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Quantitative changes in Factor II messenger RNA levels during ischemic/reperfusion injury in porcine… Donnachie, Elizabeth Mary 1988

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QUANTITATIVE CHANGES IN FACTOR II MESSENGER RNA LEVELS DURING ISCHEMIC/REPERFUSION INJURY IN PORCINE LIVER  By ELIZABETH MARY DONNACHIE B.M.L.Sc, The University of B r i t i s h Columbia, 1985  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES (Department of Pathology)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA December 1988  ©  Elizabeth Mary Donnachie, 1 9 8 8  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may department or by  his or her  be granted by the head of  my  representatives. It is understood that copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of  -?QT*Ke> locj ^  <  :  The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  ABSTRACT When organs are harvested, stored and transplanted they are subjected to a period of ischemia followed by reperfusion. This process results in s i g n i f i c a n t damage to the organ and the success of transplantation i s frequently dictated by the magnitude of this i n s u l t . It i s for t h i s reason that a high p r i o r i t y has been given to studying the pathological mechanisms underlying t h i s type of ischemic and reperfusion i n j u r y . Ischemic/reperfusion injury to the l i v e r s i g n i f i c a n t l y decreases the a b i l i t y of the organ to synthesize proteins. In l i v e r transplant recipients a decrease from pre-operative values i s seen in the levels of a l l plasma protein c l o t t i n g f a c t o r s .  In p a r t i c u l a r , Factor II levels decrease to 36%  of t h e i r pre-operative l e v e l .  Studies in ischemic rat l i v e r have indicated  that during post-ischemic recovery, the translatable levels of mRNA that code for albumin are q u a l i t a t i v e l y a l t e r e d . It i s not known whether these changes are quantitative. For these reasons, we elected to quantitate the levels of Factor II mRNA in tissue and compare them with plasma levels of Factor II in a porcine model of warm hepatic ischemic/reperfusion i n j u r y . In the model we employed, hepatic ischemia was achieved by diverting the portal blood through a shunt to the right external jugular vein and by clamping the hepatic and gastroduodenal a r t e r i e s . Reperfusion was i n i t i a t e d following 90 minutes of ischemia by removal of the shunt and clamps. Blood and tissue biopsy samples were collected prior to ischemia, following ischemia and at 90 minutes, 270 minutes, 1 day and 2 days of reperfusion. Tissue mRNA was extracted and quantitated r e l a t i v e to the total DNA content. The extraction e f f i c i e n c i e s were monitored and corrected for by means of a synthesized internal standard developed for this study. The e f f e c t of  ii  ischemic/reperfusion injury on Factor II mRNA was assessed using a Factor II cDNA probe and "dot-blot" hybridization techniques. A quantitative method for the determination of porcine Factor II in plasma during ischemic/reperfusion injury was established using a synthetic chromogenic substrate. In addition, routine plasma measurements of l i v e r function and Indocyanine Green clearance tests were performed. The changes seen in the routine plasma measurements performed were found to be s i m i l a r to those of other investigators. Plasma AST (aspartate aminotransferase)  levels rose s i g n i f i c a n t l y during the reperfusion phase  indicating that hepatocellular damage had occured. Plasma glucose and lactate levels increased s i g n i f i c a n t l y during ischemia and returned to +  normal by 90 minutes of reperfusion. Plasma K levels decreased s i g n i f i c a n t l y during the early stages of reperfusion (15 minutes) and returned to normal by 90 minutes of reperfusion. In contrast to the changing plasma levels of l a c t a t e , AST, glucose and 10, b i l i r u b i n values did not vary throughout the operative procedure. The clearance of ICG decreased s i g n i f i c a n t l y during ischemia due to the decrease of blood flow to the l i v e r . During reperfusion, the clearance of ICG was also decreased s i g n i f i c a n t l y , and i t was concluded that t h i s reduction was due to some degree of hepatocellular injury although differences in hepatic blood flow and perfusion cannot be ruled out.  At one  and two days of reperfusion, the ICG clearances returned to normal. Plasma Factor II levels decreased s i g n i f i c a n t l y during the ischemic phase. Concomitant with the decrease in plasma levels was trend in which there was an increase in the tissue levels of Factor II mRNA. However, during reperfusion, the tissue levels of Factor II mRNA decreased to control biopsy values. The decrease in the levels of Factor II mRNA may have occurred as the  iii  r e s u l t of damage i n f l i c t e d during the reperfusion phase, s p e c i f i c a l l y the production of oxygen r a d i c a l s . With continued reperfusion (two days posto p e r a t i v e l y ) , the Factor II mRNA levels remained low in some of the animals studied; in others, the levels started to r i s e again. The plasma Factor II l e v e l s , however, remained low throughout. It i s anticipated that these findings w i l l further our understanding of the pathological mechanisms underlying ischemic/reperfusion i n j u r y .  iv  TABLE OF CONTENTS PAGE ABSTRACT  i i  LIST OF TABLES  ix  LIST OF FIGURES  x  LIST OF PLATES  xii  LIST OF ABBREVIATIONS  xi i i  ACKNOWLEDGEMENTS  xv  INTRODUCTION  1  PATHOPHYSIOLOGY OF ISCHEMIC/REPERFUSION INJURY  3  MATERIALS AND METHODS I. EXPERIMENTAL DESIGN  12  A. Model Design  12  B. Surgical Protocol  13  I I . QUANTITATION OF FACTOR II mRNA IN PORCINE LIVER  18  A. Method Development for the Quantitation of Factor II mRNA from L i v e r . . . . 1. Development of an Internal Standard a) method  18 18 18  b) elimination of gel f i l t r a t i o n step  20  c) s t a b i l i t y study  .21  2. cDNA Probe Studies  21  a) preparation of the probe  21  b) hybridization of the human cDNA probe to porcine mRNA  24  c) cDNA probe binding saturation studies...27  v  d) cDNA probe binding l i n e a r i t y studies  27  3. Tissue DNA Quantitation  28  a) method  28  b) correlation of DNA to tissue protein content  28  c) buffer interference study B.  29  Quantitation of Factor II mRNA in Porcine Liver  30  1. Isolation and P u r i f i c a t i o n of Nucleotides from Liver  30  2. Selection of Poly (A+) RNA  31  3. Hybridization of Porcine Liver mRNA to the Human cDNA Probe for Factor II I I I . QUANTITATION OF FACTOR II IN PORCINE PLASMA  31 33  A. Method  33  B. Concentration of the Activator  34  C. Determination of the Substrate Reaction Time  34  D. Effect of Heparin in the Assay for Factor II  34  IV. ICG CLEARANCE STUDIES  ..36  A. ICG Clearance Protocol  36  B. Dye S t a b i l i t y Study  37  C. Dye Interference Study  37  V ROUTINE PLASMA MEASUREMENTS  38  A. AST  38  B. Glucose  38  C. Total B i l i r u b i n  38  vi  D. Potassium  39  E. Lactate  39  VI STATISTICAL ANALYSIS  40  RESULTS I EXPERIMENTAL DESIGN  41  II QUANTITATION OF FACTOR II mRNA IN PORCINE LIVER  41  A. Method Development for the Quantitation of Factor II from Liver  41  1. Development of the Internal Standard  41  2. cDNA Probe Studies  49  3. Tissue DNA Quantitation  53  B. Quantitation of Factor II mRNA in Porcine Liver..53 III QUANTITATION OF FACTOR II IN PORCINE PLASMA  59  A. Concentration of the Activator  59  B. Determination of Substrate Reaction Time  59  C. Effect of Heparin in the Assay for Factor II  62  D. Porcine Plasma Factor II Levels During Ischemic/ Reperfusion  Injury  62  IV ICG CLEARANCE STUDIES  62  A. Dye S t a b i l i t y Study  62  B. Dye Interference Study  66  C. The Clearance of ICG During Injury  Ischemic/Reperfusion 66  V ROUTINE PLASMA MEASUREMENTS  76  vi i  DISCUSSION I EXPERIMENTAL DESIGN  83  II QUANTITATION OF FACTOR II mRNA IN PORCINE LIVER  84  A. Method