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Gram-negative endotoxaemia Tuchek, John Michael 1983

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GRAM-NEGATIVE ENDOTOXAEMIA  by  JOHN MICHAEL TUCHEK B.Sc,  The University of Saskatchewan, 1969  M.Sc., The University of Saskatchewan, 1978  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DOCTOR OF PHILOSOPHY DEGREE  in  THE FACULTY OF GRADUATE STUDIES Department  of Pharmacology,  Faculty of Medicine  We accept this thesis as to the required  conforming  standard  THE UNIVERSITY OF BRITISH COLUMBIA JULY 1983  0  John Michael Tuchek,  1983  In  presenting  requirements of  British  it  freely  agree for  this for  an  available  that  I  by  understood  that  his  that  or  be  her or  shall  of  The U n i v e r s i t y o f B r i t i s h 1956 Main M a l l Vancouver, Canada V6T 1Y3  Date  y ^ n ^ W e v ^  the  shall  and  study.  I  copying  granted  by  publication be  the  University  the  of  allowed  t i ^ o /o Columbia  of  make  further this  head  representatives.  not  ^Pk^^M  at  of  Library  permission.  Department  fulfilment  the  extensive  may  copying  f i n a n c i a l gain  degree  reference  for  purposes  or  partial  agree  for  permission  scholarly  in  advanced  Columbia,  department  for  thesis  It  this  without  thesis  of  my  is  thesis my  written  (ii)  ABSTRACT  Endotoxins outer  are  membrane  1 ipopolysaccharide  of  gram  negative  0-antigen of gram-negative of  these organisms.  (LPS)  bacilli.  bacteria  with  The release of endotoxins  gram-negative  gram-negative  septic  septic  management  effective  of  shock  in  extractable  complexes  comprise  in the serological  from gram-negative  in  approaches  to the high mortality rate associahumans.  Indeed,  the  sequelae  administration of purified endotoxin.  gram-negative  septicaemia  would,  perhaps,  this  thesis  has  utilized  to obtain more insight  both  biochemical  and  into the mechanism of  endotoxin with the ultimate hope of finding an effective  of  The c l i n i -  be much more  The work  physiological  action  of  antagonist  E.  coli  for  this  substance.  In vivo studies with rats and guinea pigs have indicated that plasma  lysosomal  enzyme  severity of endotoxic shock. included  acid  phosphatase,  Interestingly,  when  include patients of  typing  i f agents capable of antagonizing the actions of endotoxin in vivo  reported  in  the  bacteria  could be used in combination with appropriate a n t i b i o t i c therapy.  toxic  from the  shock seen c l i n i c a l l y can be reproduced in experimental  animals by the parenteral cal  These  and are used  is believed to be a contributing factor ted  complexes  the  patients  significantly  the  activities  can  The lysosomal  be  elevated  as  N-acetyl-p-glucosaminidase  investigation  of  contained relative  lysosomal to  a measure  of  enzymes examined in this  these  in gram-negative septic shock, also  used  elevations  enzymes  study  cathepsin  D.  extended  to  was  i t was found that the plasma  enzyme  controls.  and  the  activities  Furthermore,  that the  were  average  (iii)  plasma a c t i v i t y  of  cathepsin  D in gram-negative septic  shock  patients  was  found to be s i g n i f i c a n t l y higher than that seen in the plasma of patients in other forms of shock.  Thus, these preliminary studies indicate that measure-  ments of plasma cathepsin nostic value  D a c t i v i t y may have diagnostic  in c l i n i c a l gram-negative septic shock.  and possibly  Other in vivo  prog-  investi-  51  gations  included  tissue  distribution  c o l i endotoxin in guinea pigs. to e x i s t between the  studies  A positive  accumulation  of  with  Cr-radiolabelled  correlation  endotoxin  tent with what is known c l i n i c a l l y regarding  (r = 0.964) was found  in lung tissue  (determined by plasma acid phosphatase a c t i v i t y ) .  E.  and  toxicity  These results are  consis-  the lung being a primary organ  involved in the pathophysiology of gram-negative septic shock. 51  In v i t r o erythrocyte membranes  studies  utilizing  membranes  revealed  in a s p e c i f i c  Cr-labelled that  manner.  E.  endotoxin  is  The binding of  cells  of  K + -p-nitrophenylphosphatase  from hypotonic l y s i s .  enzymatically  modified  activity  capable  including  On the  binding  including  and protection  of  red  erythrocytes  suggested  of  these  methylprednisolone,  study as possible endotoxin  studies,  lidocaine  and  antagonists.  certain  that  the  to was  inhiblood  Studies with chemically modified endotoxins  human  basis  of  and human  to membranes  properties  action of endotoxin involved l i p i d - l i p i d interactions membrane.  endotoxin  endotoxin  associated with measureable changes in functional bition  coli  and  stabilizing  between toxin and c e l l membrane-active  propranolol  were  drugs  selected  for  Of these agents, only propranolol 51  effectively membranes  antagonized in v i t r o .  the  binding  The effectiveness  of  Cr-endotoxin  of propranolol  as  to  erythrocyte  an endotoxin  anta-  (iv)  gonist was also demonstrated the  accumulation  phosphatase  of  activity  d-isomer was effective endotoxin-treated  in vivo by i t s  endotoxin in  in  lung  a b i l i t y to s i g n i f i c a n t l y reduce  tissue  endotoxin-treated  and  animals.  to  lower  plasma  However,  only  acid the  in vivo whereas the racemate was poorly tolerated by  animals.  These studies indicate that membrane-active drugs  (such as  propranolol)  are capable of antagonizing certain actions of endotoxin in vivo and as such may prove to  be valuable  adjuncts  c l i n i c a l management of gram-negative  to  specific  septicaemia.  antibiotic  therapy  in  the  (v) TABLE OF CONTENTS Abstract  ii  Table of Contents  v  L i s t of Figures  viii  L i s t of Tables  xi  Acknowledgements  xii  Quotation  xi i i  CHAPTER 1.  INTRODUCTION  1.1.  H i s t o r i c a l Background  1  1.2.  The Gram-negative Cell Envelope  4  1.3.  Isolation,  6  1.4.  Biological A c t i v i t i e s of Endotoxins  18  1.5.  Clinical  26  CHAPTER 2. 2.1.  2.2.  Composition and Structure of Endotoxin  Significance of Endotoxins  MATERIALS AND METHODS  Membrane Preparations  33  2.1.1.  Erythrocyte Ghosts  33  2.1.2.  Heart Sarcolemmal Membrane  34  2.1.3.  Liver Membranes  35  2.1.4.  Lung Membranes  36  2.1.5.  Preparation of Liver Lysosomes  36  Enzyme Assays  37  2.2.1.  Membrane Bound Enzymes  37  Acetylcholinesterase  37  Nitrophenylphosphatase  Adenosine Triphosphatase  Cytochrome C Oxidase  39  5'-Nucleotidase  40  (NPPase) (Na + ,K + )-ATPase  37 38  (vi)  2.2.2.  Lysosomal Enzymes  40  Acid phosphatase  40  N-Acetyl-p-glucosaminidase  41  Cathepsin D  42  2.3.  Haemolysis Experiments  43  2.4.  Enzyme Treatments of Intact Red Blood Cells  44  2.4.1.  Trypsinization  44  2.4.2.  Neuraminidase Treatment  45  2.4.3.  Phospholipase A 2 Treatment  45  2.4.4.  Removal of Cholesterol from Intact Erythrocytes  46  2.5.  2.6.  2.7.  2.8. 2.9.  Compositional Assays  48  2.5.1.  Protein  48  2.5.2.  S i a l i c Acid (N-Acetylneuraminic Acid)  48  2.5.3.  Cholesterol Analysis  50  2.5.4.  Phospholipid Analysis  50  2.5.5.  Ketodeoxyoctanoic Ac.id  51  Thin Layer Chromatography  52  2.6.1.  Intact Erythrocytes  52  2.6.2.  Membranes and Endotoxins  54  2.6.3.  Separation of Phospholipids '  54  Detoxification of Endotoxin  55  2.7.1.  Sodium Hydroxide Treatment  55  2.7.2.  Sodium Periodate Treatment  55  2.7.3.  Treatment with Hydroxylamine  56  Radioactive Labelling of Endotoxin 5l  Cr-Endotoxin Binding Studies  56 57  2.9.1.  Membrane Preparations  57  2.9.2.  Intact Erythrocytes  58  (vii)  2.10.  Liquid S c i n t i l l a t i o n Counting  58  2.11.  Preparation  59  2.12.  of Samples for S c i n t i l l a t i o n Counting  2.11.1. Plasma Membranes  59  2.11.2. Intact Red Blood Cells  59  2.11.3. Tissues  59  2.11.4. Plasma  60  Animal Studies  60  CHAPTER 3.  RESULTS  3.1.  Some Physiological  3.2.  Binding Studies with ^Cr-Endotoxin  3.3.  Effects of Endotoxin on Red Blood Cell  3.4. 3.5.  Effects of Endotoxin Administration  61 74  Membranes  82  3.3.1.  Erythrocyte Ghosts  82  3.3.2.  Intact Red Blood Cells  84  Cr-Endotoxin Binding In Vivo  99  Study of Drugs as Possible Endotoxin Antagonists  106  3.5.1.  Antagonists to Endotoxin Binding In Vitro  106  3.5.2.  Effectiveness of Endotoxin Antagonists In Vivo  107  3.6.  Effect  3.7.  V a r i a b i l i t y in Commercially Available Endotoxin Preparations  CHAPTER 4.  of Endotoxin and Gentamycin on Endotoxin Toxicity In Vivo 119 122  DISCUSSION AND CONCLUSIONS  4.1.  Gram-negative  4.2.  Host Defense Mechanisms in Bacteraemia and Endotoxaemia  126  4.3.  Pathophysiology  131  4.4.  Therapy of Gram-negative  REFERENCES  Septicaemia: A Formidable Medical Problem  of Endotoxaemia Bacteraemia  124  151  162  (vi i i) LIST OF FIGURES  Fi gure  Page  1  E. c o l i C e l l Envelope  5  2  Structure of E. c o l i Lipopolysaccharide  14  3  Structure of Lipid A  15  4  Effect  of Endotoxin and Haemorrhage on Blood Pressure and  Nutritive Flow in the Rat 5  Effect  62  of Native and Detoxified E. c o l i Endotoxins on Plasma  Acid Phosphatase A c t i v i t y in the Rat 6  Effect  65  of E. c o l i Endotoxin on Plasma Lysosomal  Enzyme A c t i v i t y  in Guinea Pigs 7  Effect  67  of Varying Doses of E. c o l i Endotoxin on Plasma Lysosomal  Enzyme A c t i v i t y in Guinea Pigs 8  69  Comparison of Plasma Cathepsin D A c t i v i t i e s  in Patients  C r i t i c a l l y 111 with Sepsis and/or Shock 9 10  Chromatography of Displacement  51  73  Cr-E. c o l i Endotoxin 51  of Bound  77  C r - E . c o l i Endotoxin from Human  Erythrocyte Membranes by Unlabelled Endotoxin 11  Comparison of ^ C r - E . Erythrocytes  c o l i Endotoxin Binding to Human  and Erythrocyte Membranes +  12  Effect  of E. c o l i  78  79 ++  Endotoxin on K - and Mg  -p-Nitrophenyl-  phosphatase A c t i v i t y in Human Erythrocyte:Membranes  83  13  Antihaemolytic Effect of E. c o l i Endotoxin on Human Red Blood C e l l s  85  14  Effect of E. c o l i Endotoxin on P r e - l y t i c Leakage of K + in Human Erythrocytes  86  (Tx)  Figure  15  Effects  Page  of Increasing Concentrations of E. co1i Endotoxin on the  Osmotic S t a b i l i t y of Rat and Human Red C e l l s 16  Temperature-Dependent Effects  87  of Endotoxin on the Osmotic  S t a b i l i t y of Erythrocytes 17  89  Comparison of the Temperature-Dependent Effects  of Endotoxin on  the Osmotic S t a b i l i t y of Erythrocytes from Various Animal Species 18  Temperature-Dependent Effects  of Endotoxin (4 mg/10^ red c e l l s )  on Modified Human Erythrocytes 19  Effects  93  95  of Detoxified Endotoxins on the Osmotic S t a b i l i t y of  Human Erythrocytes as a Function of Temperature  97  20  Fractionation of Sodium Hydroxide-Detoxified Endotoxin  98  21  Fractionation of Hydroxylamine-Detoxified Endotoxin  22  Correlation Between Accumulation of  100  51 Cr-Endotoxin in Various  Guinea Pig Organs with T o x i c i t y 23  102  Accumulation of ^ C r - L a b e l l e d Native and Detoxified Endotoxins in Guinea Pig Lung Tissue  104  51 24  Clearance of  Cr-Labelled Native and Detoxified Endotoxins  from Guinea Pig Plasma  105 51  25  Displacement of Bound  C r - E . c o l i Endotoxin from Human Eryth-  rocyte Membranes with Lidocaine 26  27  Effect of Methyl prednisolone, Pranolium and Propranolol on E. c o l i Endotoxin Binding to Membranes 51 Double Reciprocal Plot of  108 51 Cr109  Cr-E. c o l i Endotoxin Binding to  Erythrocyte Membranes in the Presence of Propranolol  110  (x)  Figure  Page  51 28  Double Reciprocal Concentrations)  29  Effect  Plot of  / Cr-E. c o l i Endotoxin Binding (High  to Membranes in the Presence of Propranolol  111  of Various Drugs on Plasma Acid Phosphatase A c t i v i t y in  Endotoxin-Treated  Rats  113 51  30  Effect  of Drug Pretreatment on Accumulation of  Cr-Endotoxin  in Guinea Pig Lung 31  Effect  114  of Drug Treatment on Mortality Rates in Mice Injected  with Endotoxin  116  32  NaOH-Detoxified Endotoxin as an Endotoxin Antagonist  33  Periodate-Detoxified  34  Effect  in vivo  Endotoxin as an Endotoxin Antagonist  in vivo  Effect  120 of Chronic Gentamycin Treatment on Mortality in Endotoxin-  Treated Mice 36  118  of Gentamycin in Combination with Endotoxin on T o x i c i t y  in Rats 35  117  Correlation Between Extent of TNBS Incorporation Endotoxin and Toxicity in Rats  121 into E. c o l i 123  (xi) LIST OF TABLES  Table  1  Page  Plasma lysosomal enzyme a c t i v i t i e s  in patients with  septicaemia  and/or shock 2  70  Comparative effects of experimental endotoxaemia and haemorrhage on lysosomal hydrolase a c t i v i t i e s  in plasma  75  51 3  Binding characteristics  of  Cr-endotoxin in intact human  erythrocytes and erythrocyte ghosts  81 51  4  Effect of temperature on the binding of  Cr-labelled lipopolysac-  charide (serotype 026:B6, lot number 669176) by human erythrocytes 5  90  Thin layer chromatographic analysis of phospholipid profiles from normal human erythrocytes, rat erythrocytes and erythrocytes from a patient with a congenital deficiency of plasma l e c i t h i n : c h o l e s terol acyltransferase (LCAT)  92  (xii)  ACKNOWLEDGEMENTS  F i r s t of a l l , I would l i k e to extend my warmest appreciation to Dr. M. C. Sutter for suggesting and i n i t i a t i n g this challenging project his  faithful,  moral  support  throughout  the  study.  as well  Secondly,  I  as for  feel  an  unfathomable depth of gratitude to Dr. D. V. Godin for his guidance, untiring encouragement laboratory.  and  supervision  The years  when  I have been  the  project  associated with  enriching experience for me in many ways. professional  expertise  was  transferred  into  his  Dr. Godin were a most  His unique a b i l i t y of combining  as a teacher and s c i e n t i s t with an unparalelled loyal  friendship has provided me a matchless Ph.D. programme. The made  superb technical assistance of Therese Ng and Maureen Garnett have  the  totally  countless  difficulties  vanquishable.  In  addition,  support w i l l never be forgotten. me the typing expertise thesis into i t s f i n a l  encountered  Also,  their  during beloved  the  research  friendship  project  and moral  I was fortunate to have available to  of Jackie Bitz and Tracy Slocombe to help put the  form.  Maureen's generous help in this regard as w e l l ,  is greatly appreciated. In addition, I would l i k e to express my gratitude to the Medical Research Council of Canada for studentship support and to my s i s t e r Frances who over the years f a i t h f u l l y sustained me through a l l t r i b u l a t i o n s .  (xiii)  "The search for truth is in one way hard and in another easy, For i t is evident that no one can master it fully,  nor miss i t wholly,  But each adds a l i t t l e to our knowledge of nature, And from a l l the facts assembled, a certain  there arises  grandeur."  Aristotle  -  1 -  CHAPTER 1 Introduction 1.1 H i s t o r i c a l Background It  has  been recognized for  from bacteria pathologist, extract  kill  reported  substances in  from putrifying  noted that water,  contain  1856  that  that  tissues  a century that  can k i l l  were  lethal  animals.  injections  to  dogs  a  chemical  Later on in the century, an  theory  in 1888,  organism  of  of  (1).  an  aaueous  Panum further  d i s t i l l e d and redissolved in  and s t i l l  possess  putrifaction  and  Gaertner was the f i r s t  (Bacteroides  extracts  Panum, a Danish  the  ability  Since no microorganisms could have survived these  Panum postulated  to  eleven hours  certain  animals.  intravenous  these extracts which were f i l t e r e d ,  could be boiled for  poisoning  more than  enteritidis)  procedures,  septic to  disease.  attribute  which  to  he  meat  isolated.  Furthermore, Gaertner found that boiled cultures of this organism were toxic when given to guinea pigs and rabbits of  a chemical entity present  disease.  (2) which supported Panum's  in bacteria  that  was  responsible  (1,3)  to denote a poison which was part of the l i v i n g  and which was released The  distinguish  cell  was  septic  before  and his  that  endotoxins  substance of  important at  endotoxins  from  that  were released  only  time because this  "exotoxins"  which  were  bacteria cells.  upon l y s i s notation  known  the  colleagues  only upon the disintegration of the bacterial  notation by Pfeiffer  the bacterial to  for  However, i t was near the turn of the century, in 1892,  use of the term "endotoxin" was popularized by P f e i f f e r  postulate  to  of  served  be  toxic  substances that are synthesized and excreted by i n t a c t , multiplying b a c t e r i a . In view of the present tion  between endotoxins  knowledge of microbial toxins,  and exotoxins  can be made.  a better  Exotoxins  are  distincusually  - 2 -  proteins which are secreted ia.  by gram-positive  bacter-  They can be completely inactivated by heating at 6 0 - 8 0 ° C or they can be  converted to toxoids which are of  and some gram-negative  the  active  toxin.  inactive but s t i l l  Exotoxins  from different  maintain the antigenicity bacterial  species  exhibit  their own unique pharmacological and biological actions on a particular type  or  tissue  contrast that  to  are  and  these  exotoxins,  actions  endotoxins  produced exclusively  are heat r e s i s t a n t , antibodies.  can are  be  neutralized  by  antitoxins.  1 ipopolysaccharide-protein  by gram-negative  bacteria.  complexes  These  complexes  large  difference  between  endotoxins  and exotoxins  that endotoxins precipitate the same vast array of biological actions pective of their bacterial  bacterial  cells  lysis  the  of  Pfeiffer's  theory  bacterial  endotoxins  were  cells  prevailed  postulates began to  later,  located  in  the  irres-  protoplasm  the only way they could be released  appear  the presence  of  investigators  for  several  when Ecker  realized  no sign  reported  that  of  both the  was  decades.  a heat-stable toxin  young growing cultures where there was decades  is  species of origin ( 1 , 3 , 4 , 5 ) .  that  and that  could demonstrate  In  do not form toxoids and are d i f f i c u l t to neutralize with  Another  Pfeiffer's  cell  through  Doubts  in 1917  that  in the f i l t r a t e s  autolysis toxic  (6).  of  to he of  Several  and O-antigenic  properties of gram-negative bacteria could be ascribed to one macromolecular complex (1,7,8,9). tures  Since the antigenic determinants were superficial  on the bacterium,  this  implied that  endotoxins were also  components and that they were probably located on the c e l l ative bacteria. wall  of  when Carey  and  Baron,  bacteria  came from two independent  working with  Salmonella  typhosa  superficial  wall of gram-neg-  Further proof that endotoxins were constituents  gram-negative  struc-  of the c e l l  studies  and  Ribi  in 1959 et  al.,  - 3 -  working  with  properties  Salmonella  of  the  cell  enteritidis, walls  protoplasmic extracts.  of  compared  these  organisms  and  not  in  the  further substantiated Evidence  that  gram-negative surrounding  toxic  with  and  their  antigenic  corresponding  These workers found that almost a l l of the toxic and  antigenic reactions were contained in the c e l l isms  the  protoplasmic  wall fraction of these organ-  fraction  (10,11).  These  by Rudbach and co-workers in 1969 endotoxins  bacteria  but  environment  are  that  as  not  they  well,  only can  was  readily  reported  by  were  (12).  superficial  be  results  components  released  several  into  of the  investigators  (13,14) including Crutchly and co-workers (15) who used the term "free endotoxin" to describe material found free species  of  gram-negative  chemically extracted  bacteria  in aerated  and  endotoxin (16).  which  l i q u i d cultures of  possessed  Crutchley postulated  toxin" was due to a metabolic over-production of c e l l vigorous growth in an aerated that  simple heat  release  of  up to  treatment one-half  l i q u i d medium.  (80°C) the  the  of  total  wall  several  properties  that  "free  material  of  endoduring  In addition, Roberts has shown  bacilli  in sodium chloride can cause  endotoxin content  of  cells  (17)  and  Rogers demonstrated in 1971 that the release of endotoxin from E. c o l i could be  effected  by a warm water  v i a b i l i t y of the bacterial  treatment  cells  (18).  with  0.1  M Tris  These experiments  without  loss  established  endotoxin is a readily solubi1izable component of the gram-negative al c e l l  wall  suspicions  rather than a cytoplasmic component (19)  in 1917.  "endotoxin" to signify the outer  surface  of  Pfeiffer)  implies that  It  would  therefore  seem  this  toxin  bacteria  misleading  is  since  that  bacteri-  and validated Eckers'  a r e a d i l y releasable toxic material gram-negative  of  to  use  the  term  that  resides on  the term (as  denoted by  contained within  the bacterial  proto-  - 4 -  plasm and released  only upon l y s i s  of the organism.  Nonetheless, the nota-  tion has prevailed and serves mainly as a label to identify these gram-negative toxins from exotoxins which are predominantly produced by gram-positive organisms. 1.2 The Gram-negative Cell Envelope The c e l l those of  walls  gram-negative  gram-positive  tron microscope. thick  of  Cell  structureless  teichoic  acid  or  gram-negative  bacteria  cell  walls layer  bacteria  when sections  of gram-positive composed  polysaccharide envelope is  appear  or  mostly both  quite  different  from  are observed under the  elec-  bacteria of  (20).  with  On the  hand,  a multilayered structure  Essentially,  two d i s t i n c t membranes; outer  membrane  bilayer (21,22).  gram-negative  cell  that  some the  is composed of proteins  as shown schematically  envelope  is  composed of  an inner cytoplasmic or plasma membrane (CM) and an  (OM), both  appearance  the  other  (LPS), l i p i d s ,  and usually only a small amount of peptidoglycan (20) Figure 1.  mainly of a  peptidoglycan  predominantly the lipopolysaccharide somatic antigen  in  consist  of  which  demonstrate  the  usual  double-track  of membranes when observed under the electron  The cytoplasmic  and outer  membranes  are  or  microscope  separated by an area  of  approximately 100 A which is referred to as the p e r i p l a s t i c region and which consists  of  periplasmic  approximately membranes produce  are  25  about  additional  Freeze-etching  A  in  space  thickness  75  A thick  layers  studies  (PS)  (23). (24).  located  have  plus  peptidoglycan  Both Some  externally  confirmed  gram-negative c e l l envelope (26,27).  a  this  the  cytoplasmic  gram-negative to  the  layer  outer  multilayer  (PG) and  bacteria membrane  structure  of  of  outer also (25). the  -  5  -  Figure 1 . E. c o l i C e l l  Envelope.  A schematic r e p r e s e n t a t i o n i l l u s t r a t i n g the p o s s i b l e molecular a r c h i t e c t u r e o f t h e E. c o l i c e l l e n v e l o p e . A b b r e v i a t i o n s used a r e : LPS, l i p o p o l y s a c c h a r i d e ; PL, p h o s p h o l i p i d ; OM, o u t e r membrane; PG, p e p t i d o a l y c a n ; PS p e r i p l a s m s space; and CM, c y t o p l a s m i c membrane. P o l y s a c c h a r i d e c h a i n s i n o n l y some o f t h e LPS m o l e c u l e s a r e shown (taken from r e f . 2 3 ) .  - 6 -  Although  cytoplasmic  and outer  membranes  of  gram-negative  bacteria  are  similar in the sense that both of these membranes are bilayers of l i p i d and protein,  these  differences. tic  membranes  functions  functions  (24,28).  are formed by the c e l l components  are  then  the compositional  the  as  that  makeup of  membranes (28,31).  compositional  enzymes  for  electron  devoid of biosynthetic transport Indeed,  transported  major  as  enzymes for many biosynthe-  the components of  the  outwards  (24,29,30).  cytoplasmic  the  transport  and electron  outer membrane  difficulties  compositional  involved  determining  membranes have been  in the  complete  Nonetheless,  differences  The main difference  Studies  and outer  these two membranes for biochemical analysis.  apparent  well  protoplasm and by the cytoplasmic membrane and these  hindered by the technical of  functional  including  the outer membrane is  transport  major  The cytoplasmic membrane contains  and transport  whereas  have  do  exist  separation it  in  is  quite  these  two  is that 50-60% of the outer membrane  is composed of somatic antigen which is 1ipopolysaccharide in nature whereas the  cytoplasmic  Also,  as  membrane  contains  one may expect  on the  little  basis  or  of  the  no  1ipopolysaccharide  lack  of  enzymes,  the  (20). outer  membrane contains much less  protein by weight (11—15%) than does the cyto-  plasmic  (20,32).  membrane  (70-80%)  The  phospholipid  content  of  both  cell  wall  membranes is s i m i l a r . 1.3 Isolation, Composition and Structure of Endotoxin The most  extensively  has been endotoxin. fact  that  this  The interest  macromolecular  membrane of the c e l l charide)  studied  wall,  component of  the  gram-negative  given to endotoxin is complex,  which  forms  contains the major somatic  in enteric bacteria and i s ,  therefore,  largely due to part  antigen  of  the  the  outer  (1ipopolysac-  important from the viewpoint  -  of  7 -  immunochemistry and taxonomy in addition to the fact  powerfully various  toxic  agent  methods  of  (20).  In  extracting  light  this  of  this  component  that  importance  from  advances towards the greater  of  endotoxin,  gram-negative  have been devised and this has been a s i g n i f i c a n t factor tific  endotoxin is a  bacteria  in enhancing scien-  understanding of both the biology and the  chemistry of these bacterial macromolecules. One of the e a r l i e s t described  by  Boivin  trichloroacetic  acid  "glucidolipide"  as  methods  in at  1933  used  to extract  that  4 ° C (33).  the material  endotoxin was  employed Boivin  a  precipitation  called  pid  Boivin in 1946 stated that  and a nitrogenous  (1).  Since  suggested  the  that  component  nitrogenous these  the  in  addition  component  extracted  or  With further  with  material 0-antigen  analysis  of  i t consisted of a phospholito  the  polysaccharide  moiety  of  polypeptides,  Boivin  consisted  endotoxins  step  extracted  contained most of the somatic  of the intact .organism from which i t was derived. the toxic substance,  a procedure  would  be  more  appropriately  described chemically as "glucidolipido-polypeptidiques". Extraction most  popular  greater  Westphal 1965  method  than  method of  with  Palmer  of  in 1952  for  (36).  with  aqueous  endotoxin  method  and Gerlough in  by Westphal and Oann  68°C  bacilli  obtaining  any other  and co-workers  dried bacteria at  of gram-negative  (1).  1940 (35).  phenol has  because  the  The original  (34)  was  Further  According  to  yields phenol  modified  and  minutes.  are  much  extraction improved by  improvements were made in this  method of  are treated with a mixture of phenol and water  five  become the  extraction, (45/55,  v/v)  Upon cooling, the homogenous mixture separates  into an upper (water) phase and a lower (phenol) phase.  Under these circum-  stances, the endotoxin is found in the water phase as a complex with nucleic  - 8 -  acid.  The phenol is  Since  endotoxin forms  u l t r a c e n t r i f ligation nucleic  acid  removed either large  by d i a l y s i s  aggregates  (105,000 x g)  contaminants.  or  in water,  by ether  it  can be  and thereby obtained  The y i e l d  of  extraction. sedimented by  relatively  endotoxin that  is  free  obtained  of  with  this extraction procedure varies from 1 to 4% of the dry bacterial weight. Other bacteria  effective that  methods  (38) and  et  al.  (11),  the  active  (39).  material  and l i p i d  Each that  that  is  of  from  diethylene  gram-negative  glycol  extraction  aqueous ether extraction method as  employs these  rich  endotoxins  include the  the EDTA extraction  and a procedure that Leive  extracting  have been reported  procedure of Morgan (37), by Ribi  of  procedure of  aqueous butanol extraction  as  methods  Leive and co-workers reported  yields  in 1 ipopolysaccharide with  vary according to  the particular  described  by Morrison  a biologically  amounts of protein  extraction  procedure  used  (40,41). Since  the  investigators changeably.  major use  constituent  the  terms  of  endotoxin  "endotoxin"  However, in a s t r i c t  and  chemical  is  1ipopolysaccharide,  "1ipopolysaccharide"  sense,  these two terms  many inter-  are  not  synonymous in that endotoxins are extracts that contain protein and loosely bound l i p i d s complexed with the lipopolysaccharide (1,3,42,43).  The c o n t r i -  bution that the various components of endotoxin make to the t o x i c i t y of the whole complex has been the subject of numerous investigations. bound l i p i d s , which are cephalins and designated  The loosely  as " l i p i d B" by some inves-  tigators (44,45) can be easily removed from the endotoxin complex by chloroform extraction. biological inert  Since  t o x i c i t y of  (44,45).  the removal  of  these  endotoxin, they are  In addition to  lipid  B,  lipids  regarded  endotoxins  has as  no effect  on the  being b i o l o g i c a l l y  contain a firmly bound  - 9 -  lipid  moiety,  covalently complex. the  linked  "lipid  to  both  A" by Westphal  the  components  hydrolysis.  of  Boivin  endotoxin  and  and it  protein  in  1933  and found  that  would be obtained B", Tai  a  and  (reported  protein)  in  Goebel  supernatant,  proposed  that  than that reported  alkaline  was  called  but  not  endotoxins  contained  a  "toxic  by Boivin  found to  be  of endotoxins  a much shorter  and Mesrobeanu  associated  with  the  the  component  (48).  Moreover,  these workers  toxic moiety was neither protein nor polysaccharide.  "Fraction  toxic.  lipid  investigators obtain  (lipid  (TM)  These workers  toxic  minutes) moiety of  fraction  and  upon  concluded  It was the  hydrophilic  A)  as  the  toxic  entity  of  endotoxin  that  the  investigacovalent-  (46).  These  found that by hydrolyzing endotoxin with 1 N HC1 they could  a precipitate carrier  which was soluble such  combination would display (43,46).  Later,  moiety"  tion reported by Westphal and Luderitz in 1954 that implicated the bound  at  alcohol hydrolysis, they found the toxic moiety associated with the  polysaccharide  ly  investi-  time period (35  protein  the  "Fraction A"  (48).  (4 hours)  acid  N acetic acid  antigenic  found that upon acid hydrolysis for  endotoxin  which they  These  was  which could account for the toxic effects  is the  were  and which maintained some remnants of t o x i c i t y .  which was the clear and  precipitate  of  by  42,47)  of acid hydrolysis on endotoxins.  lipid  which  entities  gators treated endotoxin from Serratia marcescens with 0.2 100°C  (46)  can only be separated from  (polysaccharide  Mesrobeanu  to study the effects  and co-workers  polysaccharide  Since l i p i d A is covalently bound,  other  first  denoted  as  protein  in chloroform and when coupled to a or  up to one-fifth  The polysaccharide-containing  zates were found to be non-toxic.  low molecular of the toxic supernatants  weight  dextran,  potency of of  the  the  endotoxin  acid  hydroly-  A study by Wober and Alaupovic  reported  -10-  in 1971 has also supported the proposal that endotoxin  complex  (49).  component of endotoxins mutants deficient  Other is  evidence  the toxic  l i p i d A is the toxophore of the  that  suggests  e n t i t y are  composed of  the  the studies with  in the O-antigenic polysaccharide  these mutants are essentially  that  chain.  lipid A bacterial  Endotoxins from  l i p i d A only and t h e i r biological  potency has been shown to be similar to polysaccharide-containing endotoxins prepared from non-mutant strains  (50,51,52).  have disputed the evidence that l i p i d A is drawing attention to the fact  that  than the endotoxins from which associates  have  demonstrated  l i p i d A preparations  that (0.1  investigators  the toxic moiety of endotoxin by  they were derived  potency by mild acid hydrolysis  endotoxins  lose  acid) (43).  through the use of different  are much less  (43,53).  can  N acetic  amount of covalently bound l i p i d was released able to demonstrate  However, other  Also, their  before  any  toxic and  Ribi  biological measurable  Furthermore, they were  extraction  procedures  that  endotoxins containing as l i t t l e as 2% l i p i d A were as b i o l o g i c a l l y active those containing as much as 30% l i p i d A (54). that structural  of b i o l o g i c a l  toxins than any particular molecular component protein  investigators  and associates  configuration and/or physical size of the  complex may be more important determinants  The  Ribi  component of  since  Westphal  endotoxins  has  and Luderitz  as  proposed  lipopolysaccharide t o x i c i t y of endo-  (43). received  little  implicated l i p i d  attention  A as  the  by  toxo-  phore in endotoxin in 1954 which stimulated research  on the l i p i d moiety of  endotoxins.  reported  find  the  Previous  protein  investigations  to  moiety  1954,  one study,  necessary  for  that  was  endotoxic  potency  reported in the mid-1950's were in consensus  in 1942,  (55).  did  However,  that the protein  moiety plays a minor i f not a benign role in the b i o l o g i c a l t o x i c i t y of the  - 11 -  endotoxin complex (56,57,58) and as  a result,  complex has  However, more recent  the  largely  been  ignored.  protein moiety of ^endotoxins  earlier  conclusion  inert.  It  ently  linked  endotoxins  that  the  have raised  protein  moiety  presence  to  the  lipid  (49,59,60,61).  thousand (62). biological  A portion  of  in the endotoxin investigations  some questions of  endotoxins  is now known that the protein moiety is  molecular weight of this  rigorous  its  the  regarding  is  a peptide  that  1ipopolysaccharide  peptide  is  is  alter  approximately  Therefore,  of  procedures  endotoxins  such  as  is  acid  difficult  hydrolysis  to  are  1 ipopolysaccharide  comparisons  moiety of  assess  because  required  of biological  the complex  to  the  twelve  extracted  as  fairly  remove  well  this  are known (42,47,49).  t o x i c i t y between protein-free  ins and normal endotoxins are d i f f i c u l t to make. moiety is  of  The exact contribution that this protein moiety makes to the  effects  the  coval-  complex  protein from the endotoxin complex and these extraction procedures to  the  biologically,  Morrison and associates have determined that l i p i d A-associated  on  endotox-  However, when the protein  from the endotoxin complex either  by phenol  extraction  or acid hydrolysis, i t is collected not as pure protein, but protein conjugated to l i p i d A and this conjugated biological  toxicity  (49).  Freedman  protein component of endotoxins to the b i o l o g i c a l i t y reaction A-associated  (63).  effects  can act  it  has  co-workers  as  have  that  the  an immunogen and can contribute  by causing been  a delayed  demonstrated  the spleen  hypersensitiv-  that  this  lipid  B lymphocytes from the C3H/HeJ mouse,  are normally unresponsive to the mitogenic effects  ide or l i p i d A (59,62).  