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Antioxidant properties of propofol in coronary artery bypass surgery Sun, Jianhang 1998

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A N T I O X I D A N T PROPERTIES O F P R O P O F O L IN C O R O N A R Y A R T E R Y B Y P A S S S U R G E R Y by JIANHANG SUN M . B . , The Second Military Medical University of P L A ,  1986  A T H E S I S S U B M I T T E D IN P A R T I A L F U L F U L M E N T O F THE REQUIREMENTS FOR T H EDEGREE OF MASTER OF SCIENCE in T H E F A C U L T Y O F G R A D U A T E STUDIES (Department of Surgery)  \ f  We accept this thesis as conforming to^he required standard  T H E U N I V E R S I T Y O F BRITISH C O L U M B I A September 1998 © l i a n h a n g Sun, 1998  ;  In  presenting  degree  at  the  this  thesis  in partial  University of  freely available for reference copying  of  department publication  this thesis or  by  British Columbia,  his  or  her  ?  /  The University of British Columbia Vancouver, Canada  DE-6 (2/88)  requirements  I agree that the  representatives.  may be It  for financial gain shall not  permission.  S ^j  the  for  an  is  granted  advanced  Library shall make it  and study. I further agree that permission for  for scholarly purposes  of this thesis  Department of  fulfilment of  by the  understood  that  extensive  head  of my  copying  or  be allowed without my written  ABSTRACT  Ischaemia  r e p e r f u s i o n i n j u r y (IRI)  of c a r d i o p u l m o n a r y Reactive  dysfunction in cardiopulmonary  o x y g e n intermediates  antioxidant  capacity,  markedly  reduced  cardiopulmonary  is c o n s i d e r e d one o f the m a j o r  with tissue  have b e e n l i n k e d to I R I .  vitamin lipid  function.  bypass  An  C,  vitamin  peroxidation intravenous  E,  M e a s u r e s to  and  and  (CPB)  surgery. increase  allopurinol,  resulted  anaesthetic,  causes  in  have  improved  propofol  has  been  f o u n d to have a n t i o x i d a n t p r o p e r t i e s i n c e l l c u l t u r e a n d i n a n i m a l s t u d i e s . T h e a n t i o x i d a n t p o t e n t i a l o f p r o p o f o l d u r i n g C P B s u r g e r y has not b e e n r e p o r t e d . Following  institutional  approval  and  informed  patient  consent,  patients s c h e d u l e d f o r C P B s u r g e r y were e n r o l l e d . P a t i e n t s w e r e with sufentanil-isoflurane (control, 2.5  m g / k g b o l u s t h e n 100  n=ll),  Venous blood  capacity challenge  as  was  plasma  sampled for  concentration  p g / k g / m i n continous  determination production of  of red  against  propofol.  in  Clinical  cell vitro  dose  oxidative  parameters  of  changes,  oxygenation.  Red cell antioxidant capacity propofol.  infusion;  antioxidant  interest i n c l u d e d p e r i o p e r a t i v e i n o t r o p i c r e q u i r e m e n t , h a e m o d y n a m i c and l u n g  (1.5-  p g / k g / m i n p r e C P B , 50 u g / k g / m i n i n t r a - C P B ; n = 7),  M D A (malondialdehyde)  and  anaesthetized  s u f e n t a n i l - l o w dose p r o p o f o l  or h i g h dose p r o p o f o l ( 1 . 5 - 2 . 5 m g / k g t h e n 200 n = 8).  26  This  effect  continued 2  a n d l o w dose p r o p o f o l were  increased hours  significantly only with high  after  associated  separation  from bypass.  w i t h s i g n i f i c a n t l y less  dose High  inotropic  ii  (dopamine increased  3-5 the  operatively. lipid  percentage  Improvement  peroxidation.  cardiodepresant cardiac was  pg/kg/min)  index  requirement. of  patients  of cardiac  Propofol's  Low with  dose  propofol  normal  cardiac  significantly index  f u n c t i o n d i d not p a r a l l e l the r e d u c t i o n  effects  were  l i m i t e d to  lipid  membranes.  e f f e c t o f h i g h dose p r o p o f o l was m a n i f e s t e d as r e l a t i v e l y w i t h i n the  post-  f i r s t three h o u r s  after o p e r a t i o n .  Lung  s u p e r i o r w i t h l o w dose p r o p o f o l and i s o f l u r a n e after 6 to  o p e r a t i o n , c o m p a r i n g to that w i t h i n one h o u r after o p e r a t i o n  of  The low  oxygenation  12 h o u r s  after  (P<0.05).  iii  T A B L E OF CONTENTS  Abstract  ii  Table of Contents  iv  List of Tables  viii  List of Figures  ix  Acknowledgements  x  Chapter 1 Introduction 1.1  Overview  , 1  1.2  Free Radicals  1  1.2.1  F r e e R a d i c a l s and R e a c t i v e O x y g e n I n t e r m e d i a t e s ( R O I s )  1  1.2.2  Free Radical Effects  3  1.2.3  M e c h a n i s m s o f the E n d o g e n o u s R e l e a s e o f F r e e R a d i c a l s  4  d u r i n g T i s s u e Injury 1.3  L i p i d P e r o x i d a t i o n and D e t e c t i o n  7  1.3.1  Lipid Peroxidation  7  1.3.2  A r a c h i d o n i c A c i d M e t a b o l i s m and M D A F o r m a t i o n  8  1.3.3  M D A : A M a r k e r for L i p i d Peroxidation  10  1.3.4  C o n j u g a t e d D i e n e s and P h o s p h o l i p i d M o l a r R a t i o :  10  A n o t h e r C h e m i c a l M a r k e r for Oxidative Injury 1.4  F r e e R a d i c a l G e n e r a t i o n and M y o c a r d i a l D a m a g e d u r i n g  11  iv  CAB 1.4.1  Surgery  Pattern of Systemic CAB  Free Radical Generation during  11  Surgery  1.4.2  M y o c a r d i a l Ischaemia R e p e r f u s i o n Injury C a u s e d B y R O I s  12  1.5  B o d y A n t i o x i d a n t Defense Systems  14  1.5.1  N o n e n z y m i c : V i t a m i n C and V i t a m i n E  15  1.5.2  Major Extracellular  15  1.5.3  E n z y m i c Antioxidant Defenses  16  1.6  P r e o p e r a t i v e V i t a m i n C , V i t a m i n E , and A l l o p u r i n o l  17  Protection  Mechanisms  T h e r a p y i n Patients U n d e r g o i n g C A B Surgery 1.7  E G b 761  and C a r v e d i l o l : U n c o m m o n l y U s e d A n t i o x i d a n t s  19  for C A B Surgery 1.7.1  E G b 761  19  1.7.2  Carvedilol  20  1.8  P r o p o f o l : A n Anaesthetic  1.8.1  Structure  21  1.8.2  Basic Pharmacokinetics  23  1.8.3  Mechanisms of Antioxidant A c t i v i t y  24  1.8.4  A n t i o x i d a n t A c t i v i t i e s of P r o p o f o l in A n i m a l Studies  25  1.8.5  Propofol Anaesthesia  29  Chapter 2 Objectives  and A n t i o x i d a n t  in C A B Surgery  21  31  Chapter 3 Hypotheses  32  C h a p t e r 4 M a t e r i a l and M e t h o d s  35  4.1  35  C o r o n a r y A r t e r y Bypass Surgery: A M o d e l for Ischemia R e p e r f u s i o n Injury  4.1.1  Selection Criteria  35  4.1.2  Study D e s i g n  35  4.1.3  B l o o d Sample C o l l e c t i o n  38  4.1.4  Clinical Data Collection  40  4.1.5  C a r d i o p u l m o n a r y Bypass  40  4.1.6  Data Analysis  40  4.2  M e a s u r e m e n t of in vitro R e d C e l l M D A P r o d u c t i o n : T B A A s s a y  41  4.3  M e a s u r e m e n t of Plasma P r o p o f o l C o n c e n t r a t i o n by H P L C  43  Chapter 5 Results  48  5.1  Patient P r o f i l e  48  5.2  Red C e l l M D A Production F o l l o w i n g in vitro t B H P Challenge  48  5.2.1  t B H P Dose-Response Curve  48  5.2.2  R e d C e l l M D A P r o d u c t i o n In R e s p o n s e T o In V i t r o t B H P  48  (1.5  m M ) Challenge  5.3  Plasma Concentration of P r o p o f o l  49  5.4  Inotropic Requirement during C A B Surgery  49  5.4.1  P e r c e n t a g e o f Patients G i v e n I n o t r o p i c D r u g s  49  vi  5.4.2  P e r c e n t a g e o f P a t i e n t s G i v e n D o p a m i n e (3.5  pg/kg/min)  50  5.4.3  Percentage of Patients G i v e n A d r e n a l i n e  50  5.5  H e m o d y n a m i c C h a n g e s d u r i n g 24 H o u r s P o s t - o p e r a t i o n  50  5.5.1  C a r d i a c Index  50  5.5.2  Pulmonary C a p i l l a r y Wedge Pressure  50  5.5.3  Central Venous Pressure  51  5.6  A l v e o l a r A r t e r i a l O x y g e n G r a d i e n t d u r i n g 24 H o u r s P o s t -  51  operation  Chapter 6 D i s c u s s i o n 6.1  Evaluation of Hypothesis  67  6.2  What Does Increased Red C e l l Antioxidant Capacity M e a n ?  69  6.2.1  Generalized Enhancement  69  6.2.2  Partially Improved Cardiopulmonary Function  70  6.3  Propofol Concentration  71  6.4  M D A P r o d u c t i o n , R e d C e l l Status, and T B A A s s a y  73  6.5  I n f l u e n c e o f N o r m o t h e r m i a or H y p o t h e r m i a f o r C P B o n  74  o f T i s s u e O x i d a n t Status  and Its P r o t e c t i v e E f f e c t s  R e d C e l l A n t i o x i d a n t Status and C a r d i o p u l m o n a r y  C h a p t e r 7 C o n c l u s i o n and R e c o m m e n d a t i o n s  References  Function  for Further W o r k  77  79  vii  LIST O F T A B L E S  Table  Page  1.  Patient p r o f i l e , by group  52  2.  t B H P - M D A d o s e - r e s p o n s e c u r v e f o r r e d c e l l s f r o m the  53  three e x p e r i m e n t a l 3.  groups  R e d c e l l M D A p r o d u c t i o n and p l a s m a c o n c e n t r a t i o n o f  54  p r o p o f o l at v a r i o u s time i n t e r v a l s 4.  Inotropic requirement d u r i n g C A B surgery, by group  5.  C a r d i a c index, p u l m o n a r y c a p i l l a r y wedge pressure, c e n t r a l v e n o u s p r e s s u r e d u r i n g 24 h o u r s  6.  L/min/m 7.  d u r i n g 24 h o u r s  and  56  post-operation  P e r c e n t a g e o f patients w i t h a c a r d i a c i n d e x 2  55  >  2.5  57  post-operation  A l v e o l a r arterial oxygen gradient  58  viii  LIST O F F I G U R E S Figure 1.  Page R e a c t i o n s i n v o l v e d i n the g e n e r a t i o n o f s u p e r o x i d e b y N A D P H o x i d a s e and the r e s p i r a t o r y b u r s t o f  6  phagocytes  2.  X a n t h i n e o x i d a s e and s u p e r o x i d e r a d i c a l f o r m a t i o n  6  3.  P a t h w a y by w h i c h M D A is f o r m e d as a b y - p r o d u c t i n  9  arachidonic acid metabolism 4.  Structural formulae of p r o p o f o l , butylated hydroxytoluene  22  and v i t a m i n E 5.  E f f e c t o f p r o p o f o l i n i n t r a l i p i d or i n t r a l i p i d a l o n e o n  28  h y d r o g e n p e r o x i d e - i n d u c e d c h a n g e s o f the rat heart tissue c o n c e n t r a t i o n o f M D A 6.  Schematic time-line representation of blood sampling for red  39  c e l l M D A p r o d u c t i o n and p l a s m a c o n c e n t r a t i o n o f p r o p o f o l 7.  Schematic representation of chromatograms  o f extracts  47  8.  t B H P dose-response curve for red c e l l M D A p r o d u c t i o n  59  9.  R e d c e l l M D A p r o d u c t i o n and p l a s m a c o n c e n t r a t i o n o f  60  propofol 10.  T h e r e l a t i o n s h i p o f r e d c e l l M D A p r o d u c t i o n and p l a s m a  61  c o n c e n t r a t i o n o f p r o p o f o l i n the h i g h dose p r o p o f o l g r o u p 11.  Inotropic requirement during C A B surgery  62  12.  H e m o d y n a m i c c h a n g e s d u r i n g 24 h o u r s p o s t - o p e r a t i v e l y  63  13.  C a r d i a c i n d e x d u r i n g the f i r s t 3 h o u r s p o s t - o p e r a t i v e l y  64  C a r d i a c index versus p u l m o n a r y c a p i l l a r y wedge pressure  65  14.  d u r i n g 24 h o u r s 15.  post-operatively  A l v e o l a r a r t e r i a l o x y g e n g r a d i e n t d u r i n g 24 h o u r s  66  post-operatively  ix  ACKNOWLEDGEMENTS  I am g r a t e f u l to D r . A . K . Q a y u m i f o r t a k i n g me to the p r o g r a m and f o r his c o n t i n o u s g u i d a n c e and s u p p o r t , e s p e c i a l l y i n the w e e k l y l a b o r a t o r y am  grateful  to  Dr.  D . M . Ansley  and  Dr.  D.V.  Godin  o p p o r t u n i t y to w o r k w i t h t h e m and f o r their e n t h u s i a s t i c the t i m e .  I a m also g r a t e f u l  for  meetings.  giving  me  I  the  s u p p o r t o f m y study a l l  to D r . D . R . B e v a n f o r his k i n d  concern  about  the  thesis p r o j e c t . I w o u l d l i k e to thank M a u r e e n G a r n e t t f o r t e a c h i n g me the T B A a s s a y ; Sharon  Duncan  for  teaching  me  t e a c h i n g me the b a s i c l a b o r a t o r y slides f o r m y thesis d e f e n s e .  radioimmunoassay;  techniques;  thank  David  Fong  thank for  a n d thank Janet H a i n e s f o r m a k i n g  T o my wife Jennifer and son Sean w h o c o n t i n u a l l y p r o v i d e the i n s p i r a t i o n , and to m y sister L i p i n g S u n f o r her u n l i m i t e d s u p p o r t  xi  Chapter 1 INTRODUCTION  1.1  Overview Ischaemia  major  causes  (CAB)  surgery  [2,6-8].  reperfusion of  (IRI) has b e e n  cardiopulmonary  [1-5].  Measures  conditioning  injury  Reactive  to  with  dysfunction  considered  in  coronary  oxygen intermediates  increase  antioxidant  vitamin C , vitamin E  as  one o f the  artery  bypass  h a v e b e e n l i n k e d to I R I  capacity,  such  or a l l o p u r i n o l ,  as  have  preoperative demonstrated  m a r k e d l y r e d u c e d tissue l i p i d p e r o x i d a t i o n i n b o t h a n i m a l a n d h u m a n s t u d i e s , in  association  intravenous to  have  with  anaesthetic,  antioxidant  anaesthesia  improved  cardiopulmonary  function  [1,9-13].  An  2 , 6 - d i i s o p r o p y l p h e n o l , or p r o p o f o l , has b e e n s h o w n  activity  in animal  studies  has b e e n u s e d f o r C A B s u r g e r y  [14-18].  since  Although  propofol  1 9 8 7 , its p r o t e c t i v e  effects  as an a n t i o x i d a n t d u r i n g s u r g e r y h a v e not b e e n r e p o r t e d .  1.2  Free  Radicals  1.2.1 F r e e R a d i c a l s a n d R e a c t i v e O x y g e n I n t e r m e d i a t e s Free  radicals  unpaired electrons a pair; therefore,  are  molecules  i n their outer  or  fragments  of  (ROIs)  [19]  molecules  orbitals. U n p a i r e d electrons  containing  tend to a c q u i r e  most free r a d i c a l s are c h e m i c a l l y h i g h l y r e a c t i v e  a n d , as a  result, s h o r t - l i v e d .  l  Free  radical reactions  are  chain reactions  started  by  initiators  causing  h y d r o g e n r e m o v a l , w h i c h is f o l l o w e d by s e v e r a l steps o f p r o p a g a t i o n i n w h i c h the  free  radicals  produced  interact  w i t h one  m o l e c u l e s , and the r e a c t i o n terminates  another  and  with surrounding  w h e n the free r a d i c a l s are c o n v e r t e d to  nonradical products. Reactive free  radicals  play  an  by  hydrogen products  role  peroxide, the  oxygen,  products  (singlet  and  resemble  is  of oxygen  oxygen)  or  excitation  hydrogen  those  or  a collective [20].  