Development f o r the Quantitation of Factor II mRNA in Porcine Liver  84  1. Development of the Internal Standard  84  2. cDNA Probe Studies  89  3. Tissue DNA Quantitation  90  B. Levels of Factor II mRNA in Liver and Factor II in Plasma following Ischemic/Reperfusion  Injury  91  III QUANTITATION OF FACTOR II IN PORCINE PLASMA  95  IV  97  ICG CLEARANCE STUDIES A. Clearance of ICG During  Ischemic/Reperfusion  Injury  97  B. Assay Optimization f o r the Detection of ICG in Porcine Plasma V  99  ROUTINE PLASMA MEASUREMENTS  101  SUMMARY  104  APPENDIX 1  106  APPENDIX II  107  REFERENCES  108  vi i i  LIST OF TABLES TABLE 1  2  3  4  5  PAGE  Recovery of the internal standard during the i s o l a t i o n and p u r i f i c a t i o n of mRNA from l i v e r  45  Effect of heparin on the assay f o r plasma Factor II  63  Clearance constants of ICG during ischemic/ reperfusion injury  74  Porcine plasma AST and glucose levels during ischemic/reperfusion injury  77  +  Porcine plasma K and lactate values during ischemic/reperfusion injury  ix  80  LIST OF FIGURES FIGURE 1  PAGE  Schematic representation of the time sequence f o r specimen c o l l e c t i o n  13  2  Schematic representation of the surgical procedure  16  3  Separation of the unincorporated nucleotides from the incorporated nucleotides by gel f i l t r a t i o n chromatography  42  Elution p r o f i l e of the internal standard, human and porcine mRNA from an oligo-dT-cellulose column  44  4  5  Elution p r o f i l e of the internal standard from a gel  f i l t r a t i o n column  46  6  Degradation of the internal standard  47  7  Elution p r o f i l e of the degraded internal standard from a gel f i l t r a t i o n column  8  cDNA probe saturation studies  9  cDNA probe l i n e a r i t y studies  48 .  52 54  10 Porcine l i v e r DNA content and protein content of the tissue 11 Porcine l i v e r DNA content and wet weight of the tissue  55 56  12 Correlation between the DNA standard curve prepared with guanidine-thiocyanate buffer and with phosphate-saline buffer  57  13 Porcine l i v e r Factor II mRNA levels during ischemic/ reperfusion i n j u r y . . . .  58  14 Concentration of the activator for the assay of Factor II in porcine plasma  60  15 Substrate reaction time for the assay of Factor II in porcine plasma  61  16 Correlation between standard curves with c i t r a t e d and heparinized plasma  64  17 Porcine plasma Factor II levels during ischemic/ reperfusion injury  65  x  18 The s t a b i l i t y of Indocyanirie Green  67  19 Effect of ICG on the assay f o r Factor II in porcine plasma  68  20 Effect of ICG on the method f o r glucose  69  21 Effect of ICG on the method f o r potassium  70  22 Effect of ICG on the method f o r AST  71  23 Effect of ICG on the method f o r lactate  72  24 Effect of ICG on the method f o r total b i l i r u b i n  73  25 ICG clearance curve  75  26 Plasma AST levels following ischemic/reperfusion injury  78  27 Plasma Glucose levels following ischemic/reperfusion injury  79  +  28 Plasma K levels following ischemic/reperfusion injury  81  29 Plasma Lactate levels following ischemic/reperfusion injury  82  xi  LIST OF PLATES PLATE  PAGE  1  Agarose gel electrophoresis of the plasmid following digestion with Hin d III and Pst I  2  Hybridization of the human cDNA probe f o r Factor II to porcine mRNA following formaldehyde gel electrophoresis and Northern transfer  xi i  L i s t of Abbreviations  ADP  adenosine diphosphate  AMP  adenosine monophosphate  ATP  adenosine triphosphate  BSA  bovine serum albumin  BSP  sulfobromophthalein  cDNA  complementary deoxyribonucleic acid  Ci  curie  dATP  deoxyadenosine triphosphate  dCTP  deoxycytidine triphosphate  dGTP  deoxyguanidine triphosphate  DNA  deoxyribonucleic acid  DPM  disintegrations per minute  dTTP  deoxythymidine triphosphate  EDTA  ethylene diamine tetraacetate  HEPES  N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic  MOPS  morpholinopropanesulfonic  mm  millimeter  mM  millimolar  mg  milligram  mmol  millimoles  mL  milliliter  mRNA  messenger ribonucleic acid  ng  nanogram  nm  nanometer  xi i i  acid  acid  p  s t a t i s t i c a l probability  r  correlation c o e f f i c i e n t  RNA  ribonucleic acid  S.E.M.  standard error of the mean  S.D.  standard deviation  SDS  sodium dodecyl sulfate  Tris  t r i s(hydroxymethyl)ami nomethane  Tris-HCl  tris(hydroxymethyl)aminomethane hydrochloride  tRNA  transfer ribonucleic acid  ug  microgram  w/v  weight per volume  °C  degrees centigrade  xiv  ACKNOWLEDGEMENTS  I would l i k e to thank those individuals who contributed to the successful completion of t h i s t h e s i s , especially my f r i e n d s , Mr. Allan Hustad and Mrs. Kris G i l l e s p i e . To the members of my supervisory committee for t h e i r helpful suggestions and constructive c r i t i c i s m , I express my gratitude. I am indebted to Dr. R.T.A. MacGillivray and the members of his laboratory, in p a r t i c u l a r , Dr. D. Clevland and Dr. D. Irwin, f o r supplying the Factor II cDNA probe and helping me gain proficiency with hybridization techniques.  To my supervisors, Dr. David Seccombe and Dr. Charles Scudamore, f o r t h e i r support and encouragement throughout and f o r the good times we have shared, I thank you.  The funding of this research was provided by the B r i t i s h Columbia Health Care Research Foundation.  xv  INTRODUCTION  Liver transplantation i s the only known cure for end-stage l i v e r disease (1).  Diseases of the l i v e r that t y p i c a l l y progress to end-stage include:  malignant disorders such as hepatocellular carcinoma and cholangiocarcinoma, fulminant hepatic necrosis (either drug or v i r a l induced), inborn errors of metabolism (Wilson's disease, hemachromatosis, alpha-l-antitrypsin deficiency e t c . ) ,  chronic l i v e r disease (chronic h e p a t i t i s , primary b i l i a r y  c i r r h o s i s ) and conditions of the b i l i a r y t r a c t such as b i l i a r y a t r e s i a ( 2 ) . An apparent cure for Hemophilia A has resulted following a l i v e r transplant (3).  Patients are usually referred for l i v e r transplantation when the  prognosis, with conventional treatment, i s one year of l i f e or when the symptoms are intolerable and there are no absolute contraindications f o r transplantation ( 4 ) . As l i v e r grafting i s now accepted therapy for end-stage l i v e r disease and i s no longer considered an experimental procedure, i t i s estimated that at least 5,000 hepatic transplants per year may be indicated (5). In 1987, approximately 90 centers worldwide performed a total of 3,000 l i v e r transplants with some reporting one-year survival rates of 80% or higher (6). However, one of the major l i m i t i n g factors in l i v e r transplantation (and the transplantation of other organs) i s donor a v a i l a b i l i t y  ( 7 ) . Between  1981 and 1982, one center reported that 26.5% of i t s l i v e r patients died while waiting for a transplant (8). So optimization of the limited donor pool i s c r u c i a l .  This can be achieved in two ways. F i r s t , better  preservation techniques are needed to minimize the effects of ischemic  1  damage. Current techniques l i m i t l i v e r preservation to 8-10 hours  1  (with  comparable kidney preservation being less than two days) (8). Longer preservation times would f a c i l i t a t e the sharing of organs over a wider geographic range and ultimately decrease the cost of l i v e r transplantation. It i s for this reason that the National Institutes of Health consensus report on l i v e r transplantation in 1983 gave top p r i o r i t y to the development of improved techniques for the ex vivo preservation of the l i v e r ( 9 ) . Second, the c r i t e r i a used for donor organ selection must be changed as today's guidelines result in decreased u t i l i z a t i o n of the donor pool (10). Furthermore, the t r a d i t i o n a l parameters of assessing the donor organ are notoriously unreliable predictors of ultimate graft function. It i s estimated that up to 15% of hepatic grafts are l o s t after transplantation due to acute graft dysfunction, so c a l l e d "primary non-function"  (1, 11).  