e r  catabolized lipoprotein pool  slower  their  Conservation of the  d e s p i t e normal r a t e s o f  (107-114).  2)  A b n o r m a l s t r u c t u r e o f apo  3)  Impaired  A-I  (115).  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 failure  However, no  o f apo  A-I  unequivocal  t o a s s o c i a t e w i t h HDL evidence  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 established.  Furthermore,  structural  deficiency  R e c e n t s t u d i e s o f t h e apo  p a t i e n t have f a i l e d  of a  A-I  (120,121).  has  been  gene from  our  t o demonstrate a coding abnormality  t h e r e d o e s n o t a p p e a r t o be  33  a defect i n  TD (120).  apo  transcription, et  a l have  decrease  showed  also  and  excluded of  that  molecular apo  the  the  have  apo  only  that  A-I.  a  secretion rates  Schaefer  slight (110).  In  proapolipoprotein  convertase  by  a  A-I  convertase  activity  apo  A-I  A-I  in this  (124).  Thus  and  apo  As  majority  lipolysis TG-rich result  of  be  disease.  active  the  were  apo  can  in  i s normal  in  TD  In  isolated  of  HDL  has  lipoproteins a impair  experiments  on  the  great  have a  by a  TD  They  fragments they  or  rapid Pritchard et  patient with  purified  production  from  (123).  aggregates  from  Weech  addition,  f o r the  both  precursors  be  coworkers had  the  In  studies  purified  A-I  by  CNBr-cleavage  dimers,  Recently,  to  a  and  examined  normal  responsible  yet  generated  patients  that  appear  A-I  to  different.  d e f i c i e n c y . However,  i n TD  vivo  not  A-I  l i p o p r o t e i n s would  deficiency  pi  was  of  and apo  patients  is  al  TD  plasma A-I  in  TD  a  protein.  TG-rich  i n HDL  A-I  in activating  i t would  the  apo  weight,  could  that  TD  antibodies  possibility  biologically  and  this  structure of  demonstrated  normal  have  and  TD  a p o l i p o p r o t e i n A-I  f u n c t i o n a l l y normal  LCAT is  the  normal  catabolism  was  with  demonstrated  four monoclonal  that  fragments  have  synthesis  mature  tertiary  a l using  TD  s e c r e t i o n of  patients  been  and  or  (118,119).  The  of  A-I  to  plasma  plasma  that  i t has  converted  normal  et  shown  i n apo  addition, be  translation  deal  shown  of  that  g r e a t l y reduced  are  defect  the  derived i n the  production  this  during lipolysis  of  explanation  HDL  of  and  may  f o r the  HDL  substantiated. metabolism interesting normal plasma  34  HDL  of  HDL  i n TD  patients  information.  Schaefer  injected into  residence  time  TD  (114).  The  observed studies  h y p e r c a t a b o l i s m o f HDL  i n TD was  further corroborated by  i n w h i c h l a r g e r amounts o f HDL were i n f u s e d  (ie partial  plasma exchange) (125). C u r r e n t l y , a l l t h e c u m u l a t i v e the l i t e r a t u r e catabolism of  this  suggests  o f HDL.  t h a t t h e d e f e c t i n TD l i e s  evidence i n  i n enhanced  However, t o d a t e t h e e x a c t b i o c h e m i c a l  d e f e c t i s unknown.  35  nature  1.7  Rationale  for this  Hypercatabolism either  increased  catabolism has  i n TD  o f HDL  p l a s m a may  catabolism  without c e l l u l a r  e s t a b l i s h e d t h a t the  or  be  i n v o l v e m e n t . Work by  defect  i n TD  will  of  i n d i v i d u a l HDL  examine t h e  cells  play  i n the  have f o c u s e d 1)  on  Specific 1)  incubation  To  in  To  4)  To  and  research  i n TD  TD  p a t i e n t s . In  HDL  our  catabolism  we  o f n o r m a l HDL  i n TD  plasma  vitro.  i n t e r a c t i o n o f n o r m a l HDL  with  TD  vitro.  study the  lipid  and  incubation with  study the  fibroblast 3)  d i r e c t uptake  studies:  catabolism in  by  aims  during 2)  following  Examination of the cells  o f HDL  al  could  composition  t h e mechanisms u n d e r l y i n g  Examination of the during  2)  the  studies  plasma f a c t o r ( s ) and/or  "hypercatabolism"  approach to studying  et  the  components. T h u s , t h i s  r o l e s i n w h i c h TD  of  Schaefer  results in  or the  result  plasma  from p l a s m a . However, t h e i r  factors p r i o r to catabolism  degradation  the  increased  d i f f e r e n t i a t e b e t w e e n m o d i f i c a t i o n o f HDL  plasma  1.8  o f HDL  cellular  hypercatabolism not  study  p r o t e i n changes i n normal the  plasma from a p a t i e n t w i t h  i n t e r a c t i o n of the  TD  modified  HDL  and  normal  HDL.  compare t h e i r  ability  To  c u l t u r e peripheral blood  TD  p a t i e n t and  normal  with  TD. normal  cultures.  c u l t u r e s k i n f i b r o b l a s t s f r o m c o n t r o l s and  patient  HDL  compare t h e i r  HDL. 36  to bind  and  a  TD  degrade  m o n o c y t e s from c o n t r o l s and ability  to bind  and  degrade  a  2  MATERIALS AND  2.1  Subjects  2.1.1  METHODS  C o n t r o l subj e c t s Healthy  normolipidemic  amongst l a b o r a t o r y s t a f f .  control  recruited  from  2.1.2  Tangier patient  the L i p i d  o f TD  Clinic  o r LCAT d e f i c i e n c y  a t Shaughnessy  and were  hospital.  C a s e H i s t o r y : J.K.W., a 56 y e a r  o l d male, p r e s e n t e d  w i t h a p r o g r e s s i v e weakness and  numbness and  b o t h h a n d s . T h e s e symptoms were f i r s t drooping  of h i s r i g h t  tonsillectomy circulation  lower  eyelid.  i n the  feet"  i n 1947  i n 1964.  and  e n l a r g e m e n t o f h i s n o s e and n o s e f o r r h i n o p h y m a was procedures  due  In the  out  analysis  and  was  Insulin-dependent  earlobes. A  few  Neurological t h e hands and  "bad  of the  spleen  he-noted  surgery of h i s  he  1986  is a tall  suffered  follow-up an  cholecystectomy. l i v e r done a t t h a t  I n November 1985  d i a b e t e s m e l l i t u s was He  included:  i n 19 69 w i t h numerous  i n February  Examination:  with  for  1960's  o f a wedge b i o p s y o f t h e  l o s i n g w e i g h t and  Physical  late  t r e a t e d by  showed numerous foam c e l l s .  started  examination  ear lobes. P l a s t i c  carried  along  sympathectomy f o r  t o p o o r wound h e a l i n g . I n 1980  attack of c h o l e c y s t i t i s Histological  i n 1981  splenectomy  Histological  showed numerous f a t - l a d e n c e l l s .  noted  i n March  paresthesiae of  His past history  i n childhood, b i l a t e r a l  "hypersplenism"  time  recruited  Hypertriglyceridemic subjects  p a t i e n t s w i t h homozygous f o r m  1982  s u b j e c t s were  the p a t i e n t  the diagnosis of  made. man,  (184  cm),  with  large  y e l l o w s t r e a k s a r e e v i d e n t on h i s p h a r y n x .  examination  r e v e a l e d marked w a s t i n g  feet. A right  lower  37  and  weakness  ectropion with healed  of  corneal  u l c e r s was p r e s e n t .  The f a c i a l  T h e r e was a l o s s o f s e n s i t i v i t y hands and f e e t i n a g l o v e and  a n d weak.  t o t o u c h and v i b r a t i o n o v e r h i s  and s t o c k i n g  distribution.  The  r e s p o n s e s were d o w n g o i n g .  Histology: patients  skin,  et a l (personal  Studies  were done on b i o p s i e s  obtained  s u r a l n e r v e , r e c t a l mucosa a n d l i v e r communication). A n a l y s i s  microscopy revealed  by l i g h t  from t h e  by F r o h l i c h  and e l e c t r o n  t h e p r e s e n c e o f a l a r g e number  o f foam  cells  a l l o f t h e s e t i s s u e s . T h e s e f i n d i n g s were n o t d i f f e r e n t f r o m  those described  by F r e d r i c k s o n  e t a l (91).  F a m i l y h i s t o r y : The p a t i e n t ' s p a r e n t s w e r e f i r s t His  biceps  t r i c e p s r e f l e x e s were d e p r e s s e d , t h e a n k l e j e r k s a b s e n t a n d  plantar  in  m u s c l e s were a t r o p h i c  father died  a t age 50 o f a h e a r t  i n her e a r l y f o r t i e s alive  during  and w e l l . The f a m i l y  surgery.  attack. His only  cousins.  H i s mother d i e d sister,  e m i g r a t e d t o Canada f r o m  a g e d 55, i s  Bristol,  E n g l a n d i n 1919. The p a t i e n t h a s no p r o g e n y . The f a m i l y shown i n F i g u r e  4.  38  tree i s  W  family  £9* M  <X o, Q Tv 6 rj Q j& 3  x  5  6  d aTd75  7  6, 6 , 0 , 6 ,  10  6  5  9  21  Figure  4.  F a m i l y t r e e o f p a t i e n t JKW (•) ( C o u r t e s y o f D r . M. Hayden)  39  g  7  g,  13  2.2  Materials The f o l l o w i n g c o m p a n i e s s u p p l i e d c h e m i c a l s  o r equipment as  indicated: Amersham Canada L t d . , O n t a r i o , 1 2 5  Canada:  I-NaI.  A m i c o n Canada L t d . , O n t a r i o , 0.45uM f i l t e r s , Analtech  Canada:  microconcentration  system.  I n c , Newark, D e l . , USA:  Silica  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 a n d A5.0m, B i o - R a d PAGE s y s t e m . Boehringer  Mannheim, West Germany:  Reagent k i t s Corning  Medical,  f o r t h e FC, TG, a n d CE  assays.  Ca, USA:  Agarose g e l e l e c t r o p h o r e s i s system. G i b c o Canada, O n t a r i o ,  Canada:  MEM, RPMI-1640, FCS, t r y p s i n a n d a n t i m i c r o b i a l s . NEN  Research Products, [ H]-CE, 3  Pharmacia  Quebec,  Canada:  [ H ] - c h o l e s t e r o l , and [ H ] - g l y c e r y l 3  3  (Canada) I n c . , Quebec,  trioleate.  Canada:  Sephadex G-50, F P . Tago L t d . , C a . , USA: Apo A - I r a d i a l Western S c i e n t i f i c ,  immunodiffusion p l a t e s . B.C., C a n a d a :  Ultracentrifugation N.B.  tubes,  A l l o t h e r m a t e r i a l were  tissue culture  standard  40  dishes.  laboratory  stock.  2.3  Lipoproteins  2.3.1  Isolation  2.3.1.1  Preparative  VLDL the  (d< 1.006  patients  of  ultracentrifugation  g/ml)  w i t h TD  from normal  lipoproteins  was  from t h e f a s t i n g plasma  and LCAT d e f i c i e n c y  individuals  and  (1.14 x 1 0  5  and H D L  1.006-1.063 g/ml; respectively 42,000 rpm  Total  were i s o l a t e d  1.063-1.21 g/ml  by s e r i a l (1.13 x 1 0  upper d e n s i t y  3  5  and  a t t h e same  i n the density  1.12-1.21  subjected  ultracentrifugation  a t 15°C f o r 24 h a t  were i s o l a t e d  a d j u s t e d t o d = 1.225 to ultracentrifugation removed  recovered  Lipoprotein  g/ml  ultracentrifugation  by t u b e s l i c i n g  fetal  c a l f serum  adjusted  t o d = 1.225  0.03%  against NaN  3  fraction four  (LPDS) was p r e p a r e d  g/ml  (buffer  was  (LPDS) was  changes  thereafter  (LPDP).  o f 0.15  A), s t e r i l i z e d  of the  and s u b j e c t e d  a t 15°C f o r 40 h a t 40,000 rpm.  and t h e b o t t o m  rpm.  and t h e b o t t o m  (128) . The d e n s i t y  containing t o t a l lipoproteins,  were d i a l y z e d EDTA and  w i t h s o l i d NaBr and t h e n  d e f i c i e n t human p l a s m a  deficient  c a l f serum was  of the  a t 15°C f o r 40 h a t 40,000  b y t h e methods o f Brown and G o l d s t e i n  slicing  at the  from plasma  i n t h e o r i g i n a l p l a s m a volume  d e s i g n a t e d as l i p o p r o t e i n  fraction,  range  limit.  The t o p f r a c t i o n was  fetal  at  density.  g) and washed by r e c e n t r i f u g a t i o n  plasma l i p o p r o t e i n s  f r a c t i o n was  plasma  g/ml,  a c c o r d i n g t o Rudel e t a l (127). B r i e f l y , t h e d e n s i t y p l a s m a was  of  g ) . The p a r t i c l e s were  p u r i f i e d by a s e c o n d c e n t r i f u g a t i o n  LDL and HDL  from p o o l e d  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  15°C f o r 18 h a t 42,000 rpm further  isolated  The  removed  by  to  top tube  saved. A l l f r a c t i o n s M NaCl c o n t a i n i n g by  0.45  uM  1  mM  filtration  and  stored  2.3.1.2  LDL  could  u l of  be  The  containing  2  then c e n t r i f u g e d  Iodination  2.3.2.1  Preparation  at  glycine  1-3  (1.0  ul  2500  placed min  pH  1 2 5  10  1 2 5  I-labeled  4°C  15°C.  as  with  for 3 0 The  determined  by  lipoproteins  M NaCl,  protein  cm).  vial;  I-NaI  activity varied  was  The  10  separated  following  Bilheimer et  (0.33  6 drops of b u f f e r  7.4  free  (1.0  1  3 00  2  5  x 20.0  (buffer C).  and  i  M)  by cm)  The  final  0.4 5 uM  stored  at  10-  mg  eluted  HDL.  with  specific  cpm/ng p r o t e i n .  and  1-3  All by  4°C  the  passage for  a  G-  a  diluted  and  through a  42  M  p a s s a g e down  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  3 weeks.  (131).  0.4  B and  iodinated  millipore filter  al  were t h e n a d d e d t o  s t o c k IC1  columns  monocholoride  p a s s a g e t h r o u g h a Sephadex  from t h e  between 100  iodine  equilibrated with  by  u l of  (1 m C i ) ,  1 M EDTA, pH  the  as m o d i f i e d by  ( b u f f e r B)  x 20.0  I - H D L was  maximum o f  mixed  units/ml  at  at  alpha-lipoproteins  (130)  t h r o u g h a Sephadex G-50 0.15  p l a s m a was  al  lipoproteins  of  o f HDL  screw top  10  1 2 5  mg  buffer,  column  plastic  The  f o r 10  I - l i p o p r o t e i n s were p r e p a r e d by  Briefly,  Warnick et  M N a C l and  6000 x g  of  method o f M a c F a r l a n e  fold,  0.15  u l of  by  VLDL  electrophoresis.  2.3.2  50  500  plasma  p r e c i p i t a t i o n of  t h e n v o r t e x e d and  supernatant contained only  1 2 5  from  described  preparation,  sample was  agarose g e l  LDL  i s o l a t e d from p l a s m a by  a typical 1 M MnCl  heparin. min,  used.  w i t h h e p a r i n - m a n g a n e s e as  (129). In 50  -20°C u n t i l  P r e c i p i t a t i o n o f VLDL and  HDL and  at  2.3.2.2 The  Characterization of  I-Lipoproteins  amount o f r a d i o a c t i v i t y  determined  a s s o c i a t e d w i t h apo B was  u s i n g a modified Holmquist  100 u l o f 1 0 0 - f o l d d i l u t e d  1 2 5  procedure  (132).  Briefly,  I - L D L was m i x e d w i t h 200 u l o f  u n l a b e l e d LDL (1 mg/ml) a n d 300 u l o f i s o p r o p a n o l was a d d e d t o the mixture  w h i c h was t h e n v o r t e x e d a n d s t o r e d a t room  temperature  f o r 10 m i n p r i o r t o c e n t r i f u g a t i o n  10 m i n a t 4 ° C . The p e l l e t ,  containing the precipitated  t h e s u p e r n a t a n t were s e p a r a t e d a n d c o u n t e d 1271 RiaGamma c o u n t e r f r a c t i o n was c o u n t e d percentage The  a t 13,000 x g f o r  f o r 60 s e c o n d s  (LKB).The r a d i o a c t i v i t y  each  expressed as  a s s o c i a t e d w i t h t h e apo B.  amount o f r a d i o a c t i v i t y  component was a s s a y e d  ina  associated with  (cpm). T h e r e s u l t s were t h a n  of radioactivity  a p o B, and  by l i p i d  associated with the l i p i d  extraction  o f 10 u l o f  1  2  5  I -  l i p o p r o t e i n b y t h e method o f F o l c h e t a l (133) a n d m e a s u r i n g t h e radioactivity  i n t h e c h l o r o f o r m phase.  mg/ml) was i n c l u d e d i n a l l s a m p l e s . percentage The  radioactivity  determined addition bovine act  radioactivity  10 u l o f c a r r i e r  The r e s u l t s  a s s o c i a t e d w i t h t h e p r o t e i n component was o f 10 u l o f  1 2 5  o f 200 u l o f 50% t r i c h l o r o a c e t i c  as a c a r r i e r  centrifugation radioactivity determined.  I-lipoprotein acid  (TCA).  by t h e  200 u l o f  (BSA:5 mg/ml) was a d d e d t o a l l s a m p l e s t o  protein  and t o b i n d any f r e e  lipid.  After  f o r 10 m i n a t 17,000 x g t h e amount o f associated with the p e l l e t  The r e s u l t s  associated with Iodinated  a r e e x p r e s s e d as  associated with the l i p i d .  by p r e c i p i t a t i o n  serum a l b u m i n  LDL (1  are expressed  and s u p e r n a t a n t  as percentage  were  radioactivity  protein. 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 b y 43  agarose  gel  electrophoresis using  modified  the Corning  f o r l i p o p r o t e i n s (134).  E l e c t r o p h o r e s i s System as  Iodinated  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 electophoresis or  gels according  were  polyacrylamide g e l  (SDS-PAGE) u n d e r r e d u c i n g  12.5% a c r y l a m i d e  apolipoproteins  conditions  on e i t h e r 10  t o t h e method o f Laemmli  (135). A g a r o s e g e l s and S D S - p o l y a c r y l a m i d e g e l s radiolabeled  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  autoradiography of gels into  containing i n d i r e c t l y by  f o r 1-7 d a y s o r d i r e c t l y b y s l i c i n g  0.5 cm s e c t i o n s a n d c o u n t i n g  gels  t h e gamma r a d i a t i o n .  