of  peroxide).  o f free  radicals.  Free They  By  term  of  oxygen  (superoxide  are  However,  oxygen  generated  definition,  oxygen  for  radicals are  reduction  hydroxyl radical).  reduction and  (ROIs)  i n b i o l o g y and m e d i c i n e .  excitation  of  reactions  intermediates  and r e a c t i v e  important  oxygen  singlet  oxygen  not  free their  These products,  from anion,  all  reactive  radicals  (e.g.,  reactivity  together  and  w i t h the  true free r a d i c a l s , are c a l l e d R O I s . Well-recognized oxygen( 0 ), 1  radical  2  ROIs  hydrogen  (ROO),  include  peroxide  perhydroxyl  superoxide  (H 0 ), 2  2  superoxide  anion  is  is less  reactive,  a n i o n and it c a n  longer  lived  singlet  radical(OH),  peroxyl  2  radical  (RO),  ferryl  unreactive  and  under  by d i s m u t a t i o n , to h y d r o g e n p e r o x i d e .  and m o r e  diffuse considerable  ),  (0 ~  [21].  comparatively  p h y s i o l o g i c a l c o n d i t i o n s is c o n v e r t e d , This  hydroxyl  r a d i c a l ( H O O ) , alkoxyl  h a e m p r o t e i n r a d i c a l , and n i t r i c o x i d e ( N O ) The  radical  lipophilic  distances  f r o m its  than the site  of  superoxide generation.  2  The  m a j o r d a n g e r o f i n c r e a s e d tissue c o n c e n t r a t i o n s  o f h y d r o g e n p e r o x i d e is  the p r o d u c t i o n o f a h y d r o x y l r a d i c a l b y the H a b e r W e i s s o r F e n t o n r e a c t i o n : 0 "  +  2  The  hydroxyl radical  example,  it  will  H 0 2  -->  2  has a short  rapidly  react  0  2  +  OH- +  half-life, with  OH  a n d is e x t r e m e l y  unsaturated  fatty  reactive.  acid  side  For  chains,  r e s u l t i n g i n l i p i d p e r o x i d a t i o n . F o r t u n a t e l y , the H a b e r - W e i s s r e a c t i o n is v e r y slow  under  catalysts.  physiological conditions.  It  is, however,  accelerated  by  metal  T h i s p r o c e s s is c a l l e d the F e n t o n r e a c t i o n .  Exogenous study b e c a u s e  H 0 2  has b e e n u s e d  2  (1) H 0 2  the p a t h o g e n e s i s  2  as a s o u r c e  a n d its m e t a b o l i t e  o f I R I ; (2) H 0 2  r e a c h i n g i n t r a c e l l u l a r sites  2  of ROIs  for experimental  O H are k n o w n to be i m p o r t a n t i n  c a n penetrate the c e l l m e m b r a n e ,  thereby  [15,22].  1.2.2 Free R a d i c a l E f f e c t s A l t h o u g h o x y g e n is e s s e n t i a l f o r a e r o b i c l i f e f o r m s , too m u c h o x y g e n or i n a p p r o p r i a t e m e t a b o l i s m o f o x y g e n c a n be t o x i c to the o r g a n i s m [23]. Physiologically, process  [21].  (1)  free  radicals  Superoxide  are e s s e n t i a l  c a n be g e n e r a t e d  to m a n y by a  normal biological variety  of cells  to  p e r f o r m u s e f u l f u n c t i o n s i n the b o d y : they are part o f the c a s c a d e o f events i n tissue r e s p o n s e to i n v a d i n g m i c r o o r g a n i s m s a n d f o r e i g n m a t e r i a l t h r o u g h the antimicrobial eosinophils)  action [24,25];  of (2)  enzyme-catalyzed reactions,  phagocytic They  are  cells  (neutrophils,  intermediates  monocytes,  in and/or  f o r e x a m p l e the a c t i o n o f x a n t h i n e  products  and of  oxidase; 3  (3)  They  are r e g u l a t o r y  molecules  in biochemical processes.  lymphocytes and fibroblasts constantly r a d i c a l as g r o w t h r e g u l a t o r s and l i p o x y g e n a s e  [26].  For  generate small amounts  (4) T h e y are i n v o l v e d i n  action in eicosanoid metabolism.  example,  of superoxide cyclooxygenase  (5) T h e y are i n v o l v e d i n  the s y n t h e s i s o f a d r e n o c o r t i c a l h o r m o n e s a n d i n the f u n c t i o n a l d i v i s i o n o f the c o r t e x [25]. Pathologically,  abnormal  free  radical  reactions  may  be  elicited  by  p h y s i o l o g i c a l f r e e r a d i c a l s i f the c o n t r o l m e c h a n i s m s are d e f e c t i v e , or i f there is  a  marked  increase  in  the  production  of  free  conditions of self protection. Free radical reactions biomolecule: genetic  enzymes,  material,  proteins,  membranes  carbohydrates,  are a l l p o t e n t i a l  membrane,  sites o f l i p i d  normal  may damage v i r t u a l l y any  lipids  or n u c l e i c a c i d s . T h e  m i t o c h o n d r i a , microsomes and  p e r o x i d a t i o n damage  o r g a n is s p a r e d f r o m s u c h o x i d a t i v e d a m a g e . as c a n c e r , a t h e r o s c l e r o s i s ,  under  composed predominantly o f lipids and proteins,  and s u b c e l l u l a r s t r u c t u r e s ( p l a s m a lysosomes)  radicals  [23,26]. N o  A g i n g and many d i s o r d e r s ,  such  a n d n e u r a l d i s o r d e r s , h a v e a l l b e e n r e l a t e d to s u c h  oxidative injury [26-28].  1.2.3  M e c h a n i s m s o f the e n d o g e n o u s  Injury.  release o f Free  Radicals during Tissue  [9,21,25,29]  1. P h a g o c y t e  r e c r u i t m e n t a n d a c t i v a t i o n at the site o f i n j u r y ( F i g u r e  2.  oxidase  Xanthine  a n d other  superoxide-producing  Studies of xanthine oxidase d i s t r i b u t i o n i n cardiac  enzymes  1).  (Figure  2).  tissue h a v e s h o w n that the  4  enzyme  is  localized  than i n m y o c y t e s  in oxidation-sensitive  vascular  leakage of electrons A  number  smooth  muscle  cells  rather  [30].  3. D i s r u p t e d m i t o c h o n d r i a l e l e c t r o n t r a n s p o r t ,  4.  endothelial  of  e.g.  during ischemia, allowing  onto o x y g e n d u r i n g r e p e r f u s i o n .  cell  cells,  types, have  including been  endothelial  shown  from  cells,  studies  macrophages,  in cell  culture  and to  be  capable of p r o d u c i n g superoxide radicals.  5  Oxidase NADPH +  *  2O2  NADP+ + H  + 2 O2  +  H2O2 MPO  CI  HOC1  Figure 1. Reactions involved in the generation of superoxide by NADPH oxidase and the respiratory burst of phagocytes.  NADPH, nicotinamide adenine dinucleotide phosphate;  MPO, myeloperoxidase; H O G , hypochlorous acid.  Allopurinol  I1  Xanthine oxidase Xanthine or Hypoxanthine  NADPH  •  NADP+ + H  +  • O2  •  Uric Acid  O2 -•  Figure 2. Xanthine oxidase and by-product superoxide radical formation.  6  1.3  L i p i d P e r o x i d a t i o n and D e t e c t i o n  1.3.1  Lipid The  peroxidation.  most  common  c h e m i c a l nature  target  (cholesterol fatty  (monolayers  lipoproteins,  auto-oxidation  of  acids)  the  is m e m b r a n e  unsaturation,  polyunsaturated in  of R O I s  and  of  lipids  of  conjugated  their  bilayers  in  lipids  regular  cell  living  because  of  their  unsaturated  linkages  structural  arrangement  membranes)  organisms  is  [31].  of  However,  usually  a  slow,  c o m p a r t m e n t a l i z e d p r o c e s s b e c a u s e m o l e c u l a r o x y g e n is a w e a k o x i d a n t  [19].  L i p i d p e r o x i d a t i o n is a p r o c e s s i n w h i c h a l i p i d free r a d i c a l R- is f o r m e d by  the  effect  of  a free  molecular oxygen, started,  the  initiator.  producing a peroxy  effect  pathological  radical  of  which  conditions,  is  not  damage  The  free  radical  then  reacts  with  free r a d i c a l R O O - . A c h a i n r e a c t i o n restricted  to  neighboring  lipids,  but  may,  biomolecules  is  under  (proteins,  carbohydrates,  n u c l e i c a c i d s ) [19].  M o r e o v e r , by m e d i a t i o n o f stable  products  (comparing  unstable  of  [19,32],  to  products)  the  process,  such  as  alkanes  remote m o l e c u l e s may also be d a m a g e d t h r o u g h l o c a l d i f f u s i o n or c i r c u l a t i o n . Marked  reduction  of energy  (malondialdehyde, hydroperoxides enzymes,  has  metabolism  hydroxypentenal, been  described  and [32],  by  aldehyde  hydroxynonenal), including  and r e d u c t i o n o f m i t o c h o n d r i a l r e s p i r a t i o n .  e n z y m e s are also a f f e c t e d  products and  inhibition Other  of  of  oxidation  by  various  glycolytic  membrane-bound  [33].  7  1.3.2  A r a c h i d o n i c A c i d M e t a b o l i s m and M D A F o r m a t i o n [ 2 5 , 3 4 ] MDA  fatty  acids  ( m a l o n d i a l d e h y d e ) is f o r m e d f r o m p e r o x i d a t i o n o f p o l y u n s a t u r a t e d with conjugated  double bounds,  m a i n l y v i a the  arachidonic  acid  metabolism pathway  in vivo.  M a l o n d i a l d e h y d e is f o r m e d not o n l y as a r e s u l t  of  activity  as  cyclooxygenase  synthesis,  a  but also by l i p o x y g e n a s e  fatty a c i d s ( F i g u r e  by-product activity  of  thromboxane  as a b y - p r o d u c t o f  A2  (TxA2)  hydroperoxy  3).  8  Phospholipid ^ Phospholipase A2 Arachidonic acid  Figure 3. Pathway by which MDA is formed as a by-product in arachidonic acid metabolism. Tx A2, thromboxane A2; TxB2, thromboxane B2; HHT, (12S-12-hydroxy-5Z,8E,10E)-heptadecatrienic acid; MDA, malondialdehyde.  9  1.3.3 M D A : A M a r k e r f o r L i p i d Malondialdehyde cells[38-40],  tissues  mitochondria  [16,44]  provides a measure of  the b o d y  is  Peroxidation  metastable,  [15,41-43],  and detectable and  and microsomes  subcellular [45].  in plasma  [1,35-37],  structures  such  T h e concentration  of M D A  o f the extent o f l i p i d p e r o x i d a t i o n a n d a n t i o x i d a n t  [1,15,39,45,46].  Therefore,  M D A has b e e n  as  widely  status  used  as a  marker for lipid peroxidation. In h e a l t h y p e r s o n s ,  the a v e r a g e p l a s m a M D A c o n c e n t r a t i o n  p m o l / L f o r age less than 30 y e a r s , more  than  40  thiobarbituric specific detection  1.3.4  years acid  [35].  These  ( T B A ) assay,  high-performance  a n d about levels  0.7  1.2 p m o l / L f o r i n d i v i d u a l s o f  are  undetectable  with  but c a n be d e t e c t e d b y m o r e  liquid  is about  chromatography  the  classic  s e n s i t i v e and  ( H P L C ) with  fluorescence  [47-49].  Conjugated  Dienes  and P h o s p h o l i p i d M o l a r  Ratio:  Another  Chemical  M a r k e r for O x i d a t i v e Injury In v i t r o e x p e r i m e n t s polyunsaturated  s h o w e d that free r a d i c a l s c a n c a u s e i s o m e r i z a t i o n o f  fatty a c y l c h a i n s ,  generating  products  with diene  conjugation  but w i t h o u t p e r o x i d a t i o n [ 5 0 , 5 1 ] . T h e i s o m e r i z a t i o n m o s t s t u d i e d has b e e n the conversion (18:2(9,11)). plasma,  of  linoleic  acid  (18:2(9,12))  to  9,11-octadecadienoic  T h i s a c c o u n t s f o r o v e r 90% o f the d i e n e c o n j u g a t i o n  tissue,  a n d tissue  fluids  [52].  HPLC  1 8 : 2 ( 9 , 1 1 ) a n d the p a r e n t c o m p o u n d 1 8 : 2 ( 9 , 1 2 )  can measure  both  acid  in human  the  i n p l a s m a . M o l a r ratio  isomer is the  10  ratio o f 18:(9,11) to 1 8 : 2 ( 9 , 1 2 ) fraction  is a f f e c t e d  esterified  fraction  [4,53]. T h e molar ratio  by heparin, is  but the m o l a r  not [54].  Therefore  ratio  i n the f r e e fatty a c i d i n the p h o s p h o l i p i d -  the p h o s p h o l i p i d m o l a r  ratio  is  measured.  1.4  Free R a d i c a l G e n e r a t i o n and M y o c a r d i a l Damage d u r i n g C A B Surgery  1.4.1 P a t t e r n o f S y s t e m i c F r e e R a d i c a l G e n e r a t i o n d u r i n g C A B S u r g e r y HEART.  R e p e r f u s i o n o f m y o c a r d i u m after  c l a m p i n g or c o r o n a r y of pathways blood  from  catheters  reveals,  coronary  venous  venous  accumulate during  systemic  blood  i n the  oxidative  e n d o t h e l i a l and m y o c a r d i a l c e l l s  however,  and arterial  cross-  o c c l u s i o n c a n l e a d to the r e l e a s e o f R O I s f r o m a v a r i e t y  in phagocytes,  sampling  the p e r i o d o f a o r t i c  arterial,  that  to,  or less  Serial  and coronary  sinus  i n M D A and molar than,  during  not o c c u r  early [2].  reperfusion,  Thus,  ratio  the i n c r e a s e s  E v e n though neutrophils have  myocardium  stress does  venous  the i n c r e a s e  is s i m i l a r  b l o o d [4].  mixed  [8,55-58].  in mixed  been their  in  s h o w n to activation  it seems u n l i k e l y  that the  m y o c a r d i u m is a m a j o r s o u r c e o f s y s t e m i c R O I s d u r i n g C P B . LUNG. production collapsed  A c t i v a t e d n e u t r o p h i l s w i t h i n the l u n g s m a y be i m p o r t a n t of R O I s .  during  T h e lungs  bypass.  When  are  not v e n t i l a t e d ,  pulmonary  a n d they  perfusion  is  are  Activated  complement  components  [60-62],  markers  partially  restored  c e s s a t i o n o f b y p a s s , there is w h i t e c e l l s e q u e s t r a t i o n w i t h i n the l u n g s of  i n the  at  the  [59-61].  white  cell  il  d e g r a n u l a t i o n [61-63] a n d m a r k e r s o f free r a d i c a l a c t i v i t y are a l l d e t e c t a b l e i n the p u l m o n a r y e f f l u e n t b l o o d at this t i m e . Bypass  Apparatus.  N e u t r o p h i l s are a c t i v a t e d  b y p a s s [ 6 4 - 6 6 ] . T h e y stick to the s u r f a c e s of  the  extracorporeal  circulation  i n the c i r c u l a t i o n d u r i n g  o f the tubes a n d to the  apparatus.  This  is  oxygenator  followed  a c t i v a t i o n a n d d e g r a n u l a t i o n [92]. C o m p l e m e n t is also a c t i v a t e d ,  by  probably via  the a l t e r n a t i v e p a t h w a y , due to the c o n t a c t o f the b l o o d w i t h f o r e i g n i n c l u d i n g the p u m p , t u b i n g , o x y g e n a t o r ,  rapid  a n d b l o o d gas i n t e r f a c e  surfaces,  [67-71]. T h e  a c t i v a t e d c o m p l e m e n t i n t u r n activates n e u t r o p h i l s to p r o d u c e R O I s [72]. T h e effects  of increased  oxidative  stress  related  o c c u r m a i n l y d u r i n g , r a t h e r than f o l l o w i n g ,  1.4.2  to the b y p a s s  apparatus  bypass.  M y o c a r d i a l Ischaemia R e p e r f u s i o n Injury C a u s e d B y R O I s Ischaemia  process. platelet  r e p e r f u s i o n i n j u r y ( I R I ) is a v e r y c o m p l e x p a t h o p h y s i o l o g i c a l  Factors associated activating  factor  with IRI include f o r m a t i o n o f R O I s , p r o d u c t i o n o f (PAF),  complement  activation,  platelet  leukocyte activation, production of arachidonic acid metabolites, inflammatory mediators  It  is c o n s i d e r e d  formation,  that i n the c o m p l e x  P A F production,  most i m p o r t a n t r o l e s  and  formation of  s u c h as h i s t a m i n e , s e r o t o n i n , k i n i n a n d b r a d y k i n i n as  w e l l as i m m u n o l o g i c a l l y - p r o v o k e d types o f acute r e a c t i o n s  ROI  should  mutifactorial  and c o m p l e m e n t  [72].  