The transplantation of a non-functional l i v e r graft i s disastrous, and re-grafting i s the only chance of survival (1). Patients requiring retransplantation for acute graft non-function decompensate rapidly and have a higher mortality rate than those needing new grafts for other reasons (1). The causes of acute graft non-function  include: technical/surgical  complications, acute graft rejection (both humoral and c e l l mediated) and graft damage due to ischemic/reperfusion i n j u r y , with the majority of f a i l u r e s being attributed to ischemic/reperfusion injury (11). Ischemic/reperfusion  injury i s defined as " ... a l l injury to the organ  during donor maintenance, warm ischemia, preservation, transplantation, and  1  With the recent introduction of a perfusate solution from the University of Wisconsin, l i v e r preservation intervals have been reported to be increased to 24 hours (13). 2  reperfusion" (12). But as y e t , the pathological mechanisms underlying ischemic/reperfusion injury are poorly understood.  It i s only when these  mechanisms are more f u l l y understood can the amount of damage an organ has sustained be assessed prior to transplantation.  Further benefits from  studying ischemic/reperfusion injury i s the aquisition of information needed for the development of better preservation techniques and therapeutic interventions to minimize this damage.  Pathophysiology of Ischemic/Reperfusion Injury  Ischemia i s defined as the cessation of blood flow. This diminishes substrate supply and results in tissue anoxia. Whether or not the anoxic damage i s reversible depends upon the temperature, tissue type, duration of ischemia, a v a i l a b i l i t y of stored substrate and other unknown factors (14). In the kidney and l i v e r , c e l l death from ischemia occurs 1-2 hours after injury at 37°C, whereas, in bronchial epithelium and pancreas i r r e v e r s i b l e damage occurs l a t e r (3 hours) (14). Although the reasons f o r these d i f f e r e n t rates are unknown, a l l c e l l s progress through the same series of biochemical events that result in c e l l death (14). This biochemical progression corresponds to d i s t i n c t morphological stages which have been i d e n t i f i e d by Trump et al (14). Mitochondria depend on a continuous supply of oxygen and substrate to support the production of ATP. a decreased energy charge  2  2  Ischemia disrupts t h i s process, resulting in  in the c e l l (15).  The levels of ADP and i t s  Energy charge = ATP + 1/2 ADP/ ATP + ADP + AMP (17) 3  catabolic products - adenosine, hypoxanthine, xanthine and inosine - a l l r i s e , thereby inducing phosphorylase and phosphofructokinase a c t i v i t i e s which results i n increasing the rates of glycogenolysis and anaerobic g l y c o l y s i s (16). But, because of the anaerobic state of the organ, glycolysis is inefficient.  Consequently, glycogen stores are quickly  depleted, lactate accumulates, and the i n t r a c e l l u l a r pH f a l l s , causing clumping in nuclear chromatin (14). As i n t r a c e l l u l a r lactate l e v e l s increase, phosphofructokinase and hexokinase a c t i v i t i e s decrease, further impairing ATP production (15). With the continuing deterioration of ATP l e v e l s , ATP-dependant ion pump a c t i v i t y decreases resulting in the leakage +  ++  of K and Mg  +  from the c e l l and the d i f f u s i o n of Na and Ca  ++  into the c e l l .  Due to these changes in ion homeostasis, the c e l l u l a r volume expands with water and u l t r a s t r u c t u r a l l y this results in the appearance of small "blebs" on the c e l l membrane. Concurrently, the nuclear envelope and the cisternae of the rough endoplasmic reticulum d i l a t e , as the swelling progresses. The ribosomes detach and the reticulum takes on the appearance of discontinuous vacuoles. With continued swelling, the inner matrix compartment condenses and the intralamellar space of the mitochondria enlarges. These changes are termed "hydropic degeneration" (14). To regain ion homeostasis, the c e l l attempts to enhance substrate supply for ATP generation further depleting the glycogen and accelerating gluconeogenesis (15). However, i n t r a c e l l u l a r AMP levels are s u f f i c i e n t to i n h i b i t phosphoenolpyruvate carboxykinase, an essential enzyme f o r gluconeogenesis.  In addition, the a c t i v i t i e s of both pyruvate carboxylase  and glycerol kinase, two ATP-dependant enzymes which can augment ATP production, are decreased.  Under these conditions alternative energy  4  sources (free fatty acids, amino acids) are mobilized. On entry into the c e l l , free f a t t y acids normally undergo an ATP-dependant activation process generating acyl-coenzyme A.  The acyl-coenzyme A i s then e s t e r i f i e d to L-  carnitine by carnitine palmityltransferase and transported into the mitochondria where they undergo beta-oxidation (16,17). However, during ischemia, the a v a i l a b i l i t y of ATP becomes the rate l i m i t i n g factor for the oxidation of free f a t t y acids. With anaerobic metabolism and i n t r a c e l l u l a r lactate accumulation, the carnitine e s t e r i f i c a t i o n reaction i s i n h i b i t e d , l e t t i n g free f a t t y acids and acyl-coenzyme A moieties accumulate within the cell.  Acyl-coenzyme A, a known i n h i b i t o r of adenine nucleotide translocase  a c t i v i t y , impairs e f f e c t i v e ADP/ATP mitochondrial exchange (17). a d i r e c t uncoupling effect on oxidative phosphorylation (18).  This has  By virtue of  these e f f e c t s , i n t r a c e l l u l a r acyl-coenzyme A accumulation leads to a further reduction in c e l l u l a r ATP content which, in part, i s manifested  by  margination of the nuclear chromatin and "high amplitude swelling" of the mitochondria  (14).  A l l of these ischemia-induced changes are reversible (14). However, i f ischemia continues, an undefined " point of no return" i s reached in which further damage to membranes and mitochondria becomes i r r e v e r s i b l e (14,18). Studies directed at elucidating the i n t r a c e l l u l a r pathological mechanisms underlying ischemic injury have more recently focused on the role played by calcium.  Evidence indicates that mitochondria lose t h e i r a b i l i t y to  generate an electrochemical proton gradient (17). This gradient, which drives ATP synthetase and substrate pumps on the inner mitochondrial membrane, removes the calcium from the cytosol and as the gradient i s l o s t , calcium accumulates within the c y t o s o l , activating membrane-bound lipases  5  (phospholipase, lysophospholipase) which bring about degradation of the membrane (14,17,18). This increase in c y t o s o l i c calcium i s thought to have other damaging effects.  It may interfere d i r e c t l y with mitochondrial functions by unknown  mechanisms and through the activation of normal calcium-dependent processes. Such processes include the i n h i b i t i o n of phospholipid synthesis, regulation of hormonal a c t i v i t y , and binding of prostaglandins to the c e l l membrane (14,18). O v e r a l l , calcium-initiated membrane damage results in c y t o s o l i c contents leaking into the e x t r a c e l l u l a r space. This signals c e l l death. Following death, lysosomal enzymes are released, karyolysis of the nucleus occurs and the cytoplasm becomes more eosinophilic (14). If ischemia has not progressed to the "point of no return", the reintroduction of blood flow to the area may i t s e l f be d e l e t e r i o u s . The reperfusion with oxygenated blood removes degradation by-products such as adenosine, hypoxanthine, free fatty acids and l a c t a t e .  This removal of  potential energy substrates can further retard ATP synthesis and membrane repair.  