2.3.3  [ H] 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  Labeling  3  o f VLDL w i t h  VLDL was l a b e l l e d w i t h  3  3  g l y c e r y l t r i [ H ] o l e a t e according t o  t h e method o f F i e l d i n g ( 1 3 6 ) . tri[ H]oleate  glyceryl tri[ H]oleate 3  Briefly,  (1.0 Ci/mM) was s o l u b i l i z e d  dimethylsulfoxide,  d i l u t e d t o 4 ml w i t h  s o l u t i o n was a d d e d 5 mg o f VLDL-TG w i t h incubation against  i n 1 ml o f  b u f f e r C. T o t h i s constant  The l a b e l l e d  After  VLDL was  a t d = 1.006 gm/ml b y 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 a n d a n d 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 2.3.3.2  stirring.  f o r 3 h a t 37°C, t h e r e a c t i o n m i x t u r e was d i a l y z e d  b u f f e r C a t 4°C o v e r n i g h t .  reisolated  100 u C i g l y c e r y l  Labeling  o f plasma l i p o p r o t e i n s w i t h  b u f f e r C. [ H] 3  cholesterol Aliquots the  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  p r e s e n c e o f 10 u C i [ 7 - H ] c h o l e s t e r o l (23.7 Ci/mM) a s  previously described  3  b y P r i t c h a r d e t a l (137) . I n t h i s l a b e l i n g  p r o c e d u r e more t h a n 90% o f t h e l a b e l  44  was a s s o c i a t e d w i t h t h e  different  lipoprotein  sufficient  t i m e was  the  radiolabel  was  the  fraction  permitted  w i t h the  (d<1.21 g / m l ) . M o r e o v e r , f o r the  2.3-3.3  similar to  that  the  HDL  with  were l a b e l l e d w i t h  of  Sparks et  al  p l a s m a o r HDL incubated  (1-10  f o r 14  second v i a l  h  and  vial  CE  by  linoleate  under n i t r o g e n .  4°C.  used the  The  s a m p l e was  same  One  Determination of  lipoprotein particle  a 2 ml  Biogel 4.3 0.01%  with  0.15  EDTA, and  collected  further  M  0.3%  i n 2 ml  e a c h f r a c t i o n was  NaN  of  fractions  ml/h.  of p u r i f i e d ,  1 2 5  (90  and  10  D).  the  d e t e r m i n e d . The  a n a l y z e d by  21  (buffer  3  x  a  mM  1 2 5  1.5  I-LDL  quasielastic  HDL  was into  Eluent  applied and  pH  from the  and  light  a at  7.4,  column  label present HDL  was in was  AO.5m  using buffer  1 2 5  to  eluted  peak c o r r e s p o n d i n g t o  cm)  45  sized  typical  Tris/HCl,  B o t h c o l u m n s were p r e c a l i b r a t e d  I-VLDL,  o b t a i n e d by  and  cm)  amount o f  A more a c c u r a t e d e t e r m i n a t i o n o f was  x 1.5  passage through a B i o g e l (90  mixture  l a b e l l e d p l a s m a was  NaCl c o n t a i n i n g  c h r o m a t o g r a p h i c column rate  of  A5.0m c h r o m a t o g r a p h i c c o l u m n  ml/h  of  size  i n t o VLDL, LDL  chromatography. In  aliquot  was  Lipoproteins  subfractionated  preparation,  Ci/mM)  day.  2.3.4.1  filtration  of  then transferred  of  gel  uCi  aliquots  the  Characterization  p a r t i c l e s by  FC  procedure  (100  ml  2.3.4  P l a s m a was  a  ( 1 3 8 ) . B r i e f l y , 3-7  mg/ml) were i n t r o d u c e d and at  of  CE  3  3  3  down i n a g l a s s  [ H]  [ H]  [cholesteryl-1,2,6,7- H(N)]-cholesteryl dried  specific activity  of  fractions.  L a b e l i n g o f p l a s m a and  P l a s m a o r HDL  complete e q u i l i b r a t i o n  lipoproteins  same i n a l l l i p o p r o t e i n  as  D and by  the  a  flow  elution  I-HDL. VLDL and  scattering  LDL (QLS)  particle using  a  size  a  NiComp 270 s u b m i c r o n p a r t i c l e Mayer e t a l ( 1 3 9 ) , Biochemistry, 2.3.4.2  t o t h e method o f  o f Dr. P. C u l l i s ,  Lipoprotein l i p i d  Dept o f  Columbia.  analysis  CE and TG were d e t e r m i n e d e n z y m a t i c a l l y b y  procedures  determination  according  University of B r i t i s h  Cholesterol, standard  courtesy  sizer  (140,141). PL were q u a n t i t a t e d b y  of l i p i d  Anderson e t a l (142).  phosphorus a c c o r d i n g VLDL, LDL, HDL  t o t h e method o f  and H D L  p r o t e i n were  3  m e a s u r e d b y t h e method o f Lowry e t a l (143) a s m o d i f i e d f o r lipoproteins  (144) u s i n g BSA a s a s t a n d a r d .  d e t e r m i n e d b y D r . W. Foundation,  Apo C - I I l e v e l s  McConnathy, Oklahoma M e d i c a l  by p r e v i o u s l y d e s c r i b e d  Research  immunochemical methods  Apo A - I l e v e l s were d e t e r m i n e d by r a d i a l - i m m u n o d i f f u s i o n preprepared  anti-apo  Enzyme  2.3.5.1  H y d r o l y s i s o f CE i n p l a s m a r a t e o f CE h y d r o l y s i s (CEH) i n p l a s m a was d e t e r m i n e d by [ H ] CE 3  p l a s m a 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 3 7 ° C . The r e a c t i o n was  chloroform:methanol method o f F o l c h  petroleum  ether:  stopped  ( S e c t i o n 2.3.3.3) o r  [ H ] CE, f o r 24 h a t 4 ° C 3  by t h e a d d i t i o n o f 4 ml  ( 2 : 1 ) . The l i p i d s  were e x t r a c t e d b y t h e  (133) and t h e CE's s e p a r a t e d  l a y e r chromatography  lipid  using  Assays  i n c u b a t i n g plasma p r e l a b e l e d with  and  (145).  A-I p l a t e s .  2.3.5  The  were  (TLC) on s i l i c a  ether:  was v i s u a l i z e d  acetic  acid  f r o m t h e FC by  thin  gel G plastic plates i n a (70:10:1) s o l v e n t s y s t e m .  The  by e x p o s u r e t o i o d i n e v a p o r and t h e a r e a o f  the  silica  g e l corresponding  t o c h o l e s t e r o l o r CE was  and  placed  i n a 7 ml g l a s s s c i n t i l l a t i o n  46  vial.  then  F i v e ml o f  c u t out  Omnifluor  i n toluene  sample was a s s a y e d determined total  for H  scintillation.  3  and t h e r e s u l t s  CE p e r 24  2.3.5.2  (4 g/1) were a d d e d t o e a c h v i a l  and t h e  The r a t e o f CEH  was  expressed as percentage h y d r o l y s i s o f  hour.  Bovine m i l k l i p o p r o t e i n  Bovine milk l i p o p r o t e i n skimmed m i l k by a f f i n i t y as d e s c r i b e d p r e v i o u s l y  lipase  lipase  chromatography  activity  from  bovine  on h e p a r i n - S e p h a r o s e  ( 1 4 6 ) , was a g i f t  enzyme was homogeneous a s d e t e r m i n e d enzyme h a d a s p e c i f i c  (BmLpL), p u r i f i e d  4B  f r o m R.McLeod. The  b y SDS-PAGE.  The  purified  o f 8 umoles f r e e f a t t y  acid  r e l e a s e d p e r hour p e r ml. VLDL was described  labelled with glycerol  ( S e c t i o n 2.3.3.1)  tri[ H]oleate  glycerol  e t a l (136). B r i e f l y ,  tri[ H]oleate 3  as p r e v i o u s l y  and t h e r a t e o f h y d r o l y s i s  l a b e l l e d VLDL's by BmLpL were d e t e r m i n e d by F e i l d i n g  3  essentially  of the  as d e s c r i b e d  the assay mixture contained  l a b e l l e d VLDL, a t t h e i n d i c a t e d  concentrations, p u r i f i e d  BmLpL (0.7 u g / m l ) ,  mg/ml b o v i n e serum a l b u m i n ,  TG  0.16 M N a C l ,  0.35 M T r i s - H C l ,  pH 8.2  125  (final  volume, 1.3 m l ) . The m i x t u r e s were i n c u b a t e d f o r 45 m i n a t 37°C in  a s h a k i n g water  of  isopropanol:  acid  b a t h and l i p o l y s i s was s t o p p e d b y t h e a d d i t i o n  sulphuric  acid  r e l e a s e d was d e t e r m i n e d  results  ( 4 0 : 1 ) . The amount o f [ H ] o l e i c 3  a c c o r d i n g t o S c h o t z e t a l ( 1 4 7 ) . The  a r e e x p r e s s e d as umoles o f o l e i c  acid  r e l e a s e d p e r mg  BmLpL p e r m i n . 2.4  Cell  2.4.1  Culture Isolation  a n d c u l t u r e o f human  S k i n was b i o p s i e d TD p a t i e n t  and c u l t u r e d  fibroblasts  from t h e m e d i a l p a r t i n 35 mm 47  o f the forearm of the  d i s h e s and m o d i f i e d E a g l e s  medium  (MEM) c o n t a i n i n g 20% f e t a l  penicillin fungizone  (base),4.0  calf  serum  ug/ml s t r e p t o m y c i n  and m a i n t a i n e d  (FCS),  (base)  i n a humidified C0  2  4.0 IU/ml  a n d 10.0 ng/ml  (5%) i n c u b a t o r a t  3 7 ° C . The e x p l a n t was n o t d i s t u r b e d d u r i n g t h e f i r s t culture.  On t h e s i x t h d a y t h e medium was r e p l a c e d w i t h  medium. A f t e r containing 2.5  14 d a y s i n c u l t u r e ,  diluted  fresh  1.0 ug/ml s t r e p t o m y c i n and  monolayer reached  t h e 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 % solution,  days o f  t h e medium was c h a n g e d t o MEM  5% FCS, 1.0 IU/ml p e n i c i l l i n ,  ng/ml f u n g i z o n e . When t h e c e l l  control  five  confluency EDTA  3 t i m e s w i t h medium a n d r e p l a t e d . A g e m a t c h e d  fibroblast  g e n e t i c mutant c e l l  l i n e s were o b t a i n e d  from  t h e NIGMS Human  r e p o s i t o r y and a l l t h e c e l l  l i n e s were  used  between 5 t h and 1 5 t h 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 inspection  (cytometry)  and p r o t e i n  48  determination.  by v i s u a l  2.4.2  Preparation binding Preparation  degradation et  0.05%  On  o f human s k i n f i b r o b l a s t s  On d a y 0 o f t h e e x p e r i m e n t ,  and T a n g i e r  concentration  was  experiments  cells  trypsin/0.02%  dishes  lines prior to lipoprotein  a s s a y s was p e r f o r m e d a c c o r d i n g  a l (128).  control  of cell  from s t o c k  4  cells  f l a s k s were d i s s o c i a t e d w i t h  petri  10% FCS. On d a y 3, t h e medium  2 ml o f f r e s h g r o w t h medium c o n t a i n i n g  d a y 5, e a c h m o n o l a y e r was washed w i t h saline  confluent monolayers o f  p e r d i s h i n t o 35 x 15 mm  i n 2 ml o f MEM c o n t a i n i n g  buffered  t o t h e method o f Brown  EDTA s o l u t i o n a n d were s e e d e d a t a  o f 5.0 x l O  replaced with  f o r LDL b i n d i n g a n d  2 ml s t e r i l e  10% FCS.  phosphate  ( P B S ) . A f t e r w a r d s 2 m l o f f r e s h g r o w t h 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  fetal  calf  serum  (LPDS) was  added t o t h e c u l t u r e s . E x p e r i m e n t s were p e r f o r m e d on d a y 7 a f t e r the c e l l s The  had been i n c u b a t e d  f o r 48 h i n t h e p r e s e n c e o f LPDS.  d i s h e s were a p p r o x i m a t e l y  contained  40-100 u g o f c e l l  Fibroblasts  confluency  3 days u n t i l  ( u s u a l l y 10-12 d a y s ) .  media were removed a n d t h e c e l l s 2%  BSA f o r 2 h p r i o r t o t h e s t a r t  cell  e x p e r i m e n t s were s e e d e d a t t h e  as p r e v i o u s l y d e s c r i b e d  m e d i a were c h a n g e d e v e r y  c e l l s and  protein.  f o r HDL b i n d i n g  same c o n c e n t r a t i o n The  60-75 % c o n f l u e n t w i t h  f o r LDL e x p e r i m e n t s . the cells  had reached  On t h e d a y o f t h e e x p e r i m e n t t h e were i n c u b a t e d  i n MEM  containing  o f t h e experiment t o d i s s o c i a t e  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 B l o o d Human p e r i p h e r a l b l o o d  drawn b l o o d (148).  using  m o n o c y t e s were i s o l a t e d  a method m o d i f i e d  In a t y p i c a l  Monocytes  preparation, 49  from  freshly  f r o m t h a t o f Cohn e t a l  venous b l o o d  (69 ml) was drawn  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 was  analyzed  S).  White b l o o d  cell  oil  f o r leukocyte counts (WBC)  values  counting  cell  x g f o r 10  min  b l o o d was  v o l u m e was  was  g e n t l y l a y e r e d over  centrifugation and  ml  conical  ml  o f warm PBS  f o r 30 min  The  The  an a l i q u o t  of the c e l l  then  l a y e r was  cells  1.0  approximately pellet  427  o f l e u c o c y t e s was  a d j u s t e d t o 1.0  d i s h e s were i n c u b a t e d  50  EDTA a t then  37°C.  35  i n a 50  ml ml  centrifuged at x g.  After  layer  was  transferred to a  l a s t wash, t h e c e l l  pellet  delipidated  and  2.5  15 15  was FCS,  ng/ml  determined  by  counting  u s i n g a haemocytometer. approximately  85%  f o r monocytes t h e  x 10  pipetted into  was  were washed t w i c e w i t h  ug/ml s t r e p t o m y c i n  suspension  under  were done i n  and  the top  l e u k o c y t e c o n c e n t r a t i o n was  a l i q u o t s were t h e n d i s h e s . The  w i t h PBS  s a m p l e was  number. A s s u m i n g a s i m i l a r y i e l d was  cell  o f RPMI-1640 c o n t a i n i n g 20%  fungizone.  suspension  The  at approximately  The  individual  examined  o f F i c o l l - P a q u e (FP)  (37°C). A f t e r the  IU/ml p e n i c i l l i n ,  yield  and  (Model  s m e a r s made o f  c o n t a i n i n g 0.1%  the mononuclear c e l l  s u s p e n d e d i n 10 ml  on  blood  Hospital.  o f t h e sample f o r 30 min,  c e n t r i f u g e tube.  for  centrifuged at  a d j u s t e d t o 7 0 ml  c e n t r i f u g a t i o n tube.  room t e m p e r a t u r e  The  then  15 ml  sample o f  These 2 procedures  i n an e q u a l v o l u m e PBS  final  aspirated  stain  a t room t e m p e r a t u r e .  The  conical  200  o f Hematology, Shaughnessy  remaining  resuspended  1.0  differential  w i t h a compound m i c r o s c o p e .  The  ml  u s i n g a C o u l t e r Counter  sample s t a i n e d w i t h W r i g h t s  the D i v i s i o n  321  cells  t y p e s were o b t a i n e d by  the blood  % EDTA. A one  6  1.5  of the cell  m o n o c y t e s p e r ml x 17 mm  i n a C0  0  tissue  initial  and  one  ml  culture  controlled  incubator  at  37°C f o r 1 h . T h e n o n - a d h e r e n t  c e l l s were t h e n removed a n d t h e  m o n o c y t e s washed f o u r t i m e s w i t h 1 ml a l i q u o t s were u s e d  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 t h a n 95% p u r e a s d e t e r m i n e d s t a i n i n g . Average 10  monocyte y i e l d  from  2.5  Metabolism  2.5.1  1 2 5  washed w i t h PBS.  medium c o n t a i n i n g  inhibitor  (LBTI)  ug/ml) a n d r e m a i n  1 2 5  I-LDL  trypsin  indicated  that  binding  S t u d i e s on reached  f o u r h o u r s o f i n c u b a t i o n w i t h LDL ( a d o s e constant f o r t h e next  o f 50  f o u r h o u r s . T h u s , an  i n these studies. At the  2 ml o f t h e medium was removed  into  0.5 m l o f 50% TCA. T h e T C A - s o l u b l e , n o n - i o d i d e  r a d i o a c t i v i t y was d e t e r m i n e d (128). C e l l  aliquots of ice-cold  a c c o r d i n g t o t h e method o f G o l d s t e i n  monolayers  were washed w i t h 3 x 2 ml  PBS c o n t a i n i n g  aliquots of ice-cold  lysed  (0-100 mg/ml) i n 2 ml  (0.6 mg/ml) was a d d e d t o t h e d i s h e s .  of the incubation period,  tubes c o n t a i n i n g  ml  i n vitro  t h e LPDS medium was removed  i n c u b a t i o n t i m e o f s i x h o u r s was u s e d  Brown  cells  BSA (2.0 mg/ml) a n d l i m a b e a n  time dependency o f b i n d i n g  saturation after  and  was 1-2 x  I - L D L Metabolism by F i b r o b l a s t s  and t h e m o n o l a y e r  end  69 ml o f p l a s m a  o f l i p o p r o t e i n s by c u l t u r e d  On t h e d a y o f t h e e x p e r i m e n t ,  the  by i m m u n o p e r o x i d a s e  cells.  7  of  o f PBS. The c e l l s  2 mg/ml BSA f o l l o w e d b y 3 x 2  PBS a l o n e . T h e c e l l  monolayers  i n 1.0 m l 0.1M NaOH f o r 30 m i n a t 3 7 ° C . p r o t e i n was d e t e r m i n e d  and t h e r e s u l t s  1 2 5  were t h e n  I-radioactivity  and  cell  f o r LDL b i n d i n g  and  d e g r a d a t i o n were e x p r e s s e d a s ng LDL p r o t e i n p e r mg o f c e l l  protein. The  specific  component o f b i n d i n g was d e t e r m i n e d  d i f f e r e n c e between t h e t o t a l  as t h e  b i n d i n g component a n d t h e n o n 51  specific  b i n d i n g component  determined excess  by c o n d u c t i n g  1 2 5  The n o n - s p e c i f i c component  t h e above e x p e r i m e n t  unlabelled ligand  2.5.2  (128)•  by  were t h e n  mg/ml), L B T I HDL  3  of  Fibroblasts  On t h e d a y o f t h e e x p e r i m e n t ,  The c e l l s  i n the presence  (128) ..  I - H D L Metabolism  x 1 ml a l i q u o t s o f MEM  was  f i b r o b l a s t s were washed  and p r e i n c u b a t e d w i t h  incubated with  (6 mg/ml) and e i t h e r  2 ml MEM  1 2 5  I-HDL  2 ml MEM  with 2  f o r 2 h.  c o n t a i n i n g BSA  (2  (0-100 mg/ml) o r  1  2  5  l -  (0-100 mg/ml) f o r 2 h a t 3 7 ° C . A t t h e e n d o f t h e i n c u b a t i o n  period,  HDL  performed  cell  and d e g r a d a t i o n  a c c o r d i n g t o t h e methods  determinations t h e LDL  association  a r e almost  identical  assays  were  o f Oram e t a l ( 7 5 ) . T h e s e t o those  d e s c r i b e d above f o r  analyses.  2.5.3  1 2 5  I-HDL  . ...To, d e t e r m i n e  3  Metabolism 1 2 5  I-HDL  3  by Monocytes  a s s o c i a t e d w i t h a n d d e g r a d e d by  Control  and T a n g i e r m o n o c y t e s , t h e f o l l o w i n g a s s a y was  Freshly  i s o l a t e d m o n o c y t e s were  containing  20% LPDS and  37°C. C e l l v i a b i l i t y  was  1 2 5  I-HDL  Monocyte c e l l  RPMI-1640  (0-200 ug/ml) f o r 4 o r 20 h a t  examined a t t h e end o f t h e experiment  trypan blue exclusion. Visual viability.  