shown  a potent mitogen and can e l i c i t mitogenic responses  from lymphocytes, such as that  and  of endotoxins  In addition,  protein is  protein has been shown to possess some  of  1ipopolysacchar-  - 12 -  It  has  been known for  many years  that  endotoxin possesses antigenic properties the  component of  and since the peripheral portion of units  that display a wide spectrum of v a r i a b i l i t y within a single bacterial  genus,  structural  component  polysaccharide  repeating  this  polysaccharide  the  consists  v a r i a b i l i t y has  been  of  used,  serological typing of Enterobacteriaceae gen" was somatic  first  introduced by Weil  antigen  from f l a g e l l a r  oligosaccharide  as  strains  and Felix  antigens  "O-antigens", (33,64).  in 1918  the  fine  The term "0-antito  in f l a g e l l a t e d  in  distinguish  strains  of  this  Proteus.  Weil and Felix described the non-flagellated form of Proteus as the "0 form" (Ohne Hauch) and the f l a g e l l a t e d 65).  strain  as  the  "H form"  and somatic or body antigens  e l l a t e d strains  only possessed O-antigens.  proteins whose antigenic effect to  0- and  Escherichia,  H-antigens,  In contrast  possess capsular  enterobacterial  or envelope antigens  derived from the German word "Kapsel")  (33,65).  gens are  subdivided  acid  B-antigens  and  Escherichi a K-antigens  polysaccharides  that  the  and are  thermostable  contain  A-antigen  K-antigens  the H-antigens  are  strains,  are  In addi-  particularly K-antigens  (K  Biochemically, the K-antiinto  (65).  more  (65).  denoted as  In  toxic  thermolabile general,  than  L- and  strains  strains  of  without  (66).  Since v i r t u a l l y a l l of the biological a c t i v i t i e s also  flagellar  to O-antigens, which  can be destroyed by b o i l i n g some  (see  (O-antigens) whereas the non-flag-  are carbohydrate compounds and therefore heat r e s i s t a n t ,  can  Hauch)  Therefore, the H form of Proteus was found to possess both  (H-antigens)  tion  (mit  be  elicited  with  1ipopolysaccharide,  component of endotoxin, studies directed towards organization of  endotoxin have focused  attributed to endotoxin  which  is  the  elucidating the  on chemically pure  predominant structural  1ipopolysacchar-  - 13 -  ides  instead.  Basically,  one can regard  molecules with the hydrophilic  1 ipopolysaccharides  portion being polysaccharide  phobic portion comprised of l i p i d A (64). be  considered  to  consist  amphipathic  and the  hydro-  The hydrophilic portion can also the  lipid A  region which consists of oligosaccharide repeating units (0-antigen  polysac-  charide)  and a portion  referred  to  as  of  as  two parts;  proximal  the  "core" polysaccharide  repeating units usually consist (3,64).  to  (see  A region  Figure  2).  In the example given in Figure 2 of E. c o l i  amine (39,67). two,  glucose,  The number of oligosaccharide  in "semi-rough" bacterial  "smooth" strains  (39,68,69).  gen can be used  for  strain  lipid  distal  possesses  an  mutants,  to  which  The  of three or four different  repeating unit consists of galactose,  as  a portion  hexose units each  0111:B4,  the 0-antigen  colitose  and N-acetylglucos-  repeating  units can be as few  or as  many as  ten  in native  structure cea  0-antigen  typing of of  unique  bacterial  strains  composition.  within  a bacterial  genus  are only seen when comparing different  (70).  The core polysaccharide  In  because each contrast,  contains  genuses of  in core  Enterobacteria-  a trisaccharide  phosphorylethanolamine and several  the  and composi-  and variations  unique eight carbon sugar acid, 2-keto-3-deoxy-octulosonic seven carbon heptose,  or  As has been previously mentioned, the 0-anti-  serological  the many strains  usually  oligosaccharide  "core" polysaccharide displays much more constancy in structure tion for  is  composed of a  acid as well  hexoses  (see  as a  Figure  2). The hydrophobic region of structurally D-glucosamine  is  an  unusual  disaccharide  1ipopolysaccharide consists glycolipid units  to  that which  consists long  ( C 1 n - C i o ) are linked via both ester and amide linkages,  of of  chain  lipid  A which  e-l,6-linked fatty  acids  as shown diagram-  - 14 -  P I P  I  EtNH i P  Fa  I  I  Fa - G l c (NH ) - KDO - KDO - hept - hept - glc - gal - glc - /gal - glc - glc NAc Fa  2  I  I  Glc (NHL)  KDO  I  I  I  Fa  Fa  P  hept  I  glc N A c  I  col  I  col  EtNH  J V. lipid A  Core Polysaccharide  J n O-Antigen Polysaccharide  Figure 2. Structure of E. c o l i  Lipopolysaccharide.  Chemical structure of E. c o l i 0111:B4 lipopolysaccharide (LPS) showing the three regions of the LPS molecule. Abbreviations used are: g a l , galactose; g l c , glucose; glc NAc, N-acetylglucosamine; c o l , c o l i t o s e ; hept, heptose; EtNH, ethanolamine; KDO, 2-keto-3-deoxy-octulosonic acid; Fa, fatty acid (taken from ref. 3).  - 15 -  Figure 3. Structure of Lipid A. Structure of l i p i d A component of Salmonella lipopolysaccharides. Two l i p i d A units, each consisting of two glucosamine molecules that contain ester and amide bonded fatty acids, are shown to be linked together with a pyrophosphate bridge. The 2-keto-3-deoxy-octulosonic acid trisaccharide represents the point of attachment of the "core" polysaccharide (taken from E. T. Rietschel et a l . , in Microbiology - 1975, D. Schlessinger (ed.), p. 307). 1  - 16 -  matically in Figure 3. (64),  which  probably  A l l the fatty accounts  for  portions of 1 ipopolysaccharides  the  unsubstituted the  as  well  and,  acids  in Enterobacteriacea,  myristic acid (70) lipid  being  is  that  3-hydroxy acids  appear  saturated fatty  to  this fatty  A general  55-75% (70).  of  It  is  total  of  hydrophobic  are even numbered  can  occur  as  In comparison,  3-hydroxy  as  in most  fatty  interest  substituted  bacterial  acids  to note  a specific  linked that  feature  lipopolysac-  to  the  marker for  lipid  types  Therefore,  a  glycosidically fatty  acids  molecules  supply of f a t t y acids  lipopolysaccharide  linked  glucosamine  monomer  molecules  is  also k e t o s i d i c a l l y  charide which,  along with  linked  other  sugars  sents the "core" polysaccharide region. cally  linked to an oligosaccharide  to  fatty bacter-  bacteria  (70,74).  essentially to which  are bonded via ester and amide groups.  A are  of  of a p a r t i c u l a r  species and seem independent of the conditions, under which the  were cultured or of the external  either  acid invariably appears as 3-hydroxy-  observation  the  saturated  The ester-bound  (70,72).  be uniformly  acids bound to l i p i d A are a characteristic ial  acids  the  (71).  and  and consequently has been used  A (37,70,73).  charides  as  A are  that  from Enterobacteriacea  or 3-hydroxy substituted  amide-linked fatty  observation  display low f l u i d i t y  fatty acids of l i p i d A preparations and straight-chained  acid chains in l i p i d  consists  long-chain, One of the  of  saturated  glucosamine  a 2-keto-3-deoxyoctonate  trisac-  and phosphorylethanolamine,  repre-  This region, in turn,  repeating unit that  is g l y c o s i d i -  normally consists of  four hexoses and forms the 0-antigen portion of the lipopolysaccharide. molecular weight of the order lipid  of  A unit,  14,000,  two  The  a lipopolysaccharide monomer would t h e o r e t i c a l l y be in that  2,000 for  is,  if  one estimates  the core polysaccharide  approximately 2,000 for and another  a  10,000 for an  - 17 -  average  O-antigen  weight  of  each  known  that  weight  of  unit would be  under  normal  The  is  factors  predominantly hydrophobic forces the  lipid  A moiety  nonionic) acids,  and  (70)  alkali  since  treatment,  can cause disaggregation  in  pentasaccharide 1,000)  conditions, the  order  responsible exerted agents  for  (70). the  apparent  one  to  such  which  detergents  splits  off  and consequently  the  (both  were responsible  for  (78).  However, even of  association  various  1 ipopolysaccharide,  means the  weight is  approximately two to three times  two or three  unit  ionic  and  appar-  (70).  as  hydrophobic interac-  1 ipopolysaccharide  molecules  used  disperse  to  completely  weights  Since this  14,000),  of  the  smallest  observed molecular  the calculated  (approximately  disaggregation  molecular weight  this  suggests  that  together  to  Ultracentrifugation studies have supported the theory that  The nature  and position  includes phosphodiester adjacent  in  a reduction in the  lipopolysaccharides exist in polymeric or, more s p e c i f i c a l l y , (79,80).  acids  fatty  1ipopolysaccharide monomers are covalently linked  form a polymer.  be  ester-linked  well  molecular  range from 25,000 to 40,000  1ipopolysaccharide  of are  particles  one  to  In addition, Olins and Warner have demon-  tions  aggregates  is  million  seem  long-chain fatty  as  calcium ion binding as  after  it  molecular  twenty  that EDTA treatment can also cause 1 ipopolysaccharide  the  (molecular  However,  aggregation  by the  and they proposed that  of  of  units  lipopolysaccharides must aggregate to form  ent molecular weight (75,76,77). strated  ten  approximately  It follows then that  particles.  of  physiological  1ipopolysaccharides  (33,47,70). large  oligosaccharide  of  crosslinking that  has  trimeric units been  proposed  bonding between heptoses of core polysaccharides  1 ipopolysaccharide  chains  (81)  and  bridge diglucosamine units of l i p i d A by a l ' , 4  pyrophosphodiester  bonds  in that  linkage as shown in Figure 3  - 18 -  (72).  However, both  monomers have  been  of  these  proposed  challenged  associations  by Muhlradt and  of  1ipopolysaccharide  co-workers  (82,83).  Using  co-workers  could  31 P-nuclear  magnetic  resonance  techniques,  find no evidence of the existence diester  bic  of covalent  crosslinks  involving phospho-  and/or pyrophosphodiester bonds in either the core polysaccharide or  the l i p i d A moiety. gation  Muhlradt and  Therefore, these investigators  proposed that the aggre-  of lipopolysaccharide molecules must result  from ionic and hydropho-  interactions  (83).  The p o s s i b i l i t y  be linked by d i s u l f i d e bridges ides do not contain sulfur Although  is ruled out by the fact  sections  the  units could  lipopolysacchar-  dealt  with  the  molecular  structure  of  no mention was made, either in the text or in Figure 3,  of how the l i p i d A-associated endotoxin.  lipopolysaccharide  (12).  the foregoing  lipopolysaccharides,  that  protein f i t t e d  into the molecular stucture  of  This is mainly because i t is not known how this protein is bound  to l i p i d A other than i t seems to be a covalent bond (49).  Other compounds  known to  such  cine, Mg++  be  associated with  cadaverine, and C a + + ;  and  endotoxins  spermidine, lipid  and  include polyamines  spermine;  B (44,70).  cations  However,  since  such these  as  as  putres-  Na + ,  K ,  compounds  can  be easily removed from the lipopolysaccharide complex by techniques  such as  ion  are  exchange  chromatography  (84)  or by e l e c t r o d i a l y s i s  (85),  they  not  considered to be integral components of the lipopolysaccharide u n i t . 1.4 Biological A c t i v i t i e s of Endotoxins Although administered  it  is  known that  endotoxin  the  physiological  vary markedly  in  different  responses animal  to  parenterally  species  (86,87),  there is one common response in a l l mammalian species to a dose of endotoxin of s u f f i c i e n t magnitude and that is death (1).  As one example to  illustrate  - 19 -  the differences  in s e n s i t i v i t y to endotoxin, a dose (on a mg/kg basis)  is lethal to mice is three orders of magnitude greater toxin that would be lethal to rabbits of  this  variable  certain  response  generalizations  to  can be made which  in  are  stand the sequelae of endotoxaemia in humans. biological  effects  to humans w i l l  seen in experimental  be mentioned here.  than a dose of endo-  of comparable maturity (1).  endotoxaemia  different useful  that  In spite  animal  species,  in helping to  With this  under-  in mind, only those  endotoxaemia that may be  In the main, these effects  that are responsible for the pyrexia and hypotension that  is  applicable  comprise those seen,  and that  can proceed to shock and death, in humans a f f l i c t e d with gram-negative  endo-  toxaemia. One  of  attributed  the to  earliest  biological  endotoxins  was  responses  pyrogenicity,  (besides  when  in  death)  1875  that  was  Burdon-Sanderson  demonstrated that he could i s o l a t e a fever-inducing substance from decomposing  meat  (see  that rabbits genic  1).  In the animal kingdom, Greisman and Hornick  have approximately the same degree of s e n s i t i v i t y to the pyro-  properties  of endotoxins  of  fever  of  i t ) on the brain (89)  action  endotoxin  (90,91,92). cells  of  (93).  Cells  the  of  liver,  do human volunteers  The f i r s t  effect  of  (88).  The mechanism  endotoxin  (or  or, as most of the evidence indicates,  on  blood  leukocytes  to  the reticuloendothelial when  The secretion  process.  as  production may involve a direct  of  of  incubated leukocyte  with  form  system,  endotoxin  pyrogen  seems  (94,95,96).  such also  to  as  an indirect  stage of the process can be  pyrogens  the  produce  involve  stage following endotoxin activation  The f i r s t  a portion  endogenous  Kupffer pyrogens  a  two-stage  includes  synthesis  of the pyrogen de novo and the second stage involves secretion gen  have shown  of the pyro-  inhibited  with RNA  20  -  synthesis  inhibitors  (97)  while the  -  secretion  agents that bind to sulfhydryl groups (96). in  "activates"  Dinarello has  leukocytes  to  synthesize  Evidence  that  some tumor endogenous (99).  may support  cell  lines  this  are  pyrogens  pyrogen which  really  known but  process may involve a  de-repres-  may be  to  to  in a rabbit  (97)  biological  not  these  controversial trigger  endogenous and  some  the release of  pyrogens  can  investigators another  an  is  the  (98).  fact  constantly  that  produce  unrepressed  genome  ranging from 13,000-16,000 a c t i v i t y (1 mg is  equivalent  and t h e i r principal s i t e of action  the preoptic area of the hypothalamus (103). or  and  indicate  Leukocyte pyrogens have molecular weights  to 33,000°C fever  not  hypothesis  spontaneously  argued  (100,101,102), possess high s p e c i f i c  is  in some as yet obscure manner  de-repression  known  can be inhibited by  The mechanism by which endotox-  proposed that the activation  sion of the genome for pyrogen synthesis  process  However, the question whether  cross  the  propose  that  endogenous  is in  blood-brain leukocyte  pyrogen such as  barrier pyrogens  is may  prostaglandin E^  which can penetrate the blood-brain barrier (104). A dramatic demonstration of the biological t o x i c i t y of endotoxins is "local local  tissue r e a c t i v i t y phenomenon" o r , "Shwartzman" reaction.  as  it  is  more commonly known,  This dermal reaction was described  in the  skin  culture  filtrates  (105).  The f i r s t  tion  was  given  hours after  of of  rabbits  that  Salmonella  received  of  injection was given intradermally while the second  injec-  the intravenous  hours  twenty-four  cell-free apart  twenty-four  spaced  injections  necrotic  hours  intravenously,  typhosa  two  the  in 1928 by  Shwartzman when he noted the development of a severe haemorrhagic, lesion  the  later.  Within  two to  i n j e c t i o n , Shwartzman noted this necrotic  in the s i t e of the skin i n j e c t i o n .  The dermal  Shwartzman reaction is  six  lesion auite  - 21 -  non-specific  in  that  the  endotoxin from a genus  "provoking"  of bacterium that  used in the dermal injection (1). cal  "generalized  reaction,  cortical  glomerular  precipitate  these  lesions,  twenty-four  hours  apart  which  two  must  sus  that  there  are  given  with a variety of (108),  post-renal  the  the  conditions  generalized  the  cortical  thrombocytopenic  precipitating  necrosis  purpura  (111).  cause  lished to be due to endotoxins. exemplify  potent  effects  cardiovascular  system  and since  gram-negative  septicaemias,  in humans has  that  humoral  endotoxin  To  spaced  lesions  There is that  that  are  is  system,  mimic  this  often  is of  sepsis  toxic These  associated pregnancy (110)  these pathologies  and  that  are  in endotoxin-treated  not been  definitely  have  on  estab-  reactions  the  mammalian  a common manifestation  in human  aroused  can  after  some consen-  complications  an enormous amount of  of endotoxins with  systems, especially the cardiovascular Within the cardiovascular  (89,106).  the Shwartzman-type  endotoxins  shock  this has  into elucidating the interactions  renal  These  (109),  Although  Nonetheless,  the  by b i l a t e r a l  of  seen c l i n i c a l l y resemble the Shwartzman phenomenon seen animals,  the  Shwartzman r e a c t i o n .  and non-infectious  homotransplantation  as  rabbit  rabbit.  be  classi-  intravascular c l o t t i n g that occurs  in the  infectious  thrombotic  in  injections  to  clinical  include renal  can  from that  known  characterized  injection of endotoxin (107).  response to endotoxin seen human correlates  is  sometimes  intravenous  be  certain  injection  completely different  thrombosis  largely the result of the extensive the second intravenous  is  reaction",  "Sanarelli-Shwartzman" and  intravenous  Another related phenomenon is the  Shwartzman  necrosis  or  research  the various mammalian  system. endotoxins can interact with both the  and c e l l u l a r components of the blood.  The major humoral components  - 22 -  that  interact  with  systems.  The  classical  pathway  endotoxins  complement  the  alternate  system  which  complex to i n i t i a t e  include can  requires  the be  the  complement  activated formation  activation of C l , the f i r s t  pathway which  involves  the  and  via  the  two  of  an  coagulation  pathways;  the  antigen-antibody  component of complement and  interaction  of  an antigen  with  a  serum protein called properdin which in turn activates the terminal components of the complement system (C3, C5-C9) without the requirement for fic  antibody (112).  the  complement  Endotoxins have been shown to be capable  system  via  both  the  classical  and  activated,  (3,89,112,113).  When the complement  system  of  are  as  inflammation  generated  such  is  the  C3a  the  and  possess anaphylatoxin a c t i v i t y and enhance vascular  of  potent  promoting factors as  a result  (118).  (113,116,117).  The relevance  of complement activation  in humans has significant  include leukotactic peptides,  become increasingly  decrease in levels  Also,  gram-negative  a study reported sepsis  has  by Fearon  of  gram-negative  with  gram-negative  and associates  confirmed McCabe's e a r l i e r  gram-negative  finding strongly  suggests  the  presence  on patients  findings  of  formed sepsis  demonstrated a  and  in  sepsis with addi-  or properdin pathway  as w e l l , which can also lead to consumption of complement components latter  as  Other media-  these mediators,  t i o n , provided evidence for activation of the alternate  This  which  l y t i c - and phagocytosis-  since McCabe has  of C3 in patients  mediators  permeability as well  by endotoxin, to  apparent  pathways  fragments  smooth muscle contraction through histamine release (114,115). tors that are released  activating  alternate  C5a  speci-  endotoxins  (119).  in human  septicaemia.  One of the major  of  gram-negative  septicaemia  the frequent occurrence of disseminated  intravascular  coagulation or DIC, as  it  complications  is more commonly presented  in abbreviated  form (120).  This  in man  syndrome  is  is  - 23 -  characterized by a haemorrhagic diathesis bosis and culminates quence of  in c i r c u l a t o r y collapse.  an acute activation  deposition of f i b r i n  of  the  deposition  coagulation  (124).  system that  and  the  excessive  creating  a  fibrinolysis  condition  of  a conse-  promotes  beds causing  digests  the  ischaemia and  In conjunction with this  the f i b r i n o l y t i c system becomes activated  procoagulants Therefore,  The DIC syndrome is  thrombi within the vascular  necrosis of v i t a l tissues and organs ing process,  associated with generalized throm-  clott-  as a result of f i b r i n  fibrin,  fibrinogen  hypocoaguabi1ity  and  (121,125,126).  i t may be more appropriate to label this syndrome as "consumption  coagulopathy"  (121).  The f i r s t  indications  that  endotoxins  affected  the  coagulation system were, as previously mentioned, when Sanarelli reported in 1924  and when Shwartzman reported  properly spaced into r a b b i t s .  in 1928  the pathological  injections of culture f i l t r a t e s Although these investigators  from gram-negative  did not know at  the reaction which they described was the result with  the coagulation  investigators  (1,3).  system, It  it  was  after  that  of endotoxins  l a t e r ' shown to  is now known that  effects  be  the  organisms time that  interacting  case  by other  an infusion, or even  a single intravenous injection of endotoxin, coagulative changes are ted which result  in deposition of f i b r i n  the  and spleen  lungs,  toxins  liver  activate  the  in a variety of tissues  (3,127,128,129).  clotting  system  has  of two  after  initia-  including  The mechanism by which endo-  been  studied  extensively.  The  evidence to date indicates that endotoxins can i n i t i a t e coagulation via both intrinsic the  and extrinsic  activation  of  Rodriguez-Erdmann  pathways  factor  XII  whereby he  whole blood c l o t t i n g  time  or  (89).  The i n t r i n s i c pathway necessitates  Hageman factor  noted  that  and  endotoxin  in s i l i c o n i z e d glass  a  study  markedly  tubes  as  reported  shortened  well  as  that  by the of  - 24 -  platelet-poor  plasma implied that endotoxins could enhance c l o t t i n g via the  intrinsic  pathway  suggested  that  (122).  Hageman factor  (123) but i t wasn't until definitive  Experiments  proof was  could  was  the  activate  lipid  provided that  Hageman factor factor  A region  Hageman factor,  has  to  the  fibinolytic  the  and  activated  Melmon by  of  endotoxins  could d i r e c t l y  Furthermore, the investigators the  endotoxin  endotoxin complex  to  complex  activate  can i n i t i a t e reactions  endotoxins  that  was  activate  the  showed that required  the  (130).  Since  plasminogen  activated  proactivator  to for  Hageman of  it  the  provides the means by which endotoxins  in these important humoral systems that affect homeo-  of the coagulation  system by the  extrinsic  pathway  requires  the release of tissue thromboplastin or a c e l l - d e r i v e d procoagulant. has demonstrated a direct effect the e x t r i n s i c  1968 that factor  that  (131,132).  Activation  of  also  p r e k a l l i k r e i n of the kinin-forming system and factor XI  of the i n t r i n s i c c l o t t i n g system,  stasis  directly  Nies  presumably by providing the s i t e of attachment  ability  system,  be  by  Morrison and Cochrane reported t h e i r findings  i n t r i n s i c c l o t t i n g system (130). it  reported  rabbits  of endotoxin on any of the known proteins  coagulation pathway but Lerner and associates treated  No one  with endotoxin had s i g n i f i c a n t l y  reported in  lower levels  VII which suggested that the e x t r i n s i c coagulation pathway was  vated (133).  of  acti-  A more recent study by Garner and Evensen on dogs has corrob-  orated Lerner's e a r l i e r findings in rabbits that plasma levels of factor VII decrease after  endotoxin treatment  were able to establish  (134).  In addition, Garner and Evensen  that factor VII deficient dogs treated with endotoxin  had less thrombi and f i b r i n with endotoxin, which again  deposits in tissues than did normal dogs treated indicated strongly that  the e x t r i n s i c  coagula-  - 25 -  tion  pathway  endotoxin blood  is  activated  activates  cellular  leukocytes  in endotoxaemia  the extrinsic  components  and  as  platelets  (134).  coagulation  several  perform  The mechanism by which  pathway seems to  investigators  an  essential  have  role  in  involve the  shown  that  blood  endotoxin-induced  coagulative changes  in vivo (135,136,137,138).  These studies suggested that  endotoxins  with  components  results  interact  these blood c e l l u l a r  in the release of tissue factors  activate  the  clotting  system.  In  in a manner that  and p l a t e l e t factor 3 which in turn  vitro  investigations  have  essentially  confirmed these conclusions and have provided more insight into the types of cells  involved.  Thus, while i t is known that endotoxin can induce  platelets  to aggregate (139), to release serotonin, histamine and p l a t e l e t factor 3 in vi tro (136,140), these platelet mainly function the c e l l s  as  factors  accelerators  of  the  coagulation  substances and  process  (141).  that are p r i n c i p a l l y responsible for releasing potent  substances  that  activate  the e x t r i n s i c  of endotoxin are blood leukocytes separate research responsible Rivers  are weak procoagulant  and  procoagulant  in the  presence  (135,136,141) and, more s p e c i f i c a l l y ,  procoagulant  co-workers  (142),  a c t i v i t y (142,143).  concluded  the granulocyte was  explained by the presence  of  less  the  that  the  source  of  One of these  groups,  experimental  evidence  tissue factors  could be  than one percent monocyte contamination.  In concordance with this conclusion, Hi 11er and associates demonstrated more  endotoxin-induced  suspension  of  granulocytes/yl factor  two  groups have shown quite conclusively that the monocyte was  for this  suggesting that  coagulation pathway  Hence,  ten  monocytes/yl  (143).  originated  tissue  factor than  activity from  a  could  be  preparation  derived of  ten  that  from  a  thousand  Hi 11er and co-workers also suggested that the tissue  from the  lysosomal  fraction  of  the  monocytes.  Another  - 26 -  cell  type  that  could  play  coagulation  system  independent  investigations  thelial  cell  during  some  part  in  endotoxaemia  the  is  activation  the  of  the  endothelial  extrinsic  cell.  have reported the occurrence of  Several  extensive  endo-  damage in arteries obtained from animals treated with endotoxin  (144,145,146).  Therefore, these observations raise the p o s s i b i l i t y that the  e x t r i n s i c c l o t t i n g system could be activated either by tissue thromboplastin leaking  from  damaged  endothelial  cells  or  by  platelets  adhering  to  the  exposed underlying basement membrane and consequently activating the coagu1 at ion system (136). Therefore,  by v i r t u e  of  the  diverse  various components of blood that result cological  agents and in the  actions  endotoxins  activation of  (see  on the  in the l i b e r a t i o n of potent pharmathe  c l o t t i n g system,  more understandable how these gram-negative toxins c i r c u l a t o r y shock in animals  exert  are  capable  it of  becomes inducing  schematic representation of the pathophy-  siology of gram-negative shock in reference 147). 1.5 C l i n i c a l Since  Significance of Endotoxins  all  Enterobacteriaceae  endotoxin (28),  and most  gram-negative  bacteria  any bacteraemia due to gram-negative organisms that  produce is  seen  c l i n i c a l l y could p o t e n t i a l l y involve endotoxins as w e l l .  Bacteraemia caused  by  considered  gram-negative  organisms  other  than  Salmonella  was  a  rare  entity until  Waisbren reported twenty-nine cases,  ten of which were caused  by E. col i ,  at  in 1951,  Borden  and Hall  reported two cases of gram-negative bacteraemia that resulted from  transfus-  ing  Minneapolis  contaminated blood  gram-negative b a c i l l i in twenty years  in 1951  (149).  (148).  These  Also  reports  heralded  the  appearance  of  as the major cause of hospital-acquired infections and  following  1951,  the incidence of  gram-negative  bacteraemia  - 27 -  has increased twenty-fold (150).  Indications now are that approximately one  percent of a l l hospital patients either acquire gram-negative bacteraemia or are admitted for from t h i r t y  that  reason  to f i f t y  percent  t i v e infections with their of  the  virulence of  implicated  (150,151).  associated  these  capacity  In  isms  their  normal  host  as  when the host's  as  the  urinary,  gram-negative  defense  (152).  This  is  gram-negative Salmonell a,  respiratory or  bacilli  are  in contrast  bacilli  Brucel1 a,  genic E. c o l i .  that  flora  on the  organisms  include  Klebsiel1 a,  Entero-  from these organ-  are  impaired or when  into a s i t e susceptible to system.  known  as  normally  infection  As a r e s u l t ,  "opportunistic"  caused part  by virulent of  skin,  limited  These coli,  commonly  only  Infections  Haemophilus, Pasteurella  These organisms  gram-nega-  organisms  and possess  mechanisms  to bacteraemias not  disease vary  incidence of  (150).  vascular  sometimes  are  this  fact,  tract  Proteus, Pseudomonas and Bacteroides.  only result  for  normal  Escherichia  these bacteria are introduced d i r e c t l y such  actual  are found  mainly the Enterobacteriacea such as bacter,  This high  or gastrointestinal  for  rates  l e t h a l i t y can give a f a l s e impression  organisms.  in gram-negative sepsis  upper respiratory tract invasive  and the f a t a l i t y  the  (Yersinia)  these  pathogens strains  flora,  such  of as  and enteropatho-  cause d i s t i n c t i v e c l i n i c a l  illnesses  which  are usually not considered as " t y p i c a l " gram-negative bacteraemias where the clinical  features  organism  (150).  increased  do not d i f f e r  s i g n i f i c a n t l y regardless  Some of the reasons  incidence  of  gram-negative  that  before  and i t  is  the  have been given to  bacteraemias  enough, the very triumphs of medicine, that i s , c a l l y i l l patients  of  include,  causative  explain the interestingly  that d e b i l i t a t e d and chroni-  are kept alive for much longer periods of time than ever these  patients  with  altered  host  defenses  that  are  very  - 28 -  susceptible practices  to used  infection.  Also,  presently  Another  reason  infection.  that is  is  gram-negative  bacteraemias  coupled with  the propensity of  resistance,  conjunction with  allow bacteria  and gain access to sites of example.  in  to  bypass  this,  host  many hospital  defense  mechanisms  Venous and bladder catheters  given to  explain  the  high  the popular and extensive the host's  use  indigenous f l o r a  are an  incidence of of  antibiotics  for  the combination of which produces resistant strains  antibiotic  of  organisms  (150,153). Patients whose host  that  are  predisposed  to  gram-negative  defense mechanisms have been  underlying host  disease  (151,153,154).  Thus,  is  the major  impaired  bacteraemia  and the  are  severity  determinant of outcome in  granulocytopenic  patients  form  the  those of  the  bacteraemia  major  group  of  hospitalized patients  that have the greatest risk of becoming infected with  gram-negative  (151,155).  bacilli  have normal or even elevated which  implies  function  has  (156,157),  impaired been  leukocyte counts can develop serious  granulocyte  noted  in  also been noted that patients  patients  function with  neoplastic  diseases  (158,159),  disorders  (162,163),  surgical  immunologic burns  (165).  oids,  immunosuppressive  Patients  who receive  p a r t i c u l a r l y susceptible that  It has  commonly develop  drugs,  to gram-negative  mentation of the genitourinary t r a c t ,  or  defects (164)  treatments  extensive  corticoster-  drugs Other  include surgery  syndromes (160,161),  and  with  anti-cancer  sepsis (150,166)  infections  granulocyte  inherited  metabolic disorders  infections  Impaired  genetically  prolonged  radiotherapy  gram-negative  (151).  are  also  treatments and i n s t r u -  surgery of the gastrointestinal  and manipulation of infected wounds, especially after  that  tract  severe burns (166).  - 29 -  A major  concern with  gram-negative  progresses to c i r c u l a t o r y shock. with gram-negative these patients  bacteraemia  is  that  One estimate claims  it  frequently  that 40% of  sepsis go on to develop shock (167).  patients  Characteristically,  develop shaking c h i l l s which are closely followed by fever.  In the early stages of hypotension, some patients tation  ("warm" shock) which then proceeds  fested  by p a l l o r and a cool,  shallow ventilation is  to  clammy s k i n .  often seen.  the c i r c u l a t o r y manifestations  have peripheral  vasoconstrictive  vasodila-  shock, mani-  Respiratory distress  with rapid  Vomiting and diarrhea sometimes  precede  of shock but the development of mental confu-  sion which leads to stupor and coma along with reduced to absent urine flow are  indications  occurs (150). shock  in  of  approximately  Although syndromes  there  shock  5-10% of are  no  associated  (169), gram-positive sion of symptoms aemia,  severe  patients  really  with  bacteraemias  (except  (147,150,166,168). w-iith  syndrome  gram-negative  consistent  gram-positive  The  or  DIC  bacteraemia  differences  between  gram-negative  the  infections  tend to follow a slower stepwise  meningococcaemia) whereas  of  progres-  in gram-negative  the transition from r e l a t i v e l y good health to prostration  bacter-  can occur  within a few minutes to a few hours (167). Treatment of  gram-negative  bacteraemia  essentially  involves the prompt,  aggressive use of antimicrobial agents since more than one-half of with some types of treatments tory,  untreated  bacteraemia  die within  72 hours  (170).  patients Other  given are b a s i c a l l y designed to support and maintain the respira-  circulatory  devices, f l u i d s  and  excretory  and various  effectiveness  of  these  gram-negative  septicaemia  systems  through  pharmacological  treatments is  in  wanting.  the  use  agents (150,166).  terms More  of  reducing recent  the  of  mechanical  However, the morbidity in  developments  in  the  - 30 -  treatment ies.  of  Work  against  gram-negative  in this  death  area  bacteraemias  has  antibody t i t e r s  much  mortality rates  higher  (171).  infections  "0"  bacterial  antibodies  (171).  In  this  are  protective  study,  patients  against their infecting strain of bacteria had than patients  However, the large  species (E. c o l i  tion unwieldy except, serotypes  (153).  granulocytopenic  with  high  "0"  number of different  has 150 serotypes)  antibody  titers  perhaps, for Pseudomonas of which there are only a few  Also,  vaccinations  are  ineffective  or whose immune response  anti-cancer therapy (172).  is  ture of these two regions in  possible.  