Furthermore, reperfusion delivers calcium and oxygen to c e l l s  already compromised in t h e i r a b i l i t y to handle these substances.  As oxygen  i s reintroduced into the system, i t i s converted to p a r t i a l l y reduced forms of dioxygen or oxygen radicals (superoxide, hydrogen peroxide, hydroxyl r a d i c a l s ) (19,20,21). These free radicals are extremely reactive (22). They damage tissue by combining and reacting with a l l major classes of macromolecules: polysaccharides, l i p i d s , nucleic acids and proteins. Oxygen r a d i c a l s have been shown to fragment a major polysaccharide, hyaluronic acid (22).  Lipid peroxidation i s i n i t i a t e d by free radicals reacting with  polyunsaturated f a t t y acids to result in the addition of a covalent bond  6  across a carbon-carbon double bond (22). Free radicals also i n i t i a t e  DNA  strand breakage, causing mutagenesis and damage proteins d i r e c t l y by causing polymerization and peptide bond cleavage (20,22). Indirect damage to proteins by limited free-radical oxidation has been shown to increase t h e i r s u s c e p t i b i l i t y to enzymatic hydrolysis (20). Moreover, some products of l i p i d peroxidation are, in themselves, damaging. Maiondialdehyde and hydroxyalkenals cause errors in DNA repair mechanisms v i a base s u b s t i t u t i o n , induce DNA cross-linkage, polymerize protein and decrease the synthesis of RNA, DNA and proteins in in v i t r o systems (23). The evidence that supports the presence of free radicals i s both d i r e c t and i n d i r e c t .  Direct evidence i s given by the actual detection of free  radicals by electron spin resonance spectroscopy (24). Their existence i s strongly suggested by the beneficial effect that anti-oxidants (glutathione, vitamin E and ascorbic acid) and enzymes such as superoxide dismutase and catalase have on established models of ischemic/reperfusion injury (25,26,27). C l e a r l y , reperfusion-induced free radical production potentiates the ischemic i n s u l t .  What i s the o r i g i n , however, of free radical production  during reperfusion? Recently i t has been suggested that xanthine oxidase, an enzyme located in the cytoplasm of l i v e r c e l l s and other c e l l s , i s the major source of free r a d i c a l s .  Under normal physiological processes, the  enzyme exists in the dehydrogenase form.  Yet during ischemia, xanthine  dehydrogenase i s converted to xanthine oxidase v i a the action of a calciumdependant protease which i s thought to be activated due to the accumulation of i n t r a c e l l u l a r calcium (28). Another consequence of the ischemic injury is the accumulation of hypoxanthine, a degradation product of ATP and the  7  natural substrate f o r xanthine oxidase.  Once the organ i s reperfused,  oxygen i s reintroduced into the tissues where i t reacts with the hypoxanthine  and xanthine oxidase to produce oxygen free r a d i c a l s .  In addition to i t s e f f e c t on the d i r e c t production of oxygen r a d i c a l s , xanthine oxidase has also been shown to augment the production of free radicals indirectly.  Xanthine oxidase has the a b i l i t y to mobilize iron from  i t s storage p r o t e i n , f e r r i t i n , by both 02~-dependant and -independent pathways (29). The r o l e of iron and other t r a n s i t i o n metals as c a t a l y s t s in the production of free r a d i c a l s y_j_a the modified Haber-Weiss reaction i s well established  3  (30). Roy and McCord have studied the k i n e t i c s of the  conversion of the dehydrogenase to oxidase form (D-0) in the l i v e r and other tissues (31). During hypothermic ischemia (23°C) the D-0 conversion takes 30 minutes, which i s quite slow in comparison to the conversion in the i n t e s t i n e and heart (31). However, in the normothermic ischemic l i v e r , near-complete conversion from the dehydrogenase form to the oxidase form i s achieved in 10 minutes with a concommitant f o u r - f o l d increase in the oxidase activity  (31).  Next to the small i n t e s t i n e , the l i v e r i s the r i c h e s t source of xanthine dehydrogenase (31).  It i s also the storage organ f o r the body's trace  elements and i s the s i t e of f e r r i t i n synthesis (32).  In view of these f a c t s ,  one might conclude that i t s potential f o r free radical induced damage i s high. The l i v e r i s also highly susceptible to ischemic injury due to i t s  02"+ Fe  2+  Fe  3  + H202  Fe  2+  + 0, Fe  3+  + H0"+  HO (30) 8  architecture.  The subunit of the l i v e r , the Rappaport acinus, consists of a  three-dimensional aggregate of hepatocytes  which are organized as plates  with intervening sinusoids centered around terminal branches of the portal vein and the hepatic artery.  The blood flows from the hepatic a r t e r i o l e and  portal venule into the sinusoids then drains into the central v e i n .  This  structural organization gives r i s e to gradients in substrate and oxygen concentrations. As a r e s u l t , the hepatocytes in the acinus are divided into three d i f f e r e n t zones of metabolic a c t i v i t y , those c e l l s in zone three (perivenous hepatocytes) being most susceptible to anoxic conditions (32). Most metabolic a c t i v i t i e s of the l i v e r , including the synthesis of protein, are impaired after the onset of ischemia (33). As the synthesis of protein i s an integral part of l i v e r function, i t has been used extensively as a marker of ischemic l i v e r damage (33,34,35). Relatively mild conditions of l i v e r ischemia (hepatic artery l i g a t i o n ) have been shown to decrease protein synthesis in v i t r o and in vivo by 60% and 80%, r e s p e c t i v e l y , as judged by the incorporation of r a d i o l a b e l e d amino acids (35).  In addition,  protein synthesis has been shown to decrease progressively as the ischemic interval continues and the a b i l i t y of the l i v e r to regain i t s protein synthesizing c a p a b i l i t y i s thought to be dependant upon the duration of the ischemic event (36).  In a rat model of partial warm hepatic ischemia,  protein synthesis recovered after an ischemic interval of 60 minutes (36). Although the overall a b i l i t y of protein synthesis recovered, the production of albumin was decreased in r e l a t i o n to i t s control value and the investigators found that t h i s decrease in albumin production was a d i r e c t r e s u l t of depressed r e l a t i v e amounts of translatable albumin mRNA (37). These changes were found to occur during reperfusion, 16 hours a f t e r  9  termination of the ischemic event.  In that both ischemia-induced  injury and  free radical-induced i n j u r y , (analogous to that which i s produced during reperfusion), have been demonstrated to cause DNA strand breaks in v i t r o and in v i v o , one might hypothesize that similar strand breakage might occur to mRNA (38,39).  Although these decreases were seen during reperfusion, the  translatable levels of albumin mRNA were not investigated during ischemia. In addition, the effects of ischemia and reperfusion on mRNA for other proteins have not been established. The s p e c i f i c aim of this t h e s i s , therefore, was to use a porcine model to examine the effects of 90 minutes of complete warm hepatic ischemia followed by 2 days of reperfusion on absolute levels of Factor II (prothrombin) mRNA. Factor II was chosen as the marker of l i v e r damage for several reasons. Prothrombin i s a protein produced exclusively by the l i v e r (32). It functions as an essential enzyme in coagulation and as such, helps maintain normal hemostasis.  Liver transplant recipients are in a compromised  hemostatic condition prior to transplantation due to t h e i r existing l i v e r disease.  Furthermore, a decrease in a l l coagulation f a c t o r s , in comparison  to pre-operative values, i s noted during the transplant procedure (40). In addition, once the organ has been transplanted and the r e c i p i e n t s ' blood allowed to reperfuse the g r a f t , a coagulopathy may be i n i t i a t e d by factor(s) released from the damaged graft (41).  