incubated with  used.  inspection indicated  associated  1 2 5  I-label  was  > 90% determined  a s i m i l a r manner a s 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 2.5.1)  52  by  in  (Section  3  Results  3.1  Catabolism Table  o f n o r m a l HDL  hypoalphalipoproteinemia i n other  o f n o r m a l HDL CE c o n t e n t levels  o f t h e TD p l a s m a was  patients with  t o TD p l a s m a ,  of control  this  ( 9 0 ) . The  i n t h e FC  fraction.  The t o t a l  Apo  and  A-I  amount o f CE  plasma remained s i g n i f i c a n t l y  s a m p l e s due t o t h e l o w CE a n d h i g h  3.1.1  TG  different  content  Effect  Normal HDL  of i n v i t r o  was  incubation  TD  quantity  to  on l i p o p r o t e i n  mobility  a d d e d t o TD p l a s m a  in sufficient  o f t h i s m i x t u r e were t r a n s f e r r e d t o g l a s s t e s t and i n c u b a t e d  s a m p l e s were examined Section  2.3.2.2  n o r m a l LDL incubated  100  The described  (Figure 5).  increased  linearly  f o r 24 h. T h i s of Carlson  c o n t r o l p l a s m a was  aliquots  (13 x  by a g a r o s e g e l e l e c t r o p h o r e s i s a s  During the i n c u b a t i o n  observations  tubes  ml  a t 3 7 ° C f o r 0,4,12 a n d 24 h .  I n c o n t r o l p l a s m a t h e m o b i l i t y o f LDL was incubation.  from  of the  r a i s e t h e HDL-C l e v e l s t o a p p r o x i m a t e l y 40 m g / d l . One  sealed  and  fraction.  electrophoretic  in  addition  o f t h e TD p l a s m a were a l s o r a i s e d t o w i t h i n t h e n o r m a l  LDL and VLDL  mm),  normal  previously  disorder  p l a s m a and HDL  and  and  r e s u l t e d i n an i n c r e a s e  i n both the t o t a l  i n the Tangier  control  content  hypocholesterolemia  r a n g e by t h e a d d i t i o n o f n o r m a l HDL. TG  in vitro  and a f t e r t h e a d d i t i o n o f e x o g e n o u s  The h y p e r t r i g l y c e r i d e m i a ,  described  plasma  IV shows t h e p r o t e i n and l i p i d  o f TD p l a s m a b e f o r e HDL.  by T a n g i e r  2.7  0.7  cm t o 1.2  cm  f i n d i n g i s c o n s i s t e n t with e t a l (149).  i n samples the  The m o b i l i t y o f t h e HDL  to incubation 53  to  a t 37°C t h e m o b i l i t y o f t h e  f r o m 0.7  cm p r i o r  cm p r i o r  and r e m a i n e d  in  TABLE I V . L I P I D AND TD PLASMA  PROTEIN CONTENT OF PLASMA IN THE PRESENCE AND  AND LIPOPROTEINS IN  ABSENCE OF EXOGENOUS  HIGH DENSITY LIPOPROTEIN. FRACTION  SAMPLE  COMPOSITION  (n=3)  CE  (mg/dl)  FC  TG  APO A - I  49+9  98+78  146+7  PLASMA CONTROL  115+18  **  TANGIER TANGIER + H D L  a  45±3  27+2 **  297+15 ***  43+13  276+48***  2+0.5 *** 129+6  71+10* HDL CONTROL TANGIER  38±9 2+1 ***  9+1  18+3  1+0.5***  3+1**  N.D. N.D.  TANGIER + HDL  33+5  11±4  28+6'  N.D.  CONTROL  76±16  40+10  79+28  N.D.  TANGIER  63±4*  TANGIER + HDL  38+12'  VLDL/LDL  ** 23+3  286+16***  N.D.  248+49***  N.D.  33+15 S u f f i c i e n t n o r m a l HDL was a d d e d t o a n a l i q u o t o f TD plasma t o r a i s e t h e 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 t o t h e d e 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 V L D L / L D L f r a c t i o n s r e f e r t o h e p a r i n / m a n g a n e s e 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 , respectively. N.D.= n o t d e t e r m i n e d . A p o p r o t e i n A - I l e v e l s were n o t determined i n t h e heparin/manganese p r e c i p i t a t e d plasma. c  S i g n i f i c a n c e o f d i f f e r e n c e f r o m 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 ; TG=triglyceride  54  TANGIER  CONTROL  + jpf  -  '  I  plasma  §13  Oh 4h  1  i  ;  'E <  '  0 1 2  F i g u r e 5.  B  $  £ I  i  I  i  i  3 4 4 Mobility  electrophoresis n o r m a l HDL  t o TD  1  on  for  n o r m a l HDL  the indicated  c a t h o d e t o anode  (left  control  respectively).  control  p l a s m a were  O  following  the addition  of  plasma. patterns  (A) and c o n t r o l  times.  1  1  lipoprotein  pattern  1% a g a r o s e g e l e l e c t r o p h o r e s i s containing  1  3 2 (cm)  E f f e c t of incubation  plasma  S  Direction  plasma  o f TD  (B) i n c u b a t e d a t 3 7 °  of electrophoresis  t o r i g h t and r i g h t t o l e f t The m o b i l i t y  : LDL; 0.7  cm;  55  plasma  f o r TD and  of lipoproteins VLDL;  1.3  cm;  i s from  i n the  HDL;  2.7  cm.  unchanged d u r i n g with  previous  mobility  the incubation period. This  reports  (149).  Carlson  suggest t h a t t h e increased  i s due t o a c h a n g e i n c h a r g e o f t h e LDL p a r t i c l e when i t  becomes o x i d i z e d a n d / o r p i c k s up f r e e f a t t y The and  acids  m o b i l i t y o f t h e LDLpj-, was 1.1 cm p r i o r  increased  linearly  during  consistent with  previous  (149).  to incubation  t h e 24 h i n c u b a t i o n t o a v a l u e o f  1.4 cm. The i n c r e a s e d m o b i l i t y o f LDL,p is  i s also consistent  reports  D  c o m p a r e d t o c o n t r o l LDL  ( 9 5 , 1 0 0 ) . T h e HDL i n TD  p l a s m a h a d a m o b i l i t y o f 2.7 cm p r i o r t o t h e i n c u b a t i o n a n d t h e mobility  remained r e l a t i v e l y  p e r i o d . The r e l a t i v e dramatically  during  unchanged d u r i n g  intensity  o f t h e HDL b a n d  the incubation period  to controls indicating that catabolism occuring  E f f e c t o f i n c u b a t i o n on Previous  i n TD p l a s m a  compared  o f t h e HDL p a r t i c l e  was  T h e y showed t h a t t h e l e v e l s  decreased  HDL-cholesterol  o f HDL-C a n d t o t a l  level  that described  i n this  p l a s m a apo  r a p i d l y i n TD p l a s m a f o l l o w i n g t h e i n f u s i o n o f 3 1 o f HDL-C c o n c e n t r a t i o n  mg/dl b u t o v e r t h e 66 h a f t e r p l a s m a p h e r e s i s original  examined t h e  i n t h e TD p a t i e n t s t u d i e d  o f normal plasma. The peak v a l u e  data  diminished  work b y P r i t c h a r d and F r o h l i c h (12 4)  i n v i v o r a t e o f HDL c a t a b o l i s m  A-I  incubation  in vitro.  3 . 1 . 2  thesis.  t h e 24 h  i treturned  o f 2 m g / d l . T h i s r e s u l t was a l m o s t f o r " p a t i e n t A" b y S c h a e f e r  i n d i c a t e that both of these  was 20 to the  identical to  e t a l (12 5 ) .  TD p a t i e n t s h a d s i m i l a r  These HDL-C  turnover. Pritchard  and F r o h l i c h a l s o n o t e d r a p i d l o s s o f apo A - I f r o m  t h e p l a s m a o f t h e TD p a t i e n t followed  (124).  a b i e x p o n e n t i a l curve  with 56  The d i s a p p e a r a n c e o f apo A - I an i n i t i a l  fast  component and  a  s u b s e q u e n t s l o w component. The f a i l u r e  normal l e v e l s  and i t s r a p i d  was s i m i l a r t o t h a t  clearance  o f l a r g e amounts o f HDL  experiment examined incubation  from t h e plasma  compartment  r e p o r t e d by S c h a e f e r e t a l (125).  S i n c e HDL-C i n TD p l a s m a r a p i d l y infusion  o f apo A - I t o r e a c h  t h e changes  i n TD p l a s m a  decreases i n vivo  (107-114,  125), t h e f o l l o w i n g  i n HDL-C o f c o n t r o l  in vitro.  Exogenous  e q u a l volume  o f b u f f e r C was a d d e d  HDL-C l e v e l s  t o 40 mg/dl.  after  HDL d u r i n g  n o r m a l HDL and an  to control  plasma t o r a i s e  One ml s a m p l e s o f c o n t r o l  a n d HDL-  e n r i c h e d TD p l a s m a were i n c u b a t e d a t 37°C  f o r 0,4,8,12 a n d 24 h .  HDL-C l e v e l s were d e t e r m i n e d a s d e s c r i b e d  i n Section  results  of this  experiment a r e presented  T h i s experiment demonstrates t h a t loss of cholesterol occurs i n v i t r o .  i n Figure  2.2.5.2. The  6.  the previously observed  f r o m n o r m a l HDL i n f u s e d  i n t o TD p l a s m a  A 35% d e c r e a s e i n HDL-C o c c u r r e d  also  over the f i r s t  4 h o f t h e i n c u b a t i o n w i t h a f u r t h e r d e c r e a s e t o 50% o f i n i t i a l values after slightly  24 h . I n c o n t r o l p l a s m a , HDL-C l e v e l s  d u r i n g t h e 24 h i n c u b a t i o n  d e c r e a s e i n HDL-C i n t h e TD p l a s m a " h y p e r c a t a b o l i s m " o f HDL i n f u s e d plasma  factors  in vitro  were f o l l o w e d  this  suggests that the i s d e p e n d e n t on  involvement. In the  i n p l a s m a , HDL, a n d LDL/VLDL  o v e r a 12 h p e r i o d .  57  6 ) . Thus  i n t o TD p l a s m a  and d o e s n o t r e q u i r e c e l l u l a r  f o l l o w i n g experiment, t h e changes lipid  (Figure  increased  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  cholesterol  incubated i n control  content  o f normal  HDL  o r TD p l a s m a .  Normal HDL was a d d e d t o s a m p l e s o f TD p l a s m a a s described  i n Table  37°C ... The i n t i t a l plasma i s i n d i c a t e d cholesterol from  control  mean + S.D.  I V and i n c u b a t e d  f o r the indicated  HDL c o n c e n t r a t i o n o f t h e c o n t r o l i n Table  I V . The f i g u r e  a s s o c i a t e d w i t h HDL r e l a t i v e (A)  o r T a n g i e r plasma  f o r t h r e e independent  58  times a t  a n d TD  shows t h e amount o f  to the i n i t i a l  (H)• The d a t a experiments.  value  represent  3.1.3  Effect  of i n v i t r o  incubation  on l i p o p r o t e i n l i p i d  composition The  relative  c h a n g e s i n t h e amount o f FC, CE a n d TG i n t h e  p l a s m a , HDL and VLDL/LDL f r a c t i o n s f o l l o w i n g t h e i n v i t r o incubation  o f n o r m a l HDL w i t h  TD p l a s m a a r e shown  I n c o n t r o l p l a s m a , CE l e v e l s incubation period. the  However, t h i s  a d d i t i o n o f 5 mM  increased increase  c o u l d be i n h i b i t e d (DiFP)  inhibits  CE c o n t e n t  o f t h e TD p l a s m a r e m a i n e d u n c h a n g e d d u r i n g t h e  this patient  The o b s e r v a t i o n  r e f l e c t s t h e low LCAT a c t i v i t y  [68% o f normal  C o n t r o l plasma l e v e l s  (124)]. o f FC d e c r e a s e d b y 24 +8%  t h e r a t e o f CE p r o d u c t i o n  remained unchanged  substrate was  i n d i c a t i n g the u t i l i z a t i o n  the f i r s t  l e v e l s d e c r e a s e d by 34 +17% d u r i n g indicate that the i n i t i a l  first  of t h i s  fraction  Tangier  c h a n g e i n HDL-FC  plasma  but the  4 h. These r e s u l t s  l o s s o f c h o l e s t e r o l from normal  8 h of incubation  HDL  i s i n t h e f o r m o f CE a n d n o t FC. increased  linearly  a f t e r w h i c h no f u r t h e r  i n c r e a s e s were s e e n . By c o n t r a s t , d e c r e a s e d by 43 +4%  as a  8 h of incubation  the l a s t  I n c o n t r o l p l a s m a , HDL-CE l e v e l s the  o f TG  o v e r a 12 h  f o r t h e LCAT r e a c t i o n . However, l i t t l e  in  differences  i n b o t h c o n t r o l a n d TD p l a s m a .  s e e n i n TD p l a s m a d u r i n g  incubated  but remained  i n b o t h samples. Plasma l e v e l s  C o n t r o l HDL-FC l e v e l s d e c r e a s e d by 17 +9% incubation  that the  i n t h e plasma o f  u n c h a n g e d i n t h e TD p l a s m a w h i c h was c o n s i s t e n t w i t h in  by  which  completely  incubation period  (150).  V.  by 17 + 6% d u r i n g t h e  diisopropylfluorphosphate LCAT a c t i v i t y  i n Table  i n the f i r s t  59  HDL-CE l e v e l s  during  significant  i n TD  4 h of the incubation  plasma but d i d  TABLE V . CHANGES IN THE RELATIVE L I P I D COMPOSITION OF TD LIPOPROTEINS FOLLOWING  INCUBATION IN THE  PRESENCE OF EXOGENOUS NORMAL HIGH DENSITY LIPOPROTEINS. Samples o f c o n t r o l , TD and TD + HDL plasma ( T a b l e IV) were i n c u b a t e d f o r t h e i n d i c a t e d t i m e a t 37 C. The r e a c t i o n s were s t o p p e d by r a p i d c o o l i n g t o 0°C. The amount o f l i p i d was d e t e r m i n e d b e f o r e and a f t e r p r e c i p i t a t i o n o f VLDL and LDL by heparin/manganese. The r e s u l t s a r e e x p r e s s e d as means+1 SD o f t h e amount r e c o v e r e d r e l a t i v e t o time= Oh. The i n i t i a l v a l u e s f o r ^ e a c h sample a r e i n d i c a t e d i n T a b l e I V . S i g n i f i c a n c e o f d i f f e r e n c e o f TD from c o n t r o l : * p<0.05; * p<0.01; p<0.001 PERCENTAGE OF I N I T I A L VALUE FRACTION  JBATI0M IE (h)  CHOLESTERYL ESTER  FREE CHOLESTEROL  TRIACYLGLYCEROL  CONTROL n=4  TANGIER n=4  CONTROL n=4  TANGIER n=4  CONTROL n=3  TANGIER n=3  0  100  100  100  100  100  100  4  108+4  84+5  101+8*  99+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  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  121+14  193+31**  100  100  PLASMA  O  98+3**  HDL 100  60+17  VLDL/LDL 100  100  4  109+7  151+32*  a i+6  8  119+7  188+29*  76±10  95+11  116+9 .  174+29* • ' .  .. 75+10  116+25  12  100  100  0  101+14  100 + 1  9 5+4  9 2+2  99 + 14  91 + 5  97+7  90+2  not  decrease f u r t h e r during  the remaining  8 h . The d i f f e r e n c e  b e t w e e n TD and c o n t r o l , f o r b o t h t h e a b s o l u t e o f change, a r e h i g h l y s i g n i f i c a n t  (P<0.001)  I n c o n t r o l p l a s m a , HDL-TG l e v e l s first  8 h of incubation with  during the  the remaining  n o r m a l HDL  from t h e core  by 32 +14% changes  4 h . By c o n t r a s t , t h e r e l a t i v e  8 h. T h i s  during the  occuring TG c o n t e n t  t o 224 ± 4 % o f t h e i n i t i a l  observation  o f t h e HDL may  o f TG f r o m t h e VLDL/LDL It  (Table V ) .  no s i g n i f i c a n t  i n TD p l a s m a r o s e  over the f i r s t  rose  change and t h e r a t e  of  amount  s u g g e s t s t h a t t h e l o s s o f CE  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  fraction.  was n o t e d t h a t t h e l o s s o f HDL-CE a n d t h e i n c r e a s e i n  HDL-TG were, on a m o l a r b a s i s , e s s e n t i a l l y  t h e same f o r b o t h  control  suggest t h a t there may  be  and TD p l a s m a . T h u s , t h e s e  results  molecule of C E i n  a n e x c h a n g e o f 1 m o l e c u l e o f TG f o r e v e r y  the  core  o f t h e HDL  particle.  I n c o n t r o l p l a s m a , VLDL/LDL l e v e l s during  the f i r s t  8 h of incubation with  o f CE i n c r e a s e d b y 19 no f u r t h e r  increases  observed during the l a s t  4 h. T h i s  t r a n s f e r o f CE f r o m HDL.  The r a t e o f t h i s t r a n s f e r was  significantly  (P<0.001) g r e a t e r  h t h e r e was a n 88 +29% with  no s i g n i f i c a n t  increase  +6%  i n the f i r s t  i n TD p l a s m a . D u r i n g  changes d u r i n g  VLDL/LDL FC c h o l e s t e r o l l e v e l s 4 h followed  increase probably  i n t h e CE c o n t e n t the l a s t  decreased  +7%  r e f l e c t s the  the f i r s t  8  o f TD VLDL/LDL  4 h of incubation.  i n c o n t r o l p l a s m a by 19  by a f u r t h e r d e c r e a s e s d u r i n g t h e  s u b s e q u e n t 8 h o f i n c u b a t i o n . However, l i t t l e  c h a n g e i n VLDL/LDL  FC  8 h . VLDL/LDL TG  was o b s e r v e d  levels  i n TD p l a s m a d u r i n g  remained unchanged  the f i r s t  i n c o n t r o l p l a s m a d u r i n g t h e 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 a n  61  approximate  10% d e c r e a s e  during the f i r s t  f o l l o w e d by a s m a l l d e c r e a s e 3.1.4  during the remaining  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 The  relative  TD p l a s m a a f t e r respectively,  amount o f apo A - I r e m a i n i n g  exists  w h i c h was a p p r o x i m a t e l y  suggest  vivo probably It  t h a t t h e observed  i n t h e c o n t r o l and  95% o f t h e i n i t i a l  specific  values.  f o r n o r m a l apo A - I ,  l o s s o f apo A - I f r o m  requires cellular  these  TD p l a s m a  interaction.  h a s p r e v i o u s l y b e e n r e p o r t e d t h a t apo A - I a n d A - I I f r o m  n o r m a l HDL r e d i s t r i b u t e Therefore,  The  composition  i n t h e p l a s m a o f t h e p a t i e n t w i t h TD d i s e a s e . T h u s ,  results  of  8 h.  a 24 h i n c u b a t i o n was 108 +5 a n d 105 +6 mg/dl  T h i s d e m o n s t r a t e s t h a t no p r o t e a s e ,  in  4 h of incubation  these  among TD l i p o p r o t e i n s  i n the following investigation,  approximately  (125).  the i n vitro  a p o p r o t e i n s between t h e l i p o p r o t e i n  HDL-C l e v e l  i n vivo  transfer  c l a s s e s was s t u d i e d .  