Studies  and  in patients  the sequelae of gram-negative titers  protective  against  mechanism  a n t i t o x i n - l i k e effect  of  the  have in fact  core  the  core  is  against the core polysacchar-  shown that  of  glycolipid  endotoxin antiserum  rather than opsonization (151).  immunity  protection  in patients  /the case, then this vaccine would also be effective  struc-  v a r i a b i l i t y in a l l  cross-protective  greatest  glycolipid  are  treatment,  As mentioned previously, the  therefore  shock was  by drug  that  may be more feasible  of endotoxin show l i t t l e  Enterobacteriaceae  in patients  inhibited  A vaccine that  ide and l i p i d A portions of endotoxin.  species  O-antigens in each  makes this method of vaccina-  one which induces the formation of antibodies  antibody  immunological stud-  These "0" antibodies are IgG and IgM antibodies and they act as heat  stable opsonins.  such as  indicated that  in gram-negative  with low "0"  have come from  If this  is  against  who had high  (171,173). may  the  The  involve  an  proves to be  in patients that have an  impaired immune system. The  results  obtained  with  the  immunoprophylaxis experiments  strongly  indicate the great portent endotoxins wield in the production of the lae  of  gram-negative  bacteraemias  and  also  suggest  that  seque-  the b i o l o g i c a l l y  - 31 -  active  portion of  Analogous that  experiments  this  endotoxin (175).  endotoxin with  drug,  which  (174),  can  These results  may be a necessary  involves  the  the cationic  binds also  lipid  A region  antibiotic  suggest that  the  the  polymyxin  stoichiometrically to inhibit  of  the  biological  B have shown  lipid  actions  complex.  A region of  endotoxins  the binding of endotoxin to target  prelude to the expression  of i t s  of  cells  b i o l o g i c a l t o x i c i t y and  agents which can form complexes with endotoxin, such as polymyxin B or core g l y c o l i p i d antiserum, ing in  its this  prevent the toxic expressions  a b i l i t y to interact with various thesis  have been designed for  cells.  of endotoxin by hinderThe experiments  the primary purpose  of testing  relationship between the binding of endotoxin to target c e l l s festation  of i t s  lethal effects.  aim of  studying  the  nature  this  and the mani-  To this end, in v i t r o investigations  conducted in which the red blood c e l l the  described  of  was used as  a model target  endotoxin-cel1ular  were  cell  interactions  with  and for  examining how certain membrane components can influence the binding of endotoxins to plasma membranes.  In this regard,  various  chemical and enzymatic  treatments were employed to modify either the structure of certain constituents  of the endotoxin or  of the erythrocyte plasma membrane and the effect  these modifications on toxin-red blood c e l l  interactions was studied.  of  Also,  51 in  v i t r o binding experiments  utilizing  Cr r a d i o l a b e l e d  E. c o l i  endotox-  in were designed to test the c a p a b i l i t y of various pharmacological agents to antagonize  the binding of endotoxin to  were found to be effective to endotoxin-treated toxic  effects  of  human red blood c e l l s .  Drugs  endotoxin antagonists in v i t r o were administered  animals and assessed for t h e i r a b i l i t y to mitigate  endotoxin  that  in v i v o .  If  antagonism of endotoxin binding in v i t r o  a  positive  relationship  and a decrease  in i t s  the  between  biological  - 32 -  toxicity  in vivo was found to e x i s t ,  possibly serve as capability cal  a convenient  of preventing  actions of endotoxins.  then the  screening  the i n i t i a t i o n  in v i t r o  test for  binding tests  could  drugs that would have the  of some of the deleterious  biologi-  The use of these drugs in conjunction with a n t i -  b i o t i c s would then provide a more effective mode of therapy for gram-negative bacteraemia than is currently  employed.  - 33 -  CHAPTER 2 Materials and Methods 2.1 Membrane Preparations 2.1.1 Erythrocyte ghosts Erythrocyte membranes were prepared the Red Cross blood bank. blood c e l l s  in a series of progressively  at 800 x g for  buffy coat were removed.  The red c e l l s  isotonic  aspirating red c e l l s  all  NaCl,  each  time  NaCl  and the red c e l l s  and the  procedure was  at  800  coat.  in 10 volumes 0.08  were then resuspended  steps included washes, as above, adjustment  to 7.4 with T r i s  as  for  the  to y i e l d  pellets  were  resusupended  One hundred ml packed M NaCl,  stirred  for 10  The supernatant was  0.08  M step.  Subsequent  The membranes were then s t i r r e d for 10 min  The supernatant was  in sufficient  double  approximately 3-4 mg/ml.  sion of erythrocyte membranes was then quick frozen use.  5 min and  in 10 volumes of 0.06 M  at the 0.009 M stage.  a protein concentration of  stored at - 2 0 ° C for later  outdated  in 0.04 M, 0.02 M, and 0.009 M NaCl with pH  at 4 ° C and centrifuged at 20,000 xg for 10 min. and the  the  x g for  resuspended in 3 volumes of 10 mM Tris-HCl pH 7.4 buffer,  ded  according  were then washed twice in 5 volumes  the buffy  repeated  solutions  5 minutes and both the plasma and  min at 4 ° C and then centrifuged at 9,000 x g for 5 min. discarded  blood donated by  More s p e c i f i c a l l y ,  centrifuging  remaining traces of  were then resuspended  hypotonic NaCl  (176).  0 + blood was centrifuged  cold  0+  B a s i c a l l y , the procedure involved washing the red  to the method of Godin and Schrier  of  from outdated  discar-  d i s t i l l e d H2O This  suspen-  in acetone/dry  ice and  - 34 -  2.1.2 Heart Sarcolemmal Membranes 1.0  gm of  homogenized DTT-0.5  guinea  in  pig  ventricular  10 ml cold buffer  mM CaCl^-lO  mM T r i s  Polytron PT-10 homogenizer  tissue  was  which was  pH 7.4.  minced with  composed  The tissue  (Brinkman Instruments)  of  was for  scissors  1.25  M KC1-2  homogenized  5 sec  at  and mM  with  a  1/4 maximum  2 speed.  The  centrifuged  homogenate  was  filtered  at 1,200 x g for 10 min.  p e l l e t was resuspended  through  a  0.5  The supernatant  mm  fitting  at 500 x g for 10 min.  the pellet was resuspended DTT-10 mM T r i s pH 8.2.  six  gradient  and the  teflon  pestle using 5  The homogenate was  The supernatant  was  in 6 . 0 ml 10% w/v sucrose buffer  discarded  and  containing 2 mM  The suspension was homogenized to homogeneity with a  glass hand homogenizer. of  and  and homogenized in a  up and down strokes with a Potter-Elvehjem homogenizer. then centrifuged  mesh  was discarded  in 10 ml homogenization buffer  40 ml glass homogenization tube with a tight  nylon  1.0 ml of the homogenate was layered on top of each  tubes which contained a discontinuous  sucrose gradient  that  started on the bottom with 2.5 ml 60% sucrose and on which was layered ml portions of Tris  55%, 52.5% and 50% w/v sucrose dissolved  pH 8.2.  Beckman  The gradient  ultracentrifuge.  55-60 % sucrose interface fraction  equilibrated  fraction  was removed with  Tris  pH 7.4,  resuspended  and in  was The  sarcolemmal  at  in 2 mM DTT-10 mM  40,000 x g for  fraction  eaui1ibrated  on top of the 50 % sucrose f r a c t i o n . a Pasteur at  distilled  approximately 3-4 mg/ml.  1 hr  while the mitochondrial and sarcoplasmic  centrifuged  double  centrifuged  pipette,  washed 15  H£0 to  protein  yield  a  min.  in a  at  the  reticular  The sarcolemmal  in 5 volumes  30,000 x g for  2.5  of  10 mM  The pellet concentration  was of  The membranes were quick frozen in acetonp/dry ice  and stored at - 2 0 ° C for future  use.  - 35 -  2.1.3 Liver Membranes Liver membranes were prepared according to a procedure described by T.K. Ray (177). 0.5  Accordingly, 3.0  gm guinea pig  mM CaCl 2 -2 mM DTT-10 mM T r i s  l i v e r were homogenized in 15 ml  pH 7.4-7.5  buffer  for  30  sec  with  a  Polytron PT-10 homogenizer at 1/4 maximum speed.  The homogenate was diluted  to 2% (1:10  and f i l t e r e d  d i l u t i o n ) with homogenization buffer  through a 0.5  2 mm  nylon mesh.  The f i l t e r e d homogenate was then centrifuged  for 10 min and the supernatant 20 ml homogenization buffer izer  was resuspended  The p e l l e t was resuspended  a tight  fitting  teflon  pestle.  the previous d i l u t i o n volume ( i . e .  with homogenization buffer  pellet  1,200  x g in  and homogenized with a Potter-Elvehjem homogen-  using 5 up and down strokes with  homogenate was diluted to 1/2 ate)  was discarded.  at  and centrifuged  in approximately  The  4% homogen-  at 500 x g for 10 min.  The  30 ml of homogenization buffer  and  again homogenized 5 strokes with a Potter-Elvehjem homogenizer.  This homo-  genate was again centrifuged at 500 x g for 10 min and the p e l l e t was resuspended (w/w)  in 4.0  ml of  sucrose  To this  (made up in r^O) which resulted  tration of 48%. 6 gradient  homogenization buffer.  3.0 ml of this  was  in a final  added  14 ml 62%  sucrose  concen-  sample were layered on the bottom of each of  tubes and successive 3.0  (w/w) sucrose were added on top.  ml layers  of 45%, 41% and 2.5  The gradient was centrifuged  for 2 hrs in a Beckman ultracentrifuge.  at 90,000 x g  Liver membranes appeared  at the 37-41% sucrose interface.  The membranes were c o l l e c t e d ,  in 5 volumes  resuspended  10 mM T r i s pH 7.4,  mg/ml protein) and quick frozen in acetone/dry  in double ice.  ml 37%  as a band  washed once  distilled  b^O (3-4  - 36 -  2.1.4 Lung Membranes The method used for the preparation of same  procedure  membranes Briefly, lung  as  except  the  the  one  sucrose  diluted  2%,  filtered,  for  10 min),  the  pellet  was  sucrose.  3.0  ml of  this  gradient  tubes.  55% sample  sucrose.  liver.  at  1/4  centrifuged  the  in the  After made  gradient  rehomogenized  18 ml by the  sample were  included 3.0  Lung membranes,  were  layered  ml of  52%,  liver  changed.  gm guinea  as  pig  with a  described  step  addition  on the  the  The homogenate was  the t h i r d centrifugation up to  of  pH 7.4-7.5 buffer  maximum speed.  The remainder of the gradient layer  preparation  by homogenizing 3.0  and  This gradient was centrifuged  ultracentrifuge.  for  CaCl 2 -2 mM DTT-10 mM T r i s  section 2.1.3  the  used  prepared  homogenizer for 30 sec to  was  concentrations  a 20% homogenate was  in 15 ml 0.5  Polytron  that  lung membranes was b a s i c a l l y  (500  in  x g for  of  55%  (w/w)  bottom of  each  of 6  that  was  layered  48%  and  2.5  on top of  ml 45%  (w/w)  at 90,000 x g for 2 hrs in a Beckman  which equilibrated  at  the 48-52%  interface, were c o l l e c t e d , washed twice in 10 mM T r i s pH 7.4, double d i s t i l l e d h^O and quick frozen in acetone/dry  sucrose  resuspended  in  ice.  2.1.5 Preparation of Liver Lysosomes A 10% homogenate of 0.25  M sucrose with  l i v e r tissue was prepared  a Potter-Elvehjem homogenizer  tight f i t t i n g teflon pestle. 5 min and the  pellet  was  equalled  using 4  The homogenate was centrifuged discarded.  3,500 x g for 15 min and the pellet sucrose that  by homogenizing l i v e r in  the o r i g i n a l  The supernatant was resuspended  homogenate  used as a lysosomal-rich preparation for  with  a  at 600 x g for centrifuged  at  in a volume of 0.25 M  volume.  experiments.  was  strokes  This  supension  was  - 37 -  2.2 Enzyme Assays 2.2.1  Membrane Bound Enzymes Acetylcholinesterase Acetylcholinesterase a c t i v i t y in erythrocyte ghost membrane was determined at acetylthiocholine acid)  room temperature in the  presence  (DTNB) and 0.1 M Tris-HCl  by monitoring the hydrolysis of  0.01  buffer pH 8.0.  0.1 ml 0.01 M DTNB (made up in T r i s buffer), (approximately  thiocholine.  ml 0.1  M T r i s buffer  activity  and 0.05  ml 0.03  assayed  in  rat  erythrocyte  concentrated membrane suspension was used ( i . e . Nitrophenylphosphatase  determined  by measuring of  Mg++  consisted  of 1.0 ml 0.15  phenylphosphate, suspension  and  0.1  K+  in  hydrolysis at  37°C  of  and  M MgCl 2 ,  and double d i s t i l l e d  0.1  membrane  pH  7.4.  pH 7.4, ml 0.9  h^O in a final  The  containing  3.0  to  4.0  assayed  M KCI, 0.2  reaction  reaction was 1 hr whereas  more  were  used  and  was  in  the  mixture  M p-nitroml membrane  volume of  3.0 ml.  and the reaction  in human erythrocyte  mg protein/ml  reaction  0.1 ml 0.09  varied according to the type of membranes being assayed. enzyme a c t i v i t y was  a  preparations  p-nitrophenylphosphate  The protein concentration of the membrane suspension  the  ghosts,  3.0-4.0 mg protein/ml).  various  M imidazole buffer  ml 0.09  When acetyl-  (NPPase)  activity the  presence  M acetyl-  at 412 nanometers was recorded every minute for  was  Nitrophenylphosphatase  pH 8.0,  0.1 ml human erythrocyte ghost  five minutes following the addition of the acetylthiocholine. cholinesterase  0.03 M  The reaction was carried out  0.3-0.4 mg protein/ml)  The absorbance  of  M 5,5'-dithiobis-(2-nitrobenzoic  in a spectrophotometric cuvette by adding 2.75  membranes  preparations  times  For example, when ghosts,  the  suspensions  duration  of  the  suspensions of heart membranes containing approxi-  - 38 -  mately  1.0  mg protein/ml were reacted  for  15  minutes.  The reaction  was  stopped by the addition of 1.0 ml cold 20% TCA and the precipitated protein was centrifuged down at 10,000 x g.  A 3.0 ml aliquot of the supernatant was  taken and combined with 1.0 ml 1.5  M Tris solution.  resulting  at  solution  was  measured  component of the enzyme was  412  The absorbance  nanometers.  The  determined by subtracting  enzyme assayed in the presence  of M g + +  (basal  the  activity)  the enzyme assayed  in the  presence  of  both Mg  K + -stimulated  a c t i v i t y of  from the  the  activity  + ,  ++  of  of the  and K  (total  activ-  ity).  Adenosine Triphosphatase: (Na + ,K + )-ATPase +  +  (Na ,K )-ATPase  activity  was  assayed  in  various  membrane  prepara-  tions by incubating at 37°C a reaction mixture that consisted of 1.0 ml 165 mM Tris-HCl  buffer  pH 7.4,  0.1  ml 0.09  M MgCl 2 ,  0.1  ml 3 mM EGTA,  0.3 ml  30 mM ATP, 0.1 ml 0.6 M KCI, 0.1 ml 2.4 M NaCl, 0.2 ml membrane suspension and double  distilled  H^O to  make the  tions of the membrane suspensions  volume 3.0  ml.  Protein  concentra-  varied from 3.0-4.0 mg/ml for erythrocyte  ghosts to 0.7-0.8 mg/ml for preparations  of heart membranes.  Reaction times  also varied from 15 minutes for heart membranes to 60 minutes for erythrocyte  ghosts.  The reaction was  TCA and the mixture was aliquot  centrifuged  of the supernatant  solution which was ammonium molybdate. ted by adding 0.2  stopped at  by the addition of 1.0 10,000 x g for  was taken and to  composed of  1.4  Color development for ml Fiske-Subbarow reagent  5 min.  i t was added 1.8  ml double d i s t i l l e d  ml cold 10% A 3.0 ml  ml molybdate  H^O and 0.4  inorganic phosphorus was to the mixture  and the  ml 5% initiaabsor-  bance at 660 nanometers was determined spectrophotometrically at 15 minutes. These absorbance readings were compared to the readings  observed in prepared  - 39 -  standards  that  contained  Na + ,K + -stimulated  portion  the a c t i v i t y observed ++ when Mg  + , Na  known of  quantities  the  of  enzyme  in the presence  inorganic  was  of M g + +  phosphorus.  determined  by  The  subtracting  alone from the a c t i v i t y seen  + and K were a l l present  in the reaction mixture.  The Fiske-Subbarow reagent was prepared fresh weekly by the addition of 0.25  gm l-amino-2-naphthol-4-sulfonic  freshly  prepared 15% sodium b i s u l f i t e  of  gm anhydrous  0.5  acid  (ANS) with  (anhydrous)  sodium s u l f i t e .  stirring  to  100 ml  followed by the addition  The solution  was  stored  in  a dark  bottle. Cytochrome c Oxidase Cytochrome  c  oxidase  activity  was  assayed  accordance with the method of Rabinowitz et  membrane  in  a modifica-  tion of the procedure outlined by Cooperstein and Lazarow (192).  Basically,  solution of 0.03  805  0.03  ml  reduced  (Sigma  M Na 2 HP0 4  cytochrome  prepared  1.2  M phosphate buffer  c was  with  195  freshly  M sodium hydrosulfite  Type III)  dissolved  in 0.03  solution was shaken vigorously for sulfite.  ml  pH 7.4  0.03  prepared to  (191)  fractions  which i s  a stock  al.  in  M KH 2 P0 4 . by  a 17  adding  A  100  yM solution  M phosphate several  was prepared  buffer.  yl of  by mixing  solution of  of  freshly  cytochrome c  The cytochrome c  minutes to remove excess hydro-  3.0 ml of this reduced cytochrome c solution were then transferred  to a spectrophotometric cuvette and to this an aliquot of no more than 50 yl of  sample was  second fully  mixed.  intervals  for  The absorbance 3 minutes.  at  550  nanometers  ted from each of the  taken  The cytochrome c in the cuvette  oxidized by the addition of a few crystals  The absorbance of this f u l l y  was  30  was then  of potassium f e r r i c y a n i d e .  oxidized solution of cytochrome c was  absorbances obtained at  at  the 30 second  time  subtracintervals  - 40 -  after  the sample was added.  The logarithm of the difference  bance values when plotted against time gave a straight  of these absor-  line with a  negative  slope from which could be determined the amount of cytochrome c (in pinoles) that was oxidized per minute per mg sample protein.  5'-Nucleotidase  5'-nucleotidase  a c t i v i t y in membrane preparations  was  assayed by mixing  together  in a volume of 0.5 ml a reaction mixture which consisted  Tris-HCl  buffer  pH 9.0,  concentrations).  2.5  mM MgCl 2 ,  100 mM KCI and 8.0  reaction volume 0.5 ml). 15 minutes)  while the reaction rate was s t i l l  linear,  supernatant of  was  0.28  taken  ml double  and  assayed  distilled  for  the  (usually 10 reaction was  The protein precipitate was  centrifuged down in an Eppendorf bench-top centrifuge.  addition  (final  (making the total  After an appropriate reaction interval  stopped by the addition of 0.5 ml cold 12% TCA.  the  mM 5'-AMP  The mixture was preincubated at 37°C for 5 minutes and the  reaction was then started with 100 yl membrane suspension  or  of 50 mM  A 0.6 ml aliquot of  inorganic  H^O, 0.08  phosphorus  by  the  ml 5% ammonium molybdate  and 0.04 ml amino-naphthol-sulfonic acid (ANS) reagent (see Methods 2 . 2 . 1 . 3 ) . The color was developed at room temperature 660 nanometers  at precisely  15 minutes  which contained known quantities  of  and the  after  inorganic  absorbance  was read  the ANS was added. phosphorus  at  Standards  were also  assayed  and with reference to these absorbances the results of the samples could be expressed as ymoles Pi liberated/mg protein/hour. 2.2.2  Lysosomal Enzymes Acid Phosphatase Acid phosphatase a c t i v i t y was determined as described b u l l e t i n No. 104.  Basically,  in Sigma technical  the reaction mixture consisted of 0.5 ml 0.09 M  - 41 -  citrate 0.2  buffer  ml of  values  pH 4.8,  sample.  0.5 ml p-nitrophenylphosphate solution (4 mg/ml) and  Samples were  did not exceed  0.900.  plasma were diluted 1:3 acid  phosphatase  and then stopped  color.  The  phosphatase  of  5.0  the  solution  read  (determined  from  a  absorbance  and guinea  pig  they were assayed  for  out  N NaOH which was  that  rat  carried  of 0.3 to convert the results  activity  so  of  before  was  ml 0.1  diluted  samples  respectively  The reaction  with  absorbance  divided by a factor  For example,  and 1:5  activity.  minutes  appropriately  at  at  37°C  also 410  curve  30  developed  the  nanometers  and  into Sigma units  standard  for  as  of  acid  described  in  technical b u l l e t i n No. 104). N-Acetyl-e-Glucosaminidase N-Acetyl-e-glucosaminidase extent  of  hydrolysis  of  activity  was  determined  by  measuring  the  p-nitrophenyl-N-acetyl-B-D-glucosaminide substrate  to p-nitrophenol under acidic conditions.  The reaction mixture consisted of  0.5 ml 0.3 M c i t r a t e buffer pH 4.3, 0.5 ml p-nitrophenyl-N-acetyl-B-D-glucosaminide  (3  Samples were reaction between were  mg/ml),  ml  appropriately  did  not  exceed  absorbance  usually  0.3  double  diluted 0.300,  distilled  in order  the  diluted  1:3.  that  point  and enzyme quantity  after  became  The reaction  H20  was  and  0.2  absorbance which  carried  minutes and then terminated with 1.5 ml cold 3% TCA.  out  sample.  values  the  non-linear.  ml  the  relationship  Plasma at  of  37°C  samples for  30  The solution was then  centrifuged at 10,000 x g for 5 minutes and a 2.0 ml aliquot of the supernatant was taken. M NaHCO^-O^ was  read  at  To this  M Na^CO^ 420  a l i q o t , 1.0 ml bicarbonate buffer was  nanometers.  plasma or as absorbance/mg somal suspensions.  added  to  develop  The results  protein for  were  the  color.  expressed  other types  consisting of The as  0.5  absorbance  absorbance/ml  of samples such as  lyso-  - 42 - Cathepsin D. Assays  for  cathepsin  and Heath (177)  with  D a c t i v i t y were performed  some modifications.  as  The total  described  by  Barrett  volume of the reaction  mixture was 0.4 ml and consisted of 0.1 ml 1 M formate buffer pH 3.0, 8% (w/v)  haemoglobin  substrate  (prepared  as  sample (plasma samples were diluted 1:2).  described  60 minutes,  was stopped by the addition of 2.5 ml cold 3% TCA. stand  on ice for  30 minutes  after  complete the precipitation of the proteins fuged in a table-top  centrifuge  at 4 ° C .  and 0.2  after  Lowry protein  double d i s t i l l e d  assay.  the addition  of  the  of  it  TCA to  and then the tubes were c e n t r i -  An aliquot of the supernatant  Blanks for  H20 instead  which  The reaction mixture was  to 0.4 ml) was then assayed for small molecular weight peptides standard  ml  The reaction was started with the  haemoglobin substrate and incubated at 4 5 ° C for  allowed to  below)  0.1 ml  the cathepsin  1 M formate  buffer.  (0.2  by using the  D assay contained  Results  were  expres-  sed as mg protein liberated/hr/ml plasma. The formate formate  with  buffer  was prepared  1 M formic  acid  and  by mixing equal titrating  the  volumes  resulting  of  1 M sodium  solution  with  concentrated formic acid to pH 3.0. Haemoglobin substrate was prepared from one unit of outdated human blood obtained from the Red Cross blood bank.  The blood was centrifuged  at 3,500  x g for 10 minutes and both the plasma and buffy coat were discarded.  The  erythrocytes were resuspended  The  wash was  repeated  remnants  of  resuspended CC1/,.  the  twice buffy  in a equal  The suspension  in 500 ml 0.15  more coat  and  were  volume of was  stirred  each  M NaCl and recentrifuged.  time  discarded.  the The  double d i s t i l l e d vigorously  at  supernatants packed  cells  H20 and 0.5 4 ° C for  15  as  well  were  as then  volumes minutes  of and  - 43 -  then centrifuged  at 10,000 xg.  globin was dialyzed against to 8.0  The clear red supernatant  double d i s t i l l e d  containing haemo-  H^O at. 4 ° C and then  (w/v) on the basis of a dry weight determination.  adjusted  Ten ml  were quick frozen using acetone/dry ice and stored at -20"C until  aliquots  reauired.  2.3 Haemolysis Experiments Red  blood  cell  haemolysis  described by Machleidt et a l .  experiments (178)  heparinized blood was used for and both  the  resuspended  plasma  and buffy  in  sufficient  coat  were  Freshly drawn  The blood was  discarded.  the  suspension  For example, NaCl-Tris of  red  as  centrifuged  The red  cells  were  recentri-  along with remaining portions of buffy coat, were and the red c e l l s  0.9% NaCl-15 mM T r i s  approximately 6%.  This  essentially  with some modifications. experiments.  This wash was repeated  32 ml with  conducted  in 4 volumes cold 0.9% NaC1-15 mM Tris pH 7.0 buffer,  fuged and the supernatant, discarded.  all  were  buffer  gave  was  a haematocrit  kept  on  ice  resuspended  to give a haematocrit  2.0 ml of packed red blood c e l l s  buffer  cells  pH 7.0  were then  and  of  of  made up to  approximately  6%.  thoroughly mixed when  aliquots were removed for haemolysis t e s t s . The haemolysis test was set red  cell  buffer.  suspension  was  added  up in the following manner: to  1.3  ml of  ml of  0.9% NaCl-15 ITM T r i s  the  pH 7.0  The mixture was allowed to equilibrate for 5 minutes at the desired  temperature  of  the  test,  usually  at  room temperature.  The reaction  started by the addition of 0.2 ml 0.9% NaC1-15 mM Tris pH 7.0 contained the agent to be tested. buffer.  0.2  Control  buffer  was  which  samples contained no drug in the  The suspension was incubated for 15 minutes at the desired tempera-  ture and then was subjected to a hypotonic challenge by the addition of ml 15 mM Tris pH 7.0.  The mixture was further  2.3  incubated for 10 minutes and  - 44 -  then centrifuged and  the  at 15,000 x g for  absorbance  (without  added  at  drug)  0.2 ml red c e l l  540  was  1 minute.  nanometers  approximately  suspension  in 3.8  over 100% indicated  measured.  40% of  ml double  (with drug) were usually expressed upon values  was  The supernatant  as  lytic  Control  complete  distilled  percent  was  decanted  haemolysis  haemolysis, H^O.  Test  i.e., samples  of control haemolysis where-  a c t i v i t y while values  demonstrated anti-haemolytic a b i l i t y or red blood c e l l  less  than 100%  stabilization.  2.4 Enzyme Treatments of Intact Red Blood C e l l s 2.4.1 Trypsinization The method for trypsinization of intact red blood c e l l s was adapted from Seaman  and Uhlenbruck  (179).  Blood was  freshly  drawn into  a heparinized  syringe and divided into two portions.  Each portion was centrifuged and the  plasma and buffy coat were discarded.  The red c e l l s  0.9% NaCl-15 mM Tris pH 7.0  buffer  as  described  were washed twice in  in section  2.3  of Methods.  A 0.1%; (w/v) trypsin enzyme solution was made up in 0.9% NaC 1-15  mM T r i s pH  7.0 buffer and 3.0 ml of this enzyme solution were mixed with 1.0  ml packed  red blood c e l l s . control  The other portion of packed red blood c e l l s  and was mixed in 3.0 ml buffer  and treated  red c e l l  containing no enzyme.  of  a  Both control  The supernatants were removed and the red c e l l s were  again washed twice in 0.9% NaCl-15 mM T r i s  crit  as  suspensions were incubated at 37°C for 30 minutes and  then were centrifuged.  the red blood c e l l s  served  were resuspended  approximately  6%.  This  experiments as described in section  buffer.  After  in the NaCl-Tris  suspension 2.3.  was  then  the f i n a l  buffer used  wash,  to a haematofor  haemolysis  - 45 -  2.4.2  Neuraminidase Treatment  The procedure been adapted  for  treating  from several  intact  sources  erythrocytes  (179,180,181).  blood was divided into two portions,  a control  with  neuraminidase  Freshly drawn heparinized and enzyme treated  sample.  The blood was centrifuged and the plasma and buffy coat were discarded. red blood c e l l s  were then washed twice as  has  described  2.3  except  that isotonic saline was used instead of 0.9% NaCl-15.mM T r i s buffer.  After  the wash, the red c e l l s  were resuspended  mM NaHCOg pH 7.2 buffer  in a r a t i o of 3.0 ml buffer  erythrocytes.  The treated red c e l l  in 0.145  in section  The  M NaCl-5.0 mM CaCl 2 -0.3 for  each ml of packed  suspension received 10 units of neurami-  nidase enzyme solution (500 units/ml, Behringwerke) for each ml of red blood cell of  suspension. H 2 0.  The control red c e l l  The suspensions  centrifuged.  were  suspension  incubated  treatment  for  buffer  to  a haematocrit  removed about  70% of  30 minutes red c e l l s  and then  were washed  For haemolysis studies, the red c e l l s were  washed once more in 0.9% NaCl-15 mM Tris this  37°C  The supernatants were removed and the  twice with cold isotonic s a l i n e .  in  at  received an equal volume  of  the  pH 7.0 buffer  approximately sialic  acid  and then resuspended  6%.  The  residues  neuraminidase  from the  intact  erythrocytes. 2.4.3 Phospholipase A 2 Treatment The method for  treating  intact  based on the  procedure reported  investigators  reported the  erythrocytes  by Roelofsen  effectiveness  of  with  phospholipase  and associates  using  (182).  phopholipase  A 2 was These  A 2 prepara-  tions from Naja naja venom to hydrolyze phospholipids in intact erythrocytes (68% of the red c e l l porcine  pancreas  l e c i t h i n was degraded), whereas enzyme preparations from  were  ineffective  against  intact  red  cells.  Therefore,  - 46 -  phospholipase  Ar, preparations  our  The enzyme preparation  ml  studies. double  distilled  H^O to  from  Naj a  give  naj a venom (Sigma)  (1,000 u n i t s / v i a l ) an  activity  of  were  was dissolved  1,000  units/ml.  enzyme solution was then heated at 70°C for 10 minutes to destroy tic  in  in 1 This  proteoly-  a c t i v i t y and then stored at - 2 0 ° C until further required. Erythrocytes  were  obtained  by  centrifuging  freshly  drawn heparinized  blood and the red c e l l s were then washed twice with cold remove a l l  traces  of the buffy coat.  cytes were resuspended  After  the  isotonic saline  second wash,  packed red blood c e l l s .  of  red  cell  suspension.  Control  were then incubated at 37°C for 1 hour. and  the supernatants were discarded.  buffer  and  phospholipase  treated  suspensions  The samples were then centrifuged  The erythrocytes were washed twice in  cold isotonic saline and once with 0.9% NaCl-15 mM Tris pH 7.0 buffer. haemolysis  studies,  the red c e l l s  in  Erythrocyte suspen-  sions that were to be reacted with enzyme received 10 units of solution/ml  to  the erythro-  in 0.9% NaCl-10 mM CaC 12~5 mM T r i s pH 8.0  the r a t i o of 3.0 ml buffer/ml  A2  used  were then resuspended  For  in 0.9% NaCl-15 mM  T r i s pH 7.0 buffer to a haematocrit of 6%. 2.4.4 Removal of Cholesterol from Intact Erythrocytes The procedure that was used to extract cytes  was  requires was  based on the  centrifuged  a  from intact erythro-  by Gottlieb  (183).  This  approximately 50 ml of freshly drawn heparinized blood.  erythrocytes with  method developed  cholesterol  and  both  plasma  and  were washed twice in cold  buffer  composed  of  glucose-0.1% adenosine pH 7.4.  0.9%  red  blood  isotonic  NaCl-4  cells  saline  were  method  The blood  saved.  The  and then once more  mM KH 2 P0 4 -16  mM Na2HP04-0.4%  The red blood c e l l s were f i n a l l y  resuspended  in an equal volume of the buffer solution (usually 25 ml) and then stored in this manner at 4 ° C u n t i l the plasma was prepared.  - 47 -  The  plasma  (25  ml) was  incubated  at  37°C  for  60-72 hours  in order  to  allow e s t e r i f i c a t i o n of the lipoprotein cholesterol by the enzyme l e c i t h i n cholesterol cin  sulfate  prevent  was  added  bacterial  molecular were  acyltransferase  weight  to  (LCAT) which is present the  plasma  contamination. plasma  precipitated  proteins  by the  in  After  a  concentration  the  incubation  and some of  addition  of  in the plasma.  the  an equal  of  200  period,  low density volume  of  Gentamyyg/ml the  to  large  lipoproteins  60%  (NH^^SO^.  The mixture (50 ml) was s t i r r e d at room temperature for 30 minutes and then centrifuged  at 10,000 x g for 5 minutes.  against 2 l i t e r s 4°C.  of 0.9% NaCl-20 mM T r i s buffer  The dialysate  was diluted 1:6 described  was changed after  with  above.  lipoproteins  was then dialyzed  pH 7.4-7.5 for 24 hours  12 hours.  After  dialysis,  This  required  solution for  (300 ml), which contained the high  cholesterol  after  plasma solution for each 1.5 incubated at 37°C for a total  exchange,  was  they were centrifuged,  of 18 hours.  6 and 12 hours by centrifuging  incubated  in the r a t i o  ml packed red blood c e l l s .  at  the plasma  the sodium chloride-phosphate-glucose-adenosine  stored red blood c e l l s  after  The supernatant  buffer density  with of  the  100 ml  The suspension was  The plasma solution was changed  the mixture and then resuspending  the  erythrocytes in another 100 ml aliquot of the plasma solution which was kept stored at 4 ° C . in  100  ml  As a c o n t r o l , 1.5 ml packed red blood c e l l s were resuspended 0.9%  NaCl-4  Na 2 HP0 4 -0.4%  glucose-0.1%  adenosine pH 7.4 buffer and then incubated at 37°C for a total  of 18 hours.  The buffer was changed after  mM  KH 2 P0 4 -16  mM  6 and 12 hours of incubation.  Following these  incubations, the solutions were centrifuged and the erythrocytes were washed once in 0.9% NaCl-15 mM T r i s pH 7.0 buffer. in  the  same buffer  experiments.  to  a haematocrit  of  The c e l l s were then resuspended approximately  6% for  haemolysis  - 48 -  2.5 Compositional Assays 2.5.1  Protein  Protein  analysis  method described  of  various  by Lowry et  preparations  al.  (184).  was  done  according  More e x p l i c i t l y ,  to  the  the assay was  set  up in the manner described below: Stock  solution  Na+,K+-tartrate one l i t e r .  A was  and 4.0  prepared  by  gm NaOH in  dissolving  double  20  distilled  gm  Na^O^,  H ? 0 to  0.2  a volume  gm of  Stock solution A was stored at room temperature.  Stock  solution  (CuS0 4 .5H 2 0)  B consisted  of  a 1%  (w/v)  solution  of  cupric  sulfate  solution  B with  in double d i s t i l l e d H 2 0.  Solution C was prepared  freshly  by mixing 1 part  stock  99 parts stock solution A. The sample to  be  assayed was  made up to  0.5  H^O and to this was added 5.0 ml solution C. room temperature for 10 minutes reagent,  freshly  solution  was  minutes ters. curve  diluted 1:1  thoroughly  The mixture was  and then 0.5 ml of  with double  mixed.  distilled  The color  ml with double  was  that  albumin.  was  prepared  by assaying  The reaction was  linear  incubated  at  Folin-Ciocalteu's phenol H 2 0, was  allowed  to  added  and  develop  at room temperature and then the absorbance was read The amount of protein in each  distilled  for  the 30  at 750 nanome-  sample was determined  from a standard  known concentrations  of  up to  100  yg protein.  bovine  Blanks  serum  contained  double d i s t i l l e d H 2 0 instead of protein. 2.5.2  S i a l i c Acid (N-Acetylneuraminic Acid)  Assays (185).  for  sialic  acid  were  based  on the  method  described  The following solutions were prepared for the assay:  by Warren  - 49 -  Stock  solution A, consisting of  0.2  M NaI04  (sodium m-periodate)  M phosphoric acid, was prepared by dissolving 4.28  in 9  gm of sodium m-periodate  in 60 ml concentrated phosphoric acid (15 M) and then d i l u t i n g the solution to 100 ml with double d i s t i l l e d H 2 0. Stock  solution  Na 2 S0 4 -0.1 sodium  B consisted  N H 2 S0 4  m-arsenite  solution and  of  a  which  3.55  gm  10%  was  Na 2 S0 4  (w/v)  sodium  prepared  by  in  N  0.1  m-arsenite-0.5  dissolving H 2 S0 4  5.0  to  M gm  a  final  acid  in a  volume of 50 ml. Solution 0.5  C was prepared  M Na 2 S0 4  solution  to  fresh a  by dissolving  concentration  of  2-thiobarbituric 0.6%  (w/v).  The  solution  was heated to enhance s o l u b i l i t y . The sample to be assayed was made up to double  distilled  H20.  For  example,  when  assayed, a 50 ul aliquot of the suspension to  150  yl  double  distilled  to acidify the sample. utes.  After  H 2 0.  a total  Then  volume of 0.2  membrane  ml with  suspensions  were  (3.0-4.0 ma protein/ml) was added 20  yl  of  1.1  N H 2 S0 4  was  added  The sample was then hydrolyzed at 80°C for 30 min-  cooling, 100 yl of  stock  solution A was  the mixture was  the dark.  