pathogenesis  activation  of IRI,  m a y p l a y the  [ 7 2 - 7 5 ] . H o w e v e r , this s e c t i o n o f the thesis  will  ONLY  f o c u s o n the p a t h o p h y s i o l o g y o f I R I c a u s e d b y R O I s .  12  In the m y o c a r d i u m , pathways.  the r e d u c t i o n  The mitochondrial  of oxygen  cytochrome  to w a t e r  oxidase  proceeds  system  b y two  reduces  95%  of  o x y g e n to water b y t e t r a v a l e n t r e d u c t i o n w i t h o u t the p r o d u c t i o n o f R O I s . T h e r e m a i n i n g 5% o f o x y g e n  enters the u n i v a l e n t r e d u c t i v e  pathway  and several  R O I s , s u c h as s u p e r o x i d e a n i o n s , h y d r o g e n p e r o x i d e a n d the h y d r o x y l r a d i c a l , are p r o d u c e d The  [8].  myocardial  ischaemia.  A t this t i m e ,  of R O I s might this  period,  become  be s t i l l  reduced  formed  [76,77],  by  ischaemia  ROIs  produces  likely  available,  residual  starts  during  but a c e r t a i n  amount  molecular  o f the m i t o c h o n d r i a l leakage  of  oxygen.  electron  to  a  m a y be  dismutase  progressive  (SOD).  decline  by xanthine  uric acid and superoxide  During  onto  radicals  chain  residual  [21].  Most  R O I p r o d u c t i o n f r o m m i t o c h o n d r i a is  in  Increasing  radicals  the  S O D activity,  o f d e a l i n g w i t h the i n c r e a s e d  generated  (1)  transport  electrons  l e a d i n g to the f o r m a t i o n o f s u p e r o x i d e  m i t o c h o n d r i a less c a p a b l e  [81].  from  allowing  superoxide  leads  C A B surgery  is no l o n g e r  i n the e a r l y phase o f i s c h a e m i a ,  neutralized  (2)  during  oxygen  the c o m p o n e n t s  molecular oxygen, likely,  damage  oxidase,  leaving  radical flux  because  during ischaemia  (3) R O I s m a y be g e n e r a t e d w i t h i n m e m b r a n e s ,  duration  of the  [78-80].  cardiac  tissue  and reperfusion  in association  w i t h the  arachidonic acid cascade. During through  reperfusion,  the  mitochondria,  following  oxygen  is  available,  mechanisms:  but e l e c t r o n  egress  (1)  through  and R O I s  Reperfusion cytochrome  c a n be  produced  re-energizes oxidase  is  the  reduced  13  because ROIs  o f the l a c k  [8].  (2)  of adenosine  ROIs  again  diphosphate  m a y be p r o d u c e d  ( A D P ) causing through  pathway  [81]. (3) N e u t r o p h i l s c o u l d also be a c t i v a t e d ,  though  neutrophils  have  not  been  demonstrated  formation  the x a n t h i n e  of  oxidase  generating R O I s . E v e n to  contribute  to  ROI  g e n e r a t i o n i n the m y o c a r d i u m d u r i n g r e p e r f u s i o n [ 2 , 6 ] , there is e v i d e n c e that neutrophils  do  Furthermore, decreased  accumulate  in  the  myocardium  during  reperfusion  [2,7].  the d e p l e t i o n o f c i r c u l a t i n g n e u t r o p h i l s has b e e n a s s o c i a t e d  myocardial  damage  in models  of  ischaemia  reperfusion  with  injury  [7,82]. Thus, endogenous  it seems  that  ischaemia  defense mechanisms  induces  against  alterations  capable  R O I challenge.  appears  to l i e at the l e v e l o f m i t o c h o n d r i a l a n t i o x i d a n t  activity  being  reduced  by  50%  after  severe  of reducing  T h e prime  alteration  enzymes  with S O D  ischaemia  [8].  Furthermore,  r e p e r f u s i o n is l i k e l y to s t i m u l a t e the p r o d u c t i o n o f R O I s to a n extent greater than the n e u t r a l i z i n g c a p a c i t y If  ischaemia  is  brief  (<  30  irreversible oxidative damage are  of mitochondrial antioxidant to  60  readmission of oxygen with coronary  damage,  1.5  against flow.  w h e n the d e f e n s e m e c h a n i s m s  the b u r s t  defenses.  m a y not r e s u l t  [ 2 , 8 ] , p r o b a b l y b e c a u s e the d e f e n s e  able to p r o t e c t m y o c a r d i a l c e l l s  of ischaemia,  min), reperfusion  enzyme  in  mechanisms  o f R O I s generated  by  R e p e r f u s i o n after a l o n g e r p e r i o d are i m p a i r e d , c a u s e s m y o c a r d i a l  w h i c h m a y l e a d to i r r e v e r s i b l e loss o f c o n t r a c t i l e f u n c t i o n .  Endogenous Antioxidant Defense System  14  Cells  and  tissues  antioxidant  defenses.  appropriate  for  usually  have  Physiologically,  dealing  with  aberrant  extracellularly  are  appropriate  proteins  for  inhibiting  and  antioxidants  lipid  consumed.  those  located  generation  binding  of  metal  peroxidation  In  only  ascorbate-replete  occurs  plasma,  ROIs;  processes  when  the  and  those  [83].  urate  ascorbate  lipids  are  haem  The  ROIs  are  located  delocalised  against >  extracellular  intracellular^  ions,  ascorbate = p r o t e i n b i l i r u b i n  peroxidation  range o f a n t i o x i d a n t s  1.5.1  for  intracellular  i n h u m a n p l a s m a and their e f f e c t i v e n e s s  i n the aqueous phase a r e : Plasma  adequate  major  generated  >  tocopherol.  is  completely  protected  [84].  The  and r a d i c a l s c a v e n g e r s is s u m m a r i z e d as f o l l o w s :  N o n e n z y m i c : V i t a m i n C and V i t a m i n E These  Vitamin  E  are  chain breaking  is  highly lipophilic.  and h y d r o x y l r a d i c a l It  contains  scavenger  shielding methyl  antioxidants. groups  in  the  v i c i n i t y o f the p h e n o l i c h y d r o x y l g r o u p o f the c h r o m o p h o r i c m o i e t y ,  and it is  optimally  [85].  positioned  myocardium, mitochondrial peroxidation i n the  it  in  has  the  been  membranes  membrane identified and  by in  its both  presumably  phytyl  side  chain  myocardial protects  them  cytosolic  and  from  lipid  [ 8 6 , 8 7 ] . V i t a m i n C is water s o l u b l e . It is a c t i v e i n c y t o s o l  extracellular  fluid.  It  functions  synergistically  with vitamin E  In  and  and it  must be p r e s e n t to r e g e n e r a t e v i t a m i n E .  1.5.2  Major Extracellular Protection Mechanisms  [21]  15  1. U r i c a c i d : s c a v e n g e s  h y d r o x y l r a d i c a l and singlet o x y g e n ;  it is also a c h e l a t o r o f i r o n and c o p p e r . 2. C a e r u l o p l a s m i n : acts as an a n t i o x i d a n t by v i r t u e o f its f e r r o x i d a s e 3.  T r a n s f e r r i n : sequesters i r o n (III),  the  Haber-Weiss reaction  rendering  w h i c h initiates  lipid  it u n a v a i l a b l e f o r p e r o x i d a t i o n or  activity. catalyzing  catalyzes  the  decomposition of lipid hydroperoxides. 4. A l b u m i n : b i n d s m e t a l s , e s p e c i a l l y c o p p e r but also i r o n w e a k l y . 5. B e t a c a r o t e n e : t r a n s p o r t e d p r i m a r i l y w i t h i n l o w - d e n s i t y l i p o p r o t e i n s [88],  1.5.3  scavenges  peroxyl radicals  E n z y m i c Antioxidant Defenses  1. S u p e r o x i d e d i s m u t a s e ( S O D ) : in  [89].  mitochondria  cytoplasm. peroxide  It  and  and  in  disposes oxygen,  the of  is p r e s e n t  copper  or  superoxide  w h i c h is  10  i n the m a n g a n e s e zinc  form  radicals  times  faster  by  form  (Cu-SOD,  Zn-SOD)  dismutation  than  by  (Mn-SOD)  to  in  hydrogen  spontaneous  decay  [90]. 2.  Glutathione peroxidase  ( G P X ) : present  i n the c y t o s o l ,  detoxifies  p e r o x i d e a n d l i p i d p e r o x i d e s i n the p r e s e n c e o f r e d u c e d g l u t a t h i o n e  hydrogen (GSH).  3. C a t a l a s e : d e t o x i f i e s h y d r o g e n p e r o x i d e In  the  concentrations,  myocardium,  SOD  and  GPX  are  present  w h i l e c a t a l a s e , at v e r y l o w c o n c e n t r a t i o n .  the f i r s t l i n e o f d e f e n s e  against  R O I challenge,  at  significant  S O D was s h o w n as  a n d G P X as the  second line  [8].  16  Red  c e l l s , i n c o n t r a s t to m y o c a r d i a l c e l l s , have l a r g e a m o u n t s o f catalase  [91]  as w e l l  and  murine  protected  as  S O D a n d G P X . In a n i m a l s t u d i e s ,  red  cells,  nucleated  probably cells  ischemic,  virtue  (L1210  o x i d a t i v e c h a l l e n g e i n v i t r o [91]. ventricular  by  of  murine  it was  s h o w n that h u m a n  catalase  activity,  completely  leukemia)  against  exogenous  R e p e r f u s i o n with human red cells  f u n c t i o n and d e c r e a s e d  myocardial hydrogen  peroxide  levels  of  i s o l a t e d rat h e a r t s . R e p e r f u s i o n w i t h r e d c e l l s that l a c k e d catalase  (aminotriazole-treated) increase  ventricular  and/or glutathione function  or  (N-ethylmaleimide-treated)  decrease  myocardial  hydrogen  c o n c e n t r a t i o n as m u c h as r e p e r f u s i o n w i t h u n t r e a t e d r e d c e l l s  1.6  increased  d i d not peroxide  [92].  P r e o p e r a t i v e V i t a m i n C , V i t a m i n E , and A l l o p u r i n o l T h e r a p y i n Patients  Undergoing C A B Surgery According follow  diets  to  that  a  public  meet  the  survey,  only  recommended  vitamin E  and v i t a m i n C . F u r t h e r m o r e ,  for  patients  some  antioxidants  may  exacerbate  any  9%  of  the  guidelines  A m e r i c a n population for  the l o w c h o l e s t e r o l prior  Its  surgery  prescribed  of  lipid-phase  [93].  concentration  i n m y o c a r d i u m was  s h o w n to  i n the l i p i d  decrease"  phase  during C A B  [95].  H i g h dose CPB  antioxidants  diets  insufficiency  V i t a m i n E p r o b a b l y is the most i m p o r t a n t a n t i o x i d a n t [94].  the  v i t a m i n C (250  and at the t i m e o f a o r t i c  mg/kg),  given intravenously  d e c l a m p i n g (125  30  m g / k g each time),  m i n before has  shown  17  protective levels  effects  on myocardium,  of M D A , C P K , C P K - M B ,  c a r d i a c i n d e x after o p e r a t i o n  manifested  as s i g n i f i c a n t l y l o w e r  a n d L D H as w e l l  as  plasma  significantly  higher  [36].  A p o p u l a r r e g i m e n is v i t a m i n C a n d v i t a m i n E ( 1 0 0 0 m g a n d 100 to 800 IU  daily,  up  to  preoperatively resulted  one  week  [96-99].  vitamin  atherosclerosis, progression  this  E  selective  of  to  give  patients  in  plasma  [96].  regimen  has  [100] or b y a n g i o g r a p h y  is a p u r i n e  IRI although In  the  it  has not  prevented  treatment  significantly  reduced  analogue.  [101].  It has b e e n  [102,103].  used  Its a n t i o x i d a n t  oxidase.  effectively  effects  i n the  likely  include  It m a y also attenuate the i n c r e a s e  activity i n IRI [9,104,105], presumably by decreasing during  of  confirmed morphologically by high resolution  i n h i b i t i o n of xanthine  neutrophils  reducing  depletion  of hyperuricemia  myeloperoxidase  combined  in myocardial  supplementation  B-mode ultrasonography  treatment  reduction  of atherosclerosis  Allopurinol  surgery)  U n f o r t u n a t e l y , this r e g i m e n f o r C A B s u r g e r y  any m e a s u r a b l e  post-surgical  before  reperfusion.  IRI independent o f xanthine  Other oxidase  mechanisms  of  of  influx  allopurinol  in  inhibition may mainly include  r e d u c t i o n i n l y s o s o m a l e n z y m e r e l e a s e f r o m n e u t r o p h i l s [106] a n d f a c i l i t a t i o n of m i t o c h o n d r i a l electron transport In a n i m a l m o d e l s tissue  antioxidant  [107].  o f I R I , a l l o p u r i n o l has e f f e c t i v e l y  capacity,  reduced  lipid  peroxidation,  increased  cell and  and significantly  improved cardiopulmonary function [9,10,108-110].  18  In  C A B surgery,  preoperative  conditioning with  allopurinol  alone  or  t o g e t h e r w i t h v i t a m i n C a n d v i t a m i n E has s h o w n [ 1 , 1 1 - 1 3 , 1 1 1 - 1 1 6 ] i m p r o v e d myocardial  contractility,  sinus r h y t h m , r e d u c e d  increased  metabolic  incidence  changes  of  spontaneous  reversion  to  (e.g. plasma M D A , C P K - M B ) [ 1 ] ,  and d e c r e a s e d h o s p i t a l m o r t a l i t y r a t e .  1.7  E G b 761  and C a r v e d i l o l :  Uncommonly  Used  Antioxidants  for C A B  Surgery EGb  761  and C a r v e d i l o l  are f o u n d  to h a v e  antioxidant  activity.  They  have r a r e l y b e e n t r i e d as a n t i o x i d a n t s i n C B P s u r g e r y out o f N o r t h A m e r i c a . H e r e is o n l y a b r i e f d e s c r i p t i o n o f the t w o d r u g s .  1.7.1 E G b 761 EGb Biloba.  is a titrated  and standardized  extract  of green  leaves  It is a c o m p l e x m i x t u r e c o m p o s e d o f f l a v o n o i d s u b s t a n c e s ,  p r o a n t h o c y a n i d i n s , o r g a n i c a c i d s , a n d other c o n s t i t u e n t s  of  Ginkgo  terpenes,  [ 1 0 2 ] . T h e e x t r a c t is  s t a n d a r d i z e d at 6 a n d 24% ( w / w ) o f terpenes  and f l a v o n o i d heterosides.  free  have been demonstrated  radical scavenger,  mitochondrial decreased  its p r o t e c t i v e  [103,117,118],  M D A production  effects  cellular [119,120], [119,121],  a d h e s i o n to e n d o t h e l i u m [ 1 2 2 ] , d e c r e a s e d  inhibited  a n d tissue  [119]  As a  o n the  levels by  neutrophil  f u n c t i o n and  retinal [123], neural  [117,120,124]  and m y o c a r d i a l d a m a g e c a u s e d b y R O I s .  19  In  patients  associated  with  undergoing  C A B surgery,  decreased  pretreatment  TBA-reactive  species  with  in  m y o c a r d i a l i m p r o v e m e n t (not s i g n i f i c a n t l y ) i n p o s t o p e r a t i v e  1.7.2  E G b 761  myocardium, recovery  was and  [121].  Carvedilol C a r v e d i l o l is a v a s o d i l a t i n g , b e t a - a d r e n o c e p t o r a n t a g o n i s t c u r r e n t l y  f o r the t r e a t m e n t o f m i l d to m o d e r a t e h y p e r t e n s i o n  [125-127].  some  potent  of  its  hydroxylated  metabolites  are  C a r v e d i l o l and  antioxidants.  