o f 700 u l o f TD p l a s m a was a d j u s t e d t o  45 mg/dl b y t h e 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 t h e sample was i n c u b a t e d a t 4 ° C a n d t h e o t h e r h a l f was i n c u b a t e d a t 3 7 ° C . A f t e r  i n c u b a t i n g f o r 24 h t h e p l a s m a  l i p o p r o t e i n s were s e p a r a t e d b y g e l f i l t r a t i o n t h e amount o f l a b e l determined.  recovered  More t h a n  i n each l i p o p r o t e i n  95% o f t h e ^ -  2 5  i  of this  At  after  are presented  24 h o u r s .  •  I n c u b a t i o n a t 37°C g r e a t l y  1  2  5  I )  91% o f a c t i v i t y  LDL and l e s s  62  The  i n F i g u r e 7.  among t h e l i p o p r o t e i n s w i t h  w i t h t h e HDL, 7% w i t h  applied to  fractions.  4 ° C , apo A - I a n d A - I I ( r e p r e s e n t e d b y t o t a l  redistributed remaining  experiment  class  radioactivity  t h e c o l u m n was r e c o v e r e d w i t h t h e l i p o p r o t e i n results  on B i o g e l A5m and  than  1% w i t h  increased the  VLDL  40  50  60  70  Fraction number  F i g u r e 7.  Gel f i l t r a t i o n  chromatography  apoproteins following or  added  HDL-C c o n c e n t r a t i o n o f 45 m g / d l . (H)  on a B i o g e l  i n the c o l l e c t e d  A5m  After  column.  63  1 2 5  aliquots  The amount o f i s shown. The  eluted  o f t h e column w i t h  i n c u b a t i o n a t 4°C  i n 2 mL  fractions  w h i c h n o r m a l VLDL, LDL and HDL precalibration  i n TD p l a s m a a t 4 ° G  t o TD p l a s m a t o g i v e a  f o r 24 h, t h e l i p o p r o t e i n s  were r e s o l v e d recovered  incubation  37°C.  125 I-HDL (100 cpm/ng) was  37°C  o f 125 I-HDL-  (£)  and  o f plasma 1  2  5  i  fractions  were d e t e r m i n e d  I-lipoprotiens.  final  by  in  redistribution the  of r a d i o a c t i v i t y  r a d i o a c t i v i t y was  t h e VLDL. The from  and  incubation. 37°C t h a n of  of the  Since redistribution this  apoproteins  composition  of  HDL  T D  o f HDL  are temperature  and  was  LDL  2%  of  with  recovered  during  apoprotein  apoprotein occurs  i s greater at  t h a t r a t e o f movement  that occurred during the  Thus,  may  be  significant  independently  (Table  during the  4°C  changes i n the  t h a t some of the  lipid  incubation  redistributed  i t appears  from  in vitro  dependent changes i n t h e  of the r a d i o a c t i v i t y  composition.  T D  d e p e n d e n t and  i n c u b a t i o n e v e n t h o u g h t h e r e were no lipid  fraction  VLDL. 18%  apoproteins dissociate  observation suggests  o f t h e HDL  However, 8%  HDL  and  I-apoprotem  X £ d  i n d i c a t e t h a t HDL  a s s o c i a t e d w i t h the temperature  V).  t o LDL  i n t h e LDL  reassociate with VLDL  a t 4°C  these  79%  HDL  fraction.  These r e s u l t s n o r m a l HDL  recovered  remaining  t h e HDL  from  redistribution  lipid  exchange  process. In  a d d i t i o n to the t r a n s f e r of  HDL  D  - ^-'I was  also recovered  L  ,  (fraction  68  label  of t h i s (free  1  2  Schaefer and  apo  f r a c t i o n we 5  we do  have been u n a b l e know t h i s  i e l u t e s much l a t e r ;  et a l  and  after after  t o c h a r a c t e r i z e the  does not  unevenly  among TD  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  therefore also  more a p p a r e n t  greater than  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  The  D  represent fraction  free 100).  (110,12 5) h a v e p r e v i o u s l y shown t h a t apo  A-II d i s t r i b u t e  following  VLDLrp  i n a minor peak which e l u t e d  i n F i g u r e 7 ) . T h i s p e a k was  i n c u b a t i o n a t 37°C. While nature  I - a p o p r o t e i n to  ,  lit L D L r p  1 2 5  s t u d i e d i n our  B i o g e l A 5.0m  in vitro  column f r a c t i o n s 64  plasma  A-I  lipoprotein  redistribution " I - H D L i n TD  of  these  plasma  was  experiments. c o n t a i n i n g VLDL, LDL  and  HDL  (Figure  7)  were r e s o l v e d  were c o n c e n t r a t e d by  12.5%  are  shown i n T a b l e V I .  the  apo  A-I  and  respectively.  apo  This  apo  A-I  26.7% only  and  14.9%  results  apo  of  are  occurred  the  at the  the  A-I  was  a greater  relative  in vivo  in vitro  the  lipoproteins.  I n an  in this  observations  labelled with  [ H]  at  4°C  and  (Figure  3 7 ° C and  8). This  h  fraction. of  apo  of of  whereas These  Schaefer et a l A-II  became  l i p o p r o t e i n s t h a n d i d apo the  does not  of  possibly,  different. A total  transfer  A-I.  of  r e q u i r e the  concurrent  plasma l i p o p r o t e i n s . the  incubation  s i z e d i s t r i b u t i o n of the earlier  8%  two  transfer rates  percentage of the  and  and  VLDL/LDL f r a c t i o n s  same r a t e and,  above experiment i n d i c a t e s t h a t  m a j o r e f f e c t s on  7%  t r a n s f e r of the  recovered  lower d e n s i t y  c e l l u l a r metabolism of the The  only  i n the  Furthermore, these r e s u l t s suggest t h a t apoproteins occurs  experiment  t r a n s f e r r e d t o VLDL/LDL i n 24  s i m i l a r t o the  with the  a t 4°C,  were s i g n i f i c a n t l y  apo  i n d i v i d u a l apoproteins  r e s u l t s of t h i s  were r e c o v e r e d  A^-II was  (110,125) i n t h a t associated  A-II  A-II  the  After incubation  However, a t 37°C, t h e  apo  of the  SDS-PAGE. The  suggests t h a t  a p o p r o t e i n s a t 4°C concurrently.  and  e x p e r i m e n t TD  l i p o p r o t e i n s were  a Biogel A  experiment demonstrates t h a t  changes i n l i p o p r o t e i n p a r t i c l e  no  various  cholesterol after incubation t h e n s e p a r a t e on  has  size during  with normal 5.0m  there  HDL  column were no  incubation  at  major  4°C  or  37°C. The  e f f e c t of  f u r t h e r examined HDL  peak  incubation  i n the  on  the  s i z e of  f o l l o w i n g s t u d y . The  ( F r a c t i o n s 51-72) was  pooled,  65  t h e HDL  particle  Biogel A  concentrated  5.0m  and  re-  was  column  applied  t o an B i o g e l  A 0.5m c o l u m n . T h e r e s u l t s o f t h i s  experiment a r e presented  i n F i g u r e 9. T h e s e r e s u l t s  indicate  the  s i z e t h e HDL p a r t i c l e was n o t s i g n i f i c a n t l y c h a n g e d  the  e x p e r i m e n t by c o m p a r i s o n w i t h  column c a n s e p a r a t e control  HDL  and TD m o d i f i e d  presence of a s i n g l e in v i t r o conversion  2  1 2 5  that  during  I - H D L . W h i l e t h e AO.5m  and HDL , t h e c o l u m n p r o f i l e o f b o t h  1 2 5  3  I - H D L showed o n l y one p e a k . T h e  subspecies o f HDL  3  o f HDL i s n o t s u r p r i s i n g  t o HDL, i s w e l l  66  as t h e  documented ( 4 7 ) .  TABLE V I . DISTRIBUTION OF  I - A P O A - I AND A - I I IN TANGIER  1 2 5  LIPOPROTEINS AFTER INCUBATION Aliquots experiment (28-32),  WITH  o f t h e column f r a c t i o n s  described i n figure  c o n c e n t r a t e d by amicon f i l t r a t i o n  of. • I a s s o c i a t e d w i t h e a c h results  indicated  s a m p l e s were into  T h e b a n d s were corresponding  t h e g e l and t h e amount determined.  amount o f l a b e l t h a t  a p o p r o t e i n i n each  difference  VLDL  i n 12.5% p o l y a c l y a m i d e  lipoprotein  i s p r e s e n t e d a s mean+1 SD f o r 3 e x p e r i m e n t s . significant  as f o l l o w s :  o f t h e s e p r o t e i n s was  the relative  a s s o c c i a t e d w i t h each  from t h e  i n commassie b l u e a n d a r e a s  t o apo A - I a n d apo A - I I were c u t from  The  - I HDL.  a n d a l i q u o t s were r e s o l v e d  c o n t a i n i n g 1%SDS a n d 1% 2 - m e r c a p t o e t h a n o l .  3  5  The r e s u l t i n g  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  v i s u a l i z e d by s t a i n i n g  2  obtained  4 were p o o l e d  LDL (33-50) a n d HDL ( 5 1 - 7 2 ) .  1  was  class.  Data  *** i n d i c a t e s a  (P<0.001) b e t w e e n t h e s a m p l e s i n c u b a t e d a t  4°C a n d 3 7 ° C .  PERCENTAGE DISTRIBUTION OF 4°C  1  2  5  I  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  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***  85.1+1.7  ***  APO A - I I  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 containing  o f [ H] c h o l e s t e r o l 3  HDL f o l l o w i n g  labeled  incubation  TD p l a s m a  a t 4 ° and  37°. HDL was added t o TD p l a s m a t o g i v e a f i n a l c o n c e n t r a t i o n o f 45 m g / d l . a n d 37°C  After  (Bj) , 2 ml a l i q u o t s  cholesterol  as d e s c r i b e d  were r e s o l v e d  on a B i o g e l  t h e column w i t h  1 2 5  f o r 24 h o u r s a t 4 ° C (©)  o f p l a s m a were l a b e l l e d w i t h  i n 'Methods'  were d e t e r m i n e d  I-lipoprotiens.  68  [ H] 3  and t h e plasma l i p o p r o t e i n s :  A5.0m c o l u m n . T h e f r a c t i o n s  n o r m a l VLDL, LDL and HDL e l u t e d of  incubation  HDL-C  i n which  by p r e c a l i b r a t i o n  T  Figure  9.  1  Gel f i l t r a t i o n incubation  1  j  1  1  x 10  6  and a volume  I-HDL  following  o f t h e column  ( F i g u r e 7, f r a c t i o n s 5 1 -  o f sample  corresponding  on a B i o g e l A 0.5m  i n w h i c h n o r m a l HDL e l u t e d  precalibration  r  i n TD p l a s m a a t 4 ° a n d 3 7 ° .  cpms was t h e n r e s o l v e d  fractions  j  chromatography o f  B o t h 4 ° (©) and 3 7 ° <-(H) HDL p e a k s 72) were c o n c e n t r a t e d  1  with  1 2 5  column. The  was d e t e r m i n e d b y I-HDL.  to 1  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 n o r m a l HDL d u r i n g  3.2.1  incubation  i n TD p l a s m a  Role o f c h o l e s t e r y l e s t e r The p o s s i b i l i t y  TD p l a s m a  that  hydrolysis  t h e l o s s o f CE f r o m HDL i n c u b a t e d  i n v i t r o may r e s u l t f r o m CE h y d r o l y s i s  e x a m i n e d b y l a b e l i n g c o n t r o l a n d TD p l a s m a e x o g e n o u s HDL was i n c l u d e d ) incubating  with  with  (CEH) was  ( i n some TD  samples  [ H ] CE f o r 4 h a t 4 ° C . A f t e r 3  t h e s a m p l e s a t 37°C o r 4 ° C f o r 24 h t h e r a t e o f CEH  was d e t e r m i n e d . The r e s u l t s o f t h i s VII  the modification of  e x p e r i m e n t a r e shown i n T a b l e  ( t h e r a t e o f CEH i s e x p r e s s e d a s p e r c e n t a g e h y d r o l y s i s o f  total  CE o c c u r i n g  during  a 24 h ) .  The r e s u l t s c l e a r l y indistiguishable  show t h e r a t e o f CEH i n TD p l a s m a i s  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 ) . I n a s u b s e q u e n t e x p e r i m e n t , n o r m a l HDL was a d d e d t o the  TD p l a s m a p r i o r t o [ H ] CE l a b e l i n g and s u b s e q u e n t 3  incubation.  This  resulted i n a slight,  but i n s i g n i f i c a n t ,  decrease  i n t h e r a t e o f CEH i n TD p l a s m a  Finally,  e v e n when HDL was p r e l a b e l e d ,  (Table  before  p l a s m a , t h e r a t e o f CEH was n o t s i g n i f i c a n t l y c o n t r o l values the  (Table  hypothesis that  during  in vitro  V I I , row 4 ) .  a d d i t i o n t o t h e TD increased  over  V I I , row 5 ) . T h e s e r e s u l t s do n o t s u p p o r t  t h e l o s s o f CE f r o m n o r m a l HDL i n TD p l a s m a  incubation  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 d e t e r m i n e d  plasma by i n c u b a t i n g 4°C  i n controls  plasma p r e l a b e l l e d w i t h  (2) a n d TD  [ H ] CE f o r 24 h a t 3  a n d 3 7 ° C . I n a d d i t i o n t h e CEH a c t i v i t y was a l s o e x a m i n e d b y  incubation  o f TD p l a s m a w i t h  HDL p r e l a b e l l e d w i t h  amount o f r a d i o l a b e l l e d CE h y d r o l y z e d determined  during  [ H ] CE. T h e 3  the incubation  was  ( M e t h o d s ) . The r e s u l t s a r e e x p r e s s e d a s t h e p e r c e n t a g e  o f CE t h a t was h y d r o l y z e d  during  the incubation  period.  Percentage H y d r o l y s i s (%)  Control  1  4.8  Control  2  6.1  TD Tangier  6.1 + *HDL  * HDL p r e l a b e l l e d w i t h  3.0  [ H ] CE 3  The r e s u l t s a r e t h e mean o f d u p l i c a t e experiment.  71  incubations  from a s i n g l e  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 The p r e c e d i n g  during  incubation  differences  studies  acyltransferase  on t h e m o d i f i c a t i o n  w i t h TD p l a s m a  (Section  i n the rate of synthesis  o f HDL t h a t  occurs  3.1) d e m o n s t r a t e t h e  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 o f CE b e t w e e n TD and c o n t r o l p l a s m a . A s LCAT p l a y s central  r o l e i n t h i s process  in the modification Table VIII by 5 mM  i n c o n t r o l p l a s m a , t h e r o l e o f LCAT  o f HDL b y TD p l a s m a was  shows t h a t  examined.  complete i n h i b i t i o n  o f LCAT  activity  D i F P h a d no e f f e c t on t h e e f f l u x o f c h o l e s t e r o l f r o m  n o r m a l HDL i n TD p l a s m a . F u r t h e r m o r e , when n o r m a l HDL was t o t h e p l a s m a o f a p a t i e n t w i t h homozygous LCAT l o s s o f HDL-C was s i g n i f i c a n t l y TD p l a s m a  (Table  to the i n i t i a l  V I I I ) . The s m a l l  rates  inhibition  modification  deficiency/the  but s i g n i f i c a n t  loss i n  l o s s o f HDL-C  i s probably  related  o f CE t r a n s f e r w h i c h a r e n o t i n f l u e n c e d b y o f LCAT  ( 1 5 1 ) . A s i m i l a r l o s s o f HDL-C was  seen i n c o n t r o l plasma c o n t a i n i n g indicate that  added  lower than t h e e q u i v a l e n t  f r o m . n o r m a l HDL i n t h e LCAT d e f i c i e n t p l a s m a  chemical  a  LCAT i s n o t d i r e c t l y  5 mM  DiFP. These r e s u l t s  responsible  f o r the  o f n o r m a l HDL i n TD p l a s m a d u r i n g  in vitro  incubations.  72  TABLE V I I I .  EFFECT OF  LCAT A C T I V I T Y AND  CHOLESTEROL FROM NORMAL HDL Plasma  f r o m TD  reconstituted  w i t h HDL  mg/dL. A d d i t i o n a l supplemented incubation  and  a t 37°C  L E V E L ON  to a final  and TD  DiFP t o i n h i b i t  plasma  HDL-C (% o f i n t i t a l  Control  124  105.0  LCAT D e f i c i e n t  823  94.0  TD  284  57.4  N.D  = not  PLASMA'-'  45  were  LCAT a c t i v i t y .  f o r 24 h, HDL-C l e v e l s w e r e  OF  were  After-  determined.  value)  - DiFP  + HDL  LOSS  HDL-C c o n c e n t r a t i o n o f  of control  Plasma TG (mg/dl)  THE  INCUBATED IN TD  LCAT d e f i c i e n t p a t i e n t s  samples  w i t h 5 mM  TG  +  DiFP  85.0 N.D* 56.3  determined.  The r e s u l t s a r e t h e means o f d u p l i c a t e experiment.  73  incubations  from a  single  3.2.3  Role of hypertriglyceridemia The  in  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  the modification  In t h e s e s t u d i e s  o f HDL was e x a m i n e d  the a b i l i t y  subjects, the  studies.  hypertriglyceridemia  In hypertriglyceridemia  '  f r o m 200 t o 341 mg/dl  while  t h e p l a s m a TG l e v e l s r a n g e d  HDL-C l e v e l s r a n g e d  i n the following  of moderately  p l a s m a t o m o d i f y HDL was d e t e r m i n e d .  i n t h e TD p l a s m a  f r o m 17 t o 54 m g / d l . A f t e r i n c u b a t i o n a t  37°C f o r 12 h t h e l o s s o f HDL-C was d e t e r m i n e d a s d e s c r i b e d and  the r e s u l t s are presented  i n Figure  above  10.  I n none o f t h e s e s a m p l e s d i d t h e l o s s o f t h e HDL-C e x c e e d 20%.  F u r t h e r m o r e , when t h e HDL-C l e v e l s o f two s a m p l e s w e r e  supplemented w i t h s u f f i c i e n t  e x o g e n o u s HDL t o r a i s e t h e HDL-C  l e v e l s t o a b o v e 50 m g / d l , t h e l o s s o f HDL-C was s t i l l and loss  17% o f i n i t i a l  values  o n l y 15%  r e s p e c t i v e l y . T h e s e d a t a show t h a t t h e  o f c h o l e s t e r o l f r o m HDL i n c u b a t e d  greater  :  t h a n c a n be e x p l a i n e d  i n TD p l a s m a i s f a r  by a s i m p l e  equilibration of l i p i d  (9). Consistent  with these r e s u l t s i s the previous  observation  t h a t when LCAT d e f i c i e n t p l a s m a , s u p p l e m e n t e d w i t h e x o g e n o u s HDL, was i n c u b a t e d  f o r 24 h a t 37°C, t h e HDL-C l e v e l s d e c r e a s e d t o 94%  of the i n i t i a l [TG  = 323 mg/dl  value  despite  (Table  the presence o f hypertriglyceridemia  VIII).  74  70-  Q  I  1  I  .  Tangier  |  1  i  1  H  c  50-  a) o  30-  o  •  0)  # ®  10-  CD CO  HyperTG Normal  c  D L.  — I  -10- 1  o o »  o  i  100  i  i  200  «  1  1  300  400  Plasma TG ( m g / d l ) Figure  10.  Plasma  L o s s o f HDL-C d u r i n g  incubation  hypertriglyceridemic  subjects.  