Then 1.0 ml stock solution B was added and the solution was mixed  it  after  vortexing,  minutes.  became c o l o r l e s s .  After  the  To this  solution  was  was  placed  20 minutes  added in  3.0  at  and, following  vortexing,  until  incubated for  added  room temperature in  ml of  a boiling  solution  water  bath  the samples were cooled, the colored complex was  C and, for  15  extracted  by adding 4.0 ml cyclohexanone, vortexing and centrifuging the samples in a table-top  centrifuge  (cyclohexanone) the absorbance  to  separate  the organic  and aqueous  phases.  The top  layer, which contained the colored complex, was removed and at 549 nanometers was measured.  This absorbance reading was  - 50 -  compared  to  the  absorbance  N-acetylneuraminic  acid  obtained  (usually  from  assaying  a  5 yg br approximately  known  quantity  20 nanomoles,  of  based  on a molecular weight of 267.24). 2.5.3 Cholesterol Analysis The method  used  for  cholesterol  reported by Zak and co-workers (186). dissolving  2.5  gm FeCl-j^H^O  reagent was stored  at - 2 0 ° C .  i  25  n  analysis  was  based  on the  procedure  An iron stock reagent was prepared by ml  glacial  acetic  acid.  This  stock  A "working" Zak reagent was prepared by d i l u t -  ing the iron stock reagent 1:100 with concentrated h^SO^. Sample volumes used were 50 yl or less.  2.0 ml g l a c i a l  acetic  acid were  added to the sample and the mixture was allowed to stand at room temperature until  clear  (approximately  reagent  were  carefully  layer.  The layers  min.).  added  so  as  Then to  1.3  ml  underlay  of  the  the  "working" Zak  glacial  acetic  were then mixed thoroughly and the color was  develop for 30 minutes, determined.  30  at which time the absorbance  at  acid  allowed to  565 nanometers  was  Sample absorbances were compared to a standard absorbance where  a known quantity of cholesterol  (50 yg or 129.3 nanomoles) was assayed.  2.5.4 Phospholipid Analysis Phospholipid content was phorus as described  determined by assaying for  by Bartlett  with perchloric acid instead  of  (187)  except  sulfuric  that  acid.  phospholipid  the samples were  phos-  digested  The assay was conducted  as  follows: Stock solution A (Fiske-Subbarow reagent) was prepared by adding 0.25 gm l-amino-2-naphthol-4-sulfonic (w/v)  sodium b i s u l f i t e  sulfite.  acid  (anhydrous)  The solution was f i l t e r e d  periods of up to a week.  (ANS)  to  100  ml  freshly  and then adding 0.5 and then stored  prepared 15%  gm anhydrous sodium  in a dark  bottle  for  - 51 -  Stock solution B consisted  of a 5% (w/v) ammonium molybdate solution in  water. Sample volumes were kept as small as membrane suspension sufficient  that  possible.  contained 3.0-4.0  for this assay.  For example,  mg protein/ml was  50 yl of a found  to  be  1.5 ml of 70% perchloric acid were added to the  sample and the mixture was digested  at 230°C in a sand bath for 30 minutes.  After  ml double d i s t i l l e d  the samples were cooled,  7.6  the solution was thoroughly mixed. developed by adding 0.5  h^O were added and  A 4.5 ml aliquot was taken and color was  ml double d i s t i l l e d ^ 0 ,  0.2  ml of  stock  solution  B, 0.2 ml stock solution A, mixing and then placing the mixture in a b o i l i n g water bath for 7 minutes.  After  830 nanometers was determined.  cooling, the absorbance  of the samples at  Sample absorbances were compared to  absorb-  ances that were obtained from a standard solution containing a known quantity of inorganic phosphorus (usually 2.0 yg).  To convert ymoles phosphorus/ml  to ymoles phospholipid/ml, the conversion factor of 25 and an average molecular weight of 700 were used. 2.5.5 Ketodeoxyoctanoic Acid (KD0) Assays for the trisaccharide 2-keto-3-deoxyoctonate  in endotoxin prepar-  ations were b a s i c a l l y conducted according to the procedure of Weissbach and Hurwitz  (188)  as  modified by Osborn (189).  In more d e t a i l ,  the  assay was  organized in the following manner: Stock  solution A which consisted  of 0.025 M NaI04  (sodium  m-periodate)  was prepared by dissolving 5.35 gm NalO^ in 1 l i t e r 0.125 N h^SO^. Stock  solution B consisted  in 0.5 N HCl.  of 0.2  M NaAs02 (sodium arsenite)  dissolved  - 52 -  0.4 ml of stock consisted  of  a 2.0  solution A was added to 0.2 ml of sample solution which mg/ml suspension  of  bacterial  endotoxin  in water.  The  solutions were mixed and incubated at room temperature for 20 minutes. 0.5 ml stock solution B was added and after to  stand for  2 minutes.  2.0  were then added and after water bath for 20 minutes. ml  cyclohexanone,  separation  ml freshly  vortexing,  Then  mixing, the mixture was allowed  prepared  0.3% t h i o b a r b i t u r i c placed  in a b o i l i n g  The chromophore was then extracted  by adding 2.0  vortexing  and  the samples were  acid  centrifuging  of the aqueous and organic phases.  hexanone phase was measured at  curve  samples  The absorbance  548 nanometers.  sample was determined from a standard  the  to  facilitate  of the cyclo-  The quantity of KDO in each that  was  prepared  by assaying  2.5 to 50 nanomoles of KDO (Sigma) as described above for samples. To determine the " t o t a l " quantity of 2-keto-3-deoxyoctonate preparations,  i t was f i r s t  in endotoxin  necessary to subject the endotoxin to acid hydro-  l y s i s in order to free a l l bound KDO. This was done by incubating 0.2 ml of a  2  mg/ml  endotoxin  suspension  with  0.2  ml  0.05  double d i s t i l l e d r^O in a b o i l i n g water bath for ing the sample,  N r^SO^ and  20 minutes.  0.1  After  ml  cool-  a 0.2 ml aliquot of the acid-hydrolyzed sample was taken and  assayed for free KDO as described above. 2.6 Thin Layer Chromatography 2.6.1  Intact Erythrocytes  Phospholipids were extracted following two methods.  from intact  erythrocytes  by either  of  the  Method A, a modified procedure, gave results similar  to the more standard procedure, Method B. Method A:  To 1.0  ml packed red blood c e l l s ,  the mixture was allowed to stand  at  1.0  room temperature  ml H^O was added and for  15 minutes.  Then  - 53 -  4.0  ml methanol were added and after  stand for 1 hour.  mixing, the  solution was  6.0 ml chloroform were then added and, after  suspension was allowed to stand for another 1 hour. were then centrifuged washed 3 times  9,000 x g for  by adding 1.0  incubating for in  at  15 minutes at  a bench-top  centrifuge  layer was discarded  after  to  enhance  each wash.  and the  of 0.75%  room temperature phase  to  mixing, the  The extracted red c e l l s  5 minutes  ml aliquots  allowed  supernatant  (w/v)  NaCl,  was  vortexing,  and centrifuging the samples separation.  The top  Following the f i n a l  aqueous  wash, the organic  layer was evaporated to dryness under nitrogen and the residue was r e d i s s o l ved  in 0.2  ml chloroform:methanol  (2:1)  just  before  the  total  sample  was  applied to thin layer plates for chromatographic separation. Method B: cells  is  methanol  the same as  for  added and the  NaCl. tants fine  was  described  1.5  5 minutes  by Reed and associates  ml packed red blood c e l l s  at  red c e l l s  then centrifuged  collected. this  that  were added to  extracted  were  This method of extracting phospholipids from intact red blood  room temperature.  were extracted  at  low speed  If the supernatant  for  (3,000  and the  Then 5.0  x  g)  and  supernatant  5.0 ml  l i p i d s were  ml chloroform were  another 5 minutes.  contained appreciable  then removed by washing the  (190).  the  The  cells  supernatant  was  amounts of haemoglobin, with  1.5  ml 0.75%  (w/v)  The extraction procedure was repeated twice more and a l l the supernawere pooled. particles  of  The supernatants red  evaporated to dryness  cells  were  were f i l t e r e d  present.  through glass  The pooled  supernatants  under nitrogen and the residue was extracted  ml chloroform for 5 minutes.  This was repeated  (6.0  ml) were pooled.  The CHC1^ was  gen)  and the powder was redissolved  wool  if  were  with  2.0  twice more and the extracts  evaporated  to  dryness  (under  nitro-  in a volume of benzene that yielded a  - 54 -  c o n c e n t r a t i o n of a p p r o x i m a t e l y 20 mg p h o s p h o l i p i d / m l benzene.  Twenty t o  30  y l were then a p p l i e d t o the t h i n l a y e r p l a t e . 2.6.2  Membranes and Phospholipids  bacterial  Endotoxins were  endotoxins  extracted  from  by i n c u b a t i n g 0.5  various ml  membrane  membrane s u s p e n s i o n  p r o t e i n / m l ) or 0.5 ml of a s o l u t i o n of e n d o t o x i n with  a 5.0  hours. NaCl  ml  mixture  The m i x t u r e was  o f CHCl^:methanol  preparations  (2:1)  (8.0 mg  (3.0-4.0  endotoxin/ml  at room t e m p e r a t u r e  then washed 3 times w i t h 1.0  by c e n t r i f u g a t i o n and the aqueous l a y e r was  ml  and mg h^O)  for 2  a l i q u o t s of  0.75%  d i s c a r d e d each t i m e .  The  o r g a n i c l a y e r was then e v a p o r a t e d t o d r y n e s s under n i t r o g e n and the phosphol i p i d s were r e d i s s o l v e d i n 0.2  ml  CHCl^MeOH  (2:1)  just  before  t h e y were  a p p l i e d t o the t h i n l a y e r p l a t e . 2.6.3  S e p a r a t i o n of P h o s p h o l i p i d s P h o s p h o l i p i d s were s e p a r a t e d on aluminum s h e e t s p r e - c o a t e d  gel 60 ( B r i n k m a n ) . f o r 30 m i n u t e s . solvent volume).  system  with  silica  T h e s e s h e e t s were f i r s t " a c t i v a t e d " by h e a t i n g at The  p h o s p h o l i p i d s were s e p a r a t e d  composed  of  P h o s p h o l i p i d s such  CHCl^:MeOH:NH^  in  i n one d i m e n s i o n the  ratio  as p h o s p h a t i d y l e t h a n o l a m i n e  and  of  110°C  using a  14:6:1  (by  phosphatidyl-  s e r i n e , which  c o n t a i n p r i m a r y amino g r o u p s , were v i s u a l i z e d w i t h n i n h y d r i n  spray reagent  ( B a k e r ) w h i l e o t h e r p h o p h o l i p i d s were i d e n t i f i e d w i t h  vapor.  The  p h o s p h o l i p i d s p o t s were then  p e r c h l o r i c a c i d at 230°C f o r 30 minutes as d e s c r i b e d i n Methods s e c t i o n 2.5.4 l i p i d s i n each s p o t .  scraped,  digested  i n 1.5  and a s s a y e d f o r i n o r g a n i c t o determine  iodine ml  70%  phosphorus  the a u a n t i t y of phospho-  - 55 -  2.7  Detoxification of Endotoxin  2.7.1  Sodium Hydroxide Treatment  This method of detoxifying Neter and co-workers (82).  endotoxin  heated at 5 6 ° C for 1 hour. acetic  mixing  the  ethanol. ing  for  acid  After  in 6.0 ml 0.25  solution  lipopolysaccharide of  endotoxin  the lipopolysaccharide  several  hours  N NaOH.  lipopolysac-  The solution was then  After cooling, the solution was neutralized with  and then the  neutralized  based on the study reported by  B r i e f l y , 100 mg of Escherichia c o l i  charide (Difco) were dissolved  glacial  is  on  ice,  10,000 x g for  10 minutes.  charide p e l l e t  was  the  reprecipitated  180  ml  cold  was allowed to precipitate  ethanolic  The ethanol  resuspended  with  was  was  solution  discarded  in approximately  was  absolute by  stand-  centrifuged  and the  15 ml double  by  at  lipopolysacd i s t i l l e d H^O  and l y o p h i l i z e d . 2.7.2 It be  Sodium Periodate Treatment was  also demonstrated  detoxified  by reacting  this procedure, in 80 ml double  by Neter and associates that  them with  sodium periodate  100 mg of Escherichia c o l i distilled  H£0.  To this  during  incubated  which  time  at  the  room temperature solution  was  mixture was then dialyzed against distilled  H2O  was  changed  every  solution  distilled  10 ml 1.0  hours.  to  dissolved  N sodium acet-  solution were added.  stirred.  H2O at 4 ° C for  could  According  in the dark for 6 and 1/2  continuously  12  (82).  lipopolysaccharide were  ate buffer pH 5.0 and 10 ml 0.1 M sodium periodate mixture was  endotoxins  The  The hours  reaction  36 hours.  The  dialysis,  the  Following  endotoxin solution was lyophilized and stored at 4 ° C for future  use.  - 56 -  2.7.3 Treatment with Hydroxy]amine The method for  detoxifying  endotoxins  based on the procedure described line hydroxylamine solution was in  ethanol  solution  with a solution  was  distilled 0.375  prepared  distilled  H20  and  prepared  by f i r s t  (193).  by mixing a solution  dissolving  in ethanol.  0.5  mixed  with  first  14  ml  An alka-  The NaOH/ethanol  gm NaOH in  dissolved  2.0  in  95% ethanol.  ml  double  Similarly,  1.0  The  ml two  double ethanol  solutions were mixed together on ice and after standing for approximately minutes, the mixture was centrifuged of  this  freshly  prepared  alkaline  to remove the precipitated  hydroxylamine supernatant  mixed with 100 mg Escherichia c o l i  lipopolysaccharide.  tion  The fine was  constant minutes.  suspension  incubated stirring.  at  of endotoxin  room  in the  temperature  under  This  alkaline  The lipopolysaccharide  pellet  at  taken  and  supension  of  glass homoceni-  for  1  hour  soluwith  10,000 x g for 5  was washed once with 20 ml cold 95%  ethanol, once with 20 ml 0.01  M acetic acid in 95% ethanol  20 ml 95% ethanol.  pellet  The final  20  20 ml  hydroxylamine  nitrogen  The solution was then centrifuged  NaCl.  were  endotoxin was thoroughly homogenized manually with a ground zer.  is  of 2.5% NaOH  was mixed with 18 ml 95% ethanol.  hydrochloride was  then  with hydroxylamine  by Mclntire and co-workers  of 2.5% NH^OH.HCl  H^O and then this  gm hyroxylamine  by treating  was resuspended  and once more in  in approximately  15 ml  double d i s t i l l e d H^O, lyophilized and stored at 4 ° C for future work. 2.8 Radioactive Labelling of Endotoxin 51 CrCl^  (New England  bed by Braude  Nuclear)  and associates  was  (194).  used  to  label  The endotoxin  endotoxin  used for  as  descri-  radiolabelling  studies was lipopolysaccharide extracted from Escherichia c o l i 026:B6 by the 51 Boivin method (Difco). A " Cr-buffer solution was prepared by taking 5  - 57 -  mCi  of  CrClg  (specific  activity  50-500  mCi/mg)  dissolved  0.5 M HCl and adding to this 9.9 ml sodium phosphate buffer composed ly  of  2.6  mM NaH ? P0 4 -4.5  mM Na 2 HP0 4 ~2.9  10 mg endotoxin were dissolved  tion  and the mixture was  constant against  stirring. double  4°C for  future  The radioactive  distilled  every 12 hours.  incubated at  H^O for  After d i a l y s i s ,  room temperature  endotoxin  36  hours  solution  at  4°C.  0.1  pH 7.0  mM NaCl.  in every 1 ml of this  in  ml  that was  Approximate-  ^Cr-buffer for  36  was  then  solu-  hours  with  dialyzed  The H^O was  changed  the endotoxin was l y o p h i l i z e d and stored  experimentation.  The specific  activity  of  the  at  radioactive  endotoxin was approximately 20,000 cpm/yq t o x i n . 51 2.9  Cr-Endotoxin Binding Studies  2.9.1 Membrane Preparations Most of the r a d i o l a b e l e d erythrocyte ghost membranes. (3.0-4.0  mg protein/ml)  NaCl-15 mM T r i s resuspended  endotoxin binding studies were done on human 2.0 ml of an erythrocyte membrane preparation  were  centrifuged  pH 7.0 buffer.  After  in 8.0 ml 0.9% NaC 1-15  and  resuspended  the second wash,  mM Tris  buffer  pH 7.0  twice  in  0.9%  the membranes were to give  a protein  concentration of approximately 0.60 mg/ml. The incubation mixture, which totaled 2.0 ml, consisted of 0.5 ml membrane suspension, 0.9% NaC1-15 mM Tris  pH 7.0  buffer  and the desired  volume  (usually  0.1  ml)  of  Cr-endo-  51 toxin  dissolved  (0.5  solution was  always  was used for  all  NaCl-Tris  buffer  toxin was added.  mg/ml)  sonicated  in to  NaCl-Tris  buffer.  insure a homogeneous  The  'Cr-endotoxin  suspension  before  binding assays.  The membranes were pre-incubated  solution  for  After  incubated at 37°C for  at  37°C  5 minutes  before  the  it  in the  radiol abelled  addition of the endotoxin, the mixture was further  15 minutes.  The incubation mixture was then c e n t r i -  - 58 -  fuged  and the membrane pellet  cold NaCl-Tris buffer was  resuspended  suspension  was  in  was  washed  twice by resuspension  and centrifugation. 1.0  ml  assayed  for  NaCl-Tris  in 2.0 ml  After the second wash, the p e l l e t  buffer.  radioactivity  A 0.2  by l i q u i d  ml  aliquot  of  this  s c i n t i l l a t i o n counting  methods as described in section 2.11.1. 2.9.2  Intact Erythrocytes  Red blood  cells  obtained  from  freshly  drawn blood were  utilized  for  51 Cr-endotoxin  binding experiments.  The heparinized blood was  centrifuged  and the red blood c e l l s were washed twice in 4 volumes cold 0.9% NaCl-15 mM Tris buffer pH 7.0 to remove a l l traces of plasma and buffy coat. second wash, the erythrocytes were resuspended final  haematocrit  of approximately  20% (e.g.  After the  in the NaCl-Tris buffer  to a  2.0 ml packed red blood  cells  were resuspended in buffer to a total  volume of 10 ml). A 0.3 ml aliquot of 51 this red blood c e l l suspension was used for Cr-endotoxin binding studies as described for membranes in section 2 . 9 . 1 .  2.10 Liquid S c i n t i l l a t i o n Counting 51 Cr  is  a gamma emitting radioisotope,  gamma emission possible  if  is  liquid  only  9%.  Therefore,  scintillation  of  ^Cr  is  almost  identical  scintillation  counting was  scintillation  counter.  better  with  the  using  frequency  counting are  of  decay  efficiencies  used  to  detect  by are the  the combination of which amount to  Cr (195).  done  the  counting methods  emission of X-rays and Auger electrons, 51 80 % of the decay frequency of  but  Since the pulse spectrum  the  of  tritium  height (195),  channel  of  spectrum all  the  ^Cr liquid  - 59 -  2.11 Preparation of Samples for S c i n t i l l a t i o n Counting 2.11.1 Plasma Membranes 51 Following  the  procedure  used  for  the  Cr-endotoxin/membrane  assay (section 2 . 9 . 1 ) , a 0.2 ml aliquot of the final placed d i r e c t l y into a glass s c i n t i l l a t i o n v i a l of  a 1:2  The  mixture of  contents  minutes. were  and  after  thorough  thoroughly  mixed  counter to be counted after 2.11.2 Intact  for  and to this was added 0.6 ml  (New England Nuclear)  of the s c i n t i l l a t i o n vial  were  The  membrane suspension was  :95% ethanol  were then incubated  at  solution.  60°C  for  45  The samples were then cooled, 15 ml Biofluor (New England Nuclear)  added  samples  Protosol  binding  mixing, 0.5  ml 0.5  once  and  again  N HC1 was  placed  in  a  added.  The  scintillation  1 hour of dark and temperature ( 1 0 ° C )  adaptation.  Red Blood Cells  procedure  for  preparing  samples containing  intact  red  blood  cells  s c i n t i l l a t i o n counting was i d e n t i c a l to the procedure used for membranes  except  that  after  the 45 minute incubation at  the samples were cooled and 0.3 the  samples.  additional  The vials  30 minutes.  were  ml 30% H 2 0 2  loosely  capped  60°C  was  with  added  Protosol:ethanol,  dropwise  and incubated  at  to  bleach  60°C  for  The samples were then cooled and mixed with  an  15 ml  Biofluor and 0.5 N HC1 as described for membranes in section 2.11.1. 2.11.3 Tissues 51 Tissues  obtained  from animals  treated  with  ed for s c i n t i l l a t i o n counting by adding 1.0 ar)  to 50 mg blotted tissue  until  the  tissue was  ml Protosol  in a s c i n t i l l a t i o n v i a l  completely  samples were then cooled and 0.1 samples.  Cr-endotoxin were  solubilized ml 30% H 2 0 2  (usually was  prepar-  (New England Nucle-  and digesting several  added to  at  55°C  hours).  The  decolorize  the  The v i a l s were loosely capped and incubated at 55°C for another 30  - 60 -  minutes. (New  The samples were cooled once more and mixed with 10 ml Econofluor  England  Nuclear).  The  vials  were  allowed  to  equilibrate  in  the  s c i n t i l l a t i o n counter for 1 hour before they were counted. 2.11.4 Plasma 51 Blood plasma obtained from for  Cr-endotoxin treated  s c i n t i l l a t i o n counting by simply mixing  Biofluor  50-100  animals ul  plasma  was  prepared  with  15 ml  (New England Nuclear) and counting in a s c i n t i l l a t i o n counter using  the tritium channel. 2.12 Animal  Studies  Animals used for endotoxin studies were always anaesthetized ostomized before each experiment.  Urethane (1000 mg/kg) served as an accep-  table anaesthetic  for both guinea pigs  commonly used for  these experiments.  right carotid artery were catheterized. for  and rats which were the animals most Both the right jugular  vein  and the  The jugular vein catheter was used  injection purposes while blood samples  the carotid artery.  and trache-  were taken from the catheter in  At the end of each experiment, the animals were s a c r i -  ficed by giving an overdose of  anaesthetic.  - 61 -  CHAPTER 3 Results 3.1 Some Physiological Effects The intravenous  of Endotoxin Administration  administration  of  bacterial  endotoxin  into  animals causes marked haemodynamic changes which are dose- and In general, in  a sub-lethal  dose of endotoxin produces  blood pressure which returns  time.  to  normal  an almost  levels  after  laboratory  time-related. immediate f a l l  a short  period  of  Larger doses of endotoxin produce greater decreases in blood pressure  and after  a partial  recovery,  the mean blood pressure can decline  steadily  with time to shock l e v e l s .  Haemodynamic changes can also be demonstrated  the microcirculatory level  in various  organs following  injections  of  at  endo-  133 toxin tissue is  by monitoring the sites  of  injection.  highly d i f f u s i b l e  proportional times  clearance 133  and  its  rate of  to  as  "nutritional"  or  in larger  an  clearance  to the c a p i l l a r y flow to that  referred  of  radioisotopes  Xenon, being  from blood flow which occurs effect  of  area.  such  inert  as  l i p o p h i l i c molecule,  from an injection  site  This blood flow is  "nutritive"  flow  to  endotoxin to eleven  rats on the mean blood pressure and n u t r i t i v e flow in skeletal Blood pressure was measured  carotid artery while n u t r i t i v e flow in the quadriceps  shunts.  is  some-  distinguish  vessels or arteriovenous  administering 4.0 mg/kg E. c o l i  hind limb is shown in Figure 4.  Xenon from  it The  anaesthetized muscle of the from the right  muscle was determined  - 62 -  EH  BLOOD  PRESSURE  NUTRITIVE  IO men  I hr  a  hr  FLOW  3 hr  ENDOTOXIN  HEMORRHAGE  Figure 4. Effect of Endotoxin and Haemorrhage on Blood Pressure the Rat.  and Nutritive Flow i n  Comparison of mean carotid a r t e r i a l blood pressure and n u t r i t i v e flow in quadraceps muscle i n haemorrhaged rats (n = 10) and rats treated with 4.0 mg/kg E . c o l i endotoxin (n = 11). Nutritive flow was measured by Xe clearance. Data from haemorrhaged animals were taken at the point of i r r e v e r s i b l e shock as defined i n r e f . 247. Results are expressed as % pretreatment ( i n i t i a l ) value. Haemorrhagic shock n u t r i t i v e flow is s i g n i f i cantly greater (.01 > P > .001) than flow 3 hr post-endotoxin. i 3 6  - 63 by the al.  133  Xenon clearance  (196).  It  is  technique  and calculated  evident from Figure 4 that  according  by 10 min after  the endotoxin, the mean blood pressure had decreased 56% of  normal  average value  while of  the  nutritive  31% of  the  flow  control  was  or  to  Lassen  i n j e c t i o n of  to an average value of  more markedly  pre-endotoxin  reduced  to  an  rate.  It  is  flow  interesting to note that while the mean blood pressure showed a recovery to an average value of 80% of normal by the t h i r d endotoxin was  injected  nutritive  flow  that  apparent  was  examine model,  the  rate  (at  which time the experiment  remained  at  10 min after  behavior  haemorrhagic  essentially  of  nutritive  shock was  the  unchanged endotoxin  flow  was  from was  of  substantial  hour after  the  terminated),  the  the  reduced  given.  in a different  induced in a group  To  10  normal  reservoir  pressure.  until  The animals  their mean blood were  additional  bleedings  or reinfusions  volume was  returned  to  the  maintained of  animals.  pressure at  this  blood u n t i l At this  was  artery 1/3  blood  the  shock  urethane-anaesthe-  30% of  point  rate  further  experimental  tized rats by bleeding the heparinized animals from the femoral a pressurized  et  of  into their  pressure  the  total  remainder  of  by  bled the  haemorrhaged blood was reinfused. Nutritive  flow  determinations  made at two different  in the  time i n t e r v a l s .  haemorrhaged  The f i r s t  when the mean blood pressure had decreased  blood pressure nutritive  in the  flow rates  (data not shown). flow in skeletal  face  of  normal  be 22% and 14% of  shock. normal  Therefore, haemorrhage can markedly affect muscle.  rats  pressure  to maintain this  impending haemorrhagic  were found to  of  were  was performed at the moment  to 1/3  second when 30% of the bled volume was reinfused  group  and the decreased  The measured respectively the n u t r i t i v e  An attempt was made to measure n u t r i t i v e flow in  - 64 haemorrhaged rats under conditions more closely endotoxin-treated  animals  by reinfusing a l l  approximating  those  in the  the haemorrhaged blood into the  animals following the development of shock in order to make the blood volume and average mean blood pressure similar to that seen three hours after toxin  administration.  greater effect  Under  on skeletal  The data presented  these  circumstances  endotoxaemia  showed  in Figure 4 on haemorrhagic  the rat hind limb determined 5 min after to  animals  shock  show the  receiving  E.  total  coli  average  muscle of  reinfusion of the blood.  endotoxin,  rats  in  (.01  62%  of  the  > P > .001)  normal.  normal  flow  rate.  This  rate  is  In  haemorrhagic  shock had considerably higher n u t r i t i v e flow rates, which were on the age,  a  muscle n u t r i t i v e flow than did haemorrhagic shock.  mean blood pressure and average nutritive flow in the quadriceps  comparison  endo-  significantly  averhigher  than the 3 hr post-endotoxin mean n u t r i t i v e flow of 30% of  These results serve to emphasize the marked s e n s i t i v i t y to endotox-  in of n u t r i t i v e flow, a c r i t i c a l determinant of tissue survival  in states of  circulatory impairment. Tissue  injury from a variety of causes would be expected  the release of lysosomal hydrolases  into the plasma.  acid phosphatase a c t i v i t y at various times after (026:B6) was Figure  5.  administered  The blood  intravenously to  sampling  had  higher  10 mg/kg E.  (P < .001)  coli  plasma  endotoxin  (native  10 mg/kg E. c o l i  endotoxin  on  hr following the administration of endotoxin. enzyme continued to increase  rats  plasma  acid  is  shown in  phosphatase  However, rats which  endotoxin)  acid phosphatase levels  in  in plasma  levels in the control animals as indicated in Figure 5. received  result  The increase  anaesthetized  no effect  to  had  than control  The levels  significantly animals  of this  by 1  lysosomal  dramatically in the plasma with time u n t i l  the  - 65 -  NATIVE ENDOTOXIN  (0 h 2 D  UJ tn <  I a  U)  • I a  NaOH DETOXIFIED' PERIODATE OETOXIFIEDo  CONTROL  u T I M E  ( h r s )  Figure 5. Effect of Native and Detoxified E. c o l i Endotoxins on Plasma Acid Phosphatase A c t i v i t y in the Rat. Blood samples (1.0 ml) f o r acid phosphatase analysis were taken from each r a t at hourly intervals after injection of the native or detoxified endotoxins (10 mg/kg) for a period of 5 hr. Five animals were used in each experimental group except the native endotoxin-treated group (n = 10) and control group (n = 10).  - 66 animals  expired,  injection effects  of of  which  this  usually  large  native  dose  virtually  of  endotoxin,  E.  sodium hydroxide or periodate had  no effect  occurred between endotoxin. coli  treatment  on plasma  5 and 6 hr following the In  endotoxin  effects  tions  under these  in the plasma  acid  conditions.  level of this  which  phosphatase  These  lysosomal  contrast was  (Methods sections  Also unlike the native endotoxin, the detoxified lethal  marked  activity  results  the  detoxified  2.7.1.  endotoxins  to  by  and 2.7.2) (Figure  5).  were devoid of  suggest  that  eleva-  hydrolase may provide a useful  measure of endotoxin t o x i c i t y in vivo. To determine i f the effect ity  in the plasma  was peculiar to  than  acid phosphatase  ments  were conducted  assays for dase  and  animals  (2.0  cathepsin  D.  on  D activities  three plasma  levels period. respond  in  acid  Thus,  activities.  pigs  experimental  effects  enzymes other  comparable  with  the  in guinea pigs.  animals  these  intravenous  employed,  Significant  Also, the a c t i v i t i e s  endotoxaemia It  is  remained  by  unaltered  indicate  displaying  interesting  to  note  the  mg/kg dose of  E. c o l i  endotoxin  N-acetyl-e-glucosaminidase  enzymes were apparent  experiments  experi-  inclusion of  conditions  receiving a 2.0  of  phosphatase,  lysosomal  increase,  activ-  enzymes, namely N-acetyl-B-glucosamini-  the  the  lysosomal  elevations  by 1 hr following the  of these enzymes continued  throughout  that  guinea  the pigs,  elevated  plasma  that  patterns  the  and  in a c t i v i t y  as the condition of the animals deteriorated with time. control  to  guinea  than 5 hr after  injection of the endotoxin. to increase  and i f  be found to  lysosomal Under  plasma  the rat  anaesthetized  Figure 6 depicts  mg/kg)  all  in  survived no longer  cathepsin of  could also  two additional  endotoxin.  of endotoxaemia on lysosomal hydrolase  experimental like  lysosomal of  Enzyme  rats, enzyme  increase  in  - 67 -  7 • ACID  5  PHOSPHATASE  •  <  A-  I  5  J  a  £ 7 -  <  5  J  a _  3  <  a U) a • < CD  <  j a  E  GLUCOSAMINIDASE 25  SO  15  TIME  (hr)  Figure 6. Effect Pigs.  of  E. c o l i  Endotoxin on Plasma  Lysosomal  Enzyme A c t i v i t y  in Guinea  Blood samples (1.3 ml) were taken from each urethane anaesthetized guinea pig at hourly intervals after the injection of endotoxin (2.0 mg/kg) for a time period of 5 hr. Controls received no endotoxin. Enzyme a c t i v i t i e s are as defined in the Methods section. Data represent mean ± S.E.M. of 10 animals.  - 68 activities instance, hr after  of  the  three  plasma  lysosomal  N-acetyl-e-glucosaminidase  of  acid  different  enzymes  is  apparent  increase  in the  that  plasma  from these experiments enzymes  tial of  in c l i n i c a l  sepsis  humans in gram-negative tions  of  obtained General for  lysosomal from  in  Hospital  plasma  all  response  to  of a l l  the a c t i v i t i e s to  endotoxin  three  increasing  the enzymes.  be used  D in  lyso-  doses  of  in plasma enzyme a c t i v i t y  of  Nonetheless, several  monitor  could be septicaemia  more  accurately  the  it  is  representacondition of  also display elevated  To explore  admitted  to  the  this  phosphatase,  through episodes  assessed,  Intensive  provided  that  plasma concentra-  possibility,  (a number of whom werein gram-negative  acid  cathepsin  guinea pigs 2 hr after  where the progress of patients  enzymes.  patients  (5 h r ) .  A method such as this would have great poten-  situations  gram-negative  that  probably can  animals during endotoxaemia.  and  although the levels  and endotoxin dose is not the same for  lysosomal  of acid phospha-  up to. the point of death  endotoxin, the relationship between the elevation  tive  For  a maximum value by 3  N-acetyl-e-glucosaminidase  groups of urethane anaesthetized It  coincide.  of varying doses of endotoxin on the plasma activ-  phosphatase,  administration.  apparent  not  the injection of endotoxin whereas the a c t i v i t i e s  Figure 7 shows the effect  somal  do  a c t i v i t y reached  tase and cathepsin D continued to increase  ities  enzymes  Care  blood Unit,  samples  Vancouver  sepsis) were assayed  N-acetyl-p-glucosaminidase  and  cathepsin  D  activities. Table 1 shows the plasma a c t i v i t i e s patients  studied,  along  with  the  obtained from healthy volunteers. of  two categories;  those  patients  of each of these enzymes in the 36  normal  plasma  The patient in shock  lysosomal  enzyme  data were placed (blood  levels  into either  pressure < 90/60)  and  - 69 -  • I  '  2  •  3  E N D O T O X I N  •  4  »  5  (mg/kg )  1  B  i i i  Effect of Varying Doses A c t i v i t y in Guinea Pigs.  of  E. c o l i  Endotoxin on Plasma  Lysosomal  Enzyme  Each dose of endotoxin was administered intravenously to guinea pigs and blood samples for lysosomal enzyme analysis were taken at 2 hr following the injection of endotoxin. Each dose-group consisted of 10 animals except for the 6 mg/kg dose-group (n = 5). Data are presented as mean ± S.E.M.  Plasma lysosomal enzyme a c t i v i t i e s Acid  TABLE 1. in patients with septicaemia and/or shock  Phosphatase  Cathepsin D * *  Glucosaminidase  (units/ml plasma)  * (a) Controls (b) Gm -ve septic shock  (absorbance/ml  plasma)  0.31 ±  .01  0.20 ±  .05  0.95 ±  .06  1.35 ±  .49  5.27 ± 1.13  3.30 ±  .57  0.60 ±  .11  1.04 ±  2.24 i  .32  (n = 13; 1 survivor) (c) Other shock  .34  (n = 10; 4 survivors) Statistical  significance  N.S.  (b v_s c)  (d) Gm -ve sepsis, no shock  .0025 > p > .0005  N.S.  0.74 ±  .28  1.00 ±  .26  2.13 ±  .33  0.57 ±  .31  0.48 ±  .28  1.60 ±  .15  (n = 9; 6 survivors) (e) Gm + ve sepsis, no shock (n = 4; 3 survivors) Statistical  significance  (d vs_ e)  N.S.  N.S.  Values are given as mean ± SEM, n = number of experimental as defined in the METHODS. groups  (b-e)  Statistically  significant  for the three enzymes studied  increases  (Student's t t e s t ,  N.S.  subjects and  enzyme a c t i v i t y units  r e l a t i v e to control were present p values  ranged from < 0.05  in a l l  to < 0.001).  P values for inter-group s t a t i s t i c a l comparisons are as indicated. N.S. = No s i g n i f i c a n t difference. * n = 15, 27 and 26 for acid phosphatase, cathepsin D, and glucosaminidase data, r e s p e c t i v e l y .  **  See F i g . 3 for individual  values.  are  bacteraemic patients without shock. that the majority of the patients in  sepsis-without-shock  (9/13  is  consistent  known  that occurs  with  the  It  is  interesting to note from Table 1  in shock (13/23 or 57%)  or 69%) had gram-negative high  incidence of  in North American hospitals  today.  were generally i d e n t i f i e d by blood culturing cases  where gram-negative  manifestation  septicaemia  of c h a r a c t e r i s t i c  was  clinical  basis  of  independent evaluations  cians.  Consistent with the results  gram-negative septic patients activities  of  (.001 > P)  and cathepsin  activities  of  gram-negative  techniques. strongly  signs  patients  bacteraemia.  This  septicaemia  In our study,  bacteraemias  However,  suspected  in some  due  peculiar to t h i s  but could not be confirmed with blood cultures, on the  and the  the diagnosis  to  the  infection,  was  assigned  made by two collaborating  physi-  from our animal studies on endotoxaemia,  in shock exhibited s i g n i f i c a n t l y higher plasma  acid phosphatase  (.025  > P > .01),  N-acetyl-e-glucosaminidase  D (.001 > P) than c o n t r o l s .  