For these reasons, an immediately  functioning graft i s essential to help replace depleted coagulation factors as uncontrollable bleeding i s a s i g n i f i c a n t factor in peri-operative mortality (42). The biological h a l f - l i f e of Factor II (96-100 hours) i s such that under normal circumstances  i t s turnover i s slow in comparison to  other coagulation factors (43).  10  Factor II mRNA was quantitated by using i t s human cDNA probe previously cloned and supplied by the laboratory of Dr. R.T.A. MacGillivray (44). The established methods f o r the detection of mRNA in tissue using t h e i r DNA (or RNA) probes, are q u a l i t a t i v e procedures and f o r the purpose of t h i s thesis i t was necessary to make these methods quantitative.  The objectives of this thesis were to: 1. establish a method f o r the accurate quantitation of Factor II mRNA using a human cDNA probe; 2. making use of this method, quantitate Factor II mRNA in l i v e r biopsies from the established model of warm hepatic ischemic/reperfusion  injury in the p i g .  3. develop and characterize a method to quantitate porcine Factor II levels in porcine plasma; 4. making use of t h i s method, analyze plasma specimens obtained from the established model of warm hepatic ischemic/reperfusion  injury  in the p i g . 5. compare the results obtained above with other established parameters of hepatocellular damage.  11  MATERIALS AND METHODS PART I - EXPERIMENTAL DESIGN  A. Model Design 1. ANIMAL The domestic, outbred pig was selected because i t s hepatic architecture and vasculature i s similar to that of man  (45). The size of the animal  f a c i l i t a t e d sample c o l l e c t i o n once the chronic blood l i n e s were in place. In addition, the animal was inexpensive to purchase and maintain.  2. BLOOD and TISSUE SAMPLE HANDLING Whole blood (2 mL) was drawn into a heparinized tube, mixed by inversion, and immediately placed on i c e . The plasma was separated from the c e l l s by centrifugation at 3,000 X g (Silencer H-103NA, VWR  S c i e n t i f i c , San  Francisco, C a l i f o r n i a ) , frozen within 20 minutes of c o l l e c t i o n , and stored at -70°C until  analyzed.  Liver biopsies were processed immediately. The tissue was rinsed with s t e r i l e saline and sectioned into three segments. One segment was  placed  into 2.5% glutaraldehyde and another segment into buffered formalin. These were processed for electron and l i g h t microscopy. The remaining segment, (1 gram) was frozen in l i q u i d nitrogen and used to quantitate tissue l e v e l s of Factor II mRNA. A graphic representation of the time sequences for specimen c o l l e c t i o n (blood and tissue) i s given below (Figure 1). A more in-depth description is given in Appendix I.  12  Model pre-op  •reperfusion-  ischemia  0  1 day  2 days  post-op  post-op  90.  •it  A  A 90  A  B  180  240  D  f  •ih  A  G  Figure 1 . Schematic r e p r e s e n t a t i o n of the Lime sequence f o r specimen c o l l e c t i o n i n the model of warm hepatic i s c h e m i c / r e p e r f u s i o n i n j u r y . L i v e r b i o p s i e s and p e r i p h e r a l blood specimens f o r r o u t i n e c h e m i s t r i e s are taken at times i n d i c a t e d as B through G . I C G clearances? are performed at A , B, F , and G . Blood f o r p r e - o p e r a t i v e r o u t i n e c h e m i s t r i e s a r e drawn a t time A .  B. Surfqical Protocol a) Day 1 Following a 24-hour f a s t , outbred female white pigs weighing 15-20 kg were siedated with an intramuscular i n j e c t i o n of ketamine (11 mg/kg) given in the s o f t t i s s u e s o f the neck. The animal was anesthetized by mask using i s o f l u r a n e t o s u f f i c i e n t depth to enable endotracheal  intubation. After  i n t u b a t i o n , the animal was v e n t i l a t e d with a mixture o f i s o f l u r a n e (1-3%)  13  and 100% oxygen. A g a s t r i c tube and rectal temperature probe were introduced and the body temperature was monitored. The animal was connected to an ECG. A 20 gauge catheter was inserted into an ear vein and normal saline infusion was started. (40 mL/hour) The pig was placed supine atop a warming pad on the operating t a b l e . The entire neck and abdomen were clipped and scrubbed with an iodine solution then prepared with a 0.5% chlorohexidine in 70% alcohol mixture and the pig was then appropriately  draped. An i n c i s i o n was made p a r a l l e l and l a t e r a l to  the sternocleidomastoid muscle and the right carotid artery and internal jugular vein were exposed by careful d i s s e c t i o n . An a r t e r i a l l i n e (polyvinyl c h l o r i d e , 1.69 mm internal diameter X 3.07 mm outer diameter) was placed into the right carotid artery with the d i s t a l artery being l i g a t e d . A venous l i n e was placed into the right internal jugular vein and advanced to the right side of the heart. Following placement, both l i n e s were tunnelled subcutaneously  to the interscapular midline and e x t e r i o r i z e d . The a r t e r i a l  l i n e was used to monitor a r t e r i a l blood pressure and blood gases. Both l i n e s were used f o r acute and chronic blood sampling. The l i n e s were kept patent on a chronic basis with heparinized saline (200 units/mL). Following l i n e placement, the neck was closed with 2-0 p o l y g l y c o l i c acid sutures. The abdomen was opened through a midline i n c i s i o n from the xyphoid to the midhypogastrium. Adequate exposure was obtained by means of a Balfour s e l f retaining retractor secured with towel c l i p s . The l i v e r was mobilized and the portal structures were i s o l a t e d . The hepatic artery was dissected throughout i t s entire length beginning at the aorta below the takeoff of the splenic artery. Minor a r t e r i a l branches were cauterized  and the  gastroduodenal and l e f t g a s t r i c arteries were i d e n t i f i e d . At t h i s point,  14  Doppler flow probe cuffs of the appropriate size were placed around the hepatic artery and the portal vein. The peritoneum and f a s c i a l layers were closed with continuous 0p o l y g l y c o l i c acid sutures and the skin with 2-0 p o l y g l y c o l i c acid sutures. The anesthetic agents were discontinued and the animal was fed following recovery. Food was subsequently withdrawn for an overnight f a s t .  b) Day 2 Following an overnight f a s t , a blood sample was drawn from the carotid artery catheter and processed for Factor II and routine chemistries +  glucose, total b i l i r u b i n , lactate and K ). Subsequently, an  (AST,  indocyanine  green clearance study was performed as per protocol. Following the clearance study, the animal was f e d .  c) Day 3 A 24 hour fast i s begun.  d) Day 4 Following the 24 hour f a s t , the animal was anesthetized and prepared for surgery as previously outlined (Day 1). The a r t e r i a l l i n e was connected to a high pressure sampling port which allowed for blood pressure monitoring as well as a r t e r i a l blood sampling. The abdomen was opened as previously described (Day 1). A 16 gauge angiocath was introduced through a venotomy in the infrahepatic i n f e r i o r vena cava and positioned so that i t s sampling end was at the level of the hepatic veins. The Doppler flow probe was removed from the portal vein and a porto-jugular shunt was placed from the portal  15  vein into the r i g h t external j u g u l a r v e i n . A Bardec cannula with a s i l i c o n connector was  placed into the portal vein on the i n t e s t i n a l side with the  d i s t a l end clamped. Blood was The shunt was  allowed to f i l l  then clamped and introduced  the shunt d i s p l a c i n g any a i r .  