from h y p e r t r i g l y c e r i d e m i c  341 mg/dl) was i n c u b a t e d values  subjects  f o r 12 h a t 37°C  were d e t e r m i n e d b e f o r e  from  (TG r a n g e  200 t o  o r 4 ° C a n d t h e HDL-C  and a f t e r t h i s  75  o f plasma  incubation.  3.3  In v i t r o  3.3.1  catabolism of H D L incubated  plasma  Introduction Infusion  biexponential section  3.1  o f n o r m a l H D L i n TD p l a s m a decay o f  loss  i n c u b a t e d w i t h TD p l a s m a  in vitro.  We  from normal H D L . be due  thus t h i s isolated  H D L  The  particle  rapid  to increased  p r o p o s a l was  cellular  examined  from normal plasma  e x p e r i m e n t . The incubation  changes  that  include  occur  a general  i n H D L during documented  associated  increase  conversion  i s generally understood l i t t l e  ability  of these p a r t i c l e s  distinct  of,\HDLrj  D  study.  HDL,  f o r t h e same t i m e a s  increase  in particle  demonstrated  used as a c o n t r o l  are well  size.  to interact  HDL  f r o m TD p l a s m a i n  i n the following  was  from  t o VLDLpjj  we  catabolism  incubated  o f normal plasma  These changes  of H D L - p r o t e i n  In  that  i s chemically  D  loss  ( c o n t a i n i n g normal H D L )  plasma  showed  Further,  (HDLrp )  rapid,  from plasma.  of cholesterol  also  during the incubation.  that the r e s u l t i n g  v i v o may  i n the  amounts o f H D L - p r o t e i n were t r a n s f e r r e d  particles  T D  results  and H D L - p r o t e i n  the rapid  significant  we  HDL-C  described  LDL  i n TD  and  TD  in this  in vitro (149,  i n HDL-TG  151-161)  and  an  W h i l e t h e mechanism o f  this  i s known a b o u t t h e  with the f i b r o b l a s t  HDL  receptor. 3.3.2  Examination of the i n v i t r o metabolism of HDLTJTQ  and  H D L  3  7  T w e n t y - f i v e ml, of  samples  n o r m o l i p e m i c d o n o r s and  level  o f TD p l a s m a was  addition the  by c u l t u r e d  o f normal H D L .  control  plasma  fibroblast  o f p l a s m a was  from a p a t i e n t  increased An  skin  obtained with  TD.  The  pool  H D L - C  t o a p p r o x i m a t e l y 4 5 mg/dl b y t h e  e q u a l volume o f b u f f e r  and b o t h s a m p l e s w e r e i n c u b a t e d  76  from a  C was  added  f o r 24 h a t  to  the H D L  37°C. A f t e r i n c u b a t i o n described analyzed  was  i n s e c t i o n 2.3.1.1. The by  (Figure  S D S - P A G E  saturation inability relatively the  saturation  large non-specific binding binding  The  specific  component o f H D L  T  H D L  iodinated, binding  2.7.2. HDLrpQ  nor  H D L  i s explained  reached  7  The  by t h e  component w h i c h  obscures  component. binding  was d e t e r m i n e d a s  and  D  H D L  3  7  analysis of the s p e c i f i c  were done  (Figure  binding  13). These  component  analysis  d  for  HDLrj,  3  a d d i t i o n , t h e maximum c a p a c i t y  of ligand binding  (B  protein/2h  and  k i n e t i c parameters  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  ,  indicate that  HDLrpp  receptor  as does  in vitro  incubation  its  ability  protein  3  7  .  The r e s u l t s o f t h i s  study  indicate  o f n o r m a l H D L i n TD p l a s m a d o e s n o t  (Figure  f o r both  H D L , p  D  and  H D L  3  7  were  14) t h e s e r e s u l t s s u g g e s t t h a t  c o m p o s i t i o n do n o t e f f e c t t h e a b i l i t y  )  H D L  f o r the  of the incubation  that  As t h e  nearly changes i n core  of this particle to  medium f o r d e g r a d a t i o n  77  D  H D L  i n t e r a c t with the H D L receptor. Analysis  T  increase  t o i n t e r a c t with the f i b r o b l a s t H D Lreceptor.  composition  identical lipid  H D L  h a s e s s e n t i a l l y t h e same a f f i n i t y  for  m a x  was d e t e r m i n e d t o be 189 a n d 183 ug/mg c e l l H D L  D  respectively.  HDL 7,  In  3  (0 t o 50 u g / m l ) .  i n d i c a t e d K ' s o f 14.93 and_.12.3 5 ug/ml were o b t a i n e d and  and  i n s e c t i o n 2.7.1 a n d t h e r e s u l t s a r e p r e s e n t e d i n  12b. S c a t c h a r d  H D L  for  range used  affinity  Figure  were t h e n  s  12a, n e i t h e r  high  described  for  binding  1  i n section  i n the concentration to detect  H D L  11) a n d u s e d  d e g r a d a t i o n assays as o u t l i n e d As i n d i c a t e d i n F i g u r e  i s o l a t e d from t h e samples as  products  showed t h a t d e g r a d a t i o n p r o d u c t s higher ligand  (Figure 17).  These p r e l i m i n a r y r e s u l t s changes o b s e r v e d  t o occur  alter  suggest  that the lipid/protein  i n HDL i n c u b a t e d  the a b i l i t y  T D  i n TD p l a s m a do n o t  of the particle  t h e 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 HDL  were d e t e c t a b l e a t t h e  T 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  H D L 3 7 were d e t e c t e d  drastically  of HDL  to interact  in vitro.  While t h e  p a r t i c l e was d e g r a d e d a t a f a s t e r r a t e t h a n H D L  exact s i g n i f i c a n c e  of this  3 7  , the  o b s e r v a t i o n remains u n c l e a r .  78  with  60  j i i i | i i i | i i i [ II i [T'i'i'i  i i i j i  Distance from top of gel (cm) Figure  11.  S D S - P A G E o n 10.0% g e l s HDL  25 1% the  3  of  1 2 5  I-labelled  2-mercaptoethanol.  t o each g e l i n t h e p r e s e n c e o f  After electrophoresis  f o r 2.5 hours  p r o t e i n s were v i s u a l i s e d b y s t a i n i n g t h e g e l s  presented of t o t a l  1  a t 300V  with  The g e l s were s u b s e q u e n t l y c u t i n t o 0 . 5 cm  bromophenol b l u e . and t h e  (©) a n d  (•).  7  ug o f p r o t e i n was a p p l i e d  sections  HDL^Q  2  5  f o r HDLpQ  I radioactivity (0)  radioactivity  and H D L counted  3 7  determined. (•)  The r e s u l t s  are expressed as percentage  and a r e t h e r e s u l t s o f a s i n g l e  determination.  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.  Total binding of  1 2 5  I-HDL  T D  and  1 2 5  I-HDL  3 7  t o normal  fibroblasts Normal f i b r o b l a s t s were i n c u b a t e d  f o r 2 h o u r s a t 37°C  p r e s e n c e 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 HDL,p  D  HDL The  3 7  i n the  (•) o r  (®) i n t h e p r e s e n c e o r a b s e n c e o f e x c e s s u n l a b e l l e d HDL.  results  a r e t h e average + range o f 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  0  10  r j  1 j  20  1 1  30  1 f  40  50  HDL (ug/ml)  F i g u r e 12b.  Specific binding of normal  HDL The  3 7  (Q)  I-HDL  T D  and  1 2 5  I-HDL  3 7  were i n c u b a t e d  f o r 2 h o u r s a t 37°C  concentrations of either  HDLpQ  a r e t h e average + range o f d u p l i c a t e  81  i n the  (B) o r  i n t h e presence o r absence o f excess u n l a b e l l e d  results  to  fibroblasts  Normal f i b r o b l a s t s presence o f increasing  1 2 5  ligand.  determinations.  20-1  ,  OH  T  0  ,  • i  ,  ,  ,  r  j  1  J  1  J  - r  50  100  150  1  200  Bound (ug/m!) F i g u r e 13.  Scatchard analysis Figure  of the data presented  12b  82  in  50-r  CN  -o.E  CD Q)  Q q) _1°  £  x  cn c -j  j  1  j  10  1  20  j  1  30  j  40  r——p  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-HDL  3 7  by normal  fibroblasts. Normal f i b r o b l a s t s were i n c u b a t e d presence o f i n c r e a s i n g HDL The  3 7  (0)  total  f o r 2 h o u r s a t 37°C i n t h e  concentrations of either HDL  T D  (H) o r  i n t h e p r e s e n c e o r absence o f excess u n l a b e l l e d HDL d e g r a d a t i o n was d e t e r m i n e d a s d e s c r i b e d  methods. The r e s u l t s a r e t h e a v e r a g e + r a n g e o f determinations.  83  ligand.  i n the  duplicate  3.4  Metabolism o f  LDL^Q  by s k i n f i b r o b l a s t s  LDL f r o m TD and c o n t r o l p l a s m a were i s o l a t e d by ultracentrifugation composition.  and a n a l y z e d  for lipid  and p r o t e i n  Table IX,presents the chemical composition of the  n o r m a l LDL a n d . L D L . As h a s b e e n p r e v i o u s l y  reported  T D  is CE,  enriched  i n PL, TG and p r o t e i n a n d d e p l e t e d  and t o a l e s s e r e x t e n t  diameter  (nm)  FC, r e l a t i v e  Analysis  t o n o r m a l LDL. The mean  18.7nm).  o f TD p l a s m a by a g a r o s e g e l e l e c t r o p h o r e s i s  position  o f normal LDL). While L D L  particle  f r o m c o n t r o l LDL, l i t t l e  As TD p a t i e n t s u s u a l l y h a v e o n l y  skin  was  showed 5;  usual  distinct  i s known a b o u t i t s m e t a b o l i s m . 30% n o r m a l l e v e l s o f LDL i n that  LDLpj-j i s more r a p i d l y o f LDLrr,  examined. The c a t a b o l i s m  f i b r o b l a s t s was  (Figure  i s a chemically  T D  (90), the p o s s i b i l i t y  c l e a r e d by c e l l s cultured  i n i t scontent of  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 t h e B band  t h e i r plasma  T D  o f t h e TD LDL was n o t d i f f e r e n t f r o m n o r m a l LDL a s  d e t e r m i n e d b y Q L S (TD: 19.2nm; n o r m a l  increased  (90), L D L  studied  as d e s c r i b e d  D  by  i n section  2.5.1. Samples o f p l a s m a were o b t a i n e d  from a p o o l  d o n o r s a n d f r o m a p a t i e n t w i t h TD. LDL was in  1  described  and used f o r  and d e g r a d a t i o n a s s a y s as o u t l i n e d i n s e c t i o n  specific  binding  o f TD and c o n t r o l  f i b r o b l a s t s was d e t e r m i n e d Binding  of control  3 0 ug/ml w h i c h and  i s o l a t e d as  s e c t i o n 2.3.1.1. The L D L s were t h e n i o d i n a t e d ,  LDL b i n d i n g The  of normolipemic  Goldstein  1 2 5  1 2 5  LDL  to cultured  (Figure 15).  I - L D L was  i s s i m i l a r the value  saturated  i n t h e l i g a n d range 84  a t approximately  o f 25 ug/ml r e p o r t e d  ( 1 2 8 ) . However, t h e b i n d i n g  appear t o be s a t u r a t e d  2.7.1.  of  1 2 5  I-LDL  examined.  T D  by Brown  d i d not  Table  IX. C h e m i c a l c o m p o s i t i o n  o f LDL  (1.006 -  1.063-g/ml),  Percent weight Control Free c h o l e s t e r o l Esterified  (FC)  cholesterol  Triacylglycerol Phospholipids  (EC)  (TG)  (PL)  Protein  85  (mg.%)  TD  9.2  3.4  29 .5  3.4  7.5  26.5  20.9  29.7  32.5  37.0  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-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 i n c u b a t e d increasing  c o n c e n t r a t i o n s o f e i t h e r TD  f o r 6 h o u r s a t 37°C i n (0) o r c o n t r o l  the presence o r absence of excess u n l a b e l l e d component o f a s s o c i a t i o n methods. The  was  ligand.  (#)  LDL i n  The s p e c i f i c  d e t e r m i n e d as d e s c r i b e d i n t h e  r e s u l t s are the average + range o f  determinations.  86  duplicate  The  s p e c i f i c component o f b i n d i n g  that  o f a second  plot  analysis  was  3-5  fold  experiment  (Table  were e x a m i n e d by d o u b l e  X). In both  lower than that  was c o n s i s t e n t l y  3-10  fold  of t h i s experiment  experiments,  for control  lower than  results  i n d i c a t e t h a t major d i f f e r e n c e s  control  LDL w i t h r e s p e c t  skin  Analysis  d e g r a d a t i o n p r o d u c t s showed t h a t was  a p p r o x i m a t e l y 50% t h e r a t e The  similar, t o those previously  and  thus these r e s u l t s i n d i c a t e that  metabolically  distinct  87  for LDL  T D  LDL. T h e s e T D  and  of degradation of L D L LDL  (Figure  LDL i n t h e s e  LDL  LDL.  x  o f t h e medium f o r  reported  from n o r m a l  a  T D  t o i n t e r a c t w i t h human  of control  are  m  e x i s t between L D L  the rate  r e s u l t s obtained f o r control  for LDL  LDL. The V  to their ability  f i b r o b l a s t s LDL r e c e p t o r .  reciprocal  the  for control  and  16).  experiments  f o r control T D  T D  LDL  (128)  i s both chemically  and  TABLE  X.  K I N E T I C ANALYSIS OF LDL C E L L BINDING  v  ug/ml Expt.  DATA  max ng/mg c e l l  1 TD  13.4  1723  control  43.3  4411  TD  10.6  892  control  52.3  7590  Expt.2  88  protein/6h  50-r  i ii|iii|iii|iii|ii i|ii i|i  LDL (ug/ml)  F i g u r e 16.  D e g r a d a t i o n o f TD a n d c o n t r o l  I-LDL b y  normal  fibroblasts. Normal increasing the  f i b r o b l a s t s were i n c u b a t e d c o n c e n t r a t i o n s o f e i t h e r TD  f o r 6 h o u r s a t 37°C i n (fl) o r c o n t r o l  p r e s e n c e o r absence o f excess u n l a b e l l e d  LDL d e g r a d a t i o n was results  d e t e r m i n e d as d e s c r i b e d  a r e t h e average + range o f d u p l i c a t e  89  ligand.  (Q) LDL i n The  total  i n t h e methods. determinations.  The  3.5  Catabolism  o f VLDLj,  D  by b o v i n e  milk  lipoprotein  lipase A common f e a t u r e o f b o t h presence  TD and LCAT d e f i c i e n c y  i s the  o f h y p e r t r i g l y c e r i d e m i a , however, t h e b i o c h e m i c a l  mechanism(s) u n d e r l y i n g t h e h y p e r t r i g l y c e r i d e m i a r e m a i n u n c l e a r . While the  both  t h e TD a n d LCAT d e f i c i e n t  low-normal range a c l e a r  demonstrated  p a t i e n t s have L p l l e v e l s i n  L p l abnormality  (90). Thus, t h e p o s s i b i l i t y  has n o t been  that the TG-rich  particles  a c c u m u l a t e i n TD a n d LCAT d e f i c i e n c y a s a r e s u l t  defective  i n t e r a c t i o n between L p L and i t s l i p o p r o t e i n  was e x a m i n e d i n t h i s The  composition  lipid  and s i z e  s t u d i e d t o determine  o r t h e p r o t e i n moiety Figure  QLS.  o f t h e VLDL a c c u m u l a t i n g  from  size  of the lipoproteins  agrees  34.3 nm w i t h a r a n g e o f 31 t o 3 6  i n n o r m a l VLDL  represents contaminating  chylomicron  particles.  VLDL i s o l a t e d  from  another  (100). A minor p o p u l a t i o n chylomicrons  distribution  The  o f 44.3 nm w i t h a  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  and b r o a d e r  uncatabolized  (100). A minor  (100 t o 170 nm). T h i s  m a j o r p o p u l a t i o n i n VLDLpp h a d a mean d i a m e t e r  mean d i a m e t e r  nm.  well with that previously reported f o r  p o p u l a t i o n was a l s o p r e s e n t  range than  )  o f s i z e e s t i m a t i o n o f VLDL by  by e l e c t r o n microscopy  broader  L C A T  existed.  t h e VLDL p o o l a s d e t e r m i n e d  most l i k e l y  (VLDL  n o r m a l i n d i v i d u a l s were composed o f a m a j o r  p o p u l a t i o n o f mean d i a m e t e r This p a r t i c l e  patient  i nthe  i f any a b n o r m a l i t i e s i n e i t h e r t h e  17 p r e s e n t s t h e r e s u l t s  Particles  substrates  study.  p l a s m a o f t h e TD p a t i e n t a n d a LCAT d e f i c i e n t was f i r s t  of a  1  larger  has a l s o been r e p o r t e d f o r  p a t i e n t w i t h TD a n d a n a l y s e d b y EM (200 t o 400 nm) p r o b a b l y  corresponds t o  a s r e p o r t e d i n o t h e r s t u d i e s (99) . The  RELATIVE  VOLUME  "O  >  O o  0)  O >  o o TJ  3'  (O  g  CO  TJ  o  CO  TJ  u :  O r~ m  •3 C H  co  N  O  m  oo  O "  >  o  > < CO CO  F i g u r e 17.  D e t e r m i n a t i o n o f VLDL s i z e  VLDL were i s o l a t e d the  plasma  size/ distribution  figure  the r e l a t i v e  population  identical  distribution  and s o l i d  particles.  91  from  individuals.  submicron samples  results.  of particles  a s s a y e d . The a n a l y s i s  distribution  a t 1.006 g/ml  shows a r e p r e s e n t a t i v e  p r o f i l e f r o m a NiComp  essentially  b y QLS  (3 mg TG/ml) was u s e d f o r  P r o f i l e obtained with equivalent  f r a m e s gave to  and n o r m a l  500 u l o f e a c h f r a c t i o n  determination. This  sizer.  by u l t r a c e n t r i f u g a t i o n  o f TD LCAT d e f i c i e n t  Approximately  and d i s t r i b u t i o n  particular particle  a t o t h e r time  The p e a k h e i g h t s  refer  of within the t o t a l  assumes a non g a u s s i a n  nature  and s i g n i f i c a n c e  unknown.  V  L  DL  L  C  A  T  o f t h e p e a k s a t 36 a n d 63 nm a r e a s y e 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 o f 70 t o 100 nm  confirming the results  o f o t h e r i n v e s t i g a t o r s who  l a r g e VLDL i n LCAT d e f i c i e n t p l a s m a The  lipid  presented and  i n Table  protein  reported size  and p r o t e i n  composition  ( F i g u r e 17) ,  VLDLrp  esterification. L C A T  0.7%, r e s p e c t i v e l y .  previously  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  had e s s e n t i a l l y  D  The c o m p o s i t i o n  c o n t r o l VLDL a n d V L D L  content  of the p a r t i c l e s i s  X I . The r e l a t i v e p r o p o r t i o n s o f TG, FC, CE, P i  i n o t h e r s t u d i e s (162).  