these enzymes were also elevated  Although  in c r i t i c a l l y  with shock from causes other than gram-negative bacteraemia, were less than those seen in patients  the ill  the  with gram-negative shock.  plasma patients  activities The activ-  i t y of cathepsin D, in p a r t i c u l a r , was s i g n i f i c a n t l y higher in gram-negative shock  plasma  than  (.0025 > P > .0005).  in  plasma  of  These results  patients  with  suggest that  enzyme a c t i v i t y in plasma of patients  ative b a c i l l i ,  of  shock  in gram-negative shock is not e n t i r e l y in a l l types of shock.  such as the release of endotoxins from gram-neg-  may be responsible for much of the lysosomal enzyme a c t i v i t y  seen in patients lysosomal  types  the elevation of lysosomal  due to compromised tissue perfusion which is present Rather, additional factors,  other  enzyme  infected with these organisms. activity  is  less  markedly  The observation that plasma elevated  when  gram-negative  - 72 septicaemia  is  endotoxaemia  than when shock  is  shock, present  in Table 1 , however,  interesting perhaps  uncomplicated with  be used as  a fairly  is  the  specific  may indicate 1 ) .  (Table  What  indication  marker for  a lesser  that  is  degree  particularly  cathepsin  gram-negative  and the a c t i v i t y of t h i s protease in the plasma may be useful  of  D can  bacteraemia  in aiding the  diagnosis or evaluating the progression of a gram-negative septic episode. When the cathepsin D data from a l l as  in Figure 8,  gram-negative (i.e.  it  is  strong  evident that only two of  studied are  the  indicated  possibility  the that  presented,  thirteen patients  shock had plasma cathepsin D a c t i v i t i e s  false-negatives).  patients  the patients  in the normal  in  range  Interestingly, while blood cultures from both these presence their  of  gram-negative  shock  states  organisms,  may have  been  there  due to  was  a  factors  other than gram-negative septicaemia.  For example, one patient had endocar-  ditis  a prosthetic  from  a septic  focus  involving  other patient had severe gastrointestinal static  pancreatic carcinoma at  lysosomal  enzyme analysis.  the time when blood  With  regard  valve while  to  samples  false-positive  were taken for data,  exhibited higher than normal  patient with a septicaemia caused by $-haemolytic streptococcus,  lysis. was  (streptolysins)  Nonetheless, this patient's  still  shock patients.  had normal  elevated values.  plasma cathepsin  an organism  capable of causing c e l l u l a r  (5.27  units)  It  also  is  Figure 8 that of the nine gram-negative septic one  in a  plasma cathepsin D a c t i v i t y ( 3 . 1 6 units)  lower than the mean value  gram-negative septic  Figure 8  The highest value ( 3 . 1 6 units) was found  which can produce potent toxins  the  haemorrhage in addition to a meta-  reveals that f i v e of ten "other" shock patients plasma cathepsin D a c t i v i t y .  aortic  seen  of  in  interest  the to  plasma  of  note from  patients without shock, only  D a c t i v i t y while  the  others  had somewhat  - 73 -  O.B •  0>4  (0  <  I  s •  J  a.2  a IO  CONTROL  > r  h U  < O R A M + VE o CAROIOSENIC  to a ui z  GRAM  -VE •  GRAM  +VE  J  r  0 a i-  (J  Q U  <  2  I  L  • RAM -VI SHOCK  OTHER SHOCK  SEPSIS Nb SHOCK  Figure 8. Comparison of Plasma Cathepsin D A c t i v i t i e s in Patients Sepsis and/or Shock.  C r i t i c a l l y 111 with  Each point represents the plasma cathepsin D a c t i v i t y in one patient with sepsis and/or shock. In this study, shock was defined as a blood pressure reading of less than 90/60. Inset shows the range of plasma cathepsin D a c t i v i t y in 27 healthy volunteers.  Thus, while the information from human studies ted,  it  enzymes  does  suggest  that  the  high  plasma  ( p a r t i c u l a r l y cathepsin D) seen  be simply due to the consequences but rather  additional  factors,  are  results  with resultant somal  from animal  pressure  (2 (to  (see  of  lysosomal  septicaemia  in  studies  mg/kg) 75 ±  plasma. to  the release  of endotoxins  from the  Consistent with this hypothe-  examining  At 3 hours  guinea  12% of  Figure 6).  cannot  of a state of compromised tissue perfusion  such as  pigs,  the  effect  only  following modest  c o n t r o l , mean ± S.D.)  shown) when appreciable elevations occurred  concentrations  substantia-  of  hypotension,  impairment in tissue perfusion, on the accumulation of  hydrolases  endotoxin  to be  in gram-negative  invading organism, could also be responsible. sis  needs  the  administration  decreases were  lysoof  in mean blood  observed  (data  not  in plasma lysosomal enyzme a c t i v i t i e s had  For comparative  purposes,  guinea  pigs  were bled  from the carotid artery to 50% of their normal mean blood pressure and maintained at this pressure for 3 hours by additional withdrawals or reinfusions of blood.  Despite the fact  that the haemorrhaged animals were subjected  more severe hypotensive conditions, their plasma lysosomal were  significantly  Table 2. cells  lower than those  seen  in various  tissues,  resulting  in c e l l u l a r  lysosomal enzymes into the c i r c u l a t i o n . ing whether endotoxins are  enzyme a c t i v i t i e s  in endotoxaemia  Thus i t seems l i k e l y that endotoxins may exert death  As an i n i t i a l  to  as  indicated  in  direct  actions on  and the  leakage of  approach to determin-  indeed capable of such actions,  experiments were  devised to examine the a b i l i t y of endotoxins to bind to c e l l u l a r membranes. El  3.2 Binding Studies with Escherichia  coli  Cr-Endotoxin  lipopolysaccharide  (026:B6)  method were obtained from Difco Laboratories  extracted  (Detroit)  by  the  Boivin  and were r a d i o l a b e l -  TABLE 2. Comparative effects of experimental endotoxaemia and haemorrhage on lysosomal hydrolase a c t i v i t i e s  in plasma  Plasma lysosomal hydrolase a c t i v i t y Acid Phosphatase (units/ml plasma) Controls  (  Increase control)  Cathepsin D (units/ml plasma)  (  Increase control)  Glucosaminidase (Absorb/ml plasma)  (  Increase control)  2.05 ± .11  —  1.28 ± .05  —  14.33 ± .54  4.94 ± . 4 1 a ' b  2.4  4.76 ± . 5 0 a ' b  3.7  25.28 ± 2 . 0 1 a ' b  1.8  2.27 ± .13  1.1  2.95 ± . 3 4 a  2.3  17.70 ± 1.01 a  1.2  (n = 10) Endotoxaemia (n = 10) Haemorrhage (n = 6) Endotoxaemia  values  were  obtained  3  hr  haemorrhagic conditions were made 3 hr after mean blood pressure,  following  a  S i g n i f i c a n t l y elevated  (P < .01)  b  S i g n i f i c a n t l y elevated (P < .02)  of  E.  coli  endotoxin.  Measurements  under  guinea pigs had been bled to produce a 50 percent reduction in  n = number of experimental  significance was assessed by Student's  injection  animals.  A l l values  t test. r e l a t i v e to control group. r e l a t i v e to haemorrhage group.  are  given as mean * SEM;  statistical  led  with  CrCl^  (New England  Nuclear)  as  described  in  Methods  section  Cr radionuclide bound t i g h t l y to the endotoxin and was  associ-  51 2.8.  The  ated with the proteolipid components of  the t o x i n .  This  is  illustrated  in  51 Figure 9 where Cr-endotoxin was disrupted by hydroxylamine treatment (see Methods section 2.7.3) and separated into three fractions by Sepharose 6B chromatography. A l l three f r a c t i o n s , which contained protein and phospholipid  (the  latter  not  shown for  simplicity),  contained  radioactive  51 Cr,  with the component appearing between fractions 40 and 60 being most 51 heavily l a b e l l e d . The Cr did not bind to a 2-keto-3-deoxyoctonate (KDO)-containing component which appeared as a peak between fractions 60 and 51 80  (not  shown in Figure 9).  Thus, the  Cr seems to  preferentially  bind  to proteolipid components of endotoxin. 51 Experiments  were then designed  capable of binding to c e l l s erythrocytes used  for  2.9.2.  to determine  and c e l l  Human erythrocyte  ghosts  as  the  Cr-endotoxin was  membranes in a s p e c i f i c  provided a convenient model  the binding study was  if  system  described  for  study.  manner.  The procedure  in Methods section  (approximately  300 yg  Human  2.9.1  protein)  and  incubated  51 with 25 yg/ml Cr-endotoxin exhibited binding which could be largely prevented by the addition of cold (or unlabelled) toxin (Figure 10). 51 Approximately 75 % of the bound Cr-endotoxin was displaceable under these experimental conditions. The data in Figure 11 (bottom) indicate that haemoglobin-free  membranes  are  capable  of  binding  considerably  protein  than  greater  51 quantities cytes 5.0  of  Cr-endotoxin per mg membrane  (1.0 ml of packed red blood c e l l s was considered  mg ghost  protein).  It  also  can be  noted  from  intact  erythro-  to be equivalent  this  Figure  that  to the  51 binding of  Cr-endotoxin to red c e l l  ghosts  and  intact  cells  appeared  to  - 77 -  • iaa  so  aa  BO  BO  too  F R A C T I O N IMO.  Figure 9. Chromatography of  c o l i Endotoxin.  ^Cr-endotoxin was treated with hydroxylamine (as described in the Methods section) and 5.0 mg was placed on a Sepharose 6B column (qel = 1.6 x 90 cm) and eluted with 0.9% NaCl - 15 mm t r i s -.02% NaN3 (pH 7 . 6 ) . 2.0 ml fractions were collected and analyzed spectrophotometrically at 254 nm (closed c i r c l e s ) . 50 yl aliquots of each fraction were analyzed for radioa c t i v i t y by s c i n t i l l a t i o n counting (open c i r c l e s ) .  - 78 -  IOO  r  '  1  ioo  S O  I S O  B O O  UNLABELLED ENDOTOXIN ( u g / m l )  Figure 10. Displacement of Bound ^ C r - E . Membranes by Unlabelled Endotoxin.  co  i-j  Endotoxin  from  Human  Erythrocyte  Effect of varying concentrations of unlabelled E. c o l i endotoxin on binding of 5 1 Cr-endotoxin (25 yg/ml) to human erythrocyte membranes (300 yg protein). Results are expressed as % of 5 1 Cr-endotoxin bound in the absence of unlabelled endotoxin. Each value represents the mean ± S.D. of 3 separate experiments done in t r i p l i c a t e .  - 79 -  Figure 11. Comparison of 5 1 C r - E . Erythrocyte Membranes.  coli  Endotoxin  Binding  to  Human  Erythrocytes  and  The lower graph compares the binding of varying concentrations of ^•'•Cr-endotoxin to human erythrocyte membranes ( s o l i d line) and red c e l l s (dotted l i n e ) . The binding procedure is described in Methods section 2.9.1 and 2.9.2. 1.0 ml packed red blood c e l l s was considered to be equivalent to 5 mg membrane protein. Each value represents the mean ± S.D. of 3 separate experiments done in t r i p l i c a t e . Upper graphs show Scatchard plots of the binding data.  - 80 involve two classes (up  to  of binding sites  concentrations  ghosts.  This  latter  of  the  intact tion  step-wise cells.  from  confirmed  the  intact c e l l s (apparent were  latent  haemolysis  binding  existence  procedure  present  dissociation from  used  to  Scatchard  more than  one  prepare  of  (Figure 11, upper panels). constant  linear  Kp)  portions  and of  maximal  the  was  membranes  from  informa-  binding s i t e  plots  course  employed.  binding  the  associa-  during the  Binding  Scatchard  in  quantitative  analysis class  only  non-specific  to obtain more detailed  data,  of  and ghosts  estimated  being  membrane components exposed  In an attempt  these  endotoxin)  non-saturating component  component in ghosts may r e f l e c t  tion of endotoxin with of  400 yg/ml  with a t h i r d  This  in both  characteristics capacity  as  (Bmax)  summarized  in  Table 3. It  is  apparent  from this  Table that  at  endotoxin concentrations  less  than 200 yg/ml, the binding sites on the erythrocyte ghost membrane displayed a higher a f f i n i t y  for  the r a d i o l a b e l e d  E. c o l i  endotoxin than did the  sites on intact red c e l l s .  Secondly, the endotoxin binding capacity seen in  the ghosts was also greater  than that seen  tions of endotoxin less than 50 yg/ml. the association trations  in the red c e l l s  As mentioned e a r l i e r ,  regard  seems unlikely that these have physiological  endotoxin may well in vivo.  saturable  binding  sites  of endotoxin in excess of 50 yg/ml  even in experimental  interaction  the nature of  a non-specific type of  affinity,  for  the  likely  With  blood concentrations  to  is  adsorption.  tered  concentra-  between erythrocyte ghost membranes and endotoxin at concen-  of endotoxin exceeding 300 yg/ml  it  for  endotoxaemia.  be most  The high  representative  Our e a r l i e r  observations  of  of  intermediate  relevance because are rarely encoun-  affinity  binding  membrane s i t e s  (Figure  10)  that  of  sites toxin  binding of  - 81 -  TABLE 3. 51  Binding characteristics  of  and erythrocyte ghosts.  Cr-endotoxin in intact human erythrocytes  [Data were derived from the Scatchard plots depicted in Figure 13.]  K D (Mg/ml) [  Cr-endotoxin]  (ug/ml)  < 50 50-200 > 200  Intact red c e l l s  Bm,„ Ghost  (ug/mg membrane protein)  Intact red c e l l s  Ghost  65  15  2  6  720  125  15  16  2900  145  - 82 51  Cr-endotoxin may be effectively  cative  of a specific  3.3.1  is  indi-  characterized  present on red c e l l membranes (197).  of Endotoxin on Red Blood Cell Membranes  was  occurred  system.  interest  a result  to  of  examine  the  what  interaction  functional  consequences,  of  endotoxin with  E. c o l i  Again, the human erythrocyte ghost was used as Initial  inhibitory to  of  as  membranes.  investigations  effect  on  Na+,  indicated  that  an  presence  of  However, the results were rather d i f f i c u l t  to  of  (Difco)  a Ca + + -dependent  contain C a + +  ATPase which  To circumvent this  toxin  of  an  had  in  greatly  rocyte plasma membranes.  examined.  endotoxin  diminished  the activation  represents  a model membrane  and  was  the  activity  enzyme and  the  absence of  Mg++  inhibitor basal  portion  of  both  activity,  and K + .  also  ambiguity,  of  i.e.  the the  The a c t i v i t i e s  inhibited by approximately 50% at  It  K  resulted  present  in  in eryth-  the effect  of endo-  (K + -NPPase),  stimulated  both  because  which  enzyme,  was  is evident that endotoxin  activity of  interpret  + + Na ,K -ATPase  the  The results are shown in Figure 12.  effective  is  the  and this  K + -nitrophenylphosphatase  + K -stimulated  the  the  plasma  Na +  enzyme  so  coli  any,  of  this  K + -ATPase a c t i v i t y  commercially prepared endotoxins  on  E.  if  ability  stimulate  endotoxin.  is  toxin  Erythrocyte Ghosts  It  K+  by unlabelled  interaction which may involve previously  lipopolysaccharide receptors 3.3 Effects  antagonized  of of  component  of  the  in  the  components  was  the these  enzyme  low (50 yg/ml) concentrations  of  endotox-  ++ in.  The Mg -stimulated  sensitive  to  inhibition,  500 yg/ml inhibited this emphasize  that  component of the NPPase enzyme was very much less  endotoxin  so  that  endotoxin  a c t i v i t y to the extent binding  integrity of plasma membranes.  is  capable  concentrations of only 30%. of  modifying  as  high  as  These r e s u l t s the  functional  - 83 -  Figure 12. Effect of E. c o l i Endotoxin on K + - and A c t i v i t y in Human Erythrocyte Membranes.  Mg + + -p-Nitrophenylphosphatase  (Mg + + ,K + )-p-Nitrophenylphosphatase a c t i v i t y was determined in human erythrocyte membranes (Methods section when these were incubated o in the absence and presence of varying concentrations of endotoxin at 3 7 ° C . Reaction time was 1 hr. The results are an average of 3 separate experiments done in duplicate.  - 84 3.3.2  Intact Red Blood C e l l s  One simple and convenient method of examining functional endotoxin binding to intact  cells  is to investigate the effect  on the s t a b i l i t y of red blood c e l l s  to hypotonic challenge.  for  in Methods 2.3.  these experiments  the marked protective toxin on red c e l l occurring  in the  is  described  effects  of  increasing  haemolysis  at  37°C  absence of  consequences  expressed  endotoxin.  as  of endotoxin  The methodology  Figure  concentrations  13  illustrates  of E. c o l i  a percent  A 50% reduction  of  of  endo-  haemolysis  in haemolysis  was  Q  obtained mal  at  an endotoxin concentration  protection  occurring at  2 mg/10^ c e l l s ) .  a 4-fold  of 0.5 mg/10  greater  endotoxin  The mechanism of the protective  toxin may involve a direct membrane s t a b i l i z i n g argued  that  endotoxin  is  acting  out of the red blood c e l l s , tonic l y s i s .  This l a t t e r  red c e l l s ,  by increasing  the  or  it  14).  hypotonic  lysis  of K + from the  Therefore,  endotoxin  protects  by a mechanism other  leakage of  human  red  blood  (where  by endotoxin cells  the p r e - l y t i c  from  leakage  cells.  using Wistar r a t s , the effects cytes to hypotonic l y s i s comparing rat  of endotoxin were largely  performed  of endotoxin on the s t a b i l i t y of rat erythro-  were also investigated. and human red c e l l s  nantly l y t i c action of endotoxin in the rat ation  K+  however, because the  phase of haemolysis  than increasing  Since studies of the in vivo actions  Figure 15,  by endo-  could also be  less than 10% haemoglobin release had occurred) was unaffected (Figure  (i.e.  more resistant to hypo-  explanation can be ruled out, in the early  afforded  pre-lytic  thereby making the c e l l s  release of K + from red blood c e l l s  concentration  effect  action  with maxi-  seen in human erythrocytes.  at  The r e s u l t s summarized in 20°C,  as contrasted  revealed  a predomi-  with the  These two dramatically  opposing  stabilizeffects  - 85 -  mg  E N D O T O X I N /  I O  9  R B C  Figure 13. Antihaemolytic Effect of E . c o l i Endotoxin on Human Red Blood C e l l s . Varying concentrations of endotoxin were incubated with human red c e l l s at 37°C for 15 min before the addition of a hypotonic solution as described in Methods section 2.3. Haemolysis was determined by the absorbance of free haemoglobin at 540 nm. Results are expressed as % haemolysis seen when no endotoxin was present. The data represent the mean of three separate experiments done in t r i p l i c a t e .  - 86 -  Figure 14. Effect cytes  of  E. c o l i  Endotoxin on P r e - l y t i c Leakage  of  K + in Human Erythro-  Human erythrocytes were incubated in the presence (open c i r c l e s ) and absence (closed c i r c l e s ) of 500 pg endotoxin under condition identical to those in haemolysis experiments (Methods section 2.3) except that the NaCl concentration was varied such that control haemolysis (no endotoxin) ranged from 1% to 50% of total haemolysis (lysis in d i s t i l l e d H 2 O ) . Supernatants were analyzed spectophotometrically at 540 nM for haemoglobin and by atomic absorption spectrometry for K content. Data represent the mean of 3 experiments assayed in duplicate.  - 87 -  1  s mg  •  '  •  *  a  e  8  ia  E N D O T O X I N / l O  9  R B C  Figure 15. Effects of Increasing Concentrations S t a b i l i t y of Rat and Human Red C e l l s .  of  E. c o l i  Endotoxin on the  Osmotic  Haemolysis experiments were conducted at 20°C as described in Methods section 2.3. Results are the mean ± S.D. of t r i p l i c a t e experiments u t i l i z ing 3 different blood samples.  - 88 of  endotoxin  probably  occur  by virtue  of  the  macromolecular complex in a manner similar to amphipathic molecules To further  (e.g.  local  amphipathic  nature  the known effects  ties of endotoxin was examined.  on the s t a b i l i z i n g  small  stability.  of endotoxin on human and rat  cytes the effect of varying temperature  this  that  anaesthetics) have on red c e l l  investigate these actions  of  erythro-  and l y t i c a c t i v i -  Figure 16 shows that the degree of  stabili-  9 zation  produced by a fixed  was markedly temperature It  is  interesting  rat red blood c e l l s cells  when the  is  even at 5 ° C .  of endotoxin  that  endotoxin, which  haemolysis  test  is  conducted  red  cells)  at  37°C.  seen with increasing temperature,  A similar  of  endotoxin  although no l y s i s  in human red  on endotoxin  cells  temperature.  lysis  of  is  greater  occurred, temperature  could  involve a  However, no  binding to human erythrocytes  of  these  trend  in that  One possible explanation for this marked effect action  the  stabilize  incubated with normal human red c e l l s  reduction in toxin binding with decreasing temperature  enhances  at lower temperatures, can s i g n i f i c a n t l y  on the s t a b i l i z i n g  of  (4 mg/10  dependent.  to note  seen when endotoxin is protection  concentration  effect  could be demon-  strated (Table 4). The observation  that  E. c o l i  endotoxin protected human erythrocytes  hypotonic l y s i s to a much greater degree than rat that  certain  influence  the  compositional  antihaemolytic  from both human and rat by thin  layer  membranes lesser  action  erythrocytes  chromatography,  contained  amounts  entities  of  greater  it  of of  red  endotoxin.  became apparent  (Sph)  cell  membrane When  were analyzed for  quantities  sphingomyelin  the  red blood c e l l s  of  that  suggested  may  plasma  from  greatly  membranes  phospholipid content the  rat  phosphatidylcholine  than did human erythrocyte  erythrocyte (PC)  but  membranes  - 89 -  200  • HUMAN /\(l_CAT DEFICIENT)  in  > j • 150  Ui  I  J •  a h Z • U  ioo  RAT 50 HUMAN (CONTROL)  1  ,  ia  1  sa T E M P  1  30  a  o  ( ° C )  Figure 16. Temperature-Dependent Erythrocytes.  Effects  of Endotoxin  on the Osmotic  S t a b i l i t y of  Erythrocytes from normal humans, rats and from a patient with congenital deficiency of plasma l e c i t h i n - c h o l e s t e r o l acyltransferase (LCAT) a c t i v i t y were incubated with E. c o l i endotoxin (4 mg/io" red c e l l s ) at various temperatures. Data for human and rat erythrocytes represent the average of 3 different blood samples while the LCAT-deficiency patient results are from a single experiment.  - 90 TABLE 4. Effect of temperature on the binding of  51 Cr-labelled  lipopolysaccharide (serotype 026:B6, lot number 669176) by human erythrocytes  Temperature  Endotoxin binding  Vc)  The results  are  from three different  5  327 ±  90  10  360 ±  53  15  487 ±  92  25  330 ± 155  37  387 ±  the mean ± SD of healthy volunteers.  11  experiments  using  Concentration of  erythrocytes Cr-endo-  toxin was 25 yg/ml. *  Binding is expressed as ng toxin bound per 10  8  erythrocytes.  as  indicated  PC/Sph  in Table  ratio,  fluidity,  which  5.  is  Thus,  rat  known to  than human red c e l l  erythrocyte  be  an  important  membranes.  acyltransferase  (LCAT)  had  seen in rat red blood c e l l s . in  the  haemolysis  effects  of  normal  endotoxin  rat red blood c e l l s that  were  human erythrocytes,  the  attempt  differences rocytes  to  a variety  challenge  (Methods  different  temperatures.  and what is cells  make-up  since  approximating  that  in  was found  different  similar  to  temperature-dependent  from  those  membranes  investigate  the  is  obtained  seen with  then suggest  very  important  effects  of  compositional  of endotoxin, eryth-  in  the  presence  and  absence  of  endotoxin  Figure  17 shows  the  results  of  these  all  species  is  were  a  that  stabilized  of  subjected  the  by endotoxin  any temperature.  These  with  interaction.  membranes on the actions species  that  the  These observations  plasma  that  erythrocytes were used  animal  at  rabbit  membrane  of  none were  blood c e l l s  ratio  of endotoxin-red c e l l  further  however, that PC/Sph r a t i o endotoxin  of  immediately apparent  examined,  human red  2.3)  of  but s t r i k i n g l y similar to the results  in red blood c e l l  from  of  as shown in Figure 16.  compositional  an  results  completely  determinant of the consequences In  a membrane PC/Sph  the  it  a higher  deficiency in l e c i t h i n - c h o l e s -  When the LCAT patient's  experiments,  have  determinant  Interestingly,  erythrocytes from a patient with a congenital terol  membranes  to  to  the  a hypotonic and  experiments of  red blood  same extent  experiments  at  did  as  reveal,  alone does not determine the membrane actions of  and  cat  erythrocytes,  human c e l l s  (198),  which  exhibited  have  PC/Sph  temperature  ratios profiles  quite comparable to those of the r a t .  S i m i l a r l y , no obvious c o r r e l a t i o n was  found  of  between the  cholesterol  temperature-dependent  effects  content  the  erythrocytes  of endotoxin on osmotic  studied  fragility.  and the  TABLE 5. Thin layer chromatographic analysis  of phospholipid p r o f i l e s from normal human  erythrocytes, rat erythrocytes and erythrocytes from a patient with a congenital of plasma l e c i t h i n :  deficiency  cholesterol acyltransferase (LCAT)  Total Phospholipid Phosphorus Species  P Serine  Human (control)  28.6 ± 1.1  11.8 ± 1.0  30.6 ± 1.9  24.8 ± 2.0  1.2  Rat  27.9 ± 0.9  14.1 ± 0.5  43.3 ± 1.3  10.7 ± 1.7  4.0  Human (LCAT deficient)  18.6 ± 0.1  6.7 ± 1.1  55.6 ± 0.1  15.0 ± 0.1  3.7  Results expressed as mean ± SD.  P Choline  Sphingomyelin  P Choline r a t i o Sphingomyelin  P Ethanolamine  - 93 -  •J 10  1  1 30  ea T E M P  l  —  ao  ( °C )  Figure 17. Comparison of the Temperature-Dependent Effects of Endotoxin on the Osmotic S t a b i l i t y of Erythrocytes from Various Animal Species. The concentration of E. c o l i endotoxin used in these experiments mg/10^ red c e l l s . The data represent the average of 3 different samples from each species.  was 4 blood  - 94 In further  experiments  to  investigate  the  role  of  membrane  structural  components in influencing the a b i l i t y of endotoxin to protect human erythrocytes  from hypotonic  neuraminidase,  lysis,  red  cells  were  enzymatically  trypsin or phospholipase A (see  modified  Methods 2 . 4 . 1 ,  2.4.2,  using 2.4.3)  which altered the membrane carbohydrate, protein and phospholipid components of  the  erythrocytes,  respectively.  shown in Figure 18. appreciably cells  affected  is  the  A treatment  treatment  contrast, membrane protect  is  treatment,  these c e l l s of  the  had  lysis.  no  PC  content  at  by  on  the  phospholipid components  of  the  endotoxin s t a b i l i z e s  ability  it  is  of  60%.  In  75% of  the  endotoxin  indicate that  membrane  is  of  temperatures.  approximately  the  which  The effect  higher  These results  are  the modified red  removed approximately  influence  that  experiments  A treatment.  apparent  which  suggest  these  the only enzyme treatment  from hypotonic l y s i s .  Our results  of  endotoxin to protect  especially  neuraminidase acid,  that  phospholipase  membrane  determining how effectively tonic  was  reduces  sialic  integrity  apparent  a b i l i t y of  from hypotonic l y s i s  phospholipase This  It  The results  to the  important in  erythrocytes  to hypo-  probably the a b i l i t y  of  the  hydrophobic ( l i p i d ) component of the endotoxin to interact with the erythrocyte membrane that has been affected  by phospholipase A treatment.  To examine the influence of the l i p i d component of endotoxin on red c e l l stability,  we undertook  experiments  using  chemically modified  wherein the l i p i d region of the complex was altered hydroxylamine treatment  (Methods 2.7.1,  to hydrolyze the ester-linked fatty acids toxin complex. ide treatment  Hydroxylamine treatment in the  sense that  it  2.7.3).  endotoxins  by sodium hydroxide or  These treatments  are known  in the l i p i d A region of the endois more drastic  removes  amide as  than sodium hydroxwell  as  ester-linked  - 95 • TRE ATE  x U N T R E A T E D  •  N E U R A M I N I D A S E  0)  in > j too • £ UJ I j • a Z • CJ  ao T E M P  30  4 0  ( ° C )  Figure 18. Temperature-Dependent Effects fied Human Erythrocytes.  of  Endotoxin  (4  mg/10^  red  cells)  on  Modi-  Human erythrocytes wre modified enzymatically with neuraminidase, trypsin and phospholipase A as described in Methods section 2.4. Results represent the mean ± S.D. of 3 experiments.  - 96 fatty  acids  endotoxin ability  in l i p i d A. by  of  sodium  these  We have also modified the carbohydrate regions  periodate  (NalO^)  carbohydrate- and  treatment  (Methods  2.7.2).  1ipid-modified endotoxins  to  human red blood c e l l s was then examined at various temperatures to  the  effects  of  control  (unmodified)  experiments are shown in Figure 19. lipid  regions  of  antihaemolytic portion  endotoxin with  action  of  had no effect  treatments  (NaOH,  It  that  whatsoever. and  yield  NaOH- and  human red  blood  cells,  endotoxins  still  had a l y t i c  The  it  is  particularly effect  at  hydroxylamine-modified endotoxin  37°C,  had  stronger  is  that  these  substances  four  different  forms  are that  not  toxins  homogeneous  may be resolved  but  to  on the  all  are  these  much  less  was the obser-  enhanced  lytic  studies  NH20H-modified  that  although  37°C.  NaOH- and  these  carbohydrate  known that  low temperatures.  sodium hydroxide-treated toxin p a r t i c u l a r l y at with  the  interest  endotoxins  at  of  endotoxins  Of p a r t i c u l a r  NHgOH-treated  of  that modification of the  alteration  Incidently,  NalO^)  and compared  had a marked effect  NH2OH  The  stabilize  The results  apparent  endotoxin, whereas  NH2OH  the  is  NaOH or  toxic than native endotoxin in vivo. vation  endotoxin.  of  the  lysis  However, a  lesser  actions  of  these extent.  than  the  The interpretation  complicated  rather  by  the  of  at  consist  of  fact least  by Sepharose 6B chromatography.  Unmodified endotoxin, which normally appears as a homogeneous macromolecular complex  (Figure  2 0 , top),  weights  following  sodium hydroxide treatment. except peak III  from  four  molecular  material  ranging  yields  1 x 10  when E. c o l i  6  (peak  peaks  I)  A l l peaks  to  (bottom 1 x 10  consisted  of  4  graph) (peak  endotoxin  was  and carbohydrate. modified  with  of IV)  proteolipid  which was found to be g l y c o l i p i d in nature.  I and II consisted of protein, l i p i d obtained  discernable  Peaks  Similar r e s u l t s were hydroxylamine  (Methods  - 97 • CONTROL o DETOXIFIED  PERIOD ATE 60  40  SO  (0  Hi >  J • I  SODIUM 160  J • II h 2 • U  A CONTROL * DETOXIFIED • CONTROL • DETOXIFIED  HYDROXYL A MINE  •-_ .  W  I  HYDROXIDE  ——•  140  ISO  IOO  80  BO  aa I O  30  SO  T E M P  ao  ( ° C )  Figure 19. Effects of Detoxified Endotoxins on the Osmotic S t a b i l i t y cytes as a Function of Temperature.  of Human Erythro-  E. c o l i endotoxin was chemically modified (detoxified) by treatment with periodate, sodium hydroxide and hydroxylamine as described i n Methods section 2.7. Toxin concentration in a l l experiments was 4 mg/10^ red cells. Each point represents the mean of 3 experiments performed on 2 different blood samples.  - 98 -  PROTEIN  o-o KDO  CONTROL  E c -  If)  .3  tu  Ul L) 2 <  ID <  CD If)  h <  to a • cn  E c  •  .1  h < Ul  CJ  Z  < CD  •  •  OB  .1 S  •4  • .08  a o  ul CD <  »oa  .OS  SO  4Q  FRACTION  60  BO  IMO.  Figure 20. Fractionation of Sodium Hydroxide-Detoxified Endotoxin. Sodium hydroxide detoxified E. c o l i endotoxin (5.0 mg) was fractionated by Sepharose 6B gel f i l t r a t i o n chromatography (gel height = 1.6 x 35 cm) using 0.9% NaCl - 0.02% NaN3 - 15 mM T r i s (pH 7.0) as eluant. 1.0 ml fractions were collected and analyzed for protein, KDO and phospholipid, the l a t t e r being present in a l l fractions but not shown for sake of c l a r i t y . Upper graph shows the separation p r o f i l e of native (control) endotoxin.  - 99 -  2.7.3)  except' that  greater  quantities  of  the  were formed as shown in Figure 21 (bottom). for  l y t i c a c t i v i t y using the haemolysis  III  enhanced the l y s i s  that  the g l y c o l i p i d component (peak III) toxin,  probably  greater  lytic  explains  activity  why  than  g l y c o l i p i d material  test,  i t was found that  only  in hypotonic buffer.  contained  a  greater  hydroxylamine-modified  proportion of  sodium hydroxide-modified d e r i v a t i v e .  ty of the phospholipid components  Endotoxins red c e l l s  determinant are  capable  of of  the  of  action  exerting  depending on such factors  species from which the red c e l l s  of  E.  either as  coli  stabilizing  ambient  lytic  red  abolishes  actions  an  on  and the animal of  the antihaemoly-  of the formation of of  In  cells.  Furthermore, alteration  as the result lysis  or  on  temperature  were obtained.  g l y c o l i p i d components which enhance the  endotoxin  had  integri-  the human erythrocyte membrane is  the l i p i d portion of the endotoxin complex i t s e l f t i c action of endotoxin, possibly  endo-  endotoxin  conclusion, the studies thus far have indicated that the structural  important  peak  The fact  than did sodium hydroxide-treated  the  the  III  When a l l four peaks were tested  of human erythrocytes  hydroxy!amine-hydrolyzed endotoxin  peak  smaller  human red blood c e l l s  in  hypotonic solution. 51 3.4  Cr-Endotoxin Binding In Vivo The in v i t r o studies mentioned above have shown that endotoxin binds to  plasma  membranes  and can  influence  possibly the physical state of c e l l s .  some membrane-dependent The next step in these  activities  and  investigations  51  was to administer Cr-labelled E. c o l i endotoxin in vivo in order determine i f the endotoxin showed preferential binding to any organ  to or  tissue and i f the t o x i c i t y of endotoxin in vivo could be correlated with the 51 association  of  Cr-endotoxin with  some target  organ.  For  these  experi-  - 100 -  • — • PROTEIN  o -o KDO  CONTROL  • .5  0 in h <  ui u 2 <  m a.  HYDROX YLAMINE DETOXIFIED  o  (0  .08  o  .04  so  Figure 21. Fractionation of Hydroxylamine-Detoxified Endotoxin Hydroxylamine-detoxified E. c o l i endotoxin (5.0 mg) was fractionated by Sepharose 6B gel f i l t r a t i o n chromatography (gel height 1.6 x 35 cm) using 0.9% NaCl - 0.02% NaN3 - 15 mM T r i s (pH 7.0) as eluant. 1.0 ml fractions were collected and analyzed for protein, KDO and phospholipid, the l a t t e r being present i n a l l fractions but not shown f o r sake of c l a r i t y . Upper graph shows the separation p r o f i l e of native (control) endotoxin.  - 101 merits,  51  Cr  labelled  doses (1.0,  E.  3.0 and 6.0  thetized guinea pigs. plasma was  coli  endotoxin was  mg/kg)  given  intravenously  to three separate groups  of  at  urethane-anaes-  Blood samples were taken at hourly intervals  assayed for  acid phosphatase,  a lysosomal  three  and the  enzyme whose a c t i v i t y  in plasma has previously been shown to correlate well with endotoxin t o x i c i 51 ty  (see  Figures 5,  stration, portions  a blood  6 and 7).  Three hours following  Cr-endotoxin admini-  sample  taken,  were  (approximately  tissue  were  (Methods  removed,  2.11.3).  was 50 mg)  of  blotted  the  guinea  liver,  and  pigs  spleen,  processed  kidney,  for  The experimentally-measured  sacrificed heart  scintillation  the  administered dose of  endotoxin  and was  lung  counting  ^Cr-endotoxin binding  ng/mg wet weight) for each tissue was standardized by expressing to  and  and  correlated  it  with  (in  relative  the  plasma  acid phosphatase a c t i v i t y following toxin infusion.  (expressed as Sigma units/ml plasma) at 3 hr In the binding data depicted in Figure 22 for 51 only Cr-endotoxin binding to the lung correlated acid phosphatase a c t i v i t y (r = 0.964). Two other  three different organs, p o s i t i v e l y with plasma 51 organs  examined  results  (not  for  Cr-endotoxin binding  depicted  graphically  between endotoxin binding to  here)  guinea pig  were  liver  showed  liver  a  seen for  The spleen  correlation  did not exhibit  any obvious  spleen.  negative  and plasma  a c t i v i t y which was very similar to that  and  The  correlation  acid phosphatase  the kidney in Figure 22. between ^Cr-endotoxin  binding and plasma acid phosphatase t i t e r s . Quantitatively, binding the  the  liver  and spleen  ^Cr-endotoxin (approximately  other  organs  by macrophages  studied.  which are  This abundant  showed the  12-16  greatest  capacity  for  ng/mg tissue/dose) compared 51  may r e f l e c t in both the  the  uptake  liver  of  and the  to  Cr-endotoxin spleen.  Lung  - 102 -  KIDNEY  Ul 0) 0 5 •  LUNG  Q 3 z • z  E  .  5  HEART  ACID PHOSPHATASE ( UNITS / ml ) Figure 22. Correlation Between Accumulation of 5 lCr-Endotoxin i n Various Organs with T o x i c i t y .  Guinea Pig  The amount of Slo-endotoxin (ng/mg wet weight) in kidney, heart and lung tissues 3 hr following, the administration of either 1.0 mg/kg (n = 3 ) , 3.0 mg/kg (n = 3) or 6.0 mg/kg (n = 2) 5 * C r - E . c o l i endotoxin to guinea pigs was correlated with plasma acid phosphatase a c t i v i t y . The contribution of dosage on the tissue content of radiolabelled toxin was corrected for by dividing the amount of toxin bound in the tissues (ng/mg wet weight) by the administration dose of toxin in mg/kg.  - 103 tissue  also  contains  macrophages  and  some of  the  51 Cr-endotoxin  apparent  "binding" may be due to endotoxin uptake by lung macrophages. results  strongly suggest that  may be an important suggestion,  determinant  one would predict  native endotoxin in their was  tested  relative 0.01)  and  the  and  were  results  However,  well)  it  are  in v i v o .  endotoxins  23.  From this  may differ  lung t i s s u e .  shown in Figure  can  that  tissue.  carbohydrate  also  be  lung tissue  from  This prediction  A decreased  into  seen  as  toxins  from  of  periodate  endotoxin,  2 hr  pigs  Figure  binding  23  after  at  a  effectively  as  native  se  is  these  dose  that  E.  (which is known to detoxify  of coli  endotox-  endotoxin.  It  not sufficient  for  in v i v o , but, in addition, the endotox-  inducing some functionally  Since  portion  effect  at  guinea  binding to lung tissue per  be capable of  target  > P > 0.005)  intravenously  an endotoxin to have a lethal  crucial  detoxified  with sodium periodate  bound to  seems, therefore,  the  toxicity  a b i l i t y to bind to  injected  endotoxin treated  in must  endotoxin  that  NH 2 0H-detoxified (0.01  3.0 mg/kg.  as  of  of endotoxin with lung tissue  to native endotoxin was seen with both NaOH-detoxified (0.02 > P >  substances  in  the interaction  However, our  relevant  detoxification this  in determining these deleterious  component  modifies of  consequences  the  perturbation the  in  antigenic  molecule  seems  of endotoxin binding,  including those involving the lung. While  alterations  in  perturb target tissues  the  ability  of  endotoxin  interact  with  are undoubtably important in determining the  in vivo t o x i c i t y of  chemically-modified toxins,  assess  the  of  these  substances.  effects  to  modification  Figure  24  on  shows  the the  i t was  also of  of  altered  interest  to  properties  of  disappearance  of  pharmacokinetic rate  and  51 Cr-labelled  detoxified  toxins  from plasma  following  intravenous  admini-  -  -  104  Ul  D  •  (J) (fl  Ul  Ul >  ID  < 2  Z D J CD  Ul  ui  X  1•ui  2  0  < j >• X X •  Iui •  0  I  E Z X  •  a  2  1  0 a h 0 • z  DC K Ul u a •  I-  d ui  •  e -  X •  • • >• I  a  UJ O)  EIMDOTOXIIM  Figure 2 3 . Accumulation of Pig Lung Tissue.  51  Cr-Labelled  Native  and  Detoxified  Endotoxins  in Guinea  The accumulation of native (toxic) and chemically detoxified 5 1 C r - l a belled E. c o l i endotoxins in lung tissue (ng/mg wet weight ± S.E.M.) was compared in 4 groups of 5 guinea pigs at 2 hours following the administration of r a d i o l a b e l e d endotoxins ( 3 . 0 mg/kg). S i g n i f i c a n t l y lower quantities of NaOH-detoxif ied toxin ( . 0 2 > P > . 0 1 ) and N H J J O H - detoxified toxin ( . 0 1 > P > . 0 0 5 ) accumulated in lung tissue in comparison to native endotoxin.  - 105 -  I  2  TIME  ( hrs )  Figure 24. Clearance of Pig Plasma.  ^•'•Cr-Labelled  Native  and  Detoxified  Endotoxins  from  Guinea  Comparison of plasma concentrations of native and chemically detoxified 51cr-E. c o l i endotoxins (yg/ml plasma ± S.E.M.) in guinea pigs at 1, 30, 60 and 120 min following an intravenous injection of the radiolabelled toxins (3.0 mg/kg). Data represent an average of 5 animals for each group except native toxin (serotype 668735) which consisted of 11 animals.  - 106 stration 20,  40,  into anaesthetized 60,  guinea pigs.  and 120 min after  scintillation  counting.  injection  Based  on  the  concentration  of  and the  the  (3.0 mg/kg) and the estimate of total (25-30 ml),  Blood samples were taken at plasma  amount  of  was  1.0,  prepared  endotoxin  for  injected  blood volumes of the guinea pigs used  endotoxin  present  complete mixing would be approximately 85 ug/ml.  It  in is  the  plasma  after  interesting to note  from Figure 24 that the only endotoxins which approached this concentration at  1.0  min were  the  NaOH- and  NH20H-detoxified endotoxins.  concentrations of these endotoxins  at  1.0 min are  concentration of the periodate-treated  plasma  contrast  to the  endotoxin (approximately 25 ug/ml)  this  same time period.  fell  somewhere in between the two extremes  ins.  in sharp  The  The 1.0 min concentrations of the native  at  endotoxins  seen with the detoxified endotox-  It is clear from these preliminary experiments that chemical d e t o x i f i -  cation  may exert  different  effects  on the  ability  of  toxins  to  bind  to  target t i s s u e s , on their i n t r i n s i c a c t i v i t y once bound and on their rate of removal from the c i r c u l a t i o n . lar modified toxin is ous  Thus, the biological a c t i v i t y of any p a r t i c u -  determined by a complex interplay between these v a r i -  properties.  3.5 Study of Drugs as Possible Endotoxin Antagonists 3.5.1 Antagonists to Endotoxin Binding In Vitro The objective  of this  series of experiments was to test  the a b i l i t y of  51 various  drugs  to antagonize  the binding of  Cr-labelled E. c o l i  to membranes using the human erythrocyte ghost purpose of these experiments was to find useful  in antagonizing  membrane l e v e l .  the  effects  of  as  endotoxin  an in v i t r o model.  The  an agent that would be p o t e n t i a l l y endotoxin  in  vivo  at  the  cellular  - 107 Figure endotoxin  25  reveals  stabilizes  that  lidocaine,  a membrane  human erythrocytes  against  active  drug,  hypotonic  which  lysis,  does  appreciably affect the binding of E. c o l i endotoxin to human red c e l l even  in  concentrations  as  high  as  10 mM.  Similarly,  like not  ghosts  methylprednisolone,  which has occasionally been used in the management of certain types of shock (including  gram-negative  sepsis),  did  not  antagonize  the  binding  of  the  51 Cr-endotoxin mental  to  the  conditions  quaternary  erythrocyte  used  ammonium  membrane  (Figure  26).  0.5  and 2 mM concentrations  endotoxin to analysis, Figure  erythrocyte  propranolol  27  shows  concentrations  the  Table 2).  of  appeared effect  to  of  unlabelled propranolol  membranes  acting  antagonized  3.5.2. Effectiveness  When the  effect  on the  binding of  E.  coli  reciprocal  plot  a competitive  on endotoxin  at endotoxin concentrations  can be seen that  in both cases,  antagonist of E. c o l i at  of  antagonist.  of  of Endotoxin Antagonists several  drugs,  a measure  the results  of  of 50 to 200 ug/ml  d, 1-propranolol  s i t e s were  in v i t r o , to counteract  of these experiments  Estimated  approximately  In Vivo  including those shown to  endotoxin  appears  28).  of endotoxin in vivo was examined u t i l i z i n g elevations as  binding  endotoxin binding.  these two classes of  binding of endotoxin to membranes  summarizes  endotoxin  toxin.  as  0.4 x 10" 3 M (Figure 27) and 1.0 x 10~ 3 M (Figure  levels  its  inhi-  K.j values for propranolol  phatase  and  ranging from 5-50 ug/ml while Figure 28 shows a similar  It  propranolol  experi-  d,l-propranolol  was subjected to double be  the  toxin  to act as a competitive  The a b i l i t y  under  at  bitory action of propranolol (see  However,  analogue, pranolium, effectively  binding almost to the same degree as 0,  preparation  toxicity  antagonize  the toxic  effects  in plasma acid in  vivo.  the  phos-  Figure  29  which were performed using male  - 108 -  a.5 BO  5  7.5  IOO  ISO  Id  LIDO.(mlVl)  SOOENDO.(ug/ml)  C Q N C ,  Figure 25. Displacement of Bound Membranes with Lidocaine.  coli  Endotoxin  from  Human  Erythrocyte  A b i l i t y of lidocaine to displace 5 1 C r - E . c o l i endotoxin from human erythrocyte ghosts i s compared to the' displacement p r o f i l e obtained with cold (unlabelled) E. c o l i endotoxin. Results are expressed as % of the amount of ^Cr-endotoxin bound in the absence of drug or unlabelled toxin (100% bound). Concentration of 5 1 Cr-endotoxin was 25 pg/ml. Data represent the mean ± S.D. of 3 separate experiments.  - 109 -  Figure 26. Effect of Methyl prednisolone, Pranolium and Endotoxin Binding to Membranes.  Propranolol  on  51rjr-E.  coli  Effect of varying concentrations (0-2.0 mM) of methyl prednisolone, pranolium and d,l-propranolol on the displacement of 5 *Cr-endotoxin bound to human erythrocyte membranes is compared to unlabelled E. c o l i endotoxin. Results are expressed as percent of the amount of ^^Cr-endotoxin bound in the absence of drug or unlabelled toxin (100% bound). Concentration of SlCr-endotoxin was 25 yg/ml.. Data represent the mean ± S.D. of 3 separate experiments (S.D. for drugs not shown for sake of c l a r i t y ) .  - 110 -  Figure 27. Double Reciprocal Plot of ^ C r - E . c o -|j Membranes in the Presence of Propranolol.  Endotoxin  Binding  to  Erythrocyte  The binding of S l p - E . c o l i endotoxin (5-50 ug/ml) to human red c e l l membranes in the presence of 0, 0.5, and 2.0 mM d,l-propranolol are expressed as a double reciprocal plot. Data represent the mean of 3 separate experiments.  - Ill -  Figure 28. Double Reciprocal Plot of ^ C r - E . C0 ]-j Endotoxin Binding tions) to Membranes in the Presence of Propranolol.  (High  Concentra-  Results of 51Cr-E. c o l i endotoxin (50-200 yg/ml) binding to human erythrocyte membranes in the presence of 0, 0.5 and 2.0 mM d,l-propranolol are presented as a double reciprocal p l o t . Data represent the mean of 3 experiments.  - 112 -  01 h O DRUG  2  3 CPZ •  01  .25 mg/ kg •  _ PRANDLIUM  <  ,S mg/kg HYDROCORTISONE 35 mg/kg  I a  I  in •  I a a  d . P R O P R A N O L O L . I mg/kg  •  CONTROL  < T I M E  ( hrs  )  F i g u r e 29. E f f e c t o f V a r i o u s Drugs on Plasma A c i d Phosphatase Treated Rats.  A c t i v i t y i n Endotoxin-  Each o f t h e i n d i c a t e d drugs was g i v e n as a s i n g l e i n t r a v e n o u s i n j e c t i o n t o s e p a r a t e groups o f p e n t o b a r b i t a l a n a e s t h e t i z e d r a t s (n = 5 f o r each drug) 10 min b e f o r e E. c o l i e n d o t o x i n (10 mg/kg) was a d m i n i s t e r e d . Plasma from b l o o d samples ( 1 . 0 ml) o b t a i n e d a t h o u r l y i n t e r v a l s from each r a t was assayed f o r a c i d phosphatase a c t i v i t y . C o n t r o l a n i m a l s (n = 5) r e c e i v e d no drug o r e n d o t o x i n whereas 0 d r u g - t r e a t e d r a t s (n = 10) r e c e i v e d o n l y endot o x i n (10 mg/kg). Data r e p r e s e n t mean ± S.E.M. In comparison t o 0 drug group, s i g n i f i c a n t l y lower a c i d phosphatase a c t i v i t i e s were seen w i t h : Pranolium Hydrocortisone d-propranolol  .05 > P > .025 at 2 hr .05 > P > .025 a t 5 hr .025 > P a t 2, 3, 4, and 5 h r .  - 113 Wistar r a t s .  Drug-treated  animals  received  a single  of the drug, at the dose indicated 10 min before endotoxin was given. promazine,  a bolus of 10 mg/kg E. c o l i  pranolium and hydrocortisone were able to  stages of endotoxaemia. shortly  after  Animals pretreated  receiving  the  bolus  of  endotoxin  with the d-isomer  2 hr and for  propranolol  was  (data  of  from the  not  shown).  propranolol, which reduc-  acid phosphatase  the duration of the experiment.  indistinguishable  later mg/kg)  a highly significant  in the a b i l i t y of endotoxin to elevate plasma at  some degree of  with d,l-propranolol (0.1  is devoid of appreciable beta blocking a c t i v i t y ,  was seen  offer  of the endotoxin, p a r t i c u l a r l y during the  However, when rats were pretreated  tion  injection  It is evident from Figure 29 that drugs such as chlor-  protection from the effects  died  intravenous  racemate  as  an  levels  The d-isomer  of  antagonist  for  endotoxin binding to red c e l l membranes in v i t r o (data not shown). 51 The effects  of various  drug pretreatments  on  Cr-endotoxin binding to  guinea pig lung tissue following in vivo administration of toxin were next examined.  It was discovered that a parallelism existed  of endotoxin antagonists of  the toxin to  effective  in  efficacy  in v i t r o and their a b i l i t y to decrease the binding  lung tissue  vitro  between the  endotoxin  in vivo.  For example,  antagonist,  reduced the binding of endotoxin to than did any of the other drugs  lungs  propranolol,  significantly  (.005  the  > P > .002)  in vivo and to a greater  shown in Figure 30.  most  Experimental  extent animals  pretreated with pranolium at a dose equivalent to that used for  d-proprano-  lol  decrease in  (0.1 mg/kg),  pulmonary  also exhibited a significant  endotoxin  content seen  binding.  The  reduction  in the methylprednisol one-treated  did not achieve s t a t i s t i c a l  significance.  (0.05 > P > 0.02) 51 in  Cr-E.  coli  group of animals  endotoxin (Figure  30)  - 114 -  111  Ul D J  • a h  ID  D j  ia-  2 • 1)  E  X  •  iii • 2  (l) 2 • 111  a a J  Ul B •  _  Ul 2  •  Hi  2  UJ 2  >• I  i-  UJ  j • j • 2 <  Ul 0 <  j  • 2  2 < (J • • j  a a • a  <  LT  a  •  e  •  a • 2  Ul  2  -  0)  c  Figure 30. Effect of Pig Lung.  Drug  Pretreatment  on  Accumulation of  51  Cr-Endotoxin  in Guinea  Methyl prednisolone (35 mg/kg), pranolium (0.1 mg/kg) and d-propranolol (0.1 mg/kg) were given as a single intravenous injection to urethane anaesthetized guinea pigs 10 min before 5 1 C r - E . c o l i endotoxin (3.0 mg/kg) was administered. Lidocaine (1 mg/kg/hr) and adenosine (50 mg/kg/hr) were given as a continuous intravenous d r i p . Data ± S.E.M. were obtained at 3 hr following endotoxin i n j e c t i o n . In a l l cases, n = 5 except d-propranolol (n = 7) and controls (n = 11). Pranolium (.05 > P > .025) and d-propranolol (.005 > P > .002) were s i g n i f i c a n t l y different from controls.  - 115 The  effects  of  a  corticosteroid  pranolium,  and  d-propranolol pretreatment on E. c o l i endotoxin l e t h a l i t y when injected  into  albino Swiss mice were examined. shown in Figure 31. ments offered 18 hr, fact  protection  against  their  effects  administration  was impressive  was  E. c o l i  lethal  of  this  offered  group of animals  amount of  as  protection  throughout  the  experimental  period  these drugs  for longer periods of time. concerning possible in vivo antagonists to  antagonists was  explored.  of using chemically  To this  end, rats  injected with either NaOH-detoxified endotoxin or periodate-detoxified toxin prior to the administration of native E. c o l i effects 33).  on plasma  endo-  endotoxin (10 mg/kg) and  phosphatase a c t i v i t y were examined  However, increasing the dose of the detoxified  produced  no  additional  10 mg/kg abolished son,  were  (Figures  32 and  Some degree of protection was obtained with 2.5 mg/kg NaOH-detoxified  endotoxin. mg/kg  acid  The  of endotoxin for 18 hr although  endotoxin was performed whereby the f e a s i b i l i t y endotoxin  are  up to  had died.  and suggested that with optimal dosing regimens,  series of experiments  detoxified  study  of endotoxin for  a considerable  continued  mortality  a l l three drug pretreat-  effects  of a large bolus not  might prove more effective A final  the  the untreated  each of these drugs  against the lethal  The results  It is interesting to note that  at which time 80% of  that  (hydrocortisone),  the protective  periodate-detoxified  phosphatase increased  levels  phatase  and  activity  when  the  dose  (Figure  a measure of  whereas  seen at  pretreatment  5.0 mg/kg,  was evident as  effect  endotoxin  from 2.5 mg/kg to  phosphatase levels  protection,  of  this  a further 33).  endotoxin  a  endotoxin to  further  increase  5.0 to  lower doses.  In compari-  also  plasma  reduced  detoxified reduction  endotoxin in plasma  Thus, using plasma toxicity,  acid  acid was acid phos-  periodate-detoxified  - 116 -  Figure 31. Effect of Drug Treatment on Mortality Rates in Mice Injected with Endotoxin. Groups of Swiss mice received intraperitoneal injections of pranolium (0.5 mg/kg; n = 10), d-propranolol (0.5 mg/kg; n = 40) or hydrocortisone (35 mg/kg; n = 10) 30 min prior to and at 12 hr after an injection of E. c o l i endotoxin (40 mg/kg). No drug (n = 50) represents animal s- injected only with endotoxin (40 mg/kg).  - 117 -  NaOH DETOXIFIED I O.O  h 2  NO TREATMENT  r  ui  /  NaOH  /  U)  DETOXIFIED  I:  < < I a  2.5  mg/kg  5.0  mg/ kg  ii : //  tn  • I a  mg/kg  /  // /  a •  • U  CONTROL  TIME ( h r s )  Figure 32. NaOH-Detoxified Endotoxin as an Endotoxin Antagonist in vivo. The effect of pretreating anaesthetized rats with various doses of NaOH-detoxified endotoxin 30 min prior to an injection of native (toxic) E. c o l i endotoxin (10 mg/kg) on t o x i c i t y as determined by plasma acid phosphatase a c t i v i t y ( ± S.E.M.) i s indicated. N = 5 for a l l groups except no treatment (native endotoxin o n l y ) , n = 10. Controls received neither native nor detoxified t o x i n . Detoxified endotoxin alone had minimal effects on plasma acid phosphatase a c t i v i t y (see Figure 5).  - 118 -  s •  h  2  NO TREATMENT  Ul U) <  PERIODATE DETOXIFIED  2.5  mg/kg  < I PERIODATE  a  DETOXIFIED  5.  •  I  2  O  m g / k g  ./I  a  CONTROL  • 1  5  3  T I M E  4  ( hrs )  Figure 33. Periodate-Detoxified  Endotoxin as an Endotoxin Antagonist in v i v o .  The effect of pretreating anaesthetized rats with periodate-detoxified endotoxin 30 min prior to an injection of native (toxic) E. c o l i endotoxin (10 mg/kg) on t o x i c i t y as determined by plasma acid phosphatase a c t i v i t y (± S.E.M.) i s indicated. N = 5 for a l l groups except no treatment (native endotoxin o n l y ) , n = 10. Controls received neither native nor detoxified toxin. Pretreatment with 5.0 mg/kg periodate-detoxified toxin resulted in s i g n i f i c a n t l y (.01 > P) lower plasma acid phosphatase a c t i v i t y at 2 to 5 hr than no treatment (native endotoxin alone).  - 119 endotoxin  was  a  better  in  vivo  antagonist  of  native  endotoxin  than  was  NaOH-detoxified endotoxin. 3.6 Effect of Endotoxin and Gentamycin on Endotoxin Toxicity In Vivo The  therapeutic  management  of  gram-negative  sepsis  clinically  invariably involves the use of aminoglycoside a n t i b i o t i c s . of  interest  to study the possible  t o x i c i t y of E. c o l i  endotoxin.  effect  of  aminoglycosides  Gentamycin was selected  aminoglycoside a n t i b i o t i c on the basis of i t s septicaemia. of  Again,  plasma  endotoxin t o x i c i t y .  Figure 34. relevant modify this  It  is  dose the  evident  (1 mg/kg,  effect  same  dose  of  of  frequent  acid phosphatase levels  The results that,  of  when administered  was  use  did  representative  in  gram-negative a measure  summarized in  at  a  not  enzyme a c t i v i t y .  administered  in vivo  a  are  acutely  therefore  on the  were used as  gentamycin  endotoxin on plasma  gentamycin  as  these experiments  intravenously),  It was  almost  clinically  signficantly However, when  intraperitoneally  to  the  animals d a i l y for three days prior to endotoxin challenge (10 mg/kg, i n t r a venously),  a highly  significant  (.002  > P > .001)  elevation  in plasma  acid  phosphatase levels r e l a t i v e to rats receiving endotoxin but no prior aminoglycoside could  therapy was seen.  also  be.  An apparent  demonstrated  when  a  single  (10 mg/kg) was administered (Figure 34). alone had no effect  synergistic  effect  larger  with endotoxin  dose  of  gentamycin  It should be noted that gentamycin  on plasma acid phosphatase a c t i v i t y .  To further  inves-  tigate the influence of gentamycin on endotoxin t o x i c i t y , mortality studies were  carried  (1 mg/kg)  for  administered later  (Figure  out. 3 days  Swiss after  albino  which various  intraperitoneally 35).  mice  By 12 hr,  were  pretreated  doses of  with  E. c o l i  and  mortalities  assessed  the  gentamycin-pretreated  12  gentamycin  endotoxin were and  mice  24  hours  displayed  a  - 120 -  h  2 ui  i2  13  ID  Ul  01  > H  2  Ul U)  Ul  0 h  2  I £ a ai cn • h I a 2 3  4  -  -I •  CD  TJ \ 01  OI  JC  E  0)  •  E  01 JC  E  a i2  • U  • E N D O T O X I N  I O mg/kg  Figure 34. Effect of Gentamycin in Combination with Endotoxin on T o x i c i t y in Rats. Rats were pretreated with gentamycin either acutely (1 mg/kg or 10 mg/kg intravenously, 15 min before E. c o l i endotoxin 10 mg/kg) or chronically (1 mg/kg/day for 3 days, given as twice daily intraperitoneal injections of 0.5 mg/kg). E. c o l i endotoxin (10 mg/kg) was injected 30 min after the f i n a l gentamycin injection in the chronically treated animals. The results are expressed as mean ± S.E.M. acid phosphatase a c t i v i t y at 3 hr following endotoxin administration. Controls received no endotoxin or gentamycin whereas 0 gentamycin represents animals that received only endotoxin. N = 10 for a l l groups. Acid phosphatase in chronically (1.0 mg/kg/day) and acutely (10 mg/kg) treated rats was s i g n i f i c a n t l y (.002 > P > .001) elevated over 0 gentamycin group.  - 121 [  • 05  .1  mg ' Effect Mice.  of  | ENDOTOXIN  .2  «4  ENDOTOXIN  .6  /l5g  Figure 35. Chronic  Gentamycin Treatment  on Mortality  in Endotoxin-Treated  Swiss mice were pretreated with gentamycin (1.0 mg/kg/day given as twice d a i l y injections of 0.5 mg/kg) for 3 days after which various doses of E. c o l i endotoxin were administered intraperitoneally and mortalities were assessed 12 and 24 hr l a t e r . Mice receiving only endotoxin were pretreated twice daily with s a l i n e . N = 10 for each dose of endotoxin in both groups (total of 50 mice/treatment group).  -  higher  mortality  mortality  at  in the  all  doses  of  previous  experiments  -  endotoxin.  gentamycin-treated  the lower doses of endotoxin. the  122  animals  At 24 hr,  was  still  obvious  Thus, these mortality  suggesting  a synergistic  the  studies  action  of  increase  in  but  at  only  substantiated gentamycin on  endotoxin t o x i c i t y in vivo. 3.7 V a r i a b i l i t y in Commercially Available Endotoxin Preparations Investigators  studying experimental  variations  in  serotypes.  During the course of the present  indicating  results  that  can  be  endotoxaemia  that marked differences  commercially obtained  E. c o l i  number.  chemically  Attempts  to  obtained  with  are  well  aware of  endotoxins  of  group-modifying whose  in biological  activity  endotoxin preparations characterize  these  exist  nanometers. a greater  differing  only  endotoxins  of  endotoxin  be  by  can  trinitrobefeonesulfonic measured  in v i t r o , which  showing  the  units  plasma  acid  lot  varying  were generally  found to  incorporated  less  correlation  (r = 0.95)  phosphatase  TNBS.  activity,  acid  be more toxic  Figure between  for  (serotype 026:B6) with the indicated lot numbers.  (TNBS), at  335  incorporating TNBS to  incorporation per mg endotoxin and the t o x i c i t y in vivo, of  in  in standard-  spectrophotometrically  It was found that endotoxin preparations  than endotoxins point  probe,  extent  even among  The method involves the use of the primary amino  chromophoric  binding to  different  study, we have obtained evidence  biological potency have yielded a procedure which may be useful izing these preparations.  the  five  36 the  in vivo  illustrates extent  of  this TNBS  expressed in terms E.  coli  endotoxins  - 123 -  Figure 36. Correlation Between Extent of TNBS Incorporation T o x i c i t y in Rats.  into E. c o l i  Endotoxin and  The incorporation of trinitrobenzenesulfonic acid (TNBS) into E. c o l i endotoxin (026:B6) of various lot numbers was determined by incubating 1.0 mg toxin in a 3.0 ml reaction mixture consisting of 1.0 ml 20 mM Tris-HCl pH 8.0, 1.9 ml H 2 0 and 0.1 ml 10 mM TNBS, pH 8.0, for 35 min at 3 7 ° C . The v reaction was i n i t i a t e d by the addition of TNBS and terminated by the addition of 2.0 ml of a 1:1 mixture of 10% SDS and 1 M HCl. The absorbance was read at 335 nm. T o x i c i t y in rats was determined by measuring plasma acid phosphatase a c t i v i t y 3 hr following an injection of endotoxin (10 mg/kg). Data represent mean ± S.E.M. of 10 animals for each lot number.  - 124 CHAPTER 4 Discussion and Conclusions 4.1 Gram-Negative Septicaemia: Recently, the results  A Formidable Medical Problem  from a ten year study on gram-negative bacteraemia  involving approximately 600 patients have been reported by Kreger and associates  (199,200).  was that  One interesting  in spite  of  observation made by these  the development of  potent  antimicrobial  incidence of gram-negative bacteraemia has been continually the 1950's and, as a r e s u l t , constitutes mia presently seen in the United States  investigators agents,  the  increasing since  the majority of cases of  bacterae-  (and probably Canada as w e l l ) .  The  high f a t a l i t y rate associated with gram-negative bacteraemia has made i t one of  the  major  (201).  causes  of  death  from  infection  in  North  American  hospitals  The increasing frequency of gram-negative bacteraemia appears to be  related to a growing proportion of patients  that are more aged and have an  underlying pathological condition such as granulocytopenia, congestive heart failure,  diabetes  mellitus,  neoplasms  or  renal  insufficiency.  Also,  the  increasing use of manipulative procedures involving the urinary or r e s p i r a tory  tracts  and extensive  antimetabolites  treatments  contribute  gram-negative bacteraemia  with  significantly  (199,200).  antibiotics, to  In their  the  corticosteroids  rising  study,  frequency  were most frequently caused by Escherichia  Other  found  organisms,  Klebsiella-Enterobacter-Serratia , Only  16% of  bacteraemia  the could  bacteraemias  in  decreasing  Pseudomonas,  be determined,  it  was  most  order Proteus  were polymicrobic.  of  Kreger and co-workers  found that the bacteraemias causative  or  of  coli.  frequency, and  When the  were  Bacteroides. source  frequently found  to  of  the  be  the  - 125 urinary  tract,  However,  followed  in almost  by the  one t h i r d  gastrointestinal  (30%)  of  the  and  patients  respiratory with  tracts.  underlying  host  disease, the s i t e of origin of the bacteraemia could not be i d e n t i f i e d (199). It is well recognized that shock is tive shock  bacteraemia occurred  patients,  (147).  In the  in 441 of the  study  reported  gram-negative  by Kreger  septic  the f a t a l i t y rate was seven times greater  who did not develop shock  (200).  It  shock commonly seen in gram-negative endotoxins  a common complication of gram-nega-  (168).  This  has  patients  is  associates,  and  in  than in septic  been proposed  septicaemia  and  that  due to  postulation was made by Weil  these  patients  the cause of  the release of  and co-workers even  before assay techniques were developed to detect the presence of endotoxins in the plasma.  Certainly i t was known that most of the c l i n i c a l  manifesta-  tions of gram-negative bacteraemia could be reproduced in animals by administering endotoxin isolated from gram-negative  bacilli  (167).  Also,  experi-  ments involving the parenteral administration of endotoxin into human volunteers have been conducted and responses  similar to those seen c l i n i c a l l y in  gram-negative bacteraemia were noted (202). belief tive  that endotoxins were involved in the sequelae of c l i n i c a l bacteraemia  gram-negative into  when  Crutchley  organisms  and  from  the Limulus  endotoxin  Jorgensen  grown in cultures  the surrounding medium (16,19).  were released until  More credence was given to the  bacilli lysate  in  However,  gram-negative  that  viable  readily release endotoxin firm  proof  septicaemia  that  was  endotoxins  not obtained  technique was developed, whereby the presence of  in the plasma could be detected  picograms/ml plasma (203).  could  demonstrated  gram-nega-  in concentrations as  low as  Indeed, i t has been shown that c l i n i c a l  aemia can occur in the absence of  a well  defined septic  0.1  endotox-  focus, p a r t i c u l a r l y  - 126 when the reticuloendothelial system is  impaired to the extent that endotox-  in,  tract,  absorbed  from the gastrointestinal  (203,204). bacilli, dis,  endotoxins most  some strains  can  strain  Although  also  (205).  of  can accumulate in the plasma  commonly originate  gram-negative c o c c i ,  liberate  endotoxins  Thus,  has  it  been  in  such as  amounts  suggested  which  that  from  Neisseria vary  this  gram-negative meningiti-  from  variable  strain  release of  endotoxin according to strain may account for the different c l i n i c a l tations of meningococcal infections (205). tors may s t i l l  in  this  area  that  largely responsible for tions of  (89).  presen-  Therefore, while some investiga-  question the contention that endotoxin plays a major role in  bacteraemic shock in man (206,207), tigators  to  these  is generally accepted by most inves-  bacterial  the morbidity  As a r e s u l t ,  gram-negative  it  cell  associated  wall  with  constituents  gram-negative  infec-  most of the experimental work on various  bacteraemia  (particularly  shock)  have  utilized  are  aspects purified  endotoxin preparations. 4.2 Host Defense Mechanisms in Bacteraemia and Endotoxaemia Certain endotoxin  differences  as  difference,  are  compared to  apparent  infusions  in the host of  live  reaction to injections of"  bacteria  (207,208).  in p a r t i c u l a r , is the manner by which the host system strives  eliminate endotoxins and whole bacteria from the c i r c u l a t i o n . bacteria  One major  are  phagocytized primarily  to  It seems that  by polymorphonuclear neutrophil  leuko-  cytes while endotoxin is mainly detoxified by the c e l l s of the reticuloendothelial  system  and blood monocytes  duction section of this t h e s i s , ing  the  outcome  overstated  of  a  (151,155).  (207,209).  As mentioned in the  Intro-  the importance of granulocytes in determin-  gram-negative Certainly,  septic the  episode  prognosis  clinically for  cannot  be  granulocytopenic  - 127 patients  that develop gram-negative bacteraemia can be greatly improved with  granulocyte transfusions  (210,211).  However, i t  by Helium and Solberg that the bactericidal cytes,  has  also been  demonstrated  a c t i v i t y of neutrophil granulo-  as measured by the reduction of nitroblue tetrazolium (NBT) dye, can  be greatly inhibited in patients Secondly,  the  studies  of  with severe bacterial  Cartwright, Galbraith,  infections  and co-workers  (212,213). have shown  that the h a l f - l i f e of a mature polymorphonuclear neutrophil leukocyte in the circulation  is  seems  although  that  only  approximately  phagocytizing c e l l s function  that  (216).  gram-negative  or  polymorphonuclear  seven  hours  leukocytes  (214,215).  are  the  Thus,  most  it  immediate  during an i n f e c t i o n , they are short-lived and hence the  of macrophages  organisms  six  may serve as  an important defense  Furthermore, when consideration  is  against invading  made of  the  evidence  infections can be complicated by endotoxaemia and,  endotoxins are detoxified primarily by reticuloendothelial c e l l s role of these c e l l s ,  or macrophages,  (209),  in the defense of gram-negative  tions gains even more significance.  Therefore, the successful  since the  infec-  recovery from  a gram-negative bacteraemia depends on the phagocytic functions of c i r c u l a t ing granulocytes,  such as  the  polymorphonuclear neutrophils,  and macropha-  ges, which have the added c a p a b i l i t y of detoxifying endotoxin. Although the macrophages  form an extensive  throughout the body, c o l l e c t i v e l y known as the Kupffer c e l l s of the l i v e r  has been demonstrated  that  liver  beryllium  which  is  treated  with  these hepatotoxins  endotoxin (218).  phagocytic  the reticuloendothelial  cells system,  are the most important in clearing c i r c u l a t -  ing endotoxin from, the blood in a l l  phosphate,  network of  experimental  poisons, toxic  to  animals  studied  (217).  It  such as carbon t e t r a c h l o r i d e , and Kupffer  hypersusceptible  cells, to  the  can  make  lethal  animals  effects  of  Indeed, i t was recognized as early as 1947 by Beeson, that  - 128 blocking  the  colloidal  thorium dioxide  effects  phagocytic  activity  of  to sensitize endotoxin stimulate animals  animals  (220).  lead  resistant  such as  compound has  the subhuman primate to the  activity  to the  and this  one  of  would  expect  that  the  BC6 and glucan, which  of  yeast  animals  cell  to the  wall  extract  lethal  is  endotoxin.  of  surprising in view of the fact enhance  non-specific  certain  bacterial  explanation in  for  host  this  endotoxin  that  However, such  (224)  paradoxical  substances  exacerbate  the  been  which  agents  have  to endotoxin.  For  shown  (221,222,223).  to  a  variety  of  component of to  This  diseases  and malignant tumors hypersensitive  glucan- and BCG-treated animals  these  both  of  sensitize is  rather  these agents, glucan in p a r t i c u l a r ,  resistance  infections  have  effects  system would make  the purified polysaccharide  zymosan,  effects  to the  substances  the reticuloendothelial  effects  with  also been shown  lethal  paradoxically been shown to render animals more sensitive example,  system  Another agent which can impair hepatic  acetate,  Alternatively,  phagocytic  reticuloendothelial  (thorotrast) made animals more susceptible  of injected endotoxin (219).  phagocytic function is  the  response to  may be related  hypoglycaemia  (225).  to  can  including A possible  endotoxin  seen  the observation  that  normally  seen  in  endotoxic  shock (217,226,227). It has long been recognized that resistance to the effects (or "tolerance") sublethal  can be produced in animals  doses of endotoxin for  endotoxin displayed increased cells  (217).  