p h y s i o c h e m i c a l , b i o c h e m i c a l and cellular assays, c a r v e d i l o l and several metabolites  directly  scavenged  cardiac and b r a i n membranes, the d e p l e t i o n o f e n d o g e n o u s  ROIs  [128],  lipoprotein)  known  to  formation  activate  lipid  antioxidants,  In o f its  peroxidation  b o t h i n v i t r o a n d i n v i v o [129]  M o r e o v e r , c a r v e d i l o l a n d its m e t a b o l i t e s density  prevented  used  in  and prevented  s u c h as v i t a m i n E a n d g l u t a t h i o n e . p r e v e n t e d the o x i d a t i o n o f L D L ( l o w  to o x i d i z e d L D L , the latter b e i n g d i r e c t l y monocytes/macrophages  and  to  cytotoxic and  stimulate  foam  cell  [130].  Carvedilol experimental  has b e e n  animal  shown  models  to  (using  produce rat,  significant  cardioprotection  d o g , a n d p i g ) o f acute  i n f a r c t i o n [ 1 3 1 - 1 3 3 ] , w i t h the most d r a m a t i c  in  myocardial  effect being observed  m o d e l o f m y o c a r d i a l I R I , w h e r e the r e d u c t i o n i n i n f a r c t s i z e r e a c h e d  i n the p i g 91%.  T h e s e r e s u l t s suggest its c l i n i c a l use i n p a t i e n t s u n d e r g o i n g C A B s u r g e r y [134].  20  1.8  P r o p o f o l : A n A n e s t h e t i c and A n t i o x i d a n t Propofol  is a s h o r t - a c t i n g  soluble.  It is w i d e l y u s e d  permits  both  efficient  in both  control an  anesthetic,  ambulatory  o f anesthetic  antioxidant,  w h i c h is h i g h l y  lipid-  and h o s p i t a l i z e d patients. depth  propofol  and rapid,  recovery  [135].  protective  e f f e c t s i n attenuating m y o c a r d i a l I R I i n a n i m a l m o d e l s  1.8.1  As  intravenous  has  been  It  controllable  shown  to  have  [14,15].  Structure Propofol,  chemically  2,6-diisopropylphenol  weight  178.27),  is  s i m i l a r to p h e n o l - b a s e d free r a d i c a l s c a v e n g e r s s u c h as b u t y l a t e d  hydroxytoluene [17,136,137]. phenyl  (molecular  ring,  and  the  endogenous  antioxidant  A l l these three c o m p o u n d s which  is k n o w n to c o n f e r  carry free  vitamin  E  (Figure  a h y d r o x y l substituent radical  scavenging  4) on a  properties  [136].  21  (CH3)2CH  OH  CH(CH3)2  Propofol  (CH3)3C  OH  C(CH3)3  Butylated hydroxytoluene  CH3  CH3  HO  CH3  CI12-(CH2-CH2 - CH-CH2) - H CH3  CH3  Vitamin E CH3  Figure 4. Structural formulae of propofol (2,6-diisopropylphenol), butylated hydroxytoluene, and vitamin E (alpha tocopherol). All three compounds carry a hydroxyl substituent on their phenol rings, which is considered to conferfreeradical scavenging properties.  22  1.8.2 B a s i c P h a r m a c o k i n e t i c s  [137]  Administration of propofol, 2-2.5mg/kg produces unconsciousness and  complete  w i t h i n a b o u t 30 s e c o n d s .  with minimal  seems to be the m o s t  g i v e n i v o v e r 15 s e c o n d s or l e s s ,  residual  important  central  advantage  A w a k e n i n g is v e r y r a p i d  nervous  over  other  system  effects,  which  d r u g s u s e d to p r o d u c e  induction o f anesthesia. Clearance  of propofol  from  the  plasma  exceeds  hepatic  blood  flow,  e m p h a s i z i n g that tissue uptake as w e l l as m e t a b o l i s m is i m p o r t a n t i n r e m o v a l o f this d r u g f r o m the p l a s m a . L e s s than 0 . 3 % o f a dose is e x c r e t e d u n c h a n g e d i n the u r i n e . T h e e l i m i n a t i o n h a l f - l i f e is 0.5 to 1.5 h o u r s . Despite evidence  of  dysfunction. plasma  the  rapid  impaired Patients  clearance  clearance  of propofol  elimination  older  than  of propofol.  in  patients  60 y e a r s There  by m e t a b o l i s m , with  there  cirrhosis  o f age e x h i b i t  m a y be a m o d e s t  is no  or  renal  a reduced  rate o f  cumulative  effect,  e s p e c i a l l y i n e l d e r l y patients r e c e i v i n g c o n t i n u o u s i n t r a v e n o u s i n f u s i o n s . Plasma  Concentration  weak o r g a n i c  of Propofol during C A B Surgery:  a c i d that is b o u n d e x t e n s i v e l y  P r o p o f o l is a  to p l a s m a a l b u m i n ,  with a  free  f r a c t i o n o f o n l y 2-3% [ 1 3 8 ] . H a e m o d i l u t i o n d u r i n g c a r d i o p u l m o n a r y b y p a s s is associated in  total  w i t h d e c r e a s e s i n the c o n c e n t r a t i o n s propofol  concentrations  concentration  of plasma proteins  of plasma proteins, a decrease  disproportional [139,140],  to  the  decreases  i n the  a n d a 1.5 to 3 f o l d i n c r e a s e i n  the f r a c t i o n o f u n b o u n d p r o p o f o l [ 1 4 1 , 1 4 2 ] .  23  Disproportional during  bypass  decreases  m a y be  i n total  attributed  plasma  not  only  to  p r o p o f o l s e q u e s t r a t i o n w i t h i n the e x t r a c o r p o r e a l of  propofol  concentration  h a e m o d i l u t i o n alone  was  u p to  50%  concentration  of  propofol  h a e m o d i l u t i o n but also  to  c i r c u i t , b e c a u s e the d e c r e a s e  more  than  that  predicted  by  [139,140].  R e a s o n s f o r the i n c r e a s e i n the f r a c t i o n o f u n b o u n d p r o p o f o l : T h e use o f h e p a r i n d u r i n g b y p a s s causes a n i n c r e a s e i n n o n - e s t e r i f i e d fatty a c i d s of  activation  of  lipoprotein  lipase  [143].  thought to be r e s p o n s i b l e f o r the d e c r e a s e  Non-esterified  fatty  because  acids  are  i n d r u g b i n d i n g to p l a s m a p r o t e i n s  after h e p a r i n [ 1 4 4 ] . A n i n c r e a s e d free f r a c t i o n o f p r o p o f o l m a y c o n t r i b u t e to the p r o l o n g e d e f f e c t  of propofol  w h e n the p l a s m a c o n c e n t r a t i o n  of propofol  was l o w [ 1 4 5 ] .  1.8.3 M e c h a n i s m s o f A n t i o x i d a n t A c t i v i t y Phenol-based hydrogen  antioxidants  abstraction,  and  (R-OH)  thereby  scavenge  themselves  ROIs ( X ) become  by a process a  (less  of  reactive)  phenoxyl radical ( R - ) R-OH Spin  + X-  — >  resonance  characterizing  radical  R-0  + X H  spectroscopy, reactions  one  of  the  most  [16,136,146,147],  specific  was u s e d  methods to  for  investigate  this m e c h a n i s m . A n i n v i t r o study was c a r r i e d out i n a l i q u i d p h a s e to e x a m i n e the w a y i n w h i c h p r o p o f o l reacts w i t h R O I s a n d , i n p a r t i c u l a r , to i d e n t i f y i f a p r o p o f o l -  24  d e r i v e d p h e n o x y l r a d i c a l is g e n e r a t e d [ 1 3 6 ] . P r o p o f o l was d e m o n s t r a t e d as an a n t i o x i d a n t b y r e a c t i n g  to act  w i t h f r e e r a d i c a l s to f o r m a p h e n o x y l r a d i c a l - a  p r o p e r t y c o m m o n to a l l p h e n o l - b a s e d free r a d i c a l s c a v e n g e r s , s u c h as v i t a m i n E. Another mitochondria that 95% under  in [16].  initiators  study  'H-NMR  o f the a d d e d  conditions  spectrum  vitro  of  was  carried  using  isolated  oxidative  intact  stress.  after  The  a 30  electron  production,  showed  a  radical  that  liver  indicated  m i n incubation spin  o f p r o p o f o l , i n c u b a t e d i n the p r e s e n c e o f E D T A - F e 2 + of radical  rat  spectra o f m i t o c h o n d r i a l l i p i d extracts  propofol remained  low  out  resonance  and H 0 2  was m o s t  2  likely  as a  d e c o m p o s i t i o n p r o d u c t o f the p h e n o x y l r a d i c a l o f p r o p o f o l . It was c o n c l u d e d f r o m the study that p r o p o f o l acted as a c h a i n r e a c t i o n - b r e a k i n g f o r m i n g a stable r a d i c a l  a n t i o x i d a n t by  [16,148].  1.8.4 A n t i o x i d a n t A c t i v i t i e s of P r o p o f o l i n A n i m a l  Studies  1. P r o p o f o l I n h i b i t s L i p i d P e r o x i d a t i o n  L i v e r M i t o c h o n d r i a o f the  i n Isolated  Rat. 1.1 reactive  u M propofol substances)  produced  production  about  50%  [16,17],  inhibition  while  8  o f M D A (or T B A -  p M propofol  completely  s u p p r e s s e d the p r o d u c t i o n o f M D A [ 1 6 ] . 2. P r o p o f o l A t t e n u a t e s During coronary  flow  I R I i n the I s o l a t e d R a t H e a r t .  reperfusion  after g l o b a l  and reduced  ischemia,  L D H release  100  [14] u M propofol  a n d the hearts  showed  increased lower  left  25  ventricular in  end-diastolic pressure.  propofol-treated  hearts  H i s t o l o g i c a l l y , the  in  terms  of  i n j u r y was  interstitial  less  edema,  myofiber  degeneration,  and f o r m a t i o n o f m y o c a r d i a l h y p e r c o n t r a c t i o n b a n d s .  3.  Attenuates  Propofol  Mechanical  and  Metabolic  Damage  E x o g e n o u s H y d r o g e n P e r o x i d e i n the I s o l a t e d R a t H e a r t 25 p-M or 50 MDA left  (Figure  and m e t a b o l i c  s i g n i f i c a n t l y attenuated  end-diastolic changes  pressure,  (decreased  p h o s p h a t e ) i n d u c e d by e x o g e n o u s 4.  Induced  by  [15].  u,M p r o p o f o l c o m p l e t e l y s u p p r e s s e d the p r o d u c t i o n o f tissue  5),  ventricular  severe  mechanical  decreased  tissue  dysfunction  (increased  ventricular  pressure)  left  concentrations  h y d r o g e n p e r o x i d e (600  o f A T P and  creatine  p,M).  In a p r e v i o u s study f r o m o u r l a b o r a t o r y u s i n g a s w i n e m o d e l o f h e a r t - l u n g  transplantation,  propofol  at  plasma  concentrations  significantly increased red cell antioxidant capacity challenge  (1.5  u M t-butyl hydrogen peroxide).  of a generalized enhancement  o f tissue  p r o t e c t i o n against c a r d i o p u l m o n a r y IRI  against  T h i s effect  antioxidant  status,  10  to  in vitro  20  uM  oxidative  m a y be i n d i c a t i v e and might  confer  [18,19].  5. In a study c a r r i e d out i n aqueous s u s p e n s i o n , 50 M D A p r o d u c t i o n by 3 8 % .  of  p-g/ml p r o p o f o l  decreased  [45]  6. P r o p o f o l d i d N O T : (1)  Propofol  antioxidant  did  not  enzyme  transplantation.  It  vitro peroxidative  have  any  activity  d i d not  in  prevent  challenge  [18],  detectable a  swine  effect  on  model  of  red c e l l glutathione probably because  plasma heart  or  red and  cell lung  d e p l e t i o n against  p r o p o f o l m a i n l y acts  in at  26  the  level  of  extracellular (2)  the  plasma  membrane  as  cytosolic  or  p M or 50  p M ) d i d not c o m p l e t e l y attenuate m e c h a n i c a l  and  m e t a b o l i c c h a n g e s i n d u c e d by e x o g e n o u s or  to  the  compartment.  P r o p o f o l (25  [15]  compared  rabbit  peroxidation.  [149]  heart,  Therefore,  in  factors  spite  h y d r o g e n p e r o x i d e i n the i s o l a t e d rat of  other  important role i n m y o c a r d i a l damage  the  complete  than l i p i d  inhibition  p e r o x i d a t i o n may  i n d u c e d b y R O I s . In f a c t ,  p r o t e i n or n o n p r o t e i n s u l f h y d r y l g r o u p s has b e e n d e m o n s t r a t e d to m y o c a r d i a l d a m a g e which account  are  not  for  the  incomplete  metabolic derangements (3)  to  lipid  peroxidation  protection  i n d u c e d by R O I s  by  of  the  propofol  lipid  play  an  oxidation  of  to  i n d u c e d by R O I s [ 1 5 0 ] . T h e s e b i o c h e m i c a l  attributed  of  contribute alterations,  cell  membrane,  may  of  mechanical  and  [15].  P r o p o f o l d i d not show any r a d i c a l s c a v e n g i n g a c t i v i t y at c o n c e n t r a t i o n s  10 p g / m l i n an e x p e r i m e n t c a r r i e d out i n a q u e o u s s u s p e n s i o n i n v i t r o  <  [45].  27  £4 «  5  2 o D_ m  o  R  ^  O  I  £ 4 O« R ^ R- =5 CM  D_ CM  13  DC  PH  u  •8  a  •  a  o  .s  T3  s  ia  + cvi CQ O  X CM  I  © © V  «! &>  -  d  I  a'  <u bX)  o  1  WO ©  a ©  a  45 «  73  I  o  ft  I  Q. CT3  I o  o  o  o  o  O  OO  (D  t  CN  ( J M Ajp B/|A|u) VQIAI  o  *3  CL ^ O LO  CO  X  N  .a a  .1  ON ON  so  a .&  1  I  i  .9. o p  o a o  S  i  §  S  g I .a  •8' »  5 a 3  w)  "e3'  i  JS u  1.8.5 P r o p o f o l A n a e s t h e s i a i n C A B S u r g e r y Propofol awakening,  has  the  advantage  w h i c h results  early  of  conveying  extubation  after  very  rapid  surgery  and  complete  and early  intensive  c a r e u n i t d i s c h a r g e [ 1 5 1 , 1 5 2 ] . It has b e e n u s e d s i n c e 1987 i n C A B s u r g e r y as total i n t r a v e n o u s a n a e s t h e s i a  [151,  153-172] o r p o s t - o p e r a t i v e  sedation  [173-  178]. Hypotension 145,179].  is  major  side  effect  of  propofol  T h i s is d u e to a d e c r e a s e i n p e r i p h e r a l v a s c u l a r  by p r o p o f o l .  anaesthesia  [137,  resistance  caused  [145,180,181].  Propofol  as  compared  significant  changes  [157,180],  cardiac  perfusion  a  pressure  to  isoflurane  [152,157,166,180] output [157]  [180],  or  enflurane  i n terms  inotropic  or p e r i o p e r a t i v e  d i d not  of myocardial  requirement  myocardial  show  any  contractility  [152],  infarction  coronary [152].  In  e a r l i e r s t u d i e s , p r o p o f o l was a s s o c i a t e d w i t h d e c r e a s e d m y o c a r d i a l b l o o d f l o w (by 2 6 % ) , m y o c a r d i a l o x y g e n c o n s u m p t i o n (by 31%) [ 1 8 2 ] , a n d c a r d i a c [113,182,183]  a n d was s u g g e s t e d  with c o r o n a r y artery disease As  an antioxidant  to l e a d to m y o c a r d i a l i s c h a e m i a i n patients  [182].  the p r o t e c t i v e  effects  of propofol on myocardium in  e x p e r i m e n t a l studies w e r e a c h i e v e d at h i g h e r t h a n r e c o m m e n d e d c l i n i c a l for  C A B surgery.  activity [45]. In  Because  of propofol during  index  of this, clinical  it was s u g g e s t e d anaesthesia  that  is l i k e l y  the  doses  scavenging  to be v e r y  limited  Its p o t e n t i a l as an a n t i o x i d a n t i n C A B s u r g e r y has not b e e n i n v e s t i g a t e d .  our previous  study  i n the s w i n e  model  of ischaemia  reperfusion injury  29  [18],  p r o p o f o l was s h o w n to h a v e a n t i o x i d a n t p r o p e r t i e s at c l i n i c a l l y  plasma  concentrations,  which  encouraged  us  to  proceed  with  this  relevant clinical  study.  