cholesterol  FC  (162).  i n t h e n o r m a l p a r t i c l e s were s i m i l a r t o t h e v a l u e s  composition.  and  have d e s c r i b e d  of V L D L  reflected  and p r o t e i n  t h e absence o f  The most marked d i f f e r e n c e between  was  i n t h e CE c o n t e n t w h i c h was  5.4%  T h e r e was a l s o a t w o - f o l d i n c r e a s e i n t h e  o f t h e VLDL, a f i n d i n g (162).  L C A T  normal l i p i d  In addition,  s i m i l a r to that reported  the r e l a t i v e  amount o f apo C - I I  a s s o c i a t e d w i t h t h e VLDLpp was t h e same a s t h a t o b s e r v e d i n c o n t r o l VLDL b u t i t was Clearly,  10-fold less  the results  of this  study  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 that and  i t i s a larger p a r t i c l e ; while i t i s an even l a r g e r It  in VLDL  show t h a t  from c o n t r o l V L D L  LCAT  addition,  alter  Fielding  supplemented w i t h (163). Thus,  their  ^  s  e  V L D L r p  D  is  subjects n  r  i  c  n  e d  except  with  FC  particle.  i s p o s s i b l e that the increased size  p a r t i c l e s may  (Table X).  L C A T  ability  o f t h e VLDL  t o i n t e r a c t w i t h LpL. I n  a n d c o w o r k e r s h a v e shown t h a t n o r m a l VLDL excess  FC i n v i t r o was a p o o r s u b s t r a t e f o r L p L  i n t h e f o l l o w i n g study  the a b i l i t y  p a r t i c l e s t o a c t a s a s u b s t r a t e f o r BmLpL was  92  of these  examined.  Table XI.  Chemical  composition  o f VLDL  (d<1.006  g/ml),  Percent weight Control  TD  (mg.%) LCAT  deficiency  4.5  5.6  7.6  5.4  3.6  0.7  64.0  58.5  72.0  16.8  19.6  15.3  Protein  9.4  13 .5  4.1  Apo C - I I  0.7  0.7  14.2  10.3  Free c h o l e s t e r o l Esterified  cholesterol  Triacylglycerol Phospholipids  TG/FC  (FC)  (TG)  (PL)  (EC)  FC/PL  0.26  0.29  FC/EC  0. 83  1.26  93  0. 07 9.5 0.49 10.9  BmLpL r e s e m b l e s  peripheral  tissue  lipase  ( i . e . adipose)  w i t h r e s p e c t t o i t s c h r o m a t o g r a p h y on h e p a r i n - S e p h a r o s e s t i m u l a t i o n b y apo C - I I ( 1 6 4 ) . I t c a n be p u r i f i e d homogeneity to  (14 6)  and i t i s s t a b l e .  both  and  t o near  For these reasons  i t was  s t u d y t h e h y d r o l y s i s o f c o n t r o l VLDL, VLDLpp a n d V L D L  L C A T  used in  vitro. Figure  18a shows t h e h y d r o l y s i s  Eadie-Hofstee analysis presented  T D  ,  respectively. affinity suggest avidly  (165) o f t h e d a t a f r o m  i n F i g u r e 18b and i n d i c a t e s t h a t  VLDL, V L D L !  o f t h e VLDL's b y B m l p l .  and  V L D L  LCAT  w  e  r  e  F i g u r e 18a i s  t h e 1^ f o r t h e n o r m a l  1-13, 2.25, a n d 2.42 mg/ml TG,  Since the reciprocal  o f 1^ i s a m e a s u r e o f t h e  of the substrate f o r i t s binding s i t e  these  results  t h a t b o t h TD a n d LCAT d e f i c i e n t V L D L ' s do n o t b i n d a s t o t h e enzyme a s t h e c o n t r o l VLDL. However, a t h i g h e r  concentrations,  t h e y may be more r a p i d l y h y d r o l y z e d a s i n d i c a t e d  by t h e 3-4  i n c r e a s e i n t h e maximal v e l o c i t y  8.47; TD, 18b).  fold  25.4; LCAT d e f i c i e n t ;  appear  m a x  )  31.3 umoles/min/mg LpL)  T h u s t h e VLDL f r o m b o t h t h e TD a n d t h e LCAT  patients  (V  (Figure  deficient  t o be good s u b s t r a t e s f o r t h e enzyme  94  (Control,  Bmlpl.  14  LCAT  12  E \ —i & _J  10  a  Def.  Tangier  E TJ  e « a  9  Control  < UL  u. o  . E 3  0.5 1.0 1.5 Triglyceride (mg/ml)  Figure  18a.  The h y d r o l y s i s o f VLDL b y BmLpL  VLDL were i s o l a t e d f r o m LCAT d e f i c i e n t , TD and n o r m a l i n d i v i d u a l s and l a b e l l e d w i t h  [ H] t r i o l e i n 3  as d e s c r i b e d  m e t h o d s . The i n d i c a t e d amount o f VLDL-TG was purified  incubated  BmLpL f o r 4 5 min a t 3 7 ° C and t h e amount o f  [ H]oleic 3  represents  acid released  was d e t e r m i n e d . E a c h  t h e mean o f d u p l i c a t e  95  incubations,  point  i n the  with  40  c  LEGEND  i  • Control • L C A T Def m Tangier  30 0>  E \ \  «  \  » ©  «  20  v v \  \  \  \  \  \ x  < u. u. o E  >. 10 ** u o  >  0  5  10  15  velocity/substrato  F i g u r e 18b. The  Eadie-Hofstee  a n a l y s i s of data  f r o m F i g u r e 18a  KJJ, f o r t h e h y d r o l y s i s o f VLDL by BmLpL was  from t h e data  i n A b y an E a d i e - H o f s t e e  o f t h e curve)  and V  m a x  (intercept  o f t h e d a t a by a Hanes p l o t were w i t h i n 5% o f t h o s e  plot  calculated  (negative slope  on t h e Y - a x i s ) . A n a l y s i s  (165) g a v e v a l u e s o f 1^ and V  reported i n the figure.  96  j n a x  that  3.6  Role o f c e l l u l a r  c o m p o n e n t s i n t h e HDL d e f i c i e n c y  Recent r e p o r t s have suggested t h a t  the rapid  f r o m p l a s m a o f TD p a t i e n t s d u r i n g p l a s m a p h e r e s i s accelerated tissue  cellular  culture  w i t h TD c e l l s 3.6.1  o f TD  r e m o v a l o f HDL i s due t o an  c a t a b o l i s m o f HDL ( 1 1 0 , 1 2 5 ) . We u s e d two  s y s t e m s t o examine t h e i n t e r a c t i o n s  o f n o r m a l HDL  in vitro.  TD f i b r o b l a s t The  d a t a on i n t e r a c t i o n  fibroblasts presented  f r o m o u r TD p a t i e n t  i n Figure  appeared t o reach  1 2 5  I-labelled  saturation  a n d TD f i b r o b l a s t s .  t h e K s o f HDL d  3  1 2 5  3  with  skin  I-labelled  at concentrations  a n a l y z e d by S c a t c h a r d a n a l y s i s  analysis  HDL  a n d an age m a t c h e d c o n t r o l a r e  19. S p e c i f i c b i n d i n g o f  mg/ml f o r b o t h c o n t r o l further  of  binding to control  were  2 0 ) . From  a n d TD  this  fibroblasts  were d e t e r m i n e d t o be 10.95 a n d 5.24 ug/ml r e s p e c t i v e l y . addition,  t h e maximum amount o f HDL  fibroblasts  were e s s e n t i a l l y  protein/2 hours r e s p e c t i v e l y .  3  bound b y c o n t r o l  r e p o r t e d b y Oram e t a l f o r n o r m a l s k i n fibroblasts  In  and T a n g i e r  i d e n t i c a l , 262 a n d 257 ng/mg These r e s u l t s  3  l o w e r t h a n 25  The r e s u l t s  (Figure  HDL  are similar  fibroblasts  cell  t o those  (73).  do n o t a p p e a r t o b i n d a n d / o r i n t e r n a l i z e  Hence TD  n o r m a l HDL  abnormally. Analysis neither of  control  HDL . T h i s 3  o f t h e medium f o r d e g r a d a t i o n p r o d u c t s showed n o r TD f i b r o b l a s t 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  skin  fibroblasts  that  TD f i b r o b l a s t s  to  degraded any d e t e c t a b l e  their ability  do n o t d e g r a d e HDL behave l i k e  to interact  3  ( 7 3 , 7 4 , 7 5 ) . We  normal  fibroblasts  w i t h n o r m a l HDL.  97  that  amount  that  concluded with  respect  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  i  i  ; i  r i  |  20  40  Ki  60  80  i  100  HDL (ug/ml) 3  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 control  TD  (B)  1 2 5  I-HDL  3  t o TD a n d  fibroblasts.  and c o n t r o l  (®) f i b r o b l a s t s  37°C i n t h e p r e s e n c e o f i n c r e a s i n g  were  incubated  concentrations of  f o r 2 hours a t  control  125 I-HDL3 ligand). in  ( i n t h e p r e s e n c e o r absence  of excess  The s p e c i f i c component o f a s s o c i a t i o n  t h e m e t h o d s . The r e s u l t s  determinations  unlabeled was d e t e r m i n e d a s d e s c  a r e t h e average + range o f  from a r e p r e s e n t a t i v e  98  experiment.  duplicate  60  OH  l  I  1  i  i  i  J  I  1  i  I  E 50Tangier cells  40-  £ a* SE CP  c  30-  _  a  2010-  0-  50  100  150  200  250  300  Bound (ug/ml)  Figure  20.  Scatchard  analysis of data  99  from F i g u r e  19  3.6.2  TD  monocytes  The a b o v e s t u d i e s  o f TD c e l l s were t h e n c a r r i e d o u t u s i n g  human p e r i p h e r a l m o n o c y t e s . F i g u r e HDL3 w i t h  21 shows t h e i n t e r a c t i o n o f  f r e s h l y i s o l a t e d monocytes  f r o m TD and c o n t r o l  T h e r e s u l t s show no d i f f e r e n c e s between t h e a b i l i t y  o f TD  c o n t r o l monocytes t o i n t e r a c t w i t h  3  It HDL  binding  (77). of the  has been p r e v i o u s l y  with t h i s  associated  HDL  l i g a n d r a n g e s we  suggested that  cell  radioactivity  to  observation  examined  associated  period.  (Figure  associated  for  HDL  i s released  increase  in  differences  1 2 5  by  control  I - l a b e l l e d HDL  period  in cell  o b s e r v e d b e t w e e n TD and c o n t r o l m o n o c y t e s  do n o t h a v e a d e f e c t  132 mg/ml  ( 1 6 6 ) . However, o u r  E v e n when t h e i n c u b a t i o n  indicate that  I-labelled  2 1 ) . Assman e t a l have  w i t h TD m o n o c y t e s d u r i n g  20 h o u r s no s i g n i f i c a n t  results  d  i s the lack of saturation  by TD m o n o c y t e s  i n d i c a t e no d r a m a t i c  incubation  that the K  s e e n i n b o t h c o n t r o l a n d TD m o n o c y t e s i n  monocytes but n o t r e l e a s e d results  and  I - l a b e l l e d HDL .  t o c u l t u r e d monocytes i s a p p r o x i m a t e l y  Consistent  cell  reported  1 2 5  cells.  a 4 hour was  100  interaction.  HDL  was  22). These  i n t h e a b s e n c e o f c h o l e s t e r o l TD i n cell/HDL  increased  associated  (Figure  3  monocytes  Figure  21.  Binding during  TD  of  1 2 5  I-HDL  t o TD a n d c o n t r o l m o n o c y t e s  3  a 4 h incubation  (H) a n d c o n t r o l  (©) m o n o c y t e s were i n c u b a t e d  37°C i n t h e presence o f i n c r e a s i n g 12  5  • I-HDL ligand). described three  a t 37°.  concentrations  f o r 4 h at  of control  . 3  ( i n t h e presence o r absence o f excess The s p e c i f i c  component  of association  unlabelled  was  determined  i n t h e methods. The r e s u l t s a r e t h e mean + SD o f  seperate  duplicate  determinations.  101  as  Figure  22.  A  "l-HDL during  TD  (•)  and  3  b i n d i n g t o TD  a 20  control  37°C i n t h e p r e s e n c e o f I-HDL The in  3  i n the  specific  c o n t r o l monocytes  incubation at  37°.  (©) monocytes were i n c u b a t e d increasing concentrations  f o r 20 h  at  of c o n t r o l  p r e s e n c e o r absence o f e x c e s s u n l a b e l l e d l i g a n d .  component o f a s s o c i a t i o n was  t h e methods. The  duplicate  h  and  results  d e t e r m i n e d as  a r e t h e mean + SD  determinations.  102  of three  described seperate  4  Discussion  4.1  M o d i f i c a t i o n o f n o r m a l HDL b y TD p l a s m a Previously,  and  Assmann e t a l ( 1 0 9 ) , S c h a e f e r e t a l (110,125)  P r i t c h a r d and F r o h l i c h  infusion  of r e l a t i v e l y  patients  i s followed  protein  (124) h a v e d e m o n s t r a t e d t h a t t h e  l a r g e amounts o f n o r m a l HDL i n t o TD  b y t h e r a p i d r e m o v a l o f b o t h HDL-C and HDL-  from t h e p l a s m a compartment. Our p a t i e n t  virtually  i d e n t i c a l HDL t u r n o v e r  Schaefer's study heterogeneity turnover  (125). T h i s  (124) h a d  k i n e t i c s as p a t i e n t A i n  i n d i c a t e s t h e absence o f any major  b e t w e e n t h e s e two c a s e s w i t h r e s p e c t  t o HDL  i n v i v o . Thus, t h e r e s u l t s a r e c o n s i s t e n t w i t h t h e  h y p o t h e s i s t h a t TD i s t h e 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  h y p e r c a t a b o l i s m o f HDL. Although the studies  o f b o t h Assmann  (110,125) r e a c h e d s i m i l a r c o n c l u s i o n s , respective  roles of cellular  (109) a n d S c h a e f e r  they d i d not address the  catabolism  and p l a s m a  o f t h e HDL i n t h e o b s e r v e d h y p e r c a t a b o l i s m . it  modification  From t h e i r  studies,  i s n o t p o s s i b l e t o d e t e r m i n e w h e t h e r c h a n g e s i n HDL  composition occurred w i t h TD c e l l s initial  p r i o r t o , o r as a consequence o f , i n t e r a c t i o n  i n vivo. This  changes t h a t  study attempted t o d e s c r i b e t h e  o c c u r i n n o r m a l HDL d u r i n g  i t s exposure t o  TD p l a s m a . The CE  a d d i t i o n o f n o r m a l HDL t o TD p l a s m a  c o n t e n t o f t h e HDL t o w i t h i n  slight  increase  associated  i n HDL-TG  with a small  These o b s e r v a t i o n s proteins  that  increased  t h e normal range  (Table  (when compared t o c o n t r o l s )  but s i g n i f i c a n t  increase  103  I V ) . The  was  i n VLDL/LDL CE.  may be due t o t h e a c t i o n o f l i p i d  appear t o remain p a r t i a l l y  t h e FC and  transfer  a c t i v e a t 4.°C ( 1 5 9 , 1 6 7 ) .  A comparison of p l a s m a by  incubation  time dependent observation  was  during  c o n f i r m e d by the  Assmann e t a l  plasma I n  vitro  interesting  one  increased  (125).  n o r m a l HDL  with  observation  that  plasma occurs  plasma during experiments,  catabolism  that  1 2 5  6). et a l  i n TD  Thus, t h e TD  and  only  in vitro  plasma  24  h.  rate of  incubation,  Thus, t h e  d e c a y o f HDL-C s e e n i n v i v o may  via  catabolic patients  incubation  are  of  Schaefer's  o f n o r m a l HDL  in  TD  compartment.  i s q u a n t i t a t i v e l y the  first  and  Furthermore,  fractional  of  TD  It is  catabolized only  in vitro  supportive  relative  be  in  two-  plasma  h e t e r o z y g o u s TD  i n vivo catabolism  i n the  (168).  that the  r e s u l t s of  plasma are  the  (110,125)  i n normal  w i t h i n the  I - H D L may  i n homozygous and  Furthermore, the  cellular  c h a n g e s i n HDL-C t h a t  (Figure  a n o n p l a s m a compartment  o f HDL  the  initial  a monoexponetial decay curve.  component b e i n g  p l a s m a compartment  during  a rapid,  l o s s o f c h o l e s t e r o l f r o m HDL  f o r i n v i v o HDL  et a l reported  (FCR)  was  plasma. T h i s  period  TD  indicated  t o n o t e t h a t Blum e t a l h a v e p r o p o s e d a  exchanging with  rate  37°C, t h e r e  f i n d i n g s of Schaefer  the  followed  individuals with  the  in vivo  i n c o n t r o l and  ( F i g u r e 5)  f o l l o w i n g the  incubation  (109),  compartment m o d e l  Schaefer  of plasma a t  l o s s o f HDL-CE i n TD  S i m i l a r t o the and  m e t a b o l i s m o f HDL  agarose g e l e l e c t r o p h o r e s i s  that during  occurred  in vitro  described same as  data occur  involvement.  104  l o s s o f HDL-C f r o m i n the  the  TD  present  in vivo  studies  suggest t h a t the  initial  independently  any  of  Further ( T a b l e V) HDL  and  a n a l y s i s of the  i n d i c a t e t h a t the  identical  increase  i n HDL-CE and  activity  o f LCAT  i n the  (50).  The  This  w i t h an  observed changes i n the  to those previously decrease  data  f o r TD  lost  loss of  CE  increased  Furthermore, the  reflects  (Figure  from the  core  concentration  of the  particles.  exchange o f  of a s p e c i f i c  CE  constituents  On  ester  LTP  Previously  plasmapheresis of  lost  from t h i s  c a t a b o l i z e d as While  our  i n f u s e d HDL fraction.  in vitro.  The  total  A-I  The  reflect  the  i n VLDL/LDL  was  CE  LTP's of  the  f o r m o f CE  (Table  associated  (Table  V).  t o VLDL/LDL  content of the was  was  core  approximately  c o n s i s t e n t with the  a  involvement  h a v e shown t h a t t h e  at a f a s t e r rate than  observation  has  where 50%  a b o u t 15%  of  protein  in vitro.  i n TD  in vivo  could  o f CE  apo  was  A-I is  was not  not  l o s s o f HDL-C be  mimicked  plasma d i d not  decrease  It i s therefore  105  the  plasma.  in vivo  p r o t e a s e e x i s t s i n TD  core  surface  T h e s e r e s u l t s s u g g e s t t h a t HDL i n TD  HDL  b e e n c o n f i r m e d by  (12 4)  whereas o n l y  amount o f A - I  incubation  specific  TG  p o s s i b l e to reproduce the  l o s s o f HDL  apo  (157).  of the  i n the  i n the  (12 5)  a whole p a r t i c l e  i t was  are  majority  o f n o r m a l HDL  p a t i e n t w i t h TD  the  the  f o r TG  catabolized  in vitro,  during  6) was  of  (50).  p r o t e i n components. T h i s  from the  activity  a molar b a s i s , there  Schaefer et a l  are  lost  the  increase  t r a n s f e r o f c h o l e s t e r o l f r o m HDL  w i t h a marked i n c r e a s e  1:1  reported  i n VLDL/LDL CE  associated HDL  incubation  plasma r e v e a l t h a t the  f r o m HDL  structure  i n HDL-FC most l i k e l y  (162). In a d d i t i o n , the  HDL-TG c o n t e n t  cholesterol V).  