several  of endotoxin  by the d a i l y administration of  days.  Animals made "tolerant"  to  phagocytic a c t i v i t y of the reticuloendothelial  Thus, i t was believed that  the development of  "tolerance"  to  endotoxin was due to enhanced phagocytic a c t i v i t y of the reticuloendothelial macrophages.  However, Starzecki  and associates found that  the clearance  of  51 Cr-endotoxin from the  c i r c u l a t i o n was  the  same in normal  and endotoxin-  - 129 resistant  dogs  (228).  Furthermore,  Greisman  and  co-workers  demonstrated  that blockade of the reticuloendothelial phagocytic a c t i v i t y with did not appreciably affect endotoxin-resistant  tolerance to the pyrogenic effect  rabbits  normal, thorotrast-treated  (229).  These investigators,  rabbits  these animals were transfused  of endotoxin in  in f a c t ,  could-be made tolerant  with plasma  thorotrast  found that  to endotoxin when  from endotoxin-resistant  rabbits.  These results  suggested the presence of a humoral factor  that was  responsi-  ble  development  animals.  Although  for  the  of  tolerance  to  endotoxin  in  still  perhaps c o n t r o v e r s i a l , there is good evidence that this humoral r e s i s -  tance  factor  19S  represents  antibodies  immunoglobulin specific  endotoxin has  been demonstrated  endotoxin-tolerance formed  for  trait  against  the to  endotoxin  "core"  or  be involved  (231,232).  (230).  Certainly, a  g l y c o l i p i d region  of  the  in the transference  of  the  Freedman  has  shown that  antibodies  against the g l y c o l i p i d portion of the endotoxin complex can provide  passive, toxins  transferable (233).  This  protection is  in  against  contrast  homologous and heterologous  to  antibodies  directed  endo-  against  the  0-antigenic polysaccharide region of the endotoxin which only provides homologous  protection, or,  same serotype  (232).  anti-endotoxin death  in other words, protection against endotoxins of the Indeed,  immunoglobulins to  normally associated  strongly endotoxin  the  appreciated  by  with  clinical reduce  the  gram-negative  investigators  immunoglobulins,  value  particularly  high  having high frequency  septicaemia  (234,235). those  of  is  of  of  shock  and  becoming more  The mechanism  directed  titers  against  by which the  core  regions of the complex, decrease the t o x i c i t y of endotoxin is believed to be due to a process of opsonization as well as to an antitoxic effect Finally,  (234).  other studies have indicated that endotoxin can be inactivated  or "detoxified"  in the plasma by nonimmunoglobulin factors  that  are  largely  - 130 unidentified be  at the present  detoxified  (237,238).  in  the  (236).  plasma  Skarnes has proposed  by  two  enzymes  which  that  are  endotoxin can  both  a-globulins  One enzyme, a heat-stable esterase, causes disaggregation of the  endotoxin complex while another,  a heat-labile  gregated  endotoxin  Other  protein,  an a - g l o b u l i n , in human serum that could cause i r r e v e r s i b l e  gregation 1 ins  (237,238).  investigators  of the endotoxin complex (239).  reported  endotoxin  is  was  detoxified  Thus,  lipids  found  in the  (high density  although there  neither  a  plasma  is  by what  lipoproteins  is  likely  in nature  a  to occur  the humoral phase of  (240,241).  the blood  circulating  polymorphonuclear  reticuloendothelial immunoglobulins teins,  that  sufficient critical  reaction.  system.  against gram-negative  and other  detoxify to  prevent  stages  granulocytes humoral  factors,  endotoxin. the  which  exemplified  of  because  of  and  the  defense may  a  are  detoxifica-  bacteraemia  involve  gram-negative  of  consists  enzymes  protective  system  comprised  macrophages response  by hypotension and shock.  or  of the of  lipopro-  mechanisms  are  bacteraemia  to  However, in circum-  these defense mechanisms may be compromised,  in very aged patients or patients  immunosuppressed  phagocytes  Normally, these  progression  stances where the function of such as  The c e l l u l a r  The  is  elucidated.  the host defense mechanisms  defense  that  disaggregation  and .associated endotoxaemia p r i n c i p a l l y involve a c e l l u l a r phagocytic and a humoral  nor an  but does depend upon  in p a r t i c u l a r )  a consensus that  tion process remains to be more f u l l y  one  disag-  lipoprotein  also capable of detoxifying endotoxin, the exact nature of this  Therefore,  only  Studies by Ulevitch and associates have also indicated  process which does not seem to be enzymatic plasma  have  the disag-  This protein, unlike the ct-globu-  by Skarnes and co-workers,  esterase (239).  enzyme, detoxifies  cancer  that  are  chemotherapy,  granulocytopenic radiation  or  are  therapy  or  - 131 patients  with  liver  disease,  could have very serious therefore, effects  specific  of  and effective  endotoxin  to  development  consequences  would  measures would be the most necessary  the  understand  be  (234).  gram-negative  In circumstances  septicaemia  such as  methods of  counteracting the  required.  To  appropriate  the  of  various  determine  and effective  effects  that  to  these,  deleterious  what  treatment  undertake,  endotoxins  it  exert  in  is the  body that can lead to the development of shock. 4.3 Pathophysiology of Endotoxaemia Shock consisting confusion shock  was  popular be  a  is of  a  term  frequently  protracted  by the  nature  of  in  shock.  Now, with  pressure and cardiac  Rather, the primary defect  describe  a  cold,  moist  skin,  syndrome mental  century, the severity of  pulse.  refined  pressures,  output  are  Before  World  blood flow which  essential  substrates and oxygen (242).  is  an  undisputed  only  instrumentation  it  is  apparent  secondary  War  indicators  in turn  fact  that  impairs  the  endotoxin  a  potent  The effects  to  of  rate, shock.  or microcir-  transcapillary  is  I,  determine  that heart  However, the mechanism by which endotoxin produces  obscure.  to  in shock is a reduction in effective  culatory  agent.  the  to  use of the sphygmomanometer allowed blood pressure measurements  monitor  It  a  At the turn of this  cardiac output and intravascular blood  clinically  hypotension, p a l l o r ,  and o l i g u r i a (242). determined  used  exchange  of  shock-inducing shock  is  still  of endotoxaemia on the microcirculation have received  limited study and considerable controversy exists with regard to the actions of endotoxins on this part of the c i r c u l a t o r y system method of  studying the effects  (244).  One convenient  of endotoxin on microcirculatory or  "nutri-  133 t i v e " flow in vivo is to monitor the washout of 133 s i t e in some particular t i s s u e . Xenon, being  Xenon from an i n j e c t i o n an inert  and  lipophilic  - 132 substance, readily diffuses  across c e l l  membranes and therefore,  i t s rate of  removal from an injection s i t e closely coincides with the degree of c a p i l l ary blood flow to that area (243). E.  coli  blood  endotoxin  flow to  L-D.^Q)  persisted that  ten  caused  a marked reduction  minutes  rats.  Using this technique, we have found that  after  it  Interestingly,  in skeletal  was  given  this  effect  for the duration of the experimental  a substantial  intravenously on  of a reasonable blood pressure supports intra-arterial  pressure  shock in bacteraemia  example,  it  (245).  mines  rather is  which  to  believed  precapillary a r t e r i a l  is  observations  an unreliable  release  endotoxins to  been termed  been proposed  that  of  a manifestation  shock  is  (246).  The  be  sphincters  ing what has  shock,  low blood  as  a  by other  investigators  indicator of the severity of  irreversible  vasoactive  increase  for  not  anoxia"  levels  the  phase  of  of  of  the  relaxation  of  the  in both  Interestingly,  endotoxic, has  of  been  all  causit  the reversible but  For  catechola-  venular sphincters,  (246).  shock  (147).  constriction  which represents only  on the microvas-  substances  plasma  and postcapillary  anoxia, of  of  responsible  "ischaemic  ischaemic  result  pressure.  impaired in spite  has  phase  types  of  described  as  "stagnant anoxia" and is characterized by an increased c a p i l l a r y pressure  fact  The mechanism by which endotoxin impairs micro-  the  known that  are  flow  endotoxin), which suggested  c i r c u l a t o r y flow does not seem to be due to a direct effect but  mg/kg,  recovery in the mean blood pressure had occurred during  the reduced c a p i l l a r y flow was not simply due to  culature  (4.0  microcirculatory  This observation that microcirculatory flow can be greatly  that  capillary  time (3 hrs) despite the  this same time period (80% of normal 3 hrs after that  muscle  hydrostatic  precapillary  arteriolar  sphincters only (246).  Irreversible  shock can be produced experimentally in  a fairly  of  haemorrhaging  short  period  time  by  animals  to  a  low blood  - 133 pressure (one-third of normal) and maintaining the animals at this blood pressure by additional bleedings or reinfusions  until  such time as 30%  of the maximal bled volume has been returned to the animals. irreversible  shock  ensues,  blood is transfused  even when the  back to the animals  At this point,  remaining volume of  (247).  microcirculatory blood flow in the skeletal  low mean  haemorrhaged  Under these conditions, the  muscle of the r a t ,  as  measured  133 by  Xenon washout,  was  found  to  be  approximately  two-fold  greater  than  the flow at three hours following endotoxin administration, even though the mean blood pressures were similar greater tal  degree of vasoconstriction exists  muscle after  shock  in both groups  is  of  rats.  This  implies a  in- the microcirculation of  three hours of endotoxaemia than is seen when i r r e v e r s i b l e  induced by haemorrhage.  Alternatively, it  can  also  be  proposed  that the reduced microcirculatory flow seen in the endotox in-treated could be due to clogging of the c a p i l l a r i e s blood  viscosity  co-workers  along with  and  have  minutes after  microcoagulation  shown that  the  the animal was  authors  as  muscle vasculature  dog  as  a result  (248,249).  forelimb  However,  lost  in skeletal  muscle  animals  of an increase  weight  vascular  Weidner  ten  injected with endotoxin and this  the noted increase  explained by the skeletal  skele-  to  fifteen  resistance,  thirty-fold  of  after  catecholamines,  was  evidence for vasoconstriction occurring in the (250). responsi-  ble for the reduction in microvascular flow seen in endotoxaemia (246). levels  and  observation,  As already mentioned, catecholamines have been proposed to be  plasma  in  adrenaline  and  noradrenaline  can  increase  the administration of endotoxin (251).  other vasoconstrictor  from  ten- to  In addition to the  agents are known to be released  the c i r c u l a t i o n in response to endotoxin administration.  The  Examples  of  into some  of these substances include renin/angiotensin (252,253), 5-hydroxytryptamine  - 134 (254)  and prostaglandins,  (255,256). endotoxin  However, i t to  plasma that bradykinin fore,  it  animals  such as is  can  have potent  F 2 a , which have vasoconstrictive  also well increase  established the  vasodilatory  is  difficult  to  attribute  microcirculatory flow solely to these mediators  may exert  as the lung (261). endotoxaemia  is  concentration  actions,  (259,260) and prostaglandins the  such  as  the administration of of  effects  effects  of  substances  histamine  of the E series  the release of  important  that  actions  in  the  (257,258),  (255,256).  There-  endotoxin on peripheral  vasoactive  agents,  although  l o c a l l y in certain organs such  Probably much of the peripheral vasoconstriction seen in due  to  baroreceptor/sympathetic  nerve  stimulation  that  occurs in response to the systemic hypotension caused by endotoxin, in order that  adequate blood flow is maintained to more v i t a l  question  the relevance  organs.  One may then  of impaired microcircul atory flow in skeletal  to the pathophysiology of endotoxaemia.  muscle  It can be stated, however, that  haemodynamics of the c i r c u l a t i o n in skeletal  muscle are not responsible  the for  the systemic hypotension that occurs upon the administration of endotoxin to animals. Therefore,  to address the o r i g i n a l question  of endotoxic shock,  regarding  the  precipitation  the examination of the microcirculatory status in v i t a l  organs such as the heart, estimate of the severity  lung, kidney, e t c . ,  would probably provide a good  of shock in endotoxaemia.  A technique whereby the  degree of shock could be e f f e c t i v e l y measured would have great potential clinical  application in gram-negative  bacteraemias.  However, measurement  for of  microcirculatory blood flow in v i t a l organs is d i f f i c u l t to do experimentally and not feasible c l i n i c a l l y .  To circumvent this technical d i f f i c u l t y of  d i r e c t l y assessing the perfusion of organs in shock, i t indirectly  by assaying the plasma for  certain  is possible  substances that  are  to do so released  - 135 from tissues product of  only during states  anaerobic  accumulates  in  (243,245).  Weil  arterial  blood  a  is  an example of  during  and A f i f i  haemorrhage (262). the extent  glycolysis,  the  blood is  of compromised perfusion.  conditions  have shown that  good  indicator  of  one such substance  of  systemic  severity  of  of  enzymes these  such  enzymes  associated with extensive plasma levels  a  that  deficit  lactate in  shock  in patients  at  the  present  substances which may be used to estimate the severity of  release  oxygen  the concentration of  the  acid,  induced by  Indeed, blood lactate levels are commonly used to assess  of c i r c u l a t o r y f a i l u r e  intracellular  Lactic  as  the  into  lysosomal  the  plasma  shock are  hydrolases  occurs  Other certain  (263,264).  only  under  tissue damage and c e l l u l a r death  of lactate or lysosomal  time.  conditions  (265).  enzymes can be used as  The  Although  indicators of  the severity of shock, they cannot be used to identify the organs  in which  the c i r c u l a t i o n is primarily impaired because lactate and lysosomal  enzymes  are  enzymes  fairly  ubiquitous  substances.  however, which are peculiar to  There  cells  of  are  some  certain  non-lysosomal  organs  and therefore,  the  presence of these enzymes in the plasma would identify the particular tissue in which c e l l u l a r l y s i s was occurring. have  been  used  to  determine  circulatory  include:  creatine phosphokinase  ily  nervous  and  present which is  in l i v e r  tissue cells  ornithine  determine the severity lysosomal  enzymes  of shock,  (cathepsin  these it  of  appears that  D in p a r t i c u l a r ) ,  organs  for muscle primarwhich  intestinal  specifically  substances that  of the seriousness of the shock state (269).  various  carbamyltransferase  and alkaline phophatase  Of a l l  in  specific  i d e n t i f i e d on the basis that this enzyme is  by L-phenylalanine (268).  of  impairment  (CPK) which is  (266);  (267);  Some examples of these enzymes which  origin  inhibited  have been used  the plasma  is  to  concentrations  provide the best  Although lysosomes  are  measure present  - 136 in most types extent  of c e l l s ,  these organelles  in the splanchnic organs,  are  concentrated  l i v e r and macrophages  to  the  greatest  (270,271).  Therefore  the presence of lysosomal enzymes in the plasma during shock states usually is  an indication of c i r c u l a t o r y impairment in the hepato-splanchnic  Indeed, that,  the c i r c u l a t i o n in this  in haemorrhagic  shock  region can be deranged  for  instance,  the  to  intestinal  such  region.  an  mucosal  extent barrier  breaks down (272), permitting the systemic absorption of endotoxins from the gut to occur (273,274). The work reported in this thesis demonstrated lysosomal  enzyme a c t i v i t y as  that occurs progressively  a measure  of  the  the value of using plasma physiological  deterioration  in animals from the moment that they are  injected  with a lethal dose of endotoxin to their time of death.  The three represen-  tative  studies  lysosomal  phosphatase  enzymes  (acid  and a protease  that  were  employed  a  glucosidase  phosphatase),  (cathepsin  D).  A l l three  indicators of the toxic effects  Endotoxins  detoxified  included a  (N-acetyl-e-glucosaminidase)  enzymes  appeared  to  be  reliable  of endotoxin in vivo as demonstrated by the  manner in which the plasma a c t i v i t i e s endotoxin and with time after  in these  of these enzymes varied with dose of  a lethal  dose of endotoxin was  chemically by sodium hydroxide or  treatment had v i r t u a l l y no effect  administered.  sodium  periodate  on plasma lysosomal enzyme a c t i v i t y during  the time period in which an equivalent dose of native endotoxin was lethal to the animals.  It was interesting to note that although the plasma a c t i v i -  ties of the three lysosomal  enzymes under study increased with time and with  dosage of endotoxin, the patterns These results originating  then suggest from  administration  are  different not  the  that  of their r i s e the  tissues same.  lysosomal upon  It  is  which  in a c t i v i t y were enzymes  different.  in question  may be  the  effects  of  endotoxin  known that  tissues  do d i f f e r  in  - 137 their lysosomal enzyme content. contain  cathepsin  that the intestine  For example, muscle lysosomes predominantly  D and ribonuclease is  (270),  a major source  of  from various  organs  the lymphatic drainage  whereas  it  has  acid phosphatase  been  (275).  may be different  reported Secondly,  and since  this  is the major route by which lysosomal enzymes enter the systemic c i r c u l a t i o n (275,276),  it  is  another  factor  that  can  influence  these enzymes in the plasma.  Another possible  in  of  the plasma concentrations  the  three  the  enzymes  in endotoxaemia  these enzymes may d i f f e r .  known  largely  cathepsin  reticuloendothelial osaminidase sinusoids  are  D is  removed  the  cleared  by a process  primarily by endothelial involving  reticuloendothelial  (263,269,279,280)  from  the  is  For example, circulation  that it  by  is the  system (277) while glucosidases such as N-acetyl-e-gluc-  a cell  "recognizes" this glycoprotein (278). of  of  explanation for the variation  the clearance mechanisms for that  concentration  and  system  therefore,  a  surface  cells  lining  receptor  the  that  hepatic  specifically  It also is believed that the function can  become  lysosomal  could accumulate in the plasma to a greater  impaired  enzyme  extent  such  in  shock  as  states  cathepsin D  than perhaps N - a c e t y l - e -  glucosaminidase whose plasma clearance does not t o t a l l y depend upon r e t i c u l oendothelial  function.  This  may  explain  our  observations  in  E.  coli  endotoxin-treated guinea pigs that plasma concentrations of cathepsin D were increased several-fold not quite doubled. of E. c o l i pigs, better  at a time when N-acetyl-e-glucosaminidase  Thus, on the basis of our findings concerning the  endotoxin on plasma lysosomal  i t can be said that both cathepsin indicators  levels had  of  the  N-acetyl-e-glucosaminidase.  state  of  effect  enzyme a c t i v i t y in rats and guinea D and acid phosphatase seem to be  endotoxic  shock  in these  animals  than  - 138 It was most interesting to compare the lysosomal enzyme data obtained in the  laboratory setting  utilizing  animals  and purified E. c o l i  lipopolysac-  charide with the data that were obtained on the same enzymes from in  gram-negative  activities  of  bacteraemic  the three  shock.  lysosomal  As  enzymes  in  the  animal  studied  patients  experiments,  in the  plasma  of  the these  patients were s i g n i f i c a n t l y elevated over the values normally seen in plasma from healthy human volunteers. were differences in  individual  four-fold,  in the extent  patients.  Again,  as  in the animal experiments,  to which each of these enzymes was  On the  average,  N-acetyl-B-glucosaminidase  also  acid  phosphatase  increased  elevated  was  four-fold  elevated while  average plasma cathepsin D concentration in the gram-negative shock was approximately twenty-six times normal. patients degree  in shock with of  gram-negative  These results  bacteraemia perhaps  microcirculatory impairment  in peripheral  there  the  patients  may indicate that sustain  tissue  a  (i.e.  greater skeletal  muscle) than in the hepatosplanchnic region, since the increase in cathepsin D a c t i v i t y in the plasma of these patients were the increases ties. effects  in acid phosphatase  Supporting this  explanation  are  was proportionally greater  than  or N-acetyl-B-glucosaminidase a c t i v i experiments  that  have  examined the  of endotoxic shock on the c i r c u l a t o r y status of sub-human primates.  These experiments have demonstrated that the administration of endotoxin to monkeys and baboons does not alter  the c i r c u l a t i o n to the intestine as much  as i t does in the dog, for example, an animal in which endotoxin is known to cause splanchnic pooling (281,282). utes to the large  increases  septic shock patients dothelial disease  system is underlying  Another factor  which probably contrib-  in plasma cathepsin D a c t i v i t y in gram-negative  is the likelihood that the function depressed  the  in these patients,  gram-negative  of the r e t i c u l o e n -  many of whom had a  bacteraemia.  Thusl  both  severe  animal  and  - 139 patient  studies have indicated that plasma lysosomal  a reliable treated  measure  of  the  pathophysiological  with purified endotoxin or patients  enzyme a c t i v i t y can be  state  with  of  either  the  gram-negative  animals  bacteraemia.  In p a r t i c u l a r , plasma cathepsin D concentration showed the greatest in  gram-negative  phosphatase  septic  activities  shock  patients  were greatly  while  both  elevated  in  cathepsin  the  plasma  increase  D and  of  acid  endotoxin-  treated rats and guinea pigs. From our  observations  on the  effect  of  endotoxaemia  or  gram-negative  septicaemia on plasma lysosomal enzyme a c t i v i t y in animals and patients, question  arises as to whether lysosomal  manifestation in  enzymes in the plasma are simply a  of c e l l u l a r damage or whether these "free"  some way to  gram-negative  exacerbate  shock  septicaemia.  In  in experimental other  words,  enzymes contribute  endotoxaemia  does  the  could  additional enzymes ? free  develop  tissue Although  lysosomal  and  whereby organ  the  lysosomal  damage and  some investigators  enzymes  subsequently  or  presence  enzymes in the plasma signify that a potentially dangerous effect  the  clinical of  these  positive-feedback themselves  the  release  may have reservations  as  cause of  more  to whether  enzymes in the plasma intensify the pathophysiology of shock  (283), much of the evidence tends to support the concept that these enzymes do play an active  role  in the development of  There is c e r t a i n l y no question about the fact which  can digest  carbohydrates considerable  a wide variety  and nucleic acids. host  in shock where the  level  nearer that  the optimum for  lysosomal  infusion of fractions  such  as  proteins,  Thus, they have the potential  accumulation  rich  conditions  of  (263,284).  that lysosomes contain enzymes  substances  damage under favorable  tered  strated  of  i r r e v e r s i b l e shock  lactic  enzymes  such  acid  (285).  in lysosomal  as  It  of inducing  those  lowers has  lipids,  the  encounpH to  a  been demon-  enzymes into dogs can  - 140 produce a state of c i r c u l a t o r y shock characterized nic vasoconstriction effects  and haemorrhagic  of these lysosomal  lesions  enzyme infusions  of  by hypotension, the  function was  of lysosomal  also been shown to affect  rabbit  or  isolated  rat  mesentery  perfused  lysosomal  cat  proteases  (287)  hearts  can  and (272).  indirectly  surgically  can  cause  Some  (277,286).  The  were more pronounced in animals  whose reticuloendothelial products has  bowel  splanch-  impeded  (277).  the microcirculation in  endothelial  investigators  exacerbate  the  Infusion  proliferation also  failing  believe  in that  circulation  in  shock by causing the formation of a small molecular weight peptide (800-1000 M.W.) that d i r e c t l y depresses myocardial c o n t r a c t i l i t y and hence referred as a "myocardial depressant factor" depressant factor tial  ischaemia  (289).  been able to detect  the presence  shocked  depressed  lysosomal drial  that  of any factors  myocardial  and impairing energy  have  shown that  most  that occur in endotoxic as well vitro  However, other  formation is investigators  consequenhave not  in the plasma of endotoxin  contractility  (290).  Finally,  enzymes may intensify the shock condition by disrupting mitochon-  function  associates  The source of the myocardial  appears to be the pancreas and i t s  to pancreatic  dogs  (288,289).  to  by incubating  Thus, the general  lysosomal  progression  of as  production.  In this  the  in mitochondrial function  changes  regard,  Mela and  haemorrhagic shock can be reproduced in  hydrolases of events  with  isolated  mitochondria (291).  in endotoxic shock may be  presented  simply as a reduction in microvascular flow and related oxygen tension which causes accumulation of l a c t i c a c i d , depletion of c e l l u l a r energy, zation  of  lysosomes  These,  in t u r n ,  and  consequently  can cause a further  energy and extensive tissue injury.  the  release  of  lysosomal  reduction in blood flow,  destabilienzymes.  depletion  of  - 141 Virtually actions  of  every  tissue  administered  radiolabelled  in the  body  endotoxin  endotoxin  was  is  (292).  injected  affected  in  Studies  some way  have  intravenously  by  the  shown  that  a  variety  into  when of  animals, most of the r a d i o a c t i v i t y was found to be associated with the l i v e r (217,292).  Significant  amounts of radiolabelled  endotoxin also  in the lung, spleen and kidney, while no detectable appeared  in the brain (292).  amounts of r a d i o a c t i v i t y  V i r t u a l l y a l l of the c i r c u l a t i n g radiolabelled  endotoxin was distributed between the plasma and buffy coat Interestingly, the  i t has also been shown that  radiolabelled  leukocytes animals  endotoxin  (294).  However,  seems to  accumulate  was  bound  since in  most  the  between  the  anaesthetized  hydrolase a c t i v i t y . enzyme while kidney.  Also  the  (293).  almost  a l l of  of  platelet  the  fraction  endotoxin  it  is  and  not  administered  believed  that  a  to  large  of endotoxaemia can be attributed  to  217).  binding of  guinea  the  liver,  In our study, we have investigated ship  fraction  in the buffy coat,  to  portion of the toxic and lethal effects injury of this organ (see  accumulated  pigs  the possible  radiolabelled  endotoxin  organs  in  Acid phosphatase was used as a representative  lysosomal  utilized  in this  included study  was  determined  various  lysosomal  studied  as  to  of a r e l a t i o n -  by plasma  organs  and t o x i c i t y ,  existence  heart, E. ,coli  lung,  liver,  endotoxin  spleen  and  radiolabelled  51 with  Cr,  appropriate following  as  it  has  been previously  label for endotoxin (194, the  intravenous  shown that 294).  injection  of  this  radionuclide  We have found that ^Cr-endotoxin  (3.0  is  an  three hours mg/kg)  into  guinea pigs, the greatest approximate accumulation of toxin was found in the spleen (10  (50 ng/mg) and l i v e r (40 ng/mg)  ng/mg)  uptake  and heart  (1.0  ng/mg).  in some of these organs  followed by lung (10 ng/mg), 51  Presumably,  (spleen  much of  the  and l i v e r in particular)  kidney  Cr-endotoxin was due to  - 142 phagocytosis  by reticuloendothelial  the role of the l i v e r is is  unresolved.  cells  While  of the spleen  others  that  of endotoxin when administered  producing  that  it  is  undisputed  the role of the the  that spleen  reticuloendothelial  in detoxifying endotoxin  splenectomy had very l i t t l e  (295),  influence on the  to mice or guinea  pigs  (296,297).  concluded that the spleen plays only a minor role in  the sequelae of endotoxaemia. of  believe  play an important role  The authors, therefore,  lity  Although  important in endotoxaemia,  investigators  have demonstrated  toxicity  cells.  The spleen does, however, possess the capabi-  antibodies  against  endotoxin  (230,298),  which  may  be  important in the development of resistance to subsequent exposures of endotoxin (230). toxin  are  But under experimental  given  as  a single  spleen would be minimal.  bolus  conditions, where lethal doses of endoinjection,  The results  antibody  production  of our investigations  have  in  the  indicated  51 that  the  accumulation  correlated  poorly  of  (r=+0.24)  Cr-endotoxin with  plasma  in acid  the  spleen  phosphatase  hours following the administration of varying doses (1.0, of the radiolabelled toxin to these animals.  (Tissue  accumulation  of  Cr-endotoxin).  These  guinea  levels  3.0  pigs  at  and 6.0  binding was  ized to 1.0 mg/kg dose of endotoxin to eliminate effect 51 on tissue  of  three mg/kg)  standard-  of increasing dosage  observations  imply  that  the spleen is not the primary organ involved in the pathophysiology of endotoxic  shock  and support  the conclusions  that  other  investigators  have made regarding the function of this organ in experimental  (296,297)  endotoxaemia.  The heart was another organ in our study for which we found no c o r r e l a 51 tion  between  activity.  Cr-endotoxin  Of the f i v e  accumulation  organs  examined,  amount of  ^Cr-endotoxin per mg tissue  ted  myocardial  that  failure  occurs  and  the  plasma  heart  (wet weight).  both  in  acid  phosphatase  accumulated It  experimental  is  the  well  lowest  documen-  endotoxic  shock  - 143 (299,300,301) and in c l i n i c a l  septic  exists  responsible  over what factors  endotoxaemia  and  hypoperfusion  septic  are  shock.  consequent  Some  to systemic  blood from the endocardium (304) the  decreased  myocardial  investigators toxaemia  shock  (302,303). for  hypotension  are  factors  contractility  and in c l i n i c a l  contractility.  There are  sepsis  endotoxaemia  shown to  heart.  Additional  decreased mines does  not  and changes  appear  the  to  causes  seen  that  in  coronary  and shunting of  endotoxic  shock.  of humoral substances  for  Other in endo-  can depress myocardial  implicating certain media-  (309) which are released during  impair  the  normal  function  of  the  changes noted in the heart during endotoxaemia include a  s e n s i t i v i t y to  (310)  seems  been  (290,299)  in  studies  and vasopressin  have  believe  (306,307) that  also reported  failure  which may be responsible  seen  tors such as histamine (308) and  cardiac  investigators  have demonstrated the presence  (305)  the  However, controversy  of  have  calcium or cardiac in energy  metabolism  any direct  myocardial  stimulants  cardiac  failure  such  (311,312). depressant  as  Since effects  in endotoxaemia  catechola-  are  endotoxin (313),  it  multiple and  51 complex. to  the  This may explain our observation myocardium did not  correlate  with  that  the  systemic  Cr-endotoxin binding toxicity  in endotoxin-  treated guinea pigs. 51 Our  studies  relating  guinea pigs with activity present way,  our  in either  organs decreased  distribution  t o x i c i t y have  correlated  data  the  also  negatively  indicate  that  the  revealed  with  kidney (r=-0.83)  of  or  the liver  E. that  amount  coli plasma of  of  acid phosphatase  radiolabelled  (r=-0.76). 51  accumulation  Cr-endotoxin in  Stated  A possible  for these results may be that as the i n t e n s i t y of endotoxic shock the blood flow to the kidney and l i v e r decreased,  in another  Cr-endotoxin in  as the severity of shock increased.  toxin  these  explanation increased,  hence reducing the d e l i v -  - 144 51  ery of  Cr-endotoxin to these organs.  It  has  long  been  documented  the administration of endotoxin to animals can cause renal c o r t i c a l such as  that  section  1.4).  observed  in the generalized  The cause of the renal  glomerular c a p i l l a r i e s  by f i b r i n  in on the coagulation  system (314).  frequently tions,  develops  necrosis  is  ing this  Also,  that  in patients with urinary tract also display  Thus the effects  infections  a high t i t e r  (292).  to  the  kidney  blood flow to the kidney is be said  Our  (315).  significantly  Support-  greater when  endotoxaemia  reduced  in this  with  support  condition.  in blood flow  is  clini-  coagula-  ^Cr-endotoxin  the  concept  However, i t  primarily due to  hypotension or to c a p i l l a r y blockage by coagulation  (316).  and  involving both the  observations  during  whether the reduction  infec-  of a n t i - l i p i d A antibodies  tion  distribution  nephritis  is  of i n d i r e c t actions  systems  the  endotox-  urinary tract  of endotoxin on the kidney both experimentally  immune  of  the frequency of chronic pyelonephri-  c a l l y seem to be the result and  occlusion  immune reaction to endotoxin  giving r i s e to abacterial  theory are observations  (Introduction  in chronic pyelonephritis, which  in patients who have a history of  tissue,  these patients  due to  necrosis  aggregates formed by the action of  the cause is believed to be a localized  bound in renal  tis  Schwartzman reaction  that  that cannot  systemic  products or whether both  factors are involved. 51 As mentioned,  Cr-endotoxin accumulation  in the  liver  also  negatively with plasma acid-phosphatase a c t i v i t y in guinea pigs. suggests that of ted  hepatic  endotoxaemia in the  accumulate studied  in  liver was  blood flow decreases  these  animals.  Although less  following endotoxin  still  much  (except the spleen)  greater at  in accordance 51  equivalent  that  found  plasma  This again  with the  Cr-endotoxin  administration,  than  correlated  acid  the in  amount the  severity accumulathat  other  phosphatase  did  organs levels.  - 145 For  example,  when  guinea  pigs  were  injected  with  the  highest  dose  of  51 Cr-endotoxin  used  in  this  study  approximately 1 ng/mg tissue after  (6.0  mg/kg),  the  kidney  three hours whereas the l i v e r  contained s t i l l bound  9-10 ng toxin/mg tissue (values standardized to 1 mg/kg dose of endotoxin). 51 The large quantity of  Cr-endotoxin taken up by the  liver  the result of phagocytosis  by reticuloendothelial c e l l s .  have  much as  demonstrated  that  as  associated with parenchymal c e l l s vations  from ultrastructural  susceptible  to  manifestation  of  (317).  studies  the  not e n t i r e l y  Some  investigators  endotoxin  in  liver  can  Perhaps related to this are  revealing  injury  during  endotoxaemia  this  injury is  systemic  hallmark of the l a t t e r sepsis  75% of  is  that  the  liver  obser-  is  (217,222,284,318).  