into the r i g h t external j u g u l a r  v e i n . Subsequently, the clamps were removed. The c e l i a c a r t e r y was with a p e d i a t r i c S t a t i n s k y clamp and the l e f t g a s t r i c a r t e r y was with a D i e t r i c k clamp. The gastroduodenal a r t e r y was s i l k l i g a t u r e and the a r t e r y was branch u n t i e d . Bleeding was  clamped  clamped  t i e d d i s t a l l y with a  p a r t i a l l y d i v i d e d leaving the proximal  noted to be present or absent. I f absent, the  hepatic c i r c u l a t i o n had been adequately i s o l a t e d . The hepatic a r t e r y and other c o l l a t e r a l s to the l i v e r were clamped with atraumatic clamps, thus beginning the ischemic period.(Figure 2) Following 90 minutes of the p o r t a l vein was  ischemia,  b r i e f l y clamped and the porto-jugular shunt removed and  the i n t e g r i t y of the vessel r e - e s t a b l i s h e d . Subsequently, the clamps were removed from the portal v e i n , hepatic a r t e r y and i t s c o l l a t e r a l s - thus marking the s t a r t of the reperfusion phase.  JV  ST  Figure ?.. Schematic r e p r e s e n t a t i o n of the s u r g i c a l procedure. Ischemia i s achieved by d i v e r t i n g b l o o d , through an e x t e r n a l shunt ( S T ) , from the p o r t a l vein (PV) to the e x t e r n a l jugular vein (JV) and by clamping the hepatic a r t e r y (HA) and the gastroduodenal a r t e r y (GD) ( 4 2 ) .  16  A l l blood samples taken during the reperfusion phase were taken from the hepatic vein angiocath following a 30 second vena caval clamp to c o l l e c t hepatic venous blood. At the end of the reperfusion phase, the hepatic vein sampling catheter was removed and the Doppler flow probe was placed around the portal v e i n . The l i n e s in the carotid artery and internal jugular vein were appropriately housed. The abdomen and neck were closed as previously described (Day 1). The anesthetic agents were discontinued, the animal was allowed to recover and was fed. Food was subsequently withdrawn for an overnight  fast.  e) Dav 5 Following an overnight f a s t , a blood sample was drawn from the a r t e r i a l l i n e and processed for Factor II and routine chemistries. Subsequent to t h i s , an indocyanine green clearance study was conducted as per p r o t o c o l . The animal was anesthetized and prepared as previously described and an open biopsy of the l i v e r was taken. The animal was closed, the anesthetic agents were discontinued and the pig was allowed to recover.  f) Dav 6 The protocol as outlined for Day 5 i s followed. The animal was then euthanized.  17  PART II - QUANTITATION OF FACTOR II MRNA IN PORCINE LIVER A. Method Development f o r the Quantitation of mRNA in Porcine Liver 1. DEVELOPMENT OF AN INTERNAL STANDARD a) Method The internal standard consisted of E. c o l i tRNA that had been polyadenylated and r a d i o l a b e l e d using poly (A) polymerase according to the method of Sippell (47). B r i e f l y , the procedure was as follows. E. c o l i tRNA (30 ug), (Boehringer Mannheim, Montreal, Quebec) was incubated at 37°C in a 4  buffer (0.100 mL total volume) of 50 mM T r i s - H C l , pH 7.9, 10 mM MgCl2, 2.5 mM g  MnCl2, 250 mM NaCl, 50 ug bovine serum albumin, 9.43 X 10" mmoles (2,5',83  H )-ATP ( s p e c i f i c a c t i v i t y approximately 50-60 Ci/mmole), (Amersham, O a k v i l l e , Ontario) and 6 units of poly (A) polymerase (Pharmacia, Dorval, Quebec). Following a 24 hour incubation, 0.1 mM of unlabelled ATP was added and the mixture was further incubated for 5 hours. The reaction was terminated by the addition of 0.100 mL of a water-saturated phenol (Bethesda Research Laboratories, Gaithersberg, Maryland):chloroform:isoamyl alcohol mixture (25:24:1).  i) P u r i f i c a t i o n of the Nucleic Acids The polyadenylated RNA and unincorporated nucleotides were p u r i f i e d from the reaction buffer by extraction of the aqueous solution of nucleic acids with a phenol:chloroform:isoamyl alcohol mixture (25:24:1). An equal volume  4  Unless otherwise indicated, a l l chemical were purchased from Sigma Chemical Company, S t . Louis, Missouri. 18  of t h i s organic mixture was added to the reaction buffer, mixed vigorously and centrifuged at 14,000 X g for 30 seconds (Model 235-B, Fisher S c i e n t i f i c Co., Ottawa, Ontario). The aqueous (upper) phase was removed and transferred to a s t e r i l e tube. The organic phase was "back extracted" twice with an equal volume of s t e r i l e d i s t i l l e d water and the aqueous phases were combined. The organic extraction was repeated two more times; however, these two additional extractions were performed using an equal volume of a chloroform:isoamyl  alcohol mixture  (24:1)(48).  i i ) Separation of Unincorporated Nucleotides from Incorporated Nucleotides by Gel F i l t r a t i o n Chromatography The separation of the unincorporated nucleotides from the incorporated nucleotides was achieved by gel f i l t r a t i o n column chromatography through Sephadex-G50 (Pharmacia, Dorval, Quebec). The column was equilibrated with a buffer consisting of 10 mM T r i s , pH 8.0, and 1 mM EDTA (TE buffer, pH 8.0). The radioactive fractions in the leading peak were collected and pooled (49). The polyadenylated RNA in the sample was concentrated by p r e c i p i t a t i o n with ethanol as described below.  i i i ) Concentration of the Nucleic Acids by Ethanol P r e c i p i t a t i o n Polyadenylated RNA was concentrated by the addition of sodium acetate, pH 5.0, to a f i n a l concentration of 0.3 M, and 2 volumes of 99% ethanol. The sample was placed at either -20°C overnight or at -70°C f o r 1 hour. Subsequently, the precipitate was collected by centrifugation at 14,000 X g for 10 minutes. The p e l l e t was dried and resuspended in s t e r i l e d i s t i l l e d  19  water (50).  iv) Selection of Poly (A+)  RNA  Polyadenylated RNA was p u r i f i e d by oligo-(dT)-cellulose a f f i n i t y chromatography. The sample was heated in a 68°C water bath for 10 minutes and then immediately placed on i c e . Sodium acetate, pH 7.0, and SDS were added to a f i n a l concentration of 0.4 M and 0.1%, r e s p e c t i v e l y . The sample was then applied f i v e times to a column (1.8 cm X 2.3 cm) which had been equilibrated with a buffer containing 0.4 M sodium acetate, 1.0 mM EDTA, and 0.1% SDS.  The  column was washed with t h i s buffer until no more radioactive counts appeared. The poly (A+) RNA was then eluted from the column with 1.0 mM EDTA and 0.1% SDS  (51). The fractions with the highest r a d i o a c t i v i t y were pooled,  ethanol-precipitated overnight, resuspended in s t e r i l e d i s t i l l e d water, and stored at -70°C until needed.  b) Elimination of the Gel F i l t r a t i o n Step A study was conducted to determine an alternative method in separating the unincorporated nucleotide from the incorporated nucleotide. In this study, two simultaneous runs of r a d i o l a b e l l i n g E. c o l i tRNA and phenol:chloroform:isoamyl  alcohol extractions were performed as described  previously. Following t h i s , one was subjected to gel f i l t r a t i o n through Sephadex G-50  as described above, while the other was ethanol-precipitated  (twice), as described above, prior to the gel f i l t r a t i o n step.  20  c) S t a b i l i t v Study The internal standard was synthesized and p u r i f i e d as described above. The absorbance 260 and the amount of r a d i o a c t i v i t y present was determined (Lambda 3B UV/VIS Spectrophotometer, Perkin Elmer Co., Norwalk, Connecticut; Beckman LS-9000 Liquid S c i n t i l l a t i o n Counter, Beckman Instruments, F u l l e r t o n , C a l i f o r n i a ) . The sample was then aliquoted and stored at -20°C. At s p e c i f i c time i n t e r v a l s , samples were thawed and then applied to a Bio-Spin 6 Column" (Bio-rad, Richmond, C a l i f o r n i a ) . The absorbance260 and the r a d i o a c t i v i t y present in the eluant were determined and compared to the baseline values. Once the recovery of the internal standard f e l l below 95%, the column was further washed with four column volumes of s t e r i l e TE buffer, pH 8.0. The r a d i o a c t i v i t y and the absorbance260 present in the eluant were determined as described.  