lipoprotein fractions  VLDL/LDL i n c o n t r o l p l a s m a d u r i n g  essentially  and  p l a s m a and  u n l i k e l y that  plasma,. T h u s , t h e  loss  an of  A-I  f r o m TD p l a s m a must be due t o i t s i n c r e a s e d  T h i s may  r a t e o f removal.  i n v o l v e a number o f m e c h a n i s m s i n c l u d i n g :  1) I n c r e a s e d 2) T r a n s f e r  catabolism  of the  H D L r p  D  particle  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  3) I n c r e a s e d  clearance  of non-lipoprotein  particles  associated  apo A - I . With these p o s s i b i l i t i e s movement  o f HDL a p o p r o t e i n s  HDL w i t h  TD p l a s m a  transfer  o f HDL a p o p r o t e i n s  i n mind, we  during  s e t out t o follow the  in vitro  ( F i g u r e 7 and T a b l e  incubation  of  1  5  less  a t 3 7 ° C . The t e m p e r a t u r e  d e p e n d e n c e o f a p o p o t e i n movement  c a n be e x p l a i n e d  mechanisms. A t 37°C t h e f l u i d i t y  the l i p o p r o t e i n i s probably  greater  t h e n a t 4°C and t h u s t h e a p o p r o t e i n s  dissociate  from t h e p a r t i c l e .  t e m p e r a t u r e s may  explanation increased  more c o l l i s i o n s that  higher  o f plasma f a c t o r s t h a t  increased  transfer i s directly  energy o f t h e HDl p a r t i c l e s  ocurring at higher  agreed with  significant B containing  previous  related t o the  and hence t h e r e a r e  t e m p e r a t u r e s . The  observation  l i p o p r o t e i n s (125).  the observation  greater  findings that  i n TD p l a s m a , a  amount o f apo A - I a n d apo A - I I i s a s s o c i a t e d w i t h  a n d A - I I move i n d e p e n d e n t l y  with  i s that  a s u b s t a n t i a l amount o f p r o t e i n was t r a n s f e r r e d t o t h e LDL  fraction  I  may be more f r e e t o  d i s s o c i a t i o n . However, t h e most o b v i o u s  i t that  kinetic  by s e v e r a l  A second e x p l a n a t i o n  increase the a c t i v i t y  promote a p o p r o t e i n  I -  V I ) . W h i l e t h e r e was some  a t 4 ° C t h i s was s i g n i f i c a n t l y  than that observed a f t e r incubation  2  Further,  t h e f i n d i n g t h a t apo A-  o f each other  that during  i n vivo  i s a l s o i n agreement  incubation  amount o f apo A - I I i s a s s o c i a t e d w i t h  106  apo  experiments a  VLDL/LDL  fraction  t h a n apo In II  A-I  i n plasma  a n o t h e r s t u d y , Assmann r e p o r t e d  specific  but  no  this  radioactivity  significant  same d e n s i t y  d i f f e r e n c e may region  i n TD  isolation  apo: A - I , or  in  the  Our  c h a n g e i n apo  A-I  specific  that  in vitro  A-II,  are  data presented  are  w i t h an  low  lost  of  HDL  in  this  i n the  specific  from t h e  in this  HDL  density  following activity  was  findings that fraction  i n TD  study suggest t h a t  both  during  The  apparently  o f HDL  that  plasma. T h i s  hypothesis  initial  plasma i n v o l v e core  a c c o m p a n i e d by  modification  removed f r o m  for  to begin with that  dilution  in  i s a CE  n o r m a l d i a m e t e r . The notion  accelerates  that the  i s s u p p o r t e d by  h a v e shown t h a t t h e plasma.  107  data  results  HDL  apo  TG-rich  in  this  initial  r e m o v a l o f HDL  TG-rich  and  poor,  i t is this  the  events  lipid  a t r a n s f e r o f - a p o A-I  r e m a i n i n g HDL  c o n s i s t e n t with the  (110,125) who  concentrations  range i n v i v o  radioactivity  explanation  A-  incubation.  to B lipoproteins.  study are  A-I  density  agree with Schaefer's  m e t a b o l i s m o f HDL  particle  ( 1 0 9 ) . An  apo  results  apo  exchanges t h a t A-II  HDL  l i p o p r o t e i n s no  and  The  the  region  be  a s t e a d y d e c a y o f apo  within  p l a s m a were s o  of  detectable.  vivo  (109,110,125).  of  fraction  from  TD  others is  rapidly  4.2  Possible In  factors involved  an a t t e m p t t o i d e n t i f y  responsible  i n the modification  t h e f a c t o r ( s ) i n TD p l a s m a  f o r the modification  CE h y d r o l y s i s , Nichols  occur  the r o l e of t h i s  cholesterol  during  DiFP  that  (Table  l o s s o f CE f r o m HDL d u r i n g by a simple  (Table  VIII)  plasma  plays  incubation  incubation  that  i n TD  increase  the rate of loss of suggesting  o f t h e HDL  i n t h e r a t e o f CE  could  hypothesis. of the l i p i d  i n t o the moderately hypertriglyceridemic  f o r these p o s s i b i l i t i e s ,  to a  significant  Furthermore, t h e a d d i t i o n o f normal  movement o f TG t o t h e HDL  fraction.  TD p l a s m a may  I n an attempt t o a c c o u n t  t h e m e t a b o l i s m o f n o r m a l HDL  a more s e v e r e h y p e r t r i g l y c e r i d e m i a  TD p l a s m a ) and t h e a d d i t i o n o f a s i g n i f i c a n t n o r m a l HDL,  only  very  small  losses  108  HDL  facilitate  i n the  p l a s m a o f 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 Despite  core  o f c h o l e s t e r o l amongst t h e  . plasma l i p o p r o t e i n s r e s u l t i n g from t h e a d d i t i o n o f a amount o f e x o g e n o u s HDL.  be  i n section  i n t h e s e e x p e r i m e n t s i s due, i n p a r t ,  i n the concentration  LCAT  hydrolysis  However, t h e r e s u l t s d e s c r i b e d  i s possible that the rapid modification  disequilibrium  that  particles.  the incubation  V I I ) do n o t s u p p o r t t h i s  o f HDL d e s c r i b e d  the  LCAT  i n t h e changes  following  i n the modification  the incubation.  It  enzyme  (150), d i d n o t a l t e r  f r o m t h e HDL p o o l  was n o t i n v o l v e d  explained  reported  e x a m i n e d . I n h i b i t i o n o f LCAT i n TD p l a s m a , b y t h e  o f 5 mM  The  t h e r o l e o f LCAT,  examined.  o f HDL d u r i n g  i n t h e s t r u c t u r e o f n o r m a l HDL  addition  was  e t a l (151) h a v e p r e v i o u s l y  Therefore,  p l a s m a was  3.2.1  o f n o r m a l HDL,  and h y p e r t r i g l y c e r i d e m i a  a major r o l e i n t h e m o d i f i c a t i o n in vitro.  o f HDL  examined.  (compared t o t h e  amount o f e x o g e n o u s  i n HDL-C were o b s e r v e d  i n the  LCAT d e f i c i e n t p l a s m a a f t e r i n c u b a t i o n VIII). the  I n a d d i t i o n , we  plasma of  results  not  lipoprotein  study be  However t h e  hypertriglyceridemia  TG s  incubations increases  and  LDL  increase data  indicate control  amount o f CE  (50).  i n the  The  TD  process  indirect  first  TD  the  and  the  and  and  f r o m n o r m a l HDL  increase  in  mass t r a n s f e r  (Table  V)  CE  as  t o VLDL  total  by  this  C E / d l plasma/24h,  plasma r e s p e c t i v e l y . Obviously, rate of  TD  i n HDL-CE. A n a l y s i s  incubation  CETP  other  r a t e o f t r a n s f e r o f CE  84.72 mg  d i f f e r e n c e i n the  there  of  the  method  for  is a  t r a n s f e r i n TD  plasma  plasma.  a n a l y s i s of the  c o n t e n t . As  from  a s s a y o f CE  vitro  (63)  d i f f e r e n c e between t h e  4 h of the 38.4  in  LCAT, w h i c h  l i p o p r o t e i n core  movement o f CE  F i e l d i n g ( 1 6 9 ) . The  l o s s o f HDL-CE was TG  a consequence o f  e x c h a n g e o f HDL-CE f o r TG  compared t o c o n t r o l Further  by  the  during  a rate of  significant  r o l e of  HDL  i n p l a s m a CE  and  The  a mass e q u i l i b r a t i o n o f  known t o a c c u m u l a t e i n t h e  i s d e t e r m i n e d by  f o r the  exact  o f HDL  modification  p l a s m a i s e s s e n t i a l l y an by  modification  in  10).  HDL  w h i c h p r o m o t e s an  described  the  (Figure  plasma i n the  o f n o r m a l p l a s m a as  the  lipoproteins  (Table  explained.  are  1  i n TD  37°C  patients  s o l e l y a c c o u n t e d f o r by  lipids.  r e m a i n s t o be  indicate that  at  m e t a b o l i s m o f n o r m a l HDL  five hypertriglyceridemic  of t h i s  p l a s m a may  examined the  f o r 24h  associated  data  i n Table V  w i t h an  indicated that  equimolar  CETP r e d i s t r i b u t e s L C A T - d e r i v e d  sites  of  synthesis  i n HDL  (50),  by  m e d i a t i n g an  to the  CE  from  l e s s dense T G - r i c h  e q u i m o l a r e x c h a n g e o f CE  109  increase  and  in  the HDL-  their  lipoproteins TG  (50), i t  may  be t h a t The  simplest  increase amount  t h i s process  i s accelerated  and most o b v i o u s e x p l a n a t i o n  i n CETP a c t i v i t y w o u l d be t h a t  lipoprotein increased personal  by a p p r o x i m a t e l y  acceptor  i s derived  increased  p l a s m a may  i n vivo  t h e changes i n l i p i d binding  i s possible that  this  the increased  accelerate  TG/CE  ratio  transfer activity  the l i p i d  i n acceptor  level  associated  s e e n i n TD  l i p o p r o t e i n s (LDL o r  observation  Support that  favor  an a  l i p o p r o t e i n s ( f o r review  r e f . 50).  TG/CE  ratio  respectively.  i n c o n t r o l LDL a n d  LDLrp  i n Table D  IX i n d i c a t e s  were 0.25  t h e TG/CE  ratio  a n d 7.79,  i n c o n t r o l VLDL and  were 11.85 and 16.25, r e s p e c t i v e l y . Thus, t h e TG/CE T D  that  In a d d i t i o n , a n a l y s i s of the data presented i n  T a b l e XI i n d i c a t e t h a t  LDL  i t has  o f CETP b u t i n s t e a d  lipoproteins will  n e t t r a n s f e r o f CE i n t o T G - r i c h  TG-rich  ( 5 0 ) . Thus,  t r a n s f e r process.  Examination of the data presented the  studies  studies  o f CETP t o b o t h VLDL and HDL  h y p o t h e s i s comes f r o m t h e r e c e n t  increased  and t h e  c o m p o s i t i o n were  n o t b e due t o an i n c r e a s e d  VLDL) w h i c h may  from r e c e n t  and i n v i t r o  abnormal composition o f t h e T G - r i c h  greater  Sparks  o f LTP's a r e i n f l u e n c e d by t h e  o f b o t h t h e HDL p a r t i c l e  l i p o p r o t e i n . In both  b e e n shown t h a t  to,the  (Mr. D.  communication).  chemical composition  see  increased  o f CETP i n t h e n o n -  50% o v e r c o n t r o l s  w h i c h h a v e shown t h a t t h e a c t i v i t y  for  i s an  f r a c t i o n o f o u r TD's p l a s m a h a s b e e n shown t o be  A second p o s s i b l e e x p l a n a t i o n  it  there  f o r the observed  o f CETP i n TD p l a s m a . W h i l e we h a v e y e t t o d i r e c t l y  m e a s u r e t h e mass o f CETP, t h e a c t i v i t y  with  i n TD.  a n d V L D L j ^ a r e more  favorable  110  t o t h e CETP  ratio  catalyzed  VLDL  T D  i n both  r e a c t i o n than are these  the  f i n d i n g s are A third  observation  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,  supportive  of the  possible explanation of  Zilversmit et a l  above  of our  (170).  hypothesis.  data  comes from  the  They have d e s c r i b e d  the  p r e s e n c e o f CETP i n h i b i t o r p r o t e i n i n p l a s m a . T h u s , 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 elevated  level  o f CETP a c t i v i t y  I n summary t h e reported resulting exact  evidence f r o m an  results  of our  increase  i n the this  plasma.  experiments are  of hypercatabolism  mechanism u n d e r l y i n g  n e e d s t o be  s e e n i n TD  o f HDL  i n TD  CETP a c t i v i t y  the  first  plasma i n v i t r o  i n TD  plasma.  increase remains unclear  elucidated.  Ill  the  and  The  T h e M e t a b o l i s m o f TD a n d T D - m o d i f i e d l i p o p r o t e i n s  4.3  by  normal  4.3.1  fibroblasts  The i n v i t r o m e t a b o l i s m o f  HDLrp  by normal  D  fibroblast From t h e p r e v i o u s loss  o f a p o A - I f r o m TD p l a s m a must b e a s s o c i a t e d w i t h  increased  r a t e o f r e m o v a l . We h a v e p o s t u l a t e d  may b e e x p l a i n e d particle.  by an i n c r e a s e d  Therefore,  incubating  i t with  specific  binding  skin  using  f o r both  same. C o m p a r i s o n  that this  uptake o f  HDL HDLrp  3 7  D  D  a s a c o n t r o l . Both t o t a l and and H D L  o f t h e K s from t h i s  3 7  were e s s e n t i a l l y t h e  study  d  d i f f e r e n c e between t h e s e  affinity,  HDLrp  fibroblasts.  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 little  removal  e x p e r i m e n t s , we e x a m i n e d t h e c a t a b o l i s m o f  by f i b r o b l a s t s  T D  cellular  an  t h e u p t a k e o f HDLJIQ was e x a m i n e d b y  normal  In p r e l i m i n a r y HDL  d i s c u s s i o n i t h a s become a p p a r e n t t h a t t h e  e v e n t h o u g h HDLj,  D  ligands, with  and H D L  there  respect  was  t o receptor  had undergone i n c u b a t i o n i n  3 7  vitro. An  obvious explanation  in  the core  to  i n t e r a c t with  incubation  lipid  decreases the  t h e HDL r e c e p t o r .  findings i s that  do n o t e f f e c t  changes  its ability  A second a l t e r n a t i v e i s t h a t  the p a r t i c l e s  ability  to interact  with the  b u t t h a t t h e s e c h a n g e s a r e masked b y i n c r e a s e s o r  in affinity  fibroblast.  picked  o f a n HDL p a r t i c l e  does e f f e c t  HDL r e c e p t o r  f o rthese  f o rother  F o r example,  up apo E i t s a b i l i t y  binding  i f , during to interact  sites  on t h e s u r f a c e o f  the incubation, with  the HDL  t h e t h e LDL r e c e p t o r  would g r e a t l y be enhanced. The  data  presented  T 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 of  HDL  these  was  3 7  d e g r a d e d by  results  fibroblasts.  i s not c l e a r as t h i s  o n c e . T h u s , more e x p e r i m e n t s findings.  The  However, t h e  experiment  significance  was  o n l y done  a r e needed t o c o n f i r m t h e s e  human f i b r o b l a s t model may  be  inappropriate for  s t u d y i n g t h e c a t a b o l i s m o f HDI*^. T h e r e f o r e ,  further studies  u s i n g t h e p e r i p h e r a l b l o o d m o n o c y t e s m o d e l s h o u l d be the exact metabolic Unfortunately, myself  due  In  while the r e s u l t s of t h i s  c a r r i e d out  by  time. fate of t h i s p a r t i c l e s t u d y do  is  not c o n v i n c i n g l y  t h e 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  fibroblast  more work i s r e q u i r e d i f i t s r o l e  hypercatabolism  o f apo  A-I  and  the pathogenesis  in  o f TD  i s to  be  understood.  4.3.2  The in The  particles cultured The  described  normal  D  t h e t r a n s f e r o f apo as  LDL.  physical, the  (90)  and  confirmed  i n F i g u r e s 15 by  L D L r p  also  be  t o more r a p i d l y c a t a b o l i z e d  metabolic  abnormal chemical  L D L p p  may  and  nature  composition  ( T a b l e I X ) . The 16  are the  first  normal f i b r o b l a s t s . D  by  examined.  chemical  was  demonstrated t h a t  A-I  A-I  T h e r e f o r e , t h e c a t a b o l i s m o f LDLpp  f i b r o b l a s t s was  metabolism of  fibroblast  vitro  such  e x a m i n e d and others  m e t a b o l i s m o f LDI*j, b y  i n c r e a s e d r a t e o f r e m o v a l o f apo  e x p l a i n e d by  by  examined i f  determined.  c o u l d n o t be  summary, t h e p r e c i s e m e t a b o l i c  support  fully  experiments  t o t h e d e a r t h o f sample and  u n c l e a r and  skin  f a t e o f HDLpp i s t o be  these  initial  b i n d i n g and  113  o f LDLpp  o f LDI#p  D  was  reported  experiments t o study  The  the  results  d e g r a d a t i o n was  lower  than  those  o f normal  LDL.  T h i s o b s e r v a t i o n may protein  composition  i n the  r i c h p a r t i c l e w h i c h may apo  B,  be  e x p l a i n e d by t h e a b n o r m a l l i p i d  LDLrp . D  The  isolated  from  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) in vitro  these sources a l s o t h e LDL  a  LDLrp , D  TG-  contain additional proteins besides  i s s i m i l a r t o LDL  transfer protein  composition of  and  the plasma  o r f o r LDL  ( 1 7 2 ) . LDL  m o d i f i e d by  particles  showed a d e c r e a s e d  ability  r e c e p t o r on t h e s u r f a c e o f c u l t u r e d  of  isolated  lipid  from  to interact  with  fibroblasts  (171,172). In  summary, t h e s e e x p e r i m e n t s  LDLpjj i n t h e h y p e r c a t a b o l i s m o f apo possible that the s t u d y and from  A-I  i n TD  plasma.  