30  Chapter 2 OBJECTIVES  T h e o b j e c t i v e s o f the thesis p r o j e c t (1)  to d e t e r m i n e  were:  if propofol in recommended clinical  dosage  enhances  red cell  a n t i o x i d a n t c a p a c i t y d u r i n g c o r o n a r y artery b y p a s s ( C A B ) s u r g e r y ; (2)  to i n v e s t i g a t e  the e f f e c t  of p r o p o f o l on patient's  cardiopulmonary functions  perioperatively; (3)  to  determine  the  maximally  effective  dose  or  maximal  infusion  rate  of  p r o p o f o l as b o t h an a n t i o x i d a n t a n d a n e s t h e t i c f o r C A B s u r g e r y . B e c a u s e o f the s m a l l s a m p l e s i z e o f the s t u d y (see c h a p t e r 4, this p r o j e c t  was d e s i g n e d to s h o w s t a t i s t i c a l  section 4.1.2),  differences only in laboratory  data  (red c e l l M D A p r o d u c t i o n a g a i n s t i n y i t r o o x i d a t i v e c h a l l e n g e ) r a t h e r than that i n c l i n i c a l data ( e . g . , c a r d i o p u l m o n a r y f u n c t i o n s ) .  31  Chapter 3 HYPOTHESES  1. I s o f l u r a n e (less t h a n 2% e n d t i d a l ) w i l l not m o d i f y the r e d c e l l a n t i o x i d a n t status ( e x p e r i m e n t a l Isoflurane radicals  the  concentrations less  has b e e n  by neutrophils  enhance  is  control).  than  superoxide enough  at  u p to 2% 2%  of  inhibit  i n excess  superoxide  radicals  b u t the m a g n i t u d e  o f this  effect  cell  antioxidant  in vitro  superoxide  end tidal,  and  neutrophils  at  o f i s o f l u r a n e f o r o u r study increase  the p r o d u c t i o n o f  s h o u l d not be  status  given  the  large  marked  antioxidants.  2. H i g h dose p r o p o f o l (200 p g / k g / m i n ) w i l l against  by  slightly  red  of  o f 2%  might  the  This  protection a f f o r d e d by plasma  red cells  the p r o d u c t i o n  [184]. T h e concentration  end tidal.  influence  to  concentrations  production  radicals,  to  reported  oxidative  enhance  challenge,  antioxidant  while l o w dose  capacity  propofol  of (50  p g / k g / m i n ) may not. The  antioxidant  experiment  carried  properties  out i n a q u e o u s  10 p g / m l d i d not s h o w propofol decreased  of  propofol  7-12  pg/ml  any r a d i c a l  scavenging  M D A p r o d u c t i o n by 38%[45].  production more  and  12-25  than 25%  dose-dependent.  In  s u s p e n s i o n , p r o p o f o l at c o n c e n t r a t i o n s  swine m o d e l o f h e a r t - l u n g t r a n s p l a n t a t i o n , of  are  pg/ml  whereas  in  Thus,  50  <  pg/ml  In o u r p r e v i o u s study i n a  p r o p o f o l at p l a s m a  resulted  and 50%[18].  activity,  an  decreased  concentrations red  by continous  cell M D A  infusion  with 32  high  dose  propofol,  concentration  we  may  reach  7-12  of p r o p o f o l during bypass.  pg/ml  or  However,  more  for  the  plasma  w i t h low dose p r o p o f o l ,  the p l a s m a c o n c e n t r a t i o n  o f p r o p o f o l m a y be too l o w to p r o t e c t the r e d  against i n v i t r o o x i d a t i v e  challenge.  3. 200  cells  p g / k g / m i n is the m a x i m a l i n f u s i o n rate o f p r o p o f o l f o r C A B s u r g e r y .  The previous  antioxidant effects study  in  swine,  of p r o p o f o l appear limited  enhancement  to be d o s e - d e p e n d e n t . of  antioxidant  a c h i e v e d w i t h p l a s m a p r o p o f o l c o n c e n t r a t i o n g r e a t e r t h a n 25  In o u r  status  was  p . g / m l [18].  The  m a x i m u m r e c o m m e n d e d c l i n i c a l i n f u s i o n rate o f p r o p o f o l f o r C A B s u r g e r y 200  p . g / k g / m i n [166].  A t this  rate,  the  plasma  concentration  is  o f p r o p o f o l is  greater than 7 p g / m l . 4.  The  antioxidant  effect  of  propofol  on  red  cell  M D A production  may  c o n t i n u e after b y p a s s . Propofol  is  highly  lipophilic.  It  mainly  distributes  structures s u c h as c y t o s o l i c a n d m i t o c h o n d r i a l m e m b r a n e s . l i v e r m i t o c h o n d r i a , it was  s h o w n , by s p i n r e s o n a n c e  to  membranous  U s i n g isolated  spectroscopy,  that  rat 95%  o f the p r o p o f o l r e m a i n e d intact e v e n after 30 m i n i n c u b a t i o n u n d e r c o n d i t i o n s of  low  oxidative  stress,  m i t o c h o n d r i a i n v i t r o [16].  and  p r o p o f o l d i d not  Propofol will  to  be  metabolized  T h i s i m p l i e s that p r o p o f o l m a y r e m a i n i n the  c e l l m e m b r a n e e v e n after p r o p o f o l a n a e s t h e s i a 5.  seem  i m p r o v e the p e r f o r m a n c e  by red  is d i s c o n t i n u e d .  o f the heart after b y p a s s  and  will  decrease inotropic support required p e r i o p e r a t i v e l y .  33  Allopurinol, improved  antioxidant  myocardial  perioperatively Given oxidative  an  that  function,  when  used  and reduced  preoperatively,  the i n o t r o p i c  effectively  support  required  [1,11-13,111,112,114,116,185]. p r o p o f o l has p r o t e c t i v e  challenge,  like  allopurinol,  effects  it m i g h t  o n the m y o c a r d i u m  against  l e a d to i m p r o v e d m y o c a r d i a l  function, and decreased inotropic requirement perioperatively. 6.  P r o p o f o l anesthesia  will  have  the b e n e f i t o f e a r l y  extubation  after C A B  surgery. Very  rapid  and  nervous system effects drugs  used  to p r o d u c e  complete  awakening  with  minimal  is the most i m p o r t a n t a d v a n t a g e i n d u c t i o n o f anesthesia  anesthesia  for C A B surgery  extubation  [151,152].  has b e e n  reported  [137].  residual  of p r o p o f o l over  central other  T h e use o f p r o p o f o l  to be r e s p o n s i b l e  for early  34  Chapter 4 MATERIAL AND METHODS  4.1  Coronary  Artery  Bypass  Surgery:  A  Model  of  Ischaemia  Reperfusion  Injury Following  institutional  approval  patients s c h e d u l e d f o r C A B s u r g e r y  4.1.1  and  informed  were r a n d o m l y  patient  consent,  26  selected.  Selection Criteria  Inclusion  criteria:  M a l e or f e m a l e 35 y e a r s o f age or o l d e r H a e m o d y n a m i c a l l y stable w i t h d o c u m e n t e d c o r o n a r y H i s t o r y o f r e c e n t (less than six m o n t h s ) m y o c a r d i a l Unstable  infarction  angina  Left ventricle Exclusion  artery disease  dysfunction  criteria:  Haemodynamically  unstable,  History of evolving myocardial infarction, A c u t e l y failed percutaneous  transluminal coronary  angioplasty  or  previous C A B surgery, A S A or s t e r o i d t h e r a p y w i t h i n 7 days o f  4.1.2  surgery.  Study D e s i g n  35  Sample marker red  Size  for lipid  Determination. peroxidation  Because  M D A has b e e n  [1,15,36,39,41,43,45,55]  widely used  a n d the  c e l l M D A p r o d u c t i o n m a y be i n d i c a t i v e o f g e n e r a l i z e d  tissue  antioxidant  status,  the c a l c u l a t i o n  of sample  size  as a  suppressed  enhancement  of  f o r the study was  b a s e d o n the d i f f e r e n c e s o f r e d c e l l M D A p r o d u c t i o n b e t w e e n i s o f l u r a n e a n d propofol groups. A t present,  there a r e n o data a v a i l a b l e o n the r e l a t i o n s h i p o f  red c e l l M D A p r o d u c t i o n and p r o p o f o l concentrations  i n patients  CAB  model  surgery.  Therefore,  data  from  a  swine  undergoing  of  heart-lung  t r a n s p l a n t a t i o n [18] w e r e u s e d to c a l c u l a t e the s a m p l e s i z e f o r this p r o j e c t . In the  isoflurane  production about  group  of  (pO) is about  30 n M / g m  about 60 n M / g m  the  swine  80 n M / g m  model,  the  average  rbc with a standard  r b c . In the p r o p o f o l g r o u p ,  red  cell M D A  deviation  (CTO) o f  the M D A p r o d u c t i o n ( u l ) is  r b c w i t h a s t a n d a r d d e v i a t i o n (c?l) o f a b o u t 20 n M / g m r b c  w h e n the p l a s m a p r o p o f o l c o n c e n t r a t i o n MDA  production).  (i.e.,  test p o w e r  is about 7 p g / m l (25%  W e define T y p e I error  90%).  as 0 . 0 5  T h e n f o r this o n e - t a i l test,  inhibition of  a n d type II e r r o r Za =  1.645,  as 0.1  a n d Z[3 =  1 . 2 8 2 . A c c o r d i n g to L a c h i n ' s m e t h o d o f s a m p l e s i z e d e t e r m i n a t i o n a n d p o w e r a n a l y s i s f o r c l i n i c a l t r i a l s [ 1 8 6 ] , the total s a m p l e s i z e is N  =  [ ( Z a * a0 +  Z p * a l ) / ( u l - uO) ]  2  =  14  T h e r e f o r e , the total s a m p l e s i z e is 14. T h a t means f o r the h i g h dose p r o p o f o l g r o u p , i n w h i c h p l a s m a p r o p o f o l c o n c e n t r a t i o n was e x p e c t e d  to r e a c h c l o s e to  7 p g / m l to s i g n i f i c a n t l y s u p p r e s s r e d c e l l M D A p r o d u c t i o n , at least 7 patients s h o u l d be r e c r u i t e d f o r this g r o u p a n d f o r the i s o f l u r a n e g r o u p .  36  Patients three study aware  were  randomly selected,  groups.  T h e anesthetist  o f the e x p e r i m e n t a l  and purposely assigned  to one o f the  i n v o l v e d i n the c a r e o f the p a t i e n t was  maneuver,  w h i c h was b l i n d e d  r e s p o n s i b l e f o r the l a b o r a t o r y a n d c l i n i c a l o u t c o m e  to the r e s e a r c h e r s  analyses.  Control group (isoflurane-sufentanil): S a m p l e s i z e : 1 1 ( 7 have data o n M D A p r o d u c t i o n . ) Anesthesia: Sufentanil 0.5-10 p g / k g I s o f l u r a n e (0-2%) i n a i r / o x y g e n  <  F i O 2 = 0.5.  L o w dose p r o p o f o l g r o u p ( p r o p o f o l - s u f e n t a n i l ) Sample size: 7 Anesthesia: Propofol:  Induction  1.5-2.5 m g / k g , bolus  Pre-CPB  100 p g / k g / m i n , c o n t i n u o u s i n f u s i o n  Intra-CPB  50 p g / k g / m i n c o n t i n u o u s i n f u s i o n  Post-CPB  as a b o v e  In I C U  as a b o v e  Sufentanil: 0.3-1 p g / k g i n i t i a l l y , and then 1 p g / k g e v e r y 5 m i n u t e s i f r e q u i r e d to m a i n t a i n haemodynamics  >  20% above ward c o n t r o l levels  H i g h dose p r o p o f o l group ( p r o p o f o l - s u f e n t a n i l ) Sample size: 8 ( 7  have data o n M D A p r o d u c t i o n . )  Anesthesia:  37  Propofol:  Induction  1.5-2.5 m g / k g , bolus  Pre-CPB  200  Intra-CPB  as  Post-CPB  50 p g / k g / m i n , c o n t i n u o u s i n f u s i o n  In I C U  as  p g / k g / m i n , continuous infusion above  above  S u f e n t a n i l : the same as i n the l o w dose p r o p o f o l g r o u p  4.1.3  B l o o d Sample C o l l e c t i o n V e n o u s b l o o d s a m p l i n g (7 m l e a c h t i m e ) f o r the m e a s u r e m e n t  of red cell  M D A p r o d u c t i o n was p e r f o r m e d o v e r s e v e n t i m e i n t e r v a l s at b a s e l i n e , 30 m i n p o s t - i n d u c t i o n , 30 m i n c a r d i o p u l m o n a r y b y p a s s ( C P B ) , 5,  10 a n d 30 m i n post  aortic  (Figure  cross  samples  clamping  were  u n t i l the  (ACC),  immediately  and  120  min  h e p a r i n i z e d and  post-CPB  stored  i n the  next m o r n i n g to be a n a l y z e d f o r p e r o x i d e - i n d u c e d  Venous b l o o d for determining plasma concentration over  4  ACC,  time and  intervals 120  at  30  min post-induction,  min post-CPB  h e p a r i n i z e d and centrifuged 1.5  cold  (Figure  (xl500,  6).  for  10  These min).  6).  room  min C P B ,  samples  (4°  C)  T B A formation.  o f p r o p o f o l was  30  These  were  30  collected min  post-  immediately  T h e p l a s m a was  placed in  m l c r y o v i a l s and i m m e d i a t e l y f r o z e n and s t o r e d at - 7 0 ° C u n t i l they c o u l d  be a n a l y z e d b y  HPLC.  38  Red Cell M D A  '  Preind  30'Postind  30'CPB  5'PostACC  •  1  10'PostACC 30'PostACC 120'PostCPB  Propofol Concentration  Figure 6. Schematic time-line representation of blood sampling for red cell MDA production and plasma concentration of propofol.  39  4.1.4  C l i n i c a l Data Collection C l i n i c a l data, notably perioperative inotropic requirement,  cardiac  index  medical  records.  4.1.5  and  post-operative  time,  were  taken  patients  were  n o n p u l s a t i l e f l o w at 2.0 (Cobelaboratories, to 70  perfused  to 2.8  during  cardiopulmonary  the  The oxygenator  was  primer.  lungs  was  open  collapsed.  the  to  air,  the  Following  consisted  of  mammary  artery  was  reversed  300  mg/m ). 2  were  that  cross-clamping  sequential grafts.  h e p a r i n i z a t i o n (300  so  myocardial Patients  not  the  ventilated  lungs  of  the  were  aorta,  cooled  to  a n d the flaccid  the  revascularization  were  pressure  a polypropylene hollow fiber Cobe  L a k e w o o d , C o l o r a d o ) w i t h a 1450  bypass,  with  2  L a k e w o o d , C o l o r a d o ) , maintaining mean arterial  mmHg.  During  bypass  L / m / m i n w i t h the use o f s t a n d a r d C o b e p u m p s  Optima (Cobe Laboratories,  tube  from  Cardiopulmonary Bypass The  at 55  extubation  post-operative  32°  ml crystalloid endotracheal and  surgical  by to  vein 34°  technique  and C.  partially  internal Systemic  U / k g ) was i n s t i t u t e d s h o r t l y b e f o r e a o r t i c c a n n u l a t i o n and  immediately before  aortic  decannulation with protamine  (180-  T h e l u n g s w e r e r e v e n t i l a t e d i m m e d i a t e l y b e f o r e the c e s s a t i o n  of  bypass.  4.1.6  Data A n a l y s i s T h e p o o l e d t test is u s e d to e v a l u a t e  the  s i g n i f i c a n c e o f the  differences  40  between  two  significance The  group o f the  C h i square  between  means.  The  differences  test  is u s e d  percentages.  among  to  When  ANOVA the  evaluate  the  test means  the  smallest  is  used  to  of more  than  significance  expected  evaluate two  o f the  value  is  the  groups.  differences  less  F i s h e r ' s exact test is u s e d instead o f the C h i square test. A P v a l u e  than <  0.05  5, is  c o n s i d e r e d to be s i g n i f i c a n t .  4.2  Measurement of in vitro R e d C e l l M D A P r o d u c t i o n : T B A assay. MDA  is  formed  during  ischemia-reperfusion  through  arachidonic acid metabolism pathway  (Figure  of M D A d u r i n g C A B surgery  l o w to be d e t e c t e d b y o u r  is too  3).  