2. cDNA PROBE STUDIES a) Preparation of the Probe for Hybridization Studies i - Isolation and P u r i f i c a t i o n of the Plasmid After harvesting the E. c o l i containing the plasmid, the bacterial p e l l e t was resuspended in ice-cold buffer consisting of 10 mM T r i s , pH 8.0, 100 mM NaCl and 1 mM EDTA (STE b u f f e r ) . This mixture was centrifuged at 3,000 X g for ten minutes. The supernatant was discarded and the p e l l e t was resuspended in 5 mL of a solution of 50 mM glucose, 25 mM T r i s , pH 8.0, 10 mM EDTA and 5 mg/mL of lysozyme (added just before use). The mixture was allowed to stand at room temperature for 5 minutes. Ten m i l l i l i t e r s of a solution containing 0.2 N NaOH and 1% SDS was then added. The tube was covered, inverted sharply and placed on ice for ten minutes. Following t h i s , 7.5 mL of  21  a 5 M potassium acetate s o l u t i o n , pH 4.8, was added and the mixture was placed on ice f o r 10 minutes. The mixture was centrifuged at 10,000 X g (Beckman J2-21, Beckman Instruments, F u l l e r t o n , C a l i f o r n i a ) f o r 20 minutes at 4°C. The supernatant was divided into two equal volumes and the DNA  was  precipitated by the addition 0.6 volumes of isopropanol. The p r e c i p i t a t e was collected by centrifugation at 12,000 X g for 30 minutes at room temperature. The DNA p e l l e t was washed with 70% ethanol, dried b r i e f l y in a vacuum dessicator and resuspended in 11 mL of TE buffer. To t h i s suspension, 11 grams of CsCl and 0.006 grams of ethidium bromide was added. This mixture was protected from l i g h t and incubated at room temperature for 30 minutes. Aggregates that formed between the bacterial proteins and the ethidium bromide were removed by centrifugation at 3,000 X g f o r 10 minutes. The supernatant was centrifuged at 140,000 X g for 42 hours at 20 °C. The lower band, (visualized by u l t r a v i o l e t l i g h t ) consisting of closed, c i r c u l a r plasmid DNA was collected through an 18 gauge needle into a s t e r i l e tube. The ethidium bromide was removed by extraction (4 times) with isoamyl a l c o h o l . The aqueous phase (containing plasmid DNA) was diluted with 2 volumes of s t e r i l e d i s t i l l e d H20. The DNA was precipitated by the addition of 2 volumes of 99% ethanol and placed at -20°C overnight. The precipitate was collected by centrifugation at 10,000 X g f o r 30 minutes at 4°C (52). The p e l l e t was d r i e d , resuspended in s t e r i l e d i s t i l l e d H20 and the absorbance at 260 nm. was determined. (A map of the plasmid i s given in Appendix II.)  i i - Removal of the Insert from Plasmid DNA Plasmid DNA (0.001 mg) was digested with Hin d III (1 unit) and Pst I (1 unit) (Bethesda Research Laboratories, Gaithersberg, Maryland) in a buffer  22  of 50 mM T r i s , 10 mM MgCl2 and 50 mM NaCl (supplied by the manufacturer) at 37°C for one hour. The insert was separated from the plasmid by agarose electrophoresis. B r i e f l y , ultrapure agarose (1%) (Bethesda Research Laboratories, Gaithersberg, Maryland) was melted in a 0.04 M T r i s - a c e t a t e , 0.001 M EDTA buffer (1 X TAE). The digested plasmid was diluted 4:1 with a loading buffer of 0.25% bromophenol blue, 0.25% xylene cyanol, and 40% (w/v) sucrose in s t e r i l e d i s t i l l e d H20 and applied to the g e l . The gel was electrophoresed in 1 X TAE buffer for 1 hour at 70 V then stained with ethidium bromide (0.05 mg/mL). The resulting bands were visualized by u l t r a v i o l e t l i g h t . The band corresponding to approximately 370 base pairs was cut from the gel and placed in a small length of d i a l y s i s tubing. The tubing was f i l l e d , with the least volume p o s s i b l e , of 0.5 X TAE buffer and immersed in an electrophoresis tank f i l l e d with 0.5 X TAE. The DNA was electroeluted from the gel s l i c e by the application of 0.02 amps of current for 10 minutes. The p o l a r i t y of the current was then reversed for 10 seconds to release the DNA from the wall of the d i a l y s i s tubing. The buffer surrounding the gel s l i c e in the d i a l y s i s tubing was removed and the DNA was p u r i f i e d by extracting once with each of phenol, phenol/chloroform, and ether (53). The DNA was then precipitated with ethanol (as described previously). The resulting precipitate was pelleted by centrifugation, d r i e d , resuspended in s t e r i l e d i s t i l l e d H20 and stored at -70°C until needed.  23  i i i - Radioactive Labelling of the cDNA Probe The cDNA probe was o l i g o - l a b e l l e d using the Klenow fragment of DNA Polymerase I. A b r i e f description of the method follows. One hundred nanograms of probe was placed in a boiling water bath for 3 minutes and then transferred to a 37°C water bath for 10-30 minutes. The probe was then added 32  to a mixture which contained 20 ug BSA, 50 uCi P-dATP, ( s p e c i f i c a c t i v i t y was approximately 3000 Ci/mmole) (New England Nuclear, Lachine, Quebec) 2.5 units of enzyme (Pharmacia, Dorval, Quebec) 0.01 mL of Klenow l a b e l l i n g buffer (described below) and s u f f i c i e n t s t e r i l e d i s t i l l e d water for a f i n a l volume of 0.05 mL. The Klenow l a b e l l i n g buffer was a combination of three stock solutions (A,B,C) that were mixed in a r a t i o of 2:5:3. Stock A consisted of 1.25 M TRIS, pH 8.0, 0.125 M MgCl2, 2.49 M 2-mercaptoethanol and 4.84 mM of each of dCTP, dTTP, dGTP. Stock B was 2 M HEPES b u f f e r , pH 6.6, and Stock C was pd(N)6 (poly deoxynucleotide consisting of 6 bases) dissolved in TE, pH 7.0, to give 90 Absorbance260 units/mL. The tube was incubated in a 37°C water bath overnight and the reaction was stopped with the addition of 1% SDS, 10 mM EDTA and 25 ug tRNA in a total volume of 0.15 mL. This method i s e s s e n t i a l l y that described by Feinberg and Volgelstein (19). The unincorporated nucleotides were separated from the incorporated nucleotides by two sequential ethanol precipitations as described in a previous section.(page 19)  b) Hybridization of the Human cDNA Probe to Porcine mRNA A preliminary study was undertaken to ascertain whether or not human cDNA for Factor II would cross hybridize to porcine mRNA.  24  i - Electrophoresis of Porcine mRNA Ultrapure agarose (1%)(Bethesda Research Laboratories, Gaithersberg, Maryland) was melted in d i s t i l l e d H20.Gel running b u f f e r , at 5 X concentration, (0.2 M MOPS, pH 7.0, 50 mM sodium acetate, 5 mM EDTA, pH 8.0) and formaldehyde were added to give 1 X and 2.2 M f i n a l concentrations, r e s p e c t i v e l y . The mRNA was incubated at 55°C in a mixture containing 50% formamide, 0.5 X gel-running buffer and 2.15 M formaldehyde in a total volume of 0.02 mL. Two m i c r o l i t e r s of a s t e r i l e loading buffer (50% g l y c e r o l , 1 mM EDTA. 0.4% bromophenol blue, 0.4% xylene cyanol) was added and the samples were loaded onto the g e l . The gel was electrophoresed at 70 volts for 3 hours. Following electrophoresis, the gel was soaked for 30 minutes in several changes of d i s t i l l e d H20. It was then soaked in an excess of 50 mM NaOH and 10 mM NaCl for 45 minutes at room temperature followed by neutralization in 0.1 M T r i s , pH 7.5, for 45 minutes. Before t r a n s f e r , the gel  was soaked in 20 X SSC for 1 hour (51).  i i - Transfer of RNA to Nitrocellulose A baking dish with a glass plate placed across the top, was f i l l e d with 10 X SSC. F i l t e r paper (Whatman 3 MM) was draped across the glass plate and into the buffer such that i t acted as a wick. The gel was placed on top of the f i l t e r paper so that the original underside was uppermost. The n i t r o c e l l u l o s e membrane (Bio-rad, Richmond, C a l i f o r n i a ) , cut s l i g h t l y smaller than the gel and wettened in 10 X SSC, was placed on top of the g e l . F i l t e r papers, s l i g h t l y smaller than the n i t r o c e l l u l o s e , were placed on top of the n i t r o c e l l u l o s e . An 8 cm stack of paper towels, cut s l i g h t l y smaller than the f i l t e r papers, was placed on top of the f i l t e r papers. A weight was  25  set on top of the paper towels and the transfer was allowed to proceed overnight. Following completion of the t r a n s f e r , the n i t r o c e l l u l o s e membrane was a i r - d r i e d then baked for 4 hours at 80°C (55).  