f i b r o b l a s t model i s t h e wrong c e l l  that c e l l s  circulation  do n o t s u p p o r t a r o l e  which are a c t i v e  for  It is type  i n the c l e a r a n c e of  (monocytes o r h e p a t o c y t e s )  may  be  is  f r o m p l a s m a may  still  be  important  necessary before e x c l u s i o n of t h i s  114  and  LDL  more  a p p r o p r i a t e t o s t u d y . T h u s ; t h i s mechanism f o r t h e r e m o v a l A-I  to  of  apo  t h e r e f o r e f u r t h e r work  c l e a r a n c e mechanism.  4.4  The l i p o l y s i s The  of  VLDLrr.  b y BmLpL  D  a s s o c i a t i o n between HDL l e v e l s a n d t h e r a t e o f l i p o l y s i s  o f t h e VLDL h a s b e e n known f o r s e v e r a l y e a r s role  o f HDL  less well  i n t h e metabolism  understood  o f p l a s m a TG  ( 4 3 ) . However, t h e  (and v i c e v e r s a ) i s  i n patients with severe disorders  o f HDL  metabolism. In t h e f o l l o w i n g study t h e p h y s i c a l , chemical properties  o f VLDL, i s o l a t e d f r o m p a t i e n t s w i t h TD and LCAT  deficiency,  were s t u d i e d  the hypertriglyceridemia has  in  i n an a t t e m p t  t o e l u c i d a t e t h e cause o f  i n these disorders.  b e e n shown t h a t t h e VLDL p a r t i c l e s t h a t  deficient  and m e t a b o l i c  In t h i s  accumulate  a n d TD d i s e a s e p l a s m a a r e s u i t a b l e s u b s t r a t e s  i n LCAT f o r BmLpL  vitro. The  k i n e t i c parameters  particles  from  the apparent greater> results  V  t h e LCAT d e f i c i e n t a n d TD p a t i e n t m  a  values are approximately  x  of this  analysis also  f o r both  and L D L V  D  control  L  C  A  substrate.  f o u r and t h r e e  that times  f o r t h e c o n t r o l VLDL. The  i n d i c a t e d t h a t the apparent  T  that  IC^ was  t h e i n t e r a c t i o n between t h e  w i t h BmLpL i s w e a k e r t h a n t h a t The d e c r e a s e d  affinity  t h e TD a n d LCAT d e f i c i e n t s u b s t r a t e size  indicate  disorders.  These r e s u l t s suggest V L D L r p  o f t h e h y d r o l y s i s o f t h e VLDL  r e s p e c t i v e l y , than t h a t  increased  may  observed  o f t h e enzyme  reflect  the  chemical  composition.  increased  F i e l d i n g e t a l h a v e shown  t h e a d d i t i o n o f exogenous FC t o n o r m a l VLDL i n v i t r o 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  increased  FC c o n t e n t o f V L D L  L C A T  115  and VLDLj,  D  with  towards  o f t h e s e a b n o r m a l 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 ,  different  the  study, i t  their  that  decreased  ( 1 6 3 ) . Thus, t h e may  explain the  abnormal ratio  KJJ/S o b s e r v e d  (Table  X I ) . However, i n TD t h e FC t o PL  i s comparable t o c o n t r o l thus suggesting  factors,  such as i n c r e a s e d  d e c r e a s e d enzyme In t h i s decreased  particle  size,  that  other  i s responsible  f o r the  affinity.  study, postheparin  i n both disorders  LpL a c t i v i t y  i s moderately  (J Frohlich, unpublished  which agrees with previous  data  deficiency  ( 1 0 3 , 1 0 4 ) . However, t h e d a t a a r e  somewhat  (173,174) o r TD  difficult  of heparin  fatty  inhibitors  o f t h e r a t e o f h y d r o l y s i s . I n some e x p e r i m e n t s ,  a c i d s would have been g e n e r a t e d s i n c e of the lipase  amount o f p o s t h e p a r i n  a decrease i n the  LpL a c t i v i t y  in itself  unpublished  results).  these subjects  vitro  absolute  i s directly  (J Frohlich,  In addition, the concentration (163),  d o e s n o t a p p e a r t o be d i r e c t l y  of hypertriglyceridemia. apo C - I I r e q u i r e d  a s low  i n a LCAT d e f i c i e n t s u b j e c t  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  sufficient  postheparin  w i t h t h e development o f h y p e r t r i g l y c e r i d e m i a  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  extent  in vitro  (104) were n o t a d d e d t o t h e  l i p a s e a c t i v i t y was a l s o o b s e r v e d  the  LCAT  due t o d i f f e r e n c e s i n t h e amount  plasma. I t appears u n l i k e l y t h a t  associated  patients with  i n j e c t e d , t h e t i m e p r i o r t o s a m p l i n g a n d t h e method o f  determination free  t o evaluate  i n other  results)  T h i s may  to fully  o f apo C - I I ,  i n t h e plasma o f  r e l a t e d t o the  reflect  the presence of  activate the lipase i n  (175). The  possibility  that  the hypertriglyceridemia  i n t h e e t i o l o g y o f t h e s e two d i s o r d e r s deficiency,  t h e few p a t i e n t s who  116  plays  a role  i s i n t r i g u i n g . I n LCAT  do n o t d e v e l o p  hyperlipidemia  (176) in  do n o t h a v e t h e r e n a l c o m p l i c a t i o n s  this  renal  disorder  (46). This  suggests that  that  are often  development o f t h e  l e s i o n s may be s e c o n d a r y t o t h e p r e s e n c e o f a b n o r m a l  particles  that  patients.  On t h e o t h e r  accumulate i n t h e m a j o r i t y hand, n e i t h e r  o f t h e LCAT d e f i c i e n t  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 renal complications) are  a t a markedly increased  their  risk  of  disease  decreases  (46,96)  i n the other  reported  established.  that postprandial  cholesterol transport  lipemia  i s associated  (177). Thus,  with  i t i s possible  increased that  deficiencies  that  preliminary  i n cholesterol transport This hypothesis  observation  that there  ability  F i e l d i n g and c o w o r k e r s h a v e  overcome some o f t h e  levels.  are not  other  t r a n s f e r o f CE t o t h e s e p a r t i c l e s may  d e c r e a s e d HDL  despite  lipoprotein  o c c u r . The  c h y l o m i c r o n s and VLDL t o a c c e p t FC a n d / o r CE f r o m i s well  patients  observation  f a c t o r s s e c o n d a r y t o t h e m a j o r d e f e c t may  lipoproteins  net  of heart  n o r TD  l o w l e v e l s o f HDL. The r e a s o n s f o r t h i s  c l e a r but i t i s probable that risk  observed  are associated  with  i s f u r t h e r s u p p o r t e d by t h e  was an i n c r e a s e  i n CE t r a n s f e r  f r o m n o r m a l HDL t o TD VLDL/LDL when n o r m a l HDL was  incubated  Tangier  ( r e s u l t s not  l i p o p r o t e i n s (d<1.21 g/ml) a n d n o r m a l LPDP  with  shown). The  p o s s i b l e causes o f h y p e r t r i g l y c e r i d e m i a  deficiency  and TD may  include  i n LCAT  one o r more o f t h e f o l l o w i n g  mechanisms: 1) I n c r e a s e d  hepatic  production  2) D e c r e a s e d c a t a b o l i s m  of TG-rich  of triglyceride  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 , be  lipoproteins;  which i n t u r n  due t o a d e c r e a s e i n l i p o p r o t e i n l i p a s e mass o r an  117  may  i m b a l a n c e between i t s a c t i v a t o r s patients 3)  The  inhibitors in  the  plasma;  inability  substrate; 4)  and  of  the  lipoprotein to  act  as  an  efficient  and  decreased hepatic  clearance of TG-rich  lipoproteins  and/or t h e i r remnants. Currently, deficiency  no  o r TD  indicate that  data regarding  the  TG-rich  suitable substrates  the  in vitro  may  not  the  The  amount o f t h e  LPL  activator  regarding  presence of p o s s i b l e  p o s t h e p a r i n LPL  LCAT d e f i c i e n t and  TD  a major r o l e i n the particles  f o r LPL  In  despite  interaction with hepatic  In due  to  no  lipase  even i n  data are  be  the  available  i s somewhat d e c r e a s e d  However, t h e  the  receptors,  i n both  magnitude of  activity  a l o n e may  TG-rich  addition,  differences  r e m a i n t o be  summary, t h e  C-II)  the not  play  lipoprotein  i t has  VLDL f r o m t h e s e p a t i e n t s  T h u s , o t h e r mechanisms, s u c h as  LCAT d e f i c i e n c y  (apo  accumulation of the  the  However,  inhibitors.  c h a n g e s i n LPL  i n these patients.  demonstrated t h a t substrates  activity  patients.  decrease suggests that  in vitro.  vivo  s i t u a t i o n . While t h e r e appears to  LCAT d e f i c i e n t p a t i e n t ,  The  study  l i p o p r o t e i n s with the  plasma of the the  LCAT  accumulate i n  for lipoprotein lipase  in vivo  in either  r e s u l t s of t h i s  lipoproteins that  i n t e r a c t i o n of TG-rich  reflect  production  have been p u b l i s h e d .  are  sufficient  TG  been  are  suitable  i n s i z e and  c a t a b o l i s m by  composition. hepatic  overproduction,  lipase,  or r o l e  of  o f HDL  is  explored.  possibility  that  a d e c r e a s e p r o d u c t i o n o f HDL  118  the  deficiency  precursors  i s not  supported  by  t h e f i n d i n g t h a t VLDLpp i s a g o o d s u b s t r a t e  However,  further studies  are required  significance of t h i s hypothesised  119  f o r BmLpL.  t o further elucidate the  defect.  4.5  A Cellular Recently,  i n TD  i s due  Defect  to  a defect  but,  i n the  n o r m a l HDL  medium  Figure  21  intracellular  i s o l a t e d f r o m TD  the  and  to  release  the  shows t h e  amount o f  No  1 2 5  and  I-HDL  3  t h a t was  b e t w e e n c o n t r o l and  TD  monocytes t h u s i n d i c a t i n g t h a t  t o t h e s e c e l l s was  not  a b n o r m a l i n TD  degradation of the  occurred.  reported  T h i s was  previously  significantly  d i f f e r e n t i n TD  s i m i l a r to those reported reported freshly  that binding  by  o f HDL  was  only  i s o l a t e d m o n o c y t e s f r o m TD  receptor  the  m o n o c y t e s i n medium c o n t a i n i n g  increased for  by  i n monocytes c o u l d  H D L . The 3  up  I-HDL  the  LDL.  ug/ml a c e t y l - L D L  i n the  release  120  of  These  incubation  turnover  the  authors  only  from t h e s e c e l l s .  cellular  the  incubating  be  cultured with  Schmitz et a l reported  o f HDL  to demonstrate d e f e c t i v e  addition,  m o n o c y t e s were  p r i o r to  are  in  expression by  and  authors  increased  regulated  TD  3  (73,74,75)  (166). In  acetylated  when c o n t r o l and  most marked r e s u l t t h a t  apparent defect attempt  be  disease.  These r e s u l t s  modestly  patients  surface  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 could  10-3 0%  2 d a y s i n 100  others  cells.  shown t h a t  HDL  demonstrated t h a t  by  1 2 5  Schmitz et a l . These  t h e s e authors have p r e v i o u s l y  associated  m a j o r d i f f e r e n c e s were o b s e r v e d  Only n e g l i g i b l e c e l l - m e d i a t e d  not  back  control individuals after 4  37°.  was  HDL  (166).  at  o f HDL  of  peripheral  incubation  binding  abnormality  trafficking  p a t i e n t s , bound  failed  w i t h c u l t u r e d m o n o c y t e s f r o m TD hours of  ?  a t a r a t e comparable t o c o n t r o l monocytes  u n l i k e c o n t r o l monocytes,  i n t o the  i n TD  T h e s e i n v e s t i g a t o r s showed t h a t  monocytes,  internalized  Metabolism  Assmann e t a l h a v e s u g g e s t e d t h a t  i n t e r n a l i z e d HDL. blood  o f HDL  1  was In  o f HDL  2  5  i -  the an  in  TD  cells the  t h a t had n o t been p r e i n c u b a t e d  incubation  period  approximately  release  o f HDL  label within any  3  control  5 hours,  associated  HDL i s r e l e a s e  i t was assumed t h a t  would m a n i f e s t  the c e l l s  significant  the length of  o f t h e e x p e r i m e n t was e x t e n d e d . A s  80% o f c e l l  monocytes w i t h i n  i n acetyl-LDL,  during  itself  from  any d e f e c t  cells  t h e extended  should  i n the  by t h e a c c u m u l a t i o n o f incubation.  d e g r a d a t i o n o f t h e i n t e r n a l i z e d HDL  and T a n g i e r  cultured  3  Moreover,  i n both the  be a p p a r e n t a f t e r t h i s p e r i o d o f  incubation. Figure associated  22 shows t h a t t h e amount o f with the c e l l s  1 2 5  I - l a b e l l e d HDL  a f t e r 20 h o u r s was n o t d i f f e r e n t  b e t w e e n c o n t r o l o r t h e TD c e l l s .  Thus,  i t was c o n c l u d e d  a b n o r m a l HDL m e t a b o l i s m c a n n o t b e d e m o n s t r a t e d isolated The freshly  i n freshly  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  i s o l a t e d , monocytes a r e n o t c l e a r a t p r e s e n t . lipid  before  loading  the defect  o f t h e monocytes by a c e t y l - L D L i s c a n b e d e m o n s t r a t e d . However, i f t h i s  a l s o t h e case  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  CE  i n vivo,  It is  is  that  one s h o u l d  consider  that  origin  of the  proposed  r a p i d u p t a k e o f t h e HDL i n p a t i e n t s w i t h TD may s u p p l y t h e  w h i c h a c c u m u l a t e s a n d r e s u l t s i n foam c e l l  a l t h o u g h no e x p e r i m e n t a l e v i d e n c e hypothesis. for  that  monocytes.  possible that required  3  since  lipid.  LDL f r o m TD p a t i e n t s  composition  i s available to test  I t i s also possible that  the accumulating  In f a c t ,  LDL p r o v i d e s this  the cholesterol  and p r o t e i n  electrophoretic mobility  121  this  seems more f e a s i b l e  has abnormal l i p i d  (90) and i n c r e a s e d  d e v e l o p m e n t (166)  (95,100)  w h i c h may  result in cellular  Whether t h i s observed i n TD  i n TD  an  for their  ability  and  Dr.  Both of these  not  TD  s t u d y showed t h a t  TD  o r d e g r a d e n o r m a l HDL  release  and  shown t o be  in culture.  fibroblasts did  i n an i n our  abnormal l a b by  d e g r a d a t i o n o f HDL  o f ACAT and  n o r m a l i n TD  d e g r a d e HDL  showed t h a t t h e  lines.  subsequent s e r i e s of experiments the activity  turnover  not  fashion.  R.  McLeod  et  U n i v e r s i t y of Washington, S e a t t l e  d i f f e r e n t from c o n t r o l c e l l  intracellular  A-I  s k i n f i b r o b l a s t s were examined  independent s t u d i e s  internalization,  i n LDL-C  evaluated.  t o b i n d / i n t e r n a l i z e and  J.Oram a t t h e  decreases  k i n e t i c s o f apo  t h e s e r e s u l t s were c o n f i r m e d  by  s c a v e n g e r pathway.  significant  e x p l a i n the  a d d i t i o n a l study,  r e s u l t s of t h i s  Recently  was  and  r e m a i n s t o be  bind/internalize  al  r e s u l t i n the  (90)  patients In  The  could  uptake v i a the  binding,  by  TD f i b r o b l a s t s  Furthermore,  metabolism of  in a  LDL,  and  HMGCoA r e d u c t a s e were  f i b r o b l a s t s (R.  McLeod,  Wa.  the  also  unpublished  results). In a recent interaction was  slightly  o f HDL  involve the  i t was  e t i o l o g y of It  Frohlich,  has  h a v e shown t h a t  i s o l a t e d from our  f o r control adipocytes.  u n l i k e l y t h a t TD  adipocytes  p o s s i b l e , however, t h a t  a cellular  r o l e o f TD  (178)  TD  the  patient  Frohlich  play  a role in  TD.  is still  material  with adipocytes  l e s s than t h a t  concluded that the  paper, F r o h l i c h e t a l  defect  component. T h u s f u r t h e r s t u d i e s  liver  in this  been d e s c r i b e d  disorder  as  in hepatic  unpublished r e s u l t s ) .  p r e s e n t e d h e r e and  the  In  those reported 122  apo  A-I  should  may examine  immunoreactive  Kuppfer c e l l s  a d d i t i o n , the by  i n TD  i n TD  (J.  experiments  Schmitz et a l  (166)  investigated  t h e i n t e r a c t i o n o f TD c e l l s w i t h n o r m a l  However, i t i s p o s s i b l e t h a t the  TD c e l l s  these  t h e s i s , i t h a s b e e n d e m o n s t r a t e d t h a t when  i s incubated  w i t h TD p l a s m a , t h e CE c o r e  to the actual  observation  D  s i t u a t i o n . Despite  i n H D L metabolism  molecular defect  i n TD p a t i e n t s  i s t o be  123  3 . 1 ) . Thus,  p a r t i c l e may be more  a n d t h o s e o f S c h m i t z and c o w o r k e r s ,  investigations exact  i n vivo  HDLrj-,  normal  i s r a p i d l y exchanged  lipoproteins (Section  i s possible that hypercatabolism of  relevant  the  interacts with  i s markedly d i f f e r e n t from t h a t used i n  f o r TG f r o m t h e l o w e r d e n s i t y it  that  experiments. In t h i s  HDL  i n vivo  t h e HDL p a r t i c l e  HDL.  described.  these  more are required i f  REFERENCES 1.  R o s s R. N . E n g l . J . M e d . 1986,  2.  L e v y R I . A t h e r o s c l e r o s i s . 1981,  3.  Inkeles 123.  4.  G o r d o n T, C a s t e l l i WP, 251:351-374.  5.  Heiss  6.  L i p i d R e s e a r c h C l i n i c s P r o g r a m . The L i p i d R e s e a r c h C l i n i c s c o r o n a r y p r i m a r y p r e v e n t i o n t r i a l r e s u l t s . J Am Med A s s ' n . 1984, 251: 351-374.  7.  Brown MS,  8.  H e r b e r t PN, Assmann G, G o t t o AM, F r e d r i c k s o n D. 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