injury  H o w e v e r , the  T h e r e f o r e , we c h a l l e n g e d the r e d c e l l s i n v i t r o w i t h i n c r e a s i n g (0.5,  1.0,  1.5,  2.0,  2.5  mM)  of  t-butylhydroperoxide,  the  concentration T B A assay.  concentrations  thereby  achieving  M D A l e v e l s h i g h e n o u g h to be d e t e c t e d by this a s s a y . T h i s a p p r o a c h  measures  red  oxidative  cell  antioxidant  capacity  in  the  presence  of  an  in  vitro  challenge. M o r e s p e c i f i c a l l y , the p r o c e d u r e u s e d i n o u r T B A assays was as f o l l o w s ( m a i n l y adapted f r o m a p u b l i s h e d r e p o r t by D r . G o d i n )  [39]:  R e a g e n t s a n d s o l u t i o n s (for 35 test t u b e s ) : D o u b l e - d i s t i l l e d water was u s e d . 0.9%  NaCl  - NaN3 ( s a l i n e / a z i d e ) .  S i g m a , C a n a d a ) was d i s s o l v e d i n 500  NaCl  (4.5  g) and a z i d e (65  mg,  from  ml of distilled water.  41  28%  T C A (trichloroacetic  acid)  -  0.1  M  N a arsenite,  from  Sigma,  Canada. 0.5% T B A (thiobarbituric acid) i n 0.025 M N a O H .  105 m g T B A ( S i g m a ,  C a n a d a ) was d i s s o l v e d i n 10.5 m l 0 . 0 5 M N a O H a n d 10.5 m l d i s t i l l e d w a t e r . tBHP  (t-butylhydroperoxide).  100  uJ o f 7 0 %  tBHP  (Sigma)  was d i l u t e d  w i t h 6 . 9 m l s a l i n e / a z i d e to get 100 m M T B H , w h i c h was f u r t h e r d i l u t e d w i t h saline/azide During reacted  to d i f f e r e n t  peroxidative with  concentrations,  0.5  concentrations  challenge, ml  of  0.5  test  w h i c h are 0 . 5 ,  1.0,  of T B H (1.0,  ml tBHP  sample  to  2.0,  in different achieve  3.0,  4.0,  5.0).  concentrations  the  effective  is  tBHP  1 . 5 , 2 . 0 , 2.5 m M .  Procedure: 1) C e n t r i f u g e b l o o d at 1000 r p m f o r 5 m i n at 4 ° C . 2) R e m o v e p l a s m a a n d w h i t e c e l l s b y a s p i r a t i o n . 3) W a s h r e d c e l l s t w i c e w i t h s a l i n e / a z i d e b y c e n t r i f u g a t i o n . 4) W e i g h a l i q u o t s o f 50 p i p a c k e d r e d c e l l s i n 1.5 m l m i c r o c e n t r i f u g e t u b e s . 5) A d d 0 . 4 5 m l c o l d s a l i n e / a z i d e to the m i c r o c e n t r i f u g e t u b e s . 6) P r e i n c u b a t e f o r 5 m i n at 3 7 ° C . 7) Start p e r o x i d a t i v e c h a l l e n g e w i t h 0.5 m l t B H P ( S i g m a , C a n a d a ) i n d i f f e r e n t concentrations  a n d i n c u b a t e 30 m i n at 3 7 ° C .  8) Stop r e a c t i o n w i t h 0.5 m l c o l d 28% T C A - a r s e n i t e . 9) C e n t r i f u g e 5 m i n at 12000 x g a n d t r a n s f e r  1.0 m l a l i q u o t o f s u p e r n a t a n t to  100 m m glass t u b e s . 42  10) A d d 0.5  m l 0.5%  T B A i n 0 . 0 2 5 M N a O H to e a c h g l a s s  11) B o i l 15 m i n to d e v e l o p the c o l o r f o r 12)  Read absorbance  at 532  tube.  spectrophotometery.  n m and 453  nm with a P e r k i n E l m e r L a m b d a 6  spectrophotometer. 13) C a l c u l a t e the c o n c e n t r a t i o n o f M D A . delta = MDA  4.3  ( a b s @ 5 3 2 - b l a n k @ 5 3 2 ) - 20%  (nmoles/gm R B C ) =  (abs@453 - blank@453)  ( d e l t a - 0 . 0 0 5 3 ) / l .931  * 5 0 / R B C weight in gram  M e a s u r e m e n t o f P l a s m a P r o p o f o l C o n c e n t r a t i o n by High-performance liquid chromatography  (GC)  [48,187-189]  can  determine  propofol  HPLC  ( H P L C ) or gas concentration  chromatography in  plasma  w h o l e b l o o d . B o t h H P L C and G C m e t h o d s h a d g i v e n e q u i v a l e n t r e s u l t s Fluorescence  detection  concentration,  blood  concentrations lag  in  is  concentrations organ  differences specimens resulted  redistribution  collection  than  are  been  used  for  HPLC  determination  in  [188].  of propofol  b e c a u s e it is m o r e s e n s i t i v e t h a n c l a s s i c a l u l t r a v i o l e t d e t e c t i o n .  Significant whole  has  or  easier  in propofol were  f r o m the across  and  observed infusion  blood  more  concentration  cell  or  [188].  This  clearance  membranes.  convenient  between  for  the  plasma  and  discrepancy  in  o f p r o p o f o l , and Whole  blood  clinician,  but  the  sample plasma  are m o r e d i r e c t l y r e l a t e d to the a c t i v i t y o f a d r u g i n the target whole  blood  concentrations.  Therefore,  plasma  samples  are  p r e f e r r e d , but i m m e d i a t e c e n t r i f u g a t i o n is n e e d e d .  43  The HPLC  d e t a i l s o f the d e t e r m i n a t i o n o f p r o p o f o l c o n c e n t r a t i o n s  with fluorescence  detection were  published report by D r . P l u m m e r )  i n p l a s m a by  as f o l l o w s ( m a i n l y a d a p t e d  from a  [48]:  R e a g e n t s and s o l u t i o n s : Double-distilled propanol,  water was u s e d .  trifluoroacetic  acid  obtained f r o m Sigma (Canada). Caledon  (Canada).  and  Cyclohexane (spectroscopic  sodium d i h y d r o g e n orthophosphate  Tetramethylammonium  methanol working  (1  standard mg/ml)  were  hydroxide  (TMAH)  A  further  solution  diluted  of  with  (25%  in  Canada).  thymol  methanol  was to  prepared  an  in  appropriate  concentration.  Phosphate (13.6  solution. and  2-  A c e t o n i t r i l e ( H P L C g r a d e ) was o b t a i n e d f r o m  m e t h a n o l ) a n d t h y m o l were o b t a i n e d f r o m F l u k a ( A l d r i c h , Internal  grade),  b u f f e r s o l u t i o n (0.1  M).  Sodium  dihydrogen  orthophosphate  g) was d i s s o l v e d i n 1 1 o f d i s t i l l e d w a t e r . Dilute T M A H  2 - p r o p a n o l (18.5 HPLC acetonitrile,  solution. T M A H  i n m e t h a n o l ) (1.5  m l ) was a d d e d to  ml).  mobile phase. 400  (25%  The H P L C  m o b i l e phase  consisted  o f 600  m l o f d i s t i l l e d water and 1 m l o f t r i f l u o r o a c e t i c  ml  of  acid. The  m o b i l e p h a s e was d e g a s s e d b y the p a s s a g e o f h e l i u m p r i o r to u s e .  Apparatus:  44  The HPLC and  high-performance  liquid  chromatograph  used  consisted  of  a  S y s t e m ( B e c k m a n , C a n a d a ) set to d e l i v e r a s o l v e n t f l o w o f 1.5  RF  5000  Flurometer  (Shimaclzu,  Canada)  e x c i t a t i o n a n d e m i s s i o n w a v e l e n g t h s w e r e 276  fluorescence  and 310  Gold  ml/min,  detector.  The  nm, respectively,  and  b o t h m o n o c h r o m a t o r s l i t w i d t h s w e r e 10 n m . T h e s i g n a l s w e r e r e c o r d e d u s i n g a  Alltech  size,  Lichroshere  150  x  4.6  RP  mm I . D . ;  18  encpd  Mandel  reversed-phase  Scientific,  c o l u m n (5  C a n a d a was  pm  particle  at  ambient  used  temperature.  Procedure: To  a  sample  of  plasma  (0.5  ml),  p h o s p h a t e b u f f e r (1 m l ) and c y c l o h e x a n e then  placed  on  an  c e n t r i f u g a t i o n (1150  inversion  mixer  internal  standard  for  15  min  at  60  rpm.  x g f o r 5 m i n ) , an a l i q u o t o f the c y c l o h e x a n e  s o l v e n t was e v a p o r a t e d  to d r y n e s s  s o l u t i o n (50  at a m b i e n t t e m p e r a t u r e  T h e d r y r e s i d u e was t h e n r e d i s s o l v e d i n H P L C  p l ) and an a l i q u o t (100 T y p i c a l chromatograms  (20  pl),  (5 m l ) w e r e a d d e d . T h e m i x t u r e  m l ) was t r a n s f e r r e d to a tube c o n t a i n i n g d i l u t e T M A H  nitrogen.  solution  p l ) o f the s o l u t i o n was  was  Following layer  (4.5  pl).  The  under a stream m o b i l e phase  s u b j e c t e d to H P L C  of (200  analysis.  o f extracts c o n t a i n i n g p r o p o f o l are s h o w n i n F i g u r e 7.  A c a l i b r a t i o n g r a p h was p r e p a r e d by the a d d i t i o n o f k n o w n q u a n t i t i e s propofol  to  procedure. the  aliquots  of control blood  T h e peak-height ratio  concentration  and extracted  o f p r o p o f o l to  of propofol added.  according  t h y m o l was  The concentration  to  the  plotted  of  above against  o f p r o p o f o l i n test  45  amples  was  calculated  using  the  regression  parameters  obtained  from  the  alibration graph.  46  JULi 0  1  3  2  4  0  5  1  2  3  4  5  i  1  1  1  1  0  1  2  3  4  Times (minutes) A  B  C  Figure 7. Schematic representation of chromatograms of extractsfrom( A ) control human blood, ( B ) blood containing propofol (1 u.g/ml), and (C ) blood obtained from a patient having received propofol intravenously. Thymol was added as the internal standard. Peaks 1 and 2 are thymol and propofol, respectively.  1  5  Chapter 5 RESULTS  5.1  Patient P r o f i l e The  patients'  age,  sex,  body surface  area a n d b o d y w e i g h t are s h o w n i n  T a b l e 1. T h e h e m a t o c r i t v a l u e s p r e - C P B a n d d u r i n g C P B , the d u r a t i o n o f C P B and a o r t i c c r o s s - c l a m p i n g and e x t u b a t i o n t i m e , are a l s o i n c l u d e d i n T a b l e 1.  5.2  Red C e l l M D A Production F o l l o w i n g in vitro t B H P Challenge  5.2.1  t B H P Dose-Response Curve The  red  dependent. to 1.0 2.0  cell  F o r each  MDA  production  sample,  following  tBHP  challenge  the M D A p r o d u c t i o n b e g i n s to r i s e i n  is  dose-  response  m M t B H P c h a l l e n g e and r e a c h e s a p l a t e a u w h e n t B H P c o n c e n t r a t i o n  m M . The  between  1.0  steep  and 2.0  portion of  the  curve  m M . Therefore,  is  related  f r o m the  to  various  tBHP  is  concentration  concentrations,  1.5  m M o f t B H P was s e l e c t e d f o r the a n a l y s i s o f a n t i o x i d a n t c a p a c i t y o f r e d c e l l s . F i g u r e 8 e x e m p l i f i e s this s i g m o i d c u r v e w h i c h is b a s e d o n the a v e r a g e M D A p r o d u c t i o n o f the b a s e l i n e ( p r e - i n d u c t i o n ) (Table  5.2.2  b l o o d samples  o f the three  groups  2).  Red  Cell  M D A Production  in Response  to  in vitro  tBHP  (1.5  mM)  Challenge  48  R e d c e l l M D A p r o d u c t i o n is s u m m a r i z e d i n T a b l e 3 a n d F i g u r e 9. The pattern  o f M D A p r o d u c t i o n seems s i m i l a r i n the i s o f l u r a n e a n d l o w  dose p r o p o f o l g r o u p s . A s u s t a i n e d s i g n i f i c a n t d e c r e a s e i n M D A p r o d u c t i o n is seen  only with h i g h dose  p r o p o f o l , and this  effect  appears  to p e r s i s t  post-  operatively .  5.3 P l a s m a C o n c e n t r a t i o n o f P r o p o f o l Average summarized  plasma  concentrations  i n T a b l e 3 and F i g u r e  of 9.  propofol  measured  T h e average  plasma  by  HPLC  concentration  are of  p r o p o f o l was d e c r e a s e d b y 18% i n l o w dose p r o p o f o l a n d i n c r e a s e d b y 2 9 % i n h i g h dose p r o p o f o l at 30 m i n C P B c o m p a r e d to that b e f o r e C P B (30 m i n p o s t induction).  T h e propofol concentration  was s i g n i f i c a n t l y h i g h e r  i n the h i g h  dose p r o p o f o l g r o u p than i n the l o w d o s e p r o p o f o l g r o u p d u r i n g C P B . In the h i g h between  dose  propofol group,  there was a c o r r e l a t i o n  red c e l l M D A p r o d u c t i o n ( Y ) and plasma concentration  (X) (Figure  10):  Y =  (R = 0.78213) of propofol  35.945 - 16.75 l o g X  S u c h a c o r r e l a t i o n d i d not e x i s t i n the l o w d o s e p r o p o f o l g r o u p .  5.4 I n o t r o p i c R e q u i r e m e n t d u r i n g C A B S u r g e r y ( T a b l e 4 , F i g u r e  11)  5 . 4 . 1 P e r c e n t a g e o f Patients G i v e n I n o t r o p i c D r u g s T h e r e were no s i g n i f i c a n t d i f f e r e n c e s a m o n g the g r o u p s .  49  5.4.2  P e r c e n t a g e o f P a t i e n t s G i v e n D o p a m i n e (3-5 T h e patients  i n isoflurane needed more  dose or h i g h d o s e p r o p o f o l g r o u p s (57%  5.4.3  p/kg/min)  d o p a m i n e t h a n those i n the  v s . 14%,  P <  low  0.05).  P e r c e n t a g e o f Patients G i v e n A d r e n a l i n e T h e patients i n the i s o f l u r a n e g r o u p n e e d e d m o r e a d r e n a l i n e t h a n those i n  the  low  dose  or  high  dose  propofol  groups  (73%  H o w e v e r , the d i f f e r e n c e s d i d not attain s t a t i s t i c a l  5.5  versus  43%  or  57%).  significance.  H a e m o d y n a m i c C h a n g e s d u r i n g 24 H o u r s P o s t - o p e r a t i o n  5.5.1  C a r d i a c Index ( T a b l e 5, F i g u r e The  percentage  o f the  cardiac  12) index  >  2.5  L/min/m2  i n the  low  dose  p r o p o f o l g r o u p is s i g n i f i c a n t l y h i g h e r than that i n the i s o f l u r a n e g r o u p d u r i n g the f i r s t 3 h o u r s p o s t - o p e r a t i o n ( T a b l e 6, d i f f e r e n c e s a m o n g the g r o u p s  5.5.2  F i g u r e 13).  otherwise.  Pulmonary C a p i l l a r y Wedge Pressure In  the  isoflurane  s i g n i f i c a n t l y h i g h e r (P 2 hours p o s t - o p e r a t i o n .  group, <  0.05)  pressure,  and  the  ( T a b l e 5, F i g u r e  pulmonary  d u r i n g the 19-24  capillary  the  12)  wedge  pressure  is  h o u r p e r i o d t h a n it is at 1 or  W h e n it is p l o t t e d against c a r d i a c  one c a n see that the c a r d i a c wedge  T h e r e are no s i g n i f i c a n t  index (Figure  14),  i n d e x is l o w e v e n w i t h h i g h p u l m o n a r y c a p i l l a r y percentage  of  cardiac  index  >  3  L/min/m2  is  50  significantly  higher  (P  <  0.05)  in  the  low  dose  propofol  than  that  in  isoflurane group.  5.5.3  C e n t r a l V e n o u s P r e s s u r e ( T a b l e 5, F i g u r e The central  venous  pressure  is r e l a t i v e l y  T h e r e are no s i g n i f i c a n t d i f f e r e n c e s a m o n g the  5.6  12) stable t h r o u g h o u t  the  surgery.  groups.  A l v e o l a r A r t e r i a l O x y g e n G r a d i e n t d u r i n g 24 H o u r s P o s t - o p e r a t i o n  7,Figure  15)  T h e r e is a s i g n i f i c a n t i m p r o v e m e n t i n o x y g e n a t i o n 12 h o u r s  (Table  after s u r g e r y  i n the  (P  <  0.05)  i n the  i s o f l u r a n e or l o w dose p r o p o f o l g r o u p s .  first  There  are no s i g n i f i c a n t c h a n g e s i n the h i g h dose p r o p o f o l g r o u p .  