highly  Indeed,  hypoglycaemia which has  be  a  become a  stages of gram-negative endotoxin shock and c l i n i c a l  (319,320,321,322).  Some  of  the  factors  that  are  believed  to  be  responsible for the hypoglycaemia include diminished l i v e r blood flow (320), impaired gluconeogenesis (322)  and  the  insulin-like  (323),  release  actions  increased  from  (324).  It  peripheral  macrophages has  of  been proposed  u t i l i z a t i o n of  substances that  glucose  which  exert  one consequence of  the systemic hypoglycaemia in endotoxic shock is vasomotor f a i l u r e  resulting  in peripheral pooling of blood and i n t e n s i f i c a t i o n of the shock state (325). Thus, the importance of l i v e r function in endotoxaemia is undisputable. was mentioned in connection with the kidney, our observations  of  As  decreasing  51 Cr-endotoxin  accumulation  in the  liver  with  increasing  plasma  lysosomal  hydrolase a c t i v i t y support the idea proposed by Manson and co-workers that l i v e r  injury in endotoxaemia may be the result of impaired perfusion of  this organ. sion  in  (320)  the  Our observations may also be explained, however, by the depresphagocytic  function  of  the  reticuloendothelial  l i v e r , which is known to occur in endotoxaemia (326).  cells  Although  in  the  l i v e r func-  - 146 tion  is  undoubtably  important  in  endotoxaemia,  our  studies  toxicity  have  relating  51 Cr-endotoxin  distribution  in various  organs  however, that the tissue primarily affected It was noted forty years ted  a critical  feature  acutely  aware  that  sepsis,  particularly  suggested,  in endotoxaemia is the lung.  ago by Moon that pulmonary congestion represen-  in shock  states  pulmonary f a i l u r e in  to  (see  327).  constitutes  gram-negative  Today, a major  septicaemia  clinicians  are  complication in  (328).  McGovern  has  recently reported a study where lung lesions were found in two-thirds of a l l patients  dying of gram-negative  septicaemia,  incidence of lesions found in other organs  which was more than twice the  (329).  incidence of pulmonary f a i l u r e  in gram-negative  mortality rate that  90% (328).  describe  the  approaches  pulmonary  post-traumatic  wet  failure  lung  adult  (328,331) and shock-lung (332). of  a  pneumonitis  which  is  septicaemia  Various terms  associated  (330),  Associated with this high  with  acute  shock  lesion  ("focal can  then  atelectasis") develop  and  into  some  bronchopneumonia  syndrome  syndrome consists  diffuse  interstitial  to  including  distress  interstitial  intravascular congestion, i n f i l t r a t i o n of leukocytes, collapse  states,  respiratory  by  an alarming  have been used  In the early stage, this  characterized  is  septal  oedema,  focal  alveolar  hemorrhage.  characterized  by  This massive  protein-containing oedema, where the lung weight can be increased as much as 50  to  100%  above  peribronchial  as  normal  well  as  and,  by  hemorrhage  perivascular  regions  into of  the  the  intra-alveolar,  lung  (333).  shock-Tung condition causes a reduction in the venti1ation/perfusion or what has been termed a pulmonary "shunt", as a result unventilated strated  alveoli  that  reproduced  the  by venous  blood (333).  pathophysiology  experimentally  by  of  infusing  Vaughn  shock-lung endotoxin  The ratio,  of the perfusion of  and co-workers demon-  seen into  clinically sub-human  could  be  primates  - 147 (334).  Other investigators  shock-lung  lesions  Furthermore, can occur systemic  it  in  has  in the  have also demonstrated  animals  by  administering  been demonstrated  lung at  that  the lung is  endotoxin  injuries  endotoxin doses that  c i r c u l a t i o n or cause lesions  i t appears that  the a b i l i t y to  (333,335,336).  or functional  are  too  to other organs  sensitive  reproduce  to the effects  low to  changes  affect  (264,337,338).  the  Thus,  of endotoxin and is  a  target organ in the pathophysiology of endotoxaemia. Two  effects  failure  are  (336).  may cause  pulmonary  endotoxin  an alteration  hypertension in  of  effects  in  the  lung. or  that endotoxin can cause aggregation the  appears  development  coagulation to  be  that  system  Mechanical by  aggregates  of  the  platelets  and  its  section pulmonary  lysosomal Some  these  1.4),  the  effects  kinins  Of particular  prostanoid,  thromboxane  A^, which  Leukocytes are a rich source of  may  and  lysosomal  hista-  prostanoids  are  probably  and aggrega-  is the potent  originate  activ-  predominantly  substances  importance, perhaps,  doubt  consensus  substances such as  hydrolases, of  little  and can  from leukocytes and platelets upon their sequestration  tion in the lung.  (338).  of  of these blood elements  exerts  (261,264,333,335,336,338,339).  strictor  obstruction  However, while there is  (Introduction,  endotoxin  5-hydroxytryptamine,  released  pulmonary  permeability and pulmonary  through the formation and/or release of vasoactive mine,  of  is one possible mechanism by which endotoxin can increase pulmon-  ary vascular resistance (261,333,336).  ate  the  mechanisms have been proposed by which endotox-  microcirculation by thrombi  leukocytes  in  in pulmonary vascular  Several  these  involved  from  vasoconplatelets  proteases such as  cathep-  sin D (340), which can be very injurious to the lung (330,333,339). A relatively effect  of E . c o l i  recent  study  reported  by Demling and  endotoxin in sheep has indicated that  co-workers lysosomal  on the  hydrolase  - 148 activity  in  the  these animals.  lung  lymph paralleled  the  extent  Furthermore, these investigators  of  pulmonary  demonstrated  injury  that the  in  lung  showed signs of damage before any other signs of systemic injury were apparent of  (264).  While our observations  Demling and associates  damage in endotoxaemia,  with regard  lysosomal  lung  of  generalized  damage only.  Thus,  to  the  hydrolase  measured in lung lymph but rather a reflection  in guinea pigs e s s e n t i a l l y support lung  activity  in the plasma.  cellular although  being  injury  a major  in our  those  site  study was  of not  This would be more l i k e l y  rather  endotoxaemia  than  an  affects  t i s s u e s , the t o x i c i t y of endotoxin closely p a r a l l e l s  its  indication  many  organs  accumulation  of and  in the  lung. The question  then arises  as  to how endotoxin  accumulates  in the  lung.  51 Presumably,  some  of  the  Cr-endotoxin  phagocytosis by pulmonary macrophages. the  pulmonary macrophages  endotoxin the  in the  from the c i r c u l a t i o n  phagocytic  efficiency  of  rat  than  accumulation  is  Mori and co-workers are  the  much less liver  is  result  cells  in  clearing  (341).  dependent  Also,  upon high  oxygen tensions which contrasts with the a b i l i t y of polymorphonuclear cytes  and  aerobic  monocytes  conditions  to  phagocytose  (216,342).  particles  Therefore,  it  under is  anaerobic  quite  the  impaired. between  phagocytic  activity  of  the  pulmonary  Thus, one would not expect to see  well  as  possible  that  in  the  accumulation  of endotoxin  in the  is  macrophages  a linear,  positive  the result  of endotoxin binding to pulmonary endothelial  lung is  cells.  be  correlation  uptake of endotoxin by the lung was e n t i r e l y due to phagocytosis. in the  greatly  would  lung and endotoxicity  then inquire as to whether the presence of endotoxin  leuko-  as  conditions such as shock-lung, where the pulmonary oxygen tension reduced,  of  have shown that  effective  Kupffer  pulmonary macrophages  the  if  the  One may partly  - 149 We have attempted to answer these questions 0T  " E. c o l i  and with  endotoxin with plasma  membranes  a model tissue c e l l , isolated  from red  in,  obtainable  we could  specific  namely the red blood  blood  cells,  since  cell,  both  are  51  1  easily  by studying the interaction  in pure form.  indeed demonstrate  manner to  intact  Utilizing that  Cr-labelled E. c o l i  the  radiolabelled  human erythrocytes  toxin  endotox-  bound in a  and human erythrocyte  ghost  51 membranes. able  Approximately 75-80% of  with  toxin  unlabelled  endotoxin  did not exceed  50  the bound  Cr-endotoxin was  providing the  concentration  of  When higher  concentrations  of  ug/ml.  toxin were used, p a r t i c u l a r l y those exceeding 200 yg/ml, the  measured  possible  binding was  explanation  for  non-displaceable this  and  displace51 Cr-endo51 Cr-endo-  a large portion of  seemingly  unsaturable.  A  large  increase in apparent non-specific 51 binding is that at high concentrations, Cr-endotoxin macromolecules can interact with each other to form large micellular aggregates which may co-sediment with the c e l l s or membranes during the centrifugation step of the binding assay thereby obscuring any specific in  question.  This  studies  has  also  (343).  However, at  particular  been  noted  technical  binding to the  difficulty  and recently reported  in  structures  endotoxin  by other  binding  investigators  51 physiologically relevant  (50 pg/ml or less) we have found that B ) of endotoxin were higher for 3 max' probably is  understandable  ghosts than for  considering that  and contained fragmented  an increased  number of potential  binding to  reported by Ciznar labelled  the binding characteristics  3  not resealed  endotoxin  human red  and Shands  ^C-endotoxin from S.  Cr-endotoxin concentrations  cells  intact red c e l l s .  the ghost  preparation  membranes which would  binding s i t e s . were  who studied  in  (Kp and  Our results close  likely  This  used was possess  with E.  agreement  with  coli those  the binding of biosynthetically  typhi murium to  sheep  erythrocytes  (344).  - 150 Other investigators cells  have also shown that endotoxin can bind to a variety of  in addition to the erythrocyte, including isolated  lymphocytes  (346),  granulocytes  (347),  (348,349) and c e l l u l a r organelles  such as  demonstrated  ability  credence  the  to  of  endotoxins  possibility  that  hepatocytes  macrophages lyso.somes  to  bind  to  some  of  the  (343),  (350).  a  (345),  platelets  Therefore,  variety  of  pulmonary  cells  the  lends  accumulation  of  51 Cr-endotoxin which we noted in guinea pigs could be the  result  of  endo-  toxin binding to lung endothelial c e l l s . A question  which  endotoxin affect  evolves  from the  endotoxin  c e l l u l a r function d i r e c t l y ?  binding studies  is,  Most of the investigations  endotoxaemia reported in the l i t e r a t u r e suggest that endotoxins affect function  K -p-nitrophenylphosphatase  membranes. +  K -ATPase, homeostasis  Since  this  an enzyme (351), this  on c e l l u l a r  observation  by  (K -pNPPase)  enzyme represents crucial  in  the  endotoxin d i r e c t l y inhib-  activity  a  partial  maintenance  in  reaction  of  also  ties  in isolated  toxin  in v i t r o  again  function.  Sayeed  that  (353).  demonstrated  Our findings Na +  may be relevant  transport  was  impaired  that  (352).  both  A related  cell  the  Na ,  +  Na  +  +  and K  in vascular  endotoxin can  observation  is  it  to in  the  recent  lung  slices  + +  K -pNPPase  Furthermore,  calcium transport that  red  A recent report by Liu and Onji  and  dog myocyte membrane preparations  suggesting  of  cellular  + has  human  inhibitory action of endotoxin could have a profound  obtained from endotoxin-treated rats (336).  impair  organ,  organ perfusion or are themselves d i r e c t l y injurious to t i s s u e s .  We have shown in our in v i t r o studies that E. c o l i  effect  on  i n d i r e c t l y through the formation and release of mediators which in  turn affect  its  can  has  been  are  our finding  endotoxin can  subcellular  influence that  the  activi-  inhibitable by endo-  shown that  smooth muscle directly  Na ,K -ATPase  membranes,  cellular activities  integrity of  acid  - 151 phosphatase,  N-acetyl-B-glucosaminidase  and  cathepsin  average, higher in the plasma of gram-negative patients plasma  that were in shock due to other causes. cathepsin  significantly  D activity  elevated  in  protease  in the plasma  groups  patients  were  of  these observations tissue  effects  gram-negative  (.0025 > P > .0005)  lysosomal  quate  septic  of  other  in c i r c u l a t o r y  solely  perfusion.  on the basis This  is  patients  of  it  shock  patients  in shock. difficult  supported  by our  results  lysosomal  was  of  this  Since  both  to  explain inade-  comparing  the  enyzme a c t i v i t y  in guinea pigs and by the results of a study conducted by other  ary damage in dogs (354).  the mean  mean a c t i v i t y  is  the  than in  impaired c i r c u l a t i o n and  of haemorrhage and endotoxaemia on plasma  tors who compared the effects  on  In p a r t i c u l a r ,  the  shock,  were,  shock patients  septic  over  D  investiga-  of endotoxic and haemorrhagic shock on pulmon-  The results of this  latter  investigation  indica-  ted that the lung damage tended to be more immediate and more severe in dogs treated  with  endotoxin than  fore,  that  our results  shock  condition  by  on the plasma  death.  An interesting  potential sis  usefulness  or perhaps  suggest that  directly  actions  in the  haemorrhaged endotoxins  impairing  membrane resulting possibility  animals. add to  cellular  there-  severity  of  integrity  in lysosomal  suggested  the  We f e e l ,  through  disruption  by our c l i n i c a l  their  and  study  the  cell  is  the  of measuring plasma cathepsin D a c t i v i t y in the diagno-  even in assessing the prognosis  of  patients  with  gram-nega-  studies  in mind,  t i v e septic shock. 4.4 Therapy of Gram-negative With  the  interesting bacteraemia.  observations to  of  Bacteraemia the  above  speculate on an effective Certainly, antibiotic  t i v e i f used appropriately (199).  mentioned  mode of therapy for  therapy is  is  gram-negative  necessary and can be  However, i t is  it  also recognized that  effecante-  - 152 cedent a n t i b i o t i c treatment can be deleterious  and has  been reported to be  associated in some cases with an increased  incidence of shock in gram-nega-  t i v e septic patients  we have been able to  (199).  Interestingly,  demonstrate  that gentamycin, an a n t i b i o t i c commonly used in the management of t i v e bacteraemia, in in r a t s .  can actually enhance the toxic effects  gram-nega-  of E. c o l i  endotox-  The mechanism by which gentamycin enhances the toxic effects of  endotoxin is unknown but may possibly involve an action at the level of the mitochondrion.  It has recently been reported that  with mitochondrial functions,  such  as  gentamycin can  calcium uptake  (356).  interfere  Mitochondrial  i n t e g r i t y may also be impaired during endotoxaemia in vivo (291,357,358,359). Indeed,  McGivney  d i r e c t l y to c e l l chondrial would  and  at  have  shown  that  the  cultures can produce deleterious  function  act,  Bradley  (360).  least  Presumably because of  initially,  at  the  addition  effects its  of  endotoxin  on c e l l u l a r mito-  large  plasma membrane,  size,  endotoxin  thereby  affecting  mitochondrial function i n d i r e c t l y whereas gentamycin could interact with and modify c e l l u l a r mitochondria d i r e c t l y .  Therefore,  gentamycin and endotoxin  might act s y n e r g i s t i c a l l y on the mitochondrion r e s u l t i n g in the depletion of cellular  energy,  ultimately, gentamycin  interference  lysosomal and  in  endotoxin  bactericidal  may  are  gram-negative  have deleterious  have  This  homeostatic  proposed  clinical  bacteraemia  side-effects  (199).  since,  bacteraemia  actions causing destruction of gram-negative  septicaemia,  therapy would be desirable.  it  is  and,  effect as  antecedent  Antibiotic  in gram-negative  processes  synergistic  relevance  frequently obtained with  in the liberation of endotoxin (361). gram-negative  cellular  destabilization.  mentioned, poor results therapy  with  of  already  antibiotic  therapy  can  by virtue of bacteria  also the  resulting  Thus, with regard to the treatment of  apparent  that  some  form  of  combination  One aspect of the therapy would involve a n t i b i -  - 153 otics  to  prevent  compon-ent  the  growth  of  the  should be directed to  liberated  endotoxins.  The  antagonism  in gram-negative  the  of  endotoxins  corticosteroids  in  is  as  part  septic  antagonize  endotoxin  therapy to combat  (200).  actions  the  use  No suitable of  of  investigators  questioned  shock  other  effects  of  new and some  have  the  toxic  importance  of the  the toxic  while  of the  the not  Others  gram-negative  which would e f f e c t i v e l y  of  septicaemia  (359).  organisms  antagonism  realization  recommend the use of corticosteroids effects  invading  endotoxin  the of  agent in vivo  has been discovered to date. We believe that an effective  endotoxin antagonist  must be one that could  prevent the binding of endotoxin to c e l l u l a r components and/or attenuate the adverse consequences  of endotoxin-cel1  of agent would be p o t e n t i a l l y capable toxin to c e l l membranes, a better action  between  receptors, in  red  of  cell  binds  membranes  and  membranes and  of antagonizing the binding of endo-  understanding of the nature of the inter-  membranes  is  (197,355).  interacts  (344,345,346,347).  endotoxin-cellular  required.  Specific  interactions  by  results  of  the  (362).  As reported, we have found that  erythrocytes  thesis  from hypotonic l y s i s  ature-sensitive concentrations red  portion of  manner.  from  with  are  present  believe  phospholipid  examining  the  regions  that of  E.  coli  induced by hypotonic challenge.  The  work have E. c o l i  effects  recently  protection  lysis  are  published  endotoxin can protect human  was  seen  increasing temperature.  hypotonic  of  been  in a concentration-dependent  Greater  of toxin or with cells  investigators  endotoxin  We have taken a simple approach to the study  haemolysis  this  Many  primarily  endotoxin on red blood c e l l  protect  To determine what kind  which have been shown to be glycoprotein in nature,  blood  endotoxin  endotoxin  interaction.  capable  with  and temperincreasing  Most agents which of  increasing  the  - 154 "fluidity"  or "disorder"  of membrane l i p i d s  l y t i c and f l u i d i z i n g properties t h i c compounds such  as  etc.  endotoxin  (365).  perhaps  red  cell  it  also can s t a b i l i z e  However, the  stabilization  to temperature  The effects  changes  cytotoxic  agent r i c i n  is  red blood c e l l s  of  lidocaine, propranolol antihaemolysis  a  by analogy  temperature-sensitive  membrane alteration by the bound toxin which governs the s t a b i l i z i n g The  importance of membrane l i p i d s to the antihaemolytic effect  was  suggested  by our  studies  cytes.  The protective  cytes,  p a r t i c u l a r l y at  with  effect  of  phospholipase endotoxin was  the higher temperatures  A-treated reduced  (25°C  effect.  of endotoxin  human erythro-  in these  and 3 7 ° C ) .  erythroInterest-  i n g l y , modifying human erythrocytes with neuraminidase or trypsin had effect  on the a b i l i t y of endotoxin to s t a b i l i z e  in  by other more  on endotoxin  may r e f l e c t  it  endotoxin-induced  in toxin binding but rather,  (366),  drugs,  subunits,  not shared  such as  of temperature  to variations  amphipathic  susceptibility  amphipathic membrane s t a b i l i z e r s  cannot be attributed the  marked  of  anti-psychotic  is not too surprising that  sucrose (362).  with  steroids,  complex  conventional and  anaesthetics, a  media.  Such antihaemo-  are exhibited by a wide variety of amphipa-  is  hypotonic  Since  local  (363,364,365).  these modified  little  erythrocytes  against hypotonic l y s i s . A very interesting observation  in our studies was that the a b i l i t y of E.  c o l i endotoxin to protect red blood c e l l s ly  different  when erythrocytes  human erythrocytes.  from hypotonic l y s i s was s t r i k i n g -  from various  animals  The degree of protection offered  were  used  instead  by endotoxin was much  less when animal red blood c e l l s were used,  and at  low temperatures  lytic  became  apparent.  effect  analyses  of  of the  endotoxin on various  adequate explanation  for  these  cells  erythrocyte membranes the  greater  (362)  susceptibility  of  failed of  (5°C) a  Compositional to  provide an  human red c e l l s  to  -  the s t a b i l i z i n g ted  that  the  actions  effect  -  155  of endotoxin.  of  temperature  However, Aloni  on the osmotic  erythrocyte was much greater than that cells  (367).  lipid  matrix  inverse  It  was  of  suggested  the red c e l l  relationship  between  seen for  by these  membranes temperature  fragility  other  authors were  and co-workers of  repor-  the human  species of red blood  that  differences  responsible  and osmotic  for  in  the  fragility.  observed Since  antihaemolytic effect of endotoxin also seems to be influenced by the matrix  of  the  red  cell  membrane,  it  perhaps  characteristics  of  this  component  of  the  govern the difficult lipid  greater to  matrix  protective  effect  of  assess the physicochemical simply  on the  basis  of  determined by a combination of several tidylcholine/sphingomyelin r a t i o , However, explained  the  differences  human  be  erythrocyte  endotoxin nature  of  concluded  compositional  that  cells.  erythrocyte  assays  It  such as  human  since  surface  erythrocytes  area/volume  this  is  phospha-  could  ratio  is  membrane  factors such as cholesterol,  with  the  membrane may  in these the  the lipid  and degree of acyl-chain saturation  obtained  by other factors,  can  the  (364). also  for  be  example,  which is known to correlate p o s i t i v e l y with osmotic resistance (364,368). In regard the  to the antihaemolytic  polysaccharide  examined by testing  and  lipid  the effect  effect  components  of  endotoxin  the  toxin  whereas  mild  alkaline  hypotonic  lysis.  endotoxins  exhibited  a b i l i t y of Further,  endotoxin to  sodium  haemolytic rather  hydroxide- or than  also  on the  polysaccharide  hydrolysis  protect  was  toxins  did not influence the  sodium hydroxide or hydroxylamine, both of which alter markedly reduced the  importance of  complex  Modification of the  portion of endotoxin with sodium periodate action  of  of chemically modified E. c o l i  osmotic f r a g i l i t y of human erythrocytes.  lytic  of endotoxin, the  with  the l i p i d human red  antihaemoeither  component, cells  from  hydroxylamine-treated  antihaemolytic  effects.  Other  - 156 investigators  also noted that a l k a l i - t r e a t e d endotoxins can cause haemolysis  of red c e l l s  (344).  However, our results  have indicated that  alkali-treated  endotoxins are not homogeneous but can be separated into four fractions Sepharose 6B chromatography.  Of these four f r a c t i o n s ,  only one (peak  which was g l y c o l i p i d in nature, displayed haemolytic a c t i v i t y . comparisons unless  a  between a l k a l i - t r e a t e d  particular  Therefore,  fraction  i t is apparent  and native  of  the  endotoxins  alkali-treated  by  III),  Thus, direct  should be avoided  toxin  is  specified.  from our studies that the consequences of endotox-  in binding to c e l l u l a r membranes are l i k e l y mediated by hydrophobic interactions involving both the l i p i d A component of endotoxin and the l i p i d matrix of  the membrane.  This  other investigators Therefore, investigations interact  with  postulate  is  in  agreement  were  directed  lipid  towards  matrix  of  various  was based on the hypothesis that cell  conclusions  cellular  membranes  in  a  if  similar  endotoxin antagonist,  agents  membranes  have been shown to exhibit antihaemolytic effects  with  the  of  (344,345,369,370).  regarding the quest for a suitable  the  with  which and  in v i t r o .  certain pharmacological manner to  endotoxin,  are  known  our to  like endotoxin, The rationale agents  these  interact  agents may  compete with endotoxin for membrane binding s i t e s and thereby act as pharmacological  antagonists.  Although  a  wide  variety  of  structurally  compounds are able to protect red blood c e l l s from hypotonic l y s i s have focussed  on those which have been shown to exhibit  diverse (365), we  protective  effects  Indeed, a wide variety of substances,  includ-  ing a number of membrane-active agents, have been reported to offer  benefi-  in experimental endotoxaemia.  cial  effects  fructoside ic  (175);  in endotoxaemia or septicaemia.  These  include levan, a poly-  of high molecular weight (371); polymyxin B, a cationic a n t i b i o t lidocaine  (375,376);  naloxone  (377);  propranolol  (378,379);  - 157 nonsteroidal infusions  anti-inf1ammatory  agents  of glucose-insulin-potassiurn  tase  inhibitors  (384,385);  oids  (223,359,372,386,387).  and corticosteroids  (261,380,381); solutions  thromboxane  (383);  antagonists  We have chosen to  prostacyclin  (382);  thromboxane  synthe-  (385);  study  and  corticoster-  lidocaine,  propranolol  as possible endotoxin antagonists for our studies  these drugs are known to have membrane s t a b i l i z i n g or antihaemolytic like  endotoxin  (365,388).  In addition  to  antihaemolytic  steroids have also been shown to be effective from the l y t i c actions  of phospholipase  effects,  since actions  cortico-  in protecting red blood  C (389)  cells  or sulfhydryl group modify-  ing reagents such as N-ethylmaleimide and p-chloromercuribenzoic acid  (390).  51 Interestingly,  when the effects  of  these drugs  dotoxin  (E. c o l i )  to human erythrocyte  results  indicated  that  propranolol  toxin to the membranes almost In contrast,  on the  ghost membranes  antagonized  as effectively  the  binding of  Cr-en-  were examined, 51  binding  the  of  Cr-endo-  as cold (unlabelled)  endotoxin.  methylprednisolone and lidocaine had v i r t u a l l y no effect on the  51 binding of Cr-endotoxin. It was also interesting to note that dimethyl quaternary derivative of propranolol, pranolium, inhibited 51 membrane binding of Cr-endotoxin to approximately the same extent as propranolol.  Further, these drugs were capable of modifying the  the the did  accumula-  51 tion  of  Cr-endotoxin in the  pretreated  lungs  of  with pranolium or propranolol  guinea  pigs  in v i v o .  Guinea pigs  (0.1 mg/kg) .had s i g n i f i c a n t l y  less  51 Cr-endotoxin  present  in their  lungs  than  did  at three hours following a 3 mg/kg intravenous led t o x i n . ing  the  d-isomer was  binding  of propranolol tolerated  of  control  injection of the  Although propranolol was more effective 51 pulmonary  better  untreated  Cr-endotoxin,  endotoxin-treated  guinea  radiolabel-  than pranolium in reducit  was  found  (which is v i r t u a l l y devoid of e-blocking  by  animals  pigs  than  that  the  properties)  the  racemate  - 158 (d, 1-propranolol..  Presumably,  the deleterious  effect  of  d, 1-propranolol  largely due to the impairment of the response of the heart beneficial lamines  effects  on  d-isomer  these  of  was  and lungs to  the  of sympathetic  a c t i v i t y or endogenously-released  catecho-  organs  endotoxaemia.  and  the  in preventing  the  during  propranolol  Both  were equally e f f e c t i v e ,  the  racemate  however,  51 binding  of  Cr-endotoxin  contrast,  pretreating  infusions  of  significant small  decrease  on endotoxin used,  pulmonary  or  binding to rat,  lung t i s s u e .  treatment  with  in  methylprednisolone adenosine  accumulation  activity  in the  (391)  d-propranolol  following endotoxin administration for  the remainder of the f i v e  was  less  effective  and levels  rather  endotoxin-treated  hours  than d-propranolol  non-drug  rat.  animal  was  again  a dose of  treated  d-propranolol  animals.  treatment  obtained with r e l a t i v e l y  E.  can offer coli Thus,  acid  species was found  while  doses  .05)  effective  at  two  which  optimal  caused dosage  reduced  with pranolium  that  approximately  18  80% mortality  in  and  drugs  in the  indicated  scheduling  in our study,  (0.1 mg/kg) of  hours  to hydrocorti-  also for  be  endotox-  phosphatase a c t i v i t y  some protection  endotoxin  to  Drug pretreat-  Pretreatment  in mice have  have not been determined small  < p <  but equally  Mortality studies  of d-propranolol  against  than an action  remained s i g n i f i c a n t l y  hour experiment.  sone or chlorpromazine in lowering plasma  injections  of methyl-  than other drugs in reducing the t o x i c i t y of E. c o l i  (.02  or  of an i n h i b i t o r y effect  When a different  in decreased enzyme a c t i v i t y  In  (35 mg/kg)  lungs  in (10 mg/kg) as judged by plasma acid phosphatase a c t i v i t y . ment resulted  vitro.  (0.5 mg/kg/min) had no 51 of Cr-endotoxin. The  guinea pigs may be a r e f l e c t i o n  on pulmonary macrophage  namely the  more effective  with  membranes  Cr-endotoxin a c t i v i t y observed  prednisolone-treated of the steroid  pigs  erythrocyte  (1 mg/kg/hr)  on the 51  in  human  guinea  lidocaine effect  to  the  of  results  in animals  given  - 159 large bolus injections of endotoxin were c e r t a i n l y very encouraging. the drug may be even more effective  in situations  where smaller  Indeed  levels  of  endotoxin are introduced to the c i r c u l a t i o n in a slow, sustained manner over long  periods  of  time,  as  likely  would  occur  in  clinical  gram-negative  bacteraemi as. The results  of our studies then, demonstrate the f e a s i b i l i t y of prevent-  ing the binding of endotoxins to tissues the toxic actions of these bacterial certain drugs in  tions. tions  appeared  toxicity  of  in the  in vivo,  as  measured  by plasma  following  lung (392).  vivo coupled  This with  its  ability  which  organ  would  than drugs  such as  indirectly  by  can d i r e c t l y have  greater  steroids  stabilizing  blood flow to organs. mental  administration,  antagonize  antagonize potential  the  the  or chlorpromazine for lysosomes  (393,394)  Furthermore, the r e l a t i v e  B-adrenergic antagonism in d-propranolol  mate) should make c l i n i c a l  studies of i t s  or  to  binding of  responsible for the  binding of  in the  are  propranolol  actions of propranolol in experimental endotoxaemia.  propranolol, target  phosphatase eleva-  d i s t r i b u t i o n of to  against  that the tissue concentra-  intravenous  selective  its  acid  endotoxin to membranes are undoubtedly two major factors salutary  The a b i l i t y of  to correlate with t h e i r protective effects  d,l-propranolol,  lung in  means of reducing  c e l l wall constituents.  It has been shown by other investigators  highest the  an effective  (such as propranolol) to prevent the accumulation of endotoxin  lung tissue  endotoxin  as  therapy  Drugs such as endotoxin of  at  a  endotoxaemia  example,  which  may act  adenosine  which  affects  lack of potentially d e t r i (as  compared with the  therapeutic potential  race-  in gram-neg-  ative septicaemia more f e a s i b l e . F i n a l l y , not a l l the b i o l o g i c a l actions of endotoxins are harmful to the host.  Indeed, some beneficial  actions  of endotoxins include the enhancement  - 160 of host resistance to infections tumor  growth  actions  to  possible  humans  way of  detoxified of  using  plasma  (399,400).  toxins  is  (395,396), radiation injury (397,398), and  However,  precluded  the  by the  circumventing this (401).  detoxified  In this  toxins  as  results  have  toxic  regard,  endotoxin  of  effects  problem might  acid phosphatase a c t i v i t y as  preliminary  application  these of  be  beneficial  endotoxin.  through  the  we have examined the antagonists  in  One  use  of  possibility  vivo.  Utilizing  an indication of endotoxicity in r a t s ,  indicated  that  endotoxins  detoxified  by  sodium  periodate treatment can protect against the toxic actions of native endotoxin when the modified toxins endotoxin.  were administered t h i r t y minutes before  The observation  that  sodium  periodate-detoxified  native  toxins  were  more effective  in this regard than sodium hydroxide-modified endotoxins may  be related  our observations  to  that  sodium periodate-detoxified  accumulated in the lung to approximately the same extent toxin.  These observations  periodate-modified tissue  endotoxin devoid of  l i k e native endotoxin?  0-antigen complex  may then pose  polysaccharide  may contribute  chain to  the  the  question,  toxicity  if  it  Since sodium periodate of  endotoxin,  toxic  actions  this of  endotoxin  as did native endowhy is  the sodium  accumulates treatment  portion  endotoxin,  of  in lung  removes the  the  toxin  presumably by  causing an acute inflammatory-like or anaphylactic reaction to occur in the lung, perhaps in a manner analagous to what is believed to occur in abacterial  nephritis  (315).  Thus, i t  seems that while the  lipid  component of  the  endotoxin complex is important for binding to c e l l u l a r membranes, the immune reactions  i n i t i a t e d by the polysaccharide chain can be toxic p a r t i c u l a r l y i f  these are l o c a l i z e d and interfere with the function of a v i t a l organ such as the lung.  -  In conclusion, then,  the  value of u t i l i z i n g plasma  161 -  studies  lysosomal  reported  in this  enzyme a c t i v i t i e s  thesis  as  indicate  a measure of endo-  toxin t o x i c i t y in animal experiments and suggest that plasma levels somal hydrolases  may also provide a r e l i a b l e  shock in patients  with gram-negative  cathepsin  particular,  patients shock  D, with  in  gram-negative  and may therefore  lysosomal  hydrolase  lar  actions  to  be  septic  shock  as  compared with  some diagnostic in patients  the severity of  significantly  value.  with  elevated  other  forms  The elevated  gram-negative  to  on certain  organs.  the functional  human red  cell  vivo,  could reduce  organ  of  Experimentally,  membranes  in a specific  properties  of membranes.  ghost membranes  the  pathological  experiments  tion with appropriate  accumulation involvement  suggest that  it  of  shock  was shown that  of in  It  was shown that  by the  endotoxaemia. such as  The  lung,  endotoxin  a primary  results  d-propranolol  a n t i b i o t i c therapy may greatly  septicaemia.  coli  certain  and when administered  endotoxin  the use of drugs  E.  manner and such binding can  in v i t r o ,  ness of currently employed therapeutic modalities of gram-negative  in  plasma  septic  drugs such as d-propranolol could antagonize the binding of E. c o l i in  of  involvement of endotoxins which may have direct c e l l u -  endotoxins bind to c e l l modify  found  be of  of lyso-  Plasma concentrations  were  activities  suggest the possible  assessment of  septicaemia.  the  of  our  in combina-  improve the  effective-  in the c l i n i c a l  management  - 162 LIST OF REFERENCES 1.  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