51  < PH  C  o i  a  IH  •S J,  u u «< .9 9, ffl  &  "•os  PH  u  13  9  t a  fN  +1 Tf  +1  +1  i-H  i-H  l-H i-H  t» +1  m © i-H 00 +1  fN  i-H  r-4  ro  © i-H  +1  m  (~~  +1  +1 00 Tf ^H  l-H  2  a  ^3  l-H ©  <N  ©  i-H ©  +1  +1 Tf fN  +1 IT. fN  i-H ©  ^H  i-H ©  "•8 •9  fi  2  .8  a *a  2  <  o  a  <u  u  i-H  ©  4) i. i.  eg  "O o> N j£> OS  s a s  I  6  JS  H  .a9 9 "w O J-  w ••fi  o 08  +1  +1  l-H  i-H Tf  +1 fN Tf  •s  SO  r-  Tf  +1  +1  +1  s  o  so  B  r  a  a  +1  o  =  Tf  ON  l>  +1  +1 Tf  ON  ON  l-H  l-H  ©  v>  +1  ©  ON  l-H IT)  so  +1 IT.  +1 IT,  SO  SO  fN  i-H  ©  ON  SO  00  +1 t-~  « ii  »  1/5  l-H l-H  =  i-H  ir> 00  in  u  ©  +1  o IT) ^H  ii  cs  i-H  a s  a  a  00  PH  <s  1—1  Vi  <s "8 .ts  2 a  s  i  •13 l>  00  o  •a  o  o a  U  u << CS  9  9 Vi  &  fN  o  a  o  i  ii ii VI  o  -o Vi VI  CH  SO  CS  o  B.  I Q a  3  & a  be fi o  9 93  Table 2.  tBHP-MDA Dose-Response Curve for Red Cellsfromthe Three Experimental  Groups. MDA Production (n moles/g rbc) tBHP (mM)  0.5  1.0  1.5  2.0  2.5  Isoflurane (n = 7)  1.16 ± 0.47  45.6 ± 4.2  122 ± 5  190 ± 4  198 ± 7  Propofol-Low (n = 7)  2.73 ± 1.45  46.6 ± 7.5  122 ± 12  187 ± 10  193 ± 8  Propofol-High (n = 7)  1.91 ± 0.62  33.4 ± 3.6  106 + 5  180 ±6  196 ± 5  Note: Data arefromthe first baseline (pre-induction) samples only, used to show the tBHPMDA dose-response curve. Data are expressed as mean ± SEM.  53  u u  < I  Vi  o  PH  o  • V ©  +1 t~ fN l-H  ro i-H  +1  fN i-H  + OS  +1  l-H 00  ro  ir,  +1  +1  fN fN  Tf  ro  fN  a  a  a  so  ir,  a o  '-8 -3 2 a  VO  o  +1  1/5 i-H  +1  PH  vo  ©  ^H  ro fN i-H  vo  fN l-H  fN  in  +1  +1  os  00  oo +1 o 00  ro  U U i Vi  O |PH  Q  "53 u  •s +1  1—1 ro i-H  +1  ro fN l-H  I.  .s  +1  a.  vo Tf  s  9  I. o  ON  00 an  O  PH  ir,  P9 PH  u  o ro  +1  fN ro ^H  VO  +1  i-H  +1  fN fN l-H  ro i-H  +1  Tf  ro  2 c  VO  fN  a  a>  +1  i-H  a  Vi  Tf  o  +  +1  Tf  ro  ©  Tf  Tf  +1  vo  i-5 +1 00  a  '1  +1  •a 1  on «  -s vi Vi  I  o  •a  I  o IT)  +1 fN fN  o  00  +1  fN OS  +1  +1  l-H ir,  ^H  os fN  +1  OS  ©  Q  a lU  & O  ro  'i  -a •S  ir.  +1  fN fN i-H  fN i-H  ir,  +1  +1  Tf  00 o i-H  rs l-H  o O  •a  o  a. a Vi  ?!  PH  1I a  <2  II  <2  © I i I I o  a  V  a  o  o  '  o  a  o  a  Sf J3  6J)  s O  I  T3  2 o.  J=  a  3  o  o  o  !  13  O  o  a  a?  O  o  c v u  SS  •a V  c/3 O  o  Table 4. Inotropic Requirement during CAB Surgery, by Group Inotrope Required Any Inotropic Drug  Dopamine 3-5 u,g/kg/min  Isoflurane  10/11  (91)  4/7  (57)  Propofol-Low  7/7  (100)  1/7  Propofol-High  6/7  (86)  1/7  Note: Data are expressed as number (%).  * +  Adrenaline 8/11  (73)  (14)  3/7  (43)  (14)  4/7  (57)  * P < 0.05 the isoflurane group versus low dose propofol  group, + P < 0.05 the isoflurane group versus high dose propofol group for dopamine 3-5 p-g/kg/min.  55  VO  i  Ov  oo ro  f N  1^-  fN  fN  VO  ©  ©  ©  +1  Ov  in  fN fN  fN  ©  ©  +1  +1  oo © +1  ro  fN  00  ro  ro  ro  fN  ro  rs  ©  ©  ©  +1  Ov r i  ©  +1  +1  ro ro ©  +1  i>  r i  r i  Tf  ro ©  +1  +1  i-H  VO  ©  +1  ro  ov r i  ro ©  +1  r i  r i  fN  fN  vo  ©  ©  ©  00  +1 «/5  r i  r i  ro ©  ©  +1  +1  00 r i  +1  1/5 ro  00  VO  r i  +1  ro ro  +1  fN  l>  fN  i-H  +1  +1  +1  fN  +1  i-H  O ^H  fN  k>  1/5  v© l-H  ro ^H  i-H  fN  l-H  i-H  fN  +1  +1  +1  +1  ro  ro  O  l-H  l-H l-H  O  l-H  l-H l-H  fN  fN  fN  i-H  l-H  i-H  +1  +1  +1  +1  +1  +1  Tf  fN  i-H  fN ^H  o  o  i-H  i-H  ro  fN  ^H  n  Tf  +1  IT)  Tf  +1  l-H  ro »-H  i-H l-H  fN  fN  fN  fN  O  +1  +1  +1  +1  +1  +1  fN  ro  o  l-H l-H  i-H  +1  Tf  l-H  l-H  +1  tn  l-H  l-H  +1  l-H  l-H  +1  i-H  +l  1  © ^H  l-H  i-H  i-H  i—i  fN ^H  l-H  fN  l-H  +1  ro  fN  +1  Tf  +1  ro  +1  ro  i-H  i-H  fN  fN  l-H  +1  +1  +1  +1  fN  fN ^H  Tf ^H  fN  IT/ ^H  1/5  ^H  ro  i-H  +1  i-H  i-H  i-H  +1 fN  i-H  ©  +1 ro r i  +1  +1  Tf ^H  ro ^H  Tf  i-H  i-H  fN  l-H  fN  fN  ^H  +1  +1  +1  +1  fN  ro  fN  l-H  00 ^H  fN  +1  fN  fN  Tf  1/5  © +1  o +1  i>  Tf  o  +1  r i  +1  fN  ro ©  ^H  l-H  ro ro  Ov  l*N  +1  00 r i  fN  ro  +1  ro  ©  +1 o ro  so  Tf  © +1  ©  ro  vO  ro  r i  o  +1  •a  l-H  »H  i-H  l-H  l-H  l-H  +1  +1  +1  +1  +1  l-H  vo ^H  ro  ©  fN  T—1  o  •a  l-H  l-H  o  -J  +1  Tf  l-H  9±0  Tf fN  •a •  I  I o  I'H  o  I  a,  o  I  ©  o  c.  o  Cu  o  Table 6. Percentage of Patients with a Cardiac Index > 2.5 L/min/m during 24 hours Postoperation. 2  Time (hr) Isoflurane  1^3  4^6  9 /22 (41) *  Propofol-Low  10/13  Propofol-High  4/5  7^12  13^18  19-24  6/17  (35)  6/8  (75)  7/10  (70)  5/5  (100)  (77) *  3/11  (27)  6/7  (86)  5/5  (100)  2/3  (67)  (80)  4/7  (57)  4/4  (100)  3/4  (75)  2/2  (100)  Note: * P < 0.05 between the isoflurane and low dose propofol groups. Data are expressed as number (%).  57  Table 7. Alveolar Arterial Oxygen Gradient (mmHg) Admission  1-6 Hrs Post-operation  6- 12 Hrs Post-operation  Isoflurane  195 + 19 * #  148 + 18 *  124 ±21 #  Propofol-Low  145 ± 9 **  94 ± 6  97 ± 2 6  Propofol-High  162 ± 19  149 ± 27  **  127 ± 32  Note: In the isoflurane group, * P < 0.05 ( # P < 0.001) between admission and 1-6 hours ( 6-12 hours) postoperation. In the low dose propofol group, ** P < 0.0005 between admission and 1-6 hours postoperation. P > 0.05 between each group at any time or duration. Data are expressed as mean ± SEM.  58  Figure 8. t BHP dose-response curve for red cell MDA production. Data is exemplified onlyfromthe blood samples taken at preinduction. Data are expressed as mean ± SEM.  Y = 105.7 - 58.422 X R = 0.824  n  S) 100  S o E  S  50 0  0  0.5  Log [Propofol]  1  1.5  (ug/ml plasma)  Figure 10. The relationship of red cell MDA production and plasma concentration of propofol in the high dose propofol group.  61  • Isoflurane rj Propofol-Low • Propofol-High  100 T3 0)  k. '5  rr 0) 0)  Q. O o  c  80 604020 0Any Inotrope  Dopamine 3-5 ug/kg/min  Epinephrine  Figure 11. Inotropic requirement during CAB surgery. The requirement for dopamine 3-5 pg/kg/min is significantly less in the low dose or high dose propofol groups than that in the isoflurane group, P < 0.05.  62  .Isoflurane -Propofol-Low -Propofol-High  • Isoflurane . Propofol-Low .Propofol-High  13-  19-24  . Isoflurane • Propofol-Low • Propofol-High  -gl7 14 E  > O  0  Time ( hr, postoperative)  13-  1924  Figure 12. Hemodynamic changes during 24 hours post-operatively. For cardiac index and central venous pressure, there are no significant differences between the groups using the pooled ttest.  In the isoflurane group, the pulmonary  capillary wedge pressure is significantly higher (P < 0.05) during 19-24 hour period than it is at 1 or 2 hours post-operatively.  CI during 1st 3 Hrs Postoperation  • A o  Isoflurane Propofol-Low Propofol-High  7.5 S c  5  AA  E  d,  2.5  -  0  - ^ - ^ A ^ ^ - - A -  a  • m m  0  Figure 13. Cardiac index during the firsr 3 hours post-operatively.  The percentage of  patients with a CI > 2.5 L/min/m in the low dose propofol group is significantly higher 2  than that in the isoflurane group. There are no significant differences between the high dose and low dose propofol or isoflurane groups.  64  4  f  3.5-  ™  3  S  o  A  A  A  0  A  2.5-  °  9  10  • A o  6 o  Isoflurane Propofol-Low Propofol-High  o *  1  i  i  12  14  16  18  20  PCWP(mmHg)  Figure 14.  Cardiac index versus pulmonary capillary wedge pressure during 24 hours  post-operatively. Data are expressed as mean of different time intervals as seen in Figure 11. The CI is low in the isoflurane group, even with high PCWP. The CI is high in the low dose propofol group, in which the aortic cross-clamping time is longest among three groups. There is no difference between the high dose and low dose propofol or the isoflurane groups.  The percentage of patients with a CI>3L/min/m is significantly higher in the 2  low dose propofol group (6 put of 8) than that in the isoflurane group( 1 out of 8),  P<  0.05.  65  Alveolar Arterial Oxygen Gradient 300 250 I E 200 B 150 CS o 100 ra 50 < 0  *+  Admission  • Isoflurane • Propofol-Low • Propofol-High  +  1-6 Hrs Postoperation  an i  6-12 Hrs Postoperation  Figure 15. Alveolar arterial oxygen gradient during 24 hours post-operation. * P < 0.05 (+ P < 0.001) between 1-6 (6-12) hours post-operation and admission in the isoflurane group. **, P< 0.001  between 1-6 hours post-operation and admission in the low dose propofol  group.  66  Chapter 6 DISCUSSION  6.1  Evaluation of Hypotheses I s o f l u r a n e at a c o n c e n t r a t i o n  less than 2%  e n d t i d a l , u s e d as c o n t r o l  for  this s t u d y , d i d not m o d i f y r e d c e l l a n t i o x i d a n t status. T h e r e d c e l l a n t i o x i d a n t capacity  d i d not c h a n g e  s i g n i f i c a n t l y 30  min post-induction in comparison  to  pre-induction. R e p e r f u s i o n i t s e l f d i d not r e d u c e the r e d c e l l a n t i o x i d a n t c a p a c i t y  i n any  o f the g r o u p s . T h i s m a y be b e c a u s e R O I s p r o d u c e d f r o m heart a n d l u n g s l a r g e l y d e t o x i f i e d by p l a s m a a n t i o x i d a n t  systems  cells  implicates  to  exert  significant effects.  This  were  b e f o r e they r e a c h e d the the  bypass  machine  red as  a  m a j o r c o n t r i b u t o r to the s y s t e m i c p r o d u c t i o n o f R O I s . The  antioxidant  propofol the  Low  significantly increased  surgery.  hours  after dose  capacity  properties  This  effect  bypass propofol,  before  bypass,  the but  p r o p o f o l are  the  appears  w h e n the on  of  red cell antioxidant  to  plasma other  dose-dependent.  continue  capacity  post-surgically,  propofol concentration  hand,  increased  this  effect  was  with  high  dose  not  the  red  sustained  High  throughout  even  was cell  dose  at  very  two low.  antioxidant  during  or  after  bypass. By  continuous  concentration  infusion  of p r o p o f o l reached  while on bypass,  with resultant  7.2  propofol,  p g / m l before  the  bypass  average and  9.3  plasma pg/ml  suppression of red cell M D A production  of  67  m o r e than 5 0 % . T h i s e f f e c t o n M D A p r o d u c t i o n is g r e a t e r t h a n it was i n o u r p r e v i o u s s w i n e m o d e l [18], i n w h i c h the s w i n e r e d c e l l M D A p r o d u c t i o n was s u p p r e s s e d b y o n l y 25% w h e n the p l a s m a p r o p o f o l c o n c e n t r a t i o n was b e t w e e n 7  a n d 12  p-g/ml.  production  In the latter  o f 50%  required  swine  model,  a plasma  a decrease  in red cell M D A  propofol concentration  of  12  to  25  Lig/ml.  L o w dose p r o p o f o l h a d c a r d i o p u l m o n a r y p r o t e c t i v e significantly  higher  percentage  of  patients  with  L/min/m  2  d u r i n g the 1st 3 h o u r s p o s t - o p e r a t i v e l y  L/min/m  2  d u r i n g 24 h o u r s p o s t - o p e r a t i v e l y  (P  <  0.05), and a greater degree  after s u r g e r y did  as c o m p a r e d  not i n c r e a s e  cardiac  dose p r o p o f o l , were  associated  and p r e v e n t e d the i n c r e a s e seen  in  the  protective  isoflurane  effect  significantly  on  increased  cardiac  m a n i f e s t e d as index  and with cardiac  of recovery  in lung oxygenation <  post-operatively.  0.001). High  w i t h s i g n i f i c a n t l y less  the  Isoflurane  heart.  pulmonary  index.  dose,  >  3  capillary  it  wedge  isoflurane  show  was  1-12 h o u r s  as w e l l  inotropic  d i d not  Moreover,  However,  index  2.5  H i g h dose p r o p o f o l  w h i c h was  any  detectable  associated  pressure  is a s s o c i a t e d  as l o w  requirement,  i n p u l m o n a r y c a p i l l a r y wedge pressure group.  >  c o m p a r e d to that w i t h i s o f l u r a n e  with isoflurane (P index  effects,  with  without  any  increase  in cardiac  recovery  i n l u n g o x y g e n a t i o n 1-12 h o u r s p o s t - o p e r a t i o n c o m p a r e d w i t h that o f  the same g r o u p at a d m i s s i o n to I C U after o p e r a t i o n , P <  0.001.  with  a  greater  T h e a v e r a g e e x t u b a t i o n time i n the h i g h d o s e p r o p o f o l g r o u p was s h o r t e r than that i n the i s o f l u r a n e c o n t r o l s , significant  6.2  b u t the d i f f e r e n c e  was not  statistically  [151,152].  What Does Increased  Red Cell Antioxidant Capacity Mean?  6 . 2 . 1 G e n e r a l i z e d E n h a n c e m e n t o f T i s s u e O x i d a n t Status The capacity  relationship  antioxidant  Thus,  especially  the r e l a t i o n s h i p c a n or f r o m  experiments.  red cells  manifested MDA  antioxidative  functional performance  In a s w i n e m o d e l o f h e a r t - l u n g t r a n s p l a n t a t i o n swine  a n d tissue  human tissues,  c a n not be o b t a i n e d f o r a n a l y s i s .  be i n f e r r e d i n d i r e c t l y f r o m the o r g a n  animal  status  has not b e e n c l a r i f i e d , p a r t i a l l y b e c a u s e  heart a n d l u n g , only  of red cell  as  levels  transplanted  to  in vitro  significantly correlated  lung,  oxidative decreased  significantly  as assessed  of lung functional integrity.  challenge red cell with  the  [ 3 9 ] , the s u s c e p t i b i l i t y o f  was s i g n i f i c a n t l y M D A production. functional  b y l u n g water content In a c o m p a r a t i v e  reduced, Red cell  viability  - a convenient  study o n r e d c e l l s  o f the measure  of diabetic  rats and h u m a n d i a b e t i c s [ 1 9 2 ] , the r e d c e l l p r o d u c t i o n o f M D A was i n c r e a s e d in  response  patients,  to i n v i t r o  the extent  challenge  o f this  with hydrogen  increase  peroxide.  in susceptibility  In the  of red cell  o x i d a t i o n p a r a l l e l e d the s e v e r i t y o f d i a b e t i c c o m p l i c a t i o n s  diabetic lipids  to  present.  69  Toth radical  et a l . [190]  generating  found  systems  that p e r f u s i o n o f i s o l a t e d  caused  vascular  leakage  rat l u n g s  and edema  with  free  [190]. T h e  i n c l u s i o n o f r e d c e l l s i n this m o d e l p r e v e n t e d s u c h o x i d a n t - i n d u c e d d a m a g e . V a n A s b e c k et a l . [191] r e p o r t e d that p r i o r i n s u f f l a t i o n o f r e d c e l l s i n t o rat lungs  protected  Furthermore, present  it  i n intact  the was  animals found  against that  early  catalase  death  d u e to  [91,92]  and/or  oxygen  toxicity.  glutathione  red cells can decrease m y o c a r d i a l h y d r o g e n peroxide  [92] levels  and r e p e r f u s i o n i n j u r y [92], a n d p r e v e n t d e a t h o f s o m a t i c c e l l s r e s u l t i n g f r o m in vitro oxidative challenge Therefore,  it seems  [91].  that r e d c e l l a n t i o x i d a n t c a p a c i t y  tissue o x i d a t i v e status, a n d intact r e d c e l l s c a n p r o t e c t  is a s s o c i a t e d  with  tissues f r o m o x i d a t i v e  stress. T h i s is m o r e i m p o r t a n t f o r tissues s u c h as h u m a n h e a r t ,  w h i c h unlike  r e d c e l l s is v u l n e r a b l e to o x i d a t i v e i n j u r y due to t h e i r p o o r o x i d a t i v e r e l a t i v e to r e d c e l l s - i n c l u d i n g v e r y l o w c o n c e n t r a t i o n s  status  o f catalase [9,92].  6.2.2 Partially Improved C a r d i o p u l m o n a r y F u n c t i o n R O I s are g e n e r a t e d myocardial  injury  d u r i n g C P B a n d h a v e b e e n i m p l i c a t e d as a cause o f  [8,193,194].  A s noted  earlier,  biomolecule including lipids, proteins, carbohydrates, As  m e n t i o n e d , the heart  ROIs  may damage  and n u c l e i c acids  a n d l u n g s m a y be p r o t e c t e d  any [26]  b y the a n t i o x i d a n t  c a p a c i t y o f intact r e d c e l l s or b y a g e n e r a l i z e d a n t i o x i d a n t a c t i o n o f p r o p o f o l manifest  in oxidation-sensitive  tissues  as w e l l  propofol  improved cardiopulmonary function.  as r e d c e l l s . This  In o u r s t u d y ,  m a y be d u e to its  free  70  radical The  scavenging  enhanced  membrane  activity  tissue  against  antioxidative  integrity,  with  locally  or s y s t e m i c a l l y  produced  status w o u l d be e x p e c t e d  resultant  improved  ROIs.  to p r e s e r v e  cardiopulmonary  cell  function.  S i m i l a r r e s u l t s have b e e n a c h i e v e d i n a n i m a l m o d e l s o f I R I [ 1 4 , 1 5 , 1 4 9 ] . It  s h o u l d be n o t e d ,  however,  that the i m p r o v e m e n t  f u n c t i o n w i t h i n 24 h o u r s p o s t - o p e r a t i v e l y vitro  red  cell  lipid  peroxidation  in cardiopulmonary  d i d not p a r a l l e l the i n h i b i t i o n o f i n  in  this  study,  where  significant  c a r d i o p u l m o n a r y p r o t e c t i o n was also seen i n the l o w d o s e p r o p o f o l g r o u p i n the absence o f r e d c e l l p r o t e c t i o n . O t h e r i n v e s t i g a t o r s h a v e r e p o r t e d that e v e n complete  elimination of lipid  peroxide  only mildly  [15]  or rabbit  [149]  peroxidation  attenuated heart.  induced by exogenous  the c a r d i a c  Therefore,  hydrogen  d y s f u n c t i o n i n the i s o l a t e d  factors  other  than  lipid  rat  peroxidation  may p l a y a m o r e i m p o r t a n t r o l e [ 1 9 5 - 1 9 7 ] . O x i d a t i o n o f p r o t e i n or n o n p r o t e i n sulfhydryl potential  groups causes  multifactorial production  [195]  and nucleic  acids  of diminished myocardial  pathogenesis  o f P A F or  of  other  IRI  [196]  contractile  during  mediators  have  been  performance.  C A B surgery,  m a y also  considered  play  factors an  as  In this such  important  as role  [72,73,75].  6.3  P r o p o f o l C o n c e n t r a t i o n a n d Its P r o t e c t i v e P r o p o f o l i n p l a s m a is b o u n d e x t e n s i v e l y  f r a c t i o n o f o n l y 2-3% determine  Effects to p l a s m a a l b u m i n ,  w i t h a free  [ 1 3 8 ] . It is this s m a l l free f r a c t i o n , h o w e v e r ,  the p r o t e c t i v e  effect  of p r o p o f o l on red cells  that m a y  and tissues.  The  71  amount o f p r o p o f o l i n b l o o d cells and i n plasma accounts concentration  of propofol. There  cells and tissues,  f o r the total  blood  is a l a g i n p r o p o f o l d i s t r i b u t i o n into r e d  w h i c h e x p l a i n s w h y the p l a s m a c o n c e n t r a t i o n  is m o r e  than  the w h o l e b l o o d c o n c e n t r a t i o n d u r i n g c o n t i n u o u s i n f u s i o n . F a n et a l . r e p o r t e d that p l a s m a c o n c e n t r a t i o n s during  propofol  determine  the  were 30% h i g h e r than w h o l e b l o o d  infusion  plasma  if  the  blood  concentrations;  was  even  centrifuged  following  concentrations  immediately  storage  for  1  to  hour  b e f o r e c e n t r i f u g a t i o n , the p l a s m a c o n c e n t r a t i o n was s t i l l 10% h i g h e r t h a n that in  whole  blood  correlation  [188].  between  In  plasma  our  study,  we  were  propofol concentration  not  able  to  and red cell  establish  a  antioxidant  status i n the l o w dose p r o p o f o l g r o u p . It m a y be that the a m o u n t o f p r o p o f o l d i s t r i b u t e d i n t o r e d c e l l s f r o m the p l a s m a was too s m a l l to c a u s e a s i g n i f i c a n t change i n s u s c e p t i b i l i t y to o x i d a t i v e c h a l l e n g e . O n the other h a n d , i n the h i g h dose p r o p o f o l g r o u p , a s i g n i f i c a n t c o r r e l a t i o n was e s t a b l i s h e d , p a r a l l e l i n g the result  we o b t a i n e d i n the s w i n e m o d e l [18].  U n d e r the latter c o n d i t i o n , the  a m o u n t o f p r o p o f o l i n r e d c e l l s was s u f f i c i e n t to r e d u c e p e r o x i d e - i n d u c e d r e d cell M D A production. With  the  Interestingly, 18%  initiation the p l a s m a  of  CPB,  hematocrit  concentration  was  decreased  by  o f p r o p o f o l was d e c r e a s e d  i n the l o w dose p r o p o f o l g r o u p and was i n c r e a s e d  39%.  by only  by 29%  i n the h i g h  dose p r o p o f o l g r o u p after 30 m i n o f C P B . It has b e e n r e p o r t e d  that p l a s m a  propofol  concentration  haemodilution  alone  decreases [140].  This  more  than p r e d i c t e d  suggests  o n the b a s i s  the b i n d i n g  o f acute  of propofol  to the  72  extracorporeal  circuit.  At  this  point,  it  is  difficult  to  explain  this  discrepancy. Unlike propofol  the  in plasma  p r o p o f o l does this  free  plasma  concentration  behaves  differently  fraction  was  reported  lipoprotein  lipase  esterified  fatty  [143,198].  of  acids  various  Unfortunately, We  observed  propofol,  during  to  drugs  [141,142]. hydrolyze These  including  we d i d not m e a s u r e the s u s t a i n e d  the  free  fraction  of  fraction  of  C P B . T h e free  not d e c r e a s e d u r i n g C P B . I n d e e d ,  activates  binding  of  a 1.5  This  to 3 f o l d  m a y be  increase in  because  heparin  plasma  triglycerides  into n o n -  compounds  competitively  i n h i b i t the  propofol  the free  to  plasma  proteins  [144].  fraction of propofol in plasma.  suppression of red cell  M D A p r o d u c t i o n i n the  h i g h d o s e p r o p o f o l g r o u p e v e n at 2 h o u r s post C P B w h e n the p l a s m a p r o p o f o l concentration (or  increased)  was v e r y free  l o w . T h i s m a y p o s s i b l y be b e c a u s e  fraction  of propofol  in plasma  of an unchanged  perioperatively,  and a  cumulative effect of p r o p o f o l with continuous i n f u s i o n .  6.4  M D A P r o d u c t i o n , R e d C e l l A n t i o x i d a n t Status,  and T B A Assay  M a l o n d i a l d e h y d e is f o r m e d d u r i n g l i p i d p e r o x i d a t i o n . It has b e e n w i d e l y u s e d as a m a r k e r  for lipid peroxidation [1,15,36,39,41,43,45,55].  b a s i c a l l y t w o assays a v a i l a b l e f o r the m e a s u r e m e n t and  tissues:  reactivity  the T B A assay  o f the c o l o r l e s s  and H P L C .  T h e r e are  of M D A in plasma,  T h e T B A assay  is b a s e d  cells, o n the  malondialdehyde ( M D A ) with thiobarbituric  acid  ( T B A ) to p r o d u c e a r e d a d d u c t , w h i c h is m e a s u r a b l e b y s p e c t r o p h o t o m e t r y .  In  73  c o n t r a s t to H P L C , cost-effective  the T B A assay is a c o n v e n i e n t , easy to p e r f o r m , r a p i d , a n d  test.  It i s , h o w e v e r ,  not v e r y  sensitive  a n d not v e r y  specific  [ 7 6 , 1 9 9 - 2 0 2 ] . N o r m a l l e v e l s o f M D A i n p l a s m a o r c e l l s o r tissues are too l o w to be d e t e c t e d b y this test. B e s i d e s M D A , there is the g e n e r a t i o n o f n o n - l i p i d related,  malondialdehyde-like,  overestimation not c l e a r ,  TBA-reactive  o f the M D A p r o d u c t i o n [ 2 0 3 ] . T h e nature  but sialic a c i d , g l y c o c o n j u g a t e s ,  have  been  For  example,  demonstrated  to be able  auto-oxidation  of  Fluorometric assay  detection  [207].  recommended  a  method  to  compounds  during  interfering  compounds  are  peroxidation glutathione. equivalent nm.  but  are  derived  Interference to 2 0 %  acid  errors  cell  lipid  from  caused  the  by  at  absorption  HPLC.  T h i s m e t h o d f o r m e d the basis f o r o u r T B A a s s a y .  6.5  Influence  o f the  [208].  of The  of  and  absorbance  Hypothermia  [206].  generation  products  was c o r r o b o r a t e d  nine  G i l b e r t et a l .  hemolysate  o f the m a x i m u m  or  least  the s p e c i f i c i t y  T B A assay,  as  carryover  T h e i m p r o v e d a c c u r a c y o f the m e t h o d  at  peroxidation  erythrocyte  from  of Normothermia  aldehydes  to the T B A r e a c t i o n  for  considered  o f the i n t e n s i t y  is  generates  red  results  o f this substance  linoleic  to the r e d c e l l  not  to  with T B A [202,204,205].  correct  interfering  lead  to c r o s s - r e a c t  has b e e n u s e d to i m p r o v e  W i t h respect  that  sugars a n d , i n g e n e r a l ,  b r e a k d o w n p r o d u c t s that g i v e p o s i t i v e r e s p o n s e s  TBA  substances  lipid  reduced 532  peak  nm,  at 453  with a specific  for C P B on Red  Cell  A n t i o x i d a n t Status a n d C a r d i o p u l m o n a r y F u n c t i o n  74  Hypothermia  is c o m m o n l y u s e d f o r C P B w i t h r e l a t i v e l y  long periods of  p e r f u s i o n , w h i l e n o r m o t h e r m i c C P B is e m p l o y e d f o r r e l a t i v e l y short procedures  and otherwise  uncomplicated  cases.  In this  study,  operative  we h a d o n e  n o r m o t h e r m i c C P B patient i n each g r o u p . I n d u c e d h y p o t h e r m i a f o r C P B i t s e l f is b e n e f i c i a l f o r the p a t i e n t apparently does  does  greatly  not have  depress  any a d v e r s e  body  physiological consequences  metabolism  [210],  production of R O I s because of decreased the  ischemic  and reperfused  heart  which  might  not e x p e r i e n c e  antioxidant  IRI during  and lungs,  defense  C P B as  is r e l a t i v e l y  e n h a n c e d to some d e g r e e ,  [210].  reduce  It the  oxygen consumption, especially for i n which antioxidant  may be i m p a i r e d d u r i n g I R I [ 7 8 - 8 0 ] . R e d c e l l s are s o m e w h a t do  [ 2 0 9 ] . It  do the heart  intact.  Their  different.  and lungs,  antioxidant  systems  status  They  a n d their might  be  because o f reduced body m e t a b o l i s m with possibly  r e d u c e d total p r o d u c t i o n o f R O I s . However,  i n this  study  we d i d not n o t i c e  b e t w e e n h y p o t h e r m i c and n o r m o t h e r m i c  any s i g n i f i c a n t  differences  C P B i n terms o f r e d c e l l  antioxidant  status, p l a s m a c o n c e n t r a t i o n  o f p r o p o f o l , or c a r d i o p u l m o n a r y  may  of hypothermia  be  because  negligible machine.  the  effect  in comparison In f a c t ,  even  function.  on R O I production  to  that  of  ischemia-reperfusion  with  deep  hypothermia  ( 1 5 ° to  might  a n d the  20°C),  This be  bypass  tissue  lipid  p e r o x i d a t i o n is s t i l l p r e v a l e n t [ 2 1 1 ] . U n f o r t u n a t e l y , no study has b e e n done to compare  hypothermic  and  normothermic  C P B in  terms  of  oxidative  or  75  a n t i o x i d a t i v e status, and o u r s m a l l s a m p l e s i z e m a k e s it too e a r l y to m a k e any c o n c l u s i o n s o n this i m p o r t a n t m a t t e r .  76  Chapter 7 CONCLUSION A N D RECOMMENDATIONS FOR F U R T H E R WORK  The antioxidant dose  effect  of p r o p o f o l on red cells  p r o p o f o l significantly enhanced  concentrations  o f p r o p o f o l i n the range  M D A p r o d u c t i o n by more than 50%. cardiac  red  function compared  inotropic  requirement  capillary  wedge  to  capacity.  Plasma  o f 7 to 9 p g / m l s u p p r e s s e d  red cell  i s o f l u r a n e , as  (In  antioxidant  High  T h i s e f f e c t was a s s o c i a t e d w i t h i m p r o v e d  (dopamine  pressure  cell  is d o s e - d e p e n d e n t .  3.5  the  manifested by s i g n i f i c a n t l y  pg/kg/min),  isoflurane  and  group,  it  stable was  less  pulmonary significantly  i n c r e a s e d at about 24 h o u r s c o m p a r e d to 1 or 2 h o u r s after s u r g e r y w i t h o u t an increase  in cardiac  index.)  with normal cardiac  and/or significantly higher percentage of  i n d e x p o s t - s u r g i c a l l y . H o w e v e r , the s u p p r e s s i o n o f l i p i d  p e r o x i d a t i o n d i d not p a r a l l e l the Because  of  structures  its  high  lipid  such  as  plasma  damaging effects the and  contractility. negative  acids,  solubility, and  which  propofol  distributes  mitochondrial  s u c h as have  in cardiopulmonary function.  However, p r o t e i n or  been  to  membranes,  membranous reducing  it p r o b a b l y c o u l d not nonprotein sulfhydryl  considered  important  in  i n o t r o p i c effects  o n the  w i t h a l o w (not  heart.  C o m p a r e d to  statistically  o f s m a l l s a m p l e size) c a r d i a c  low dose  the  prevent groups  myocardial  M e a n w h i l e , h i g h dose p r o p o f o l , l i k e other a n e s t h e t i c s ,  was a s s o c i a t e d because  improvement  by R O I s on them.  oxidation of structures nucleic  patients  also  has  p r o p o f o l , it  s i g n i f i c a n t i n o u r study p r o b a b l y  index d u r i n g early  (1  to 3 h o u r s  post77  o p e r a t i v e ) p e r i o d o f time after the o p e r a t i o n , lung  oxygenation  effect  of  high  w i t h i n 12 dose  hours  propofol  and not s i g n i f i c a n t r e c o v e r y  post-operatively.  on  However,  cardiopulmonary  the  function  in  long-term  could  not  be  propofol  on  d e t e r m i n e d b y the s t u d y . 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