i i - Hybridization of cDNA to mRNA The n i t r o c e l l u l o s e membrane was placed in heat-seal able p l a s t i c bags (Dazey Appliances, Industrial A i r p o r t , Kansas, Missouri) and 10 mL of prehybridization buffer, consisting of 50% formamide, 3 X SSC, 1 mM EDTA, 0.1% SDS,  10 mM TRIS, pH 7.5, 100 ug/mL Salmon testes DNA, 10 X Denhart's  (100 X Denhart's i s 2% BSA, 2% f i c o l , 2% polyvinylpyrrolidone) 0.05% sodium pyrophosphate, 2.5 ug/mL poly A and 50 ug/mL tRNA was added. The bags were then double sealed with a Micro-Seal Model 6011 (Dazey Appliances, Industrial A i r p o r t , Kansas, Missouri). The membrane was prehybridized overnight at 37°C. Following prehybridization, the buffer was removed and replaced with the hybridization buffer. The hybridization buffer was identical to the prehybridization buffer except that the hybridization buffer contained the radiolabelled probe and the total volume was 5 mL. The probe was hybridized to the mRNA for 24 hours at 37°C (55). Following hybridization, the membrane was washed once in 2 X SSC and IX Denhart's for 1 hour at room temperature, then three times in 6 X SSC and 0.1% SDS at 60°C for 30 minutes. The membrane was then rinsed four times at room temperature in 1 X SSC. Autoradiography was performed at -70°C for 3 days to locate the hybridized mRNA-cDNA bands.  26  c) cDNA Probe Binding Saturation Studies To determine the saturating concentration of Factor II cDNA, identical amounts of poly (A+) RNA were applied to n i t r o c e l l u l o s e f i l t e r s , moistened R  in 20 X SSC, using a Bio-Dot m i c r o f i l t r a t i o n apparatus, (Biorad, Richmond, C a l i f o r n i a 94804) then washed under vacuum with 1 M ammonium acetate. The membrane was baked at 80°C f o r 2 hours. These f i l t e r s were then hybridized with d i f f e r e n t concentrations of labelled cDNA probe ranging from 25 ng to 100 ng. The total amount of poly (A+) RNA spotted per f i l t e r was 2 ug. The method f o r hybridization i s that described above except that autoradiography was performed at -70°C overnight to locate the bands complementary to the cDNA probe. The portions of the membrane containing the hybridized bands were cut out and placed in l i q u i d s c i n t i l l a t i o n v i a l s . One m i l l i l i t e r of Protosol (New England Nuclear, Boston, Massachusetts 02118) and 0.1 mL of 30% H202 was added and the v i a l s were incubated at 55°C for 3 hours (56). Following t h i s , 10 mL of s c i n t i l l a n t ( B i o f l u o r , New England Nuclear, Boston, Massachusetts) and 0.05 mL of 1 N HC1 was added and the samples were dark-adapted overnight prior to determining the amount of r a d i o a c t i v i t y present.  d) cDNA Probe Binding Linearity Studies The relationship between the amounts of Factor II mRNA detected and the amount of poly (A+) RNA applied per spot to the membrane was examined. Increasing amounts of poly (A+) RNA per spot were hybridized (as described) with saturating concentrations of cDNA f o r Factor I I .  27  3. TISSUE DNA QUANTITATION a) Method DNA was determined by a fluorometric method described by Labarca et al (57). In summary, the method i s as follows. Approximately 1 gram of porcine l i v e r tissue was disrupted by a polytron tissue homogenizer (Brinkmann Instruments, Rexdale, Ontario) in 15 mL of guanidine-thiocyanate buffer, (4 M guanidine thiocyanate, 25 mM sodium c i t r a t e , pH 7.0, 0.5% s a r c o s y l , 0.1 M 2-mercaptoethanol). The homogenate was then sonicated (Sonifier Cell Disruptor 350, Branson Ultrasonics, Scarboro, Ontario) f o r 10 seconds pausing at 5 second intervals to place the homogenate in an ice water bath. Following sonication, a 0.200 mL aliquot was removed and stored at -70°C until analysis was performed. The remainder was used f o r mRNA extraction. From t h i s a l i q u o t , 0.020 mL was added to a test tube containing 1.960 mL of phosphate-saline buffer (2.0 M NaCl, 0.05 M Na2HP0J and 0.020 mL of 100 ug/mL Bisbenzimidazole (Hoecht 33258). The sample was then centrifuged at 14,000 X g f o r 5 minutes and the fluorescence was determined at an excitation wavelength of 356 nm and emission wavelength of 458 nm (Aminco Bowman Spectrofluorometer, American Instruments Co., S i l v e r Springs, Maryland). The DNA was quantitated from a standard curve constructed from c a l f thymus DNA in phosphate-saline buffer. By using this method, i t was found that the amount of DNA per gram of wet l i v e r tissue was close to that reported in the 1 iterature (57).  b) Correlation of DNA to Tissue Protein Content Because t h i s assay was to serve as a r e f l e c t i o n of the amount of tissue that was o r i g i n a l l y a v a i l a b l e , a study was undertaken to determine i f the  28  amount of DNA correlated with the protein content of the t i s s u e . Liver tissue from a normal 20 kg pig was cut into pieces approximately 1 gram each and kept at -70°C until needed. The tissue was homogenized and sonicated in phosphate-saline buffer and an aliquot was removed for DNA and protein quantitation. The DNA method used i s described above. The protein method was as follows. To a tube containing 0.010 mL of sample, 1.0 mL of a reagent consisting of a 1:1 mixture of 2% Na2C03 in 0.1 N NaOH: 0.5% CuS04 X 5 H20 in 1% sodium potassium t a r t r a t e was added and mixed. One hundred m i c r o l i t e r s of a commercially available Folin phenol reagent (made 1 N in acid) was added while the tube was being vigorously mixed. The reaction was allowed to proceed f o r 30 minutes at room temperature. The absorbance at 500 nm was then determined spectrophotometrically. This method i s that described by Lowry et a l . (57). The amount of protein was determined from a standard curve of bovine serum albumin.  c) Buffer Interference Study The homogenization buffer of choice in the extraction of RNA from tissue was guanidine thiocyanate buffer. Due to the fact that t h i s buffer i s not the same as that described in the original DNA method, a study was conducted to ensure that t h i s buffer would not interfere with the fluorescence. This was done by constructing two separate standard curves (in t r i p l i c a t e ) . The f i r s t standard curve was the normal standard curve as used in the above procedure and the second standard curve was identical to the f i r s t with the exception of the addition of 0.020 mL of guanidine-thiocyanate buffer. The fluorescence was determined in the same manner as described above.  29  B. Quantitation of Factor II mRNA in Porcine Liver a)  Isolation and P u r i f i c a t i o n of Nucleotides from Tissue The RNA was extracted from the tissue using the method as described by  Chomczynski et al (59). In summary, the method i s as follows. The tissue was homogenized in 15 mL of guanidine thiocyanate buffer (4.0 M guanidine thiocyanate, 25 mM sodium c i t r a t e , pH 7.0, 0.5% sarcosyl, 0.1 M 2mercaptoethanol). The homogenate was sonicated for 10 seconds pausing at 5 second intervals to place the homogenate in an ice water bath. A 0.100  mL  aliquot was then removed for DNA quantitation. Following t h i s , a known amount of internal standard was added to the homogenate. To the remaining homogenate, 1.5 ml of 2.0 M sodium acetate, pH 4.0, 15 ml of phenol (water saturated) and 3.0 ml of a chloroform:isoamyl  alcohol (24:1) was added with  thorough mixing after each addition. The f i n a l mixture was placed on ice for 10 minutes then centrifuged at 10,000 X g for 20 minutes at 4°C. Following c e n t r i f u g a t i o n , the aqueous phase was removed and re-extracted as described above. The aqueous phase was then placed in a s t e r i l e tube, mixed with 10 ml of isopropanol and placed at -20°C for 1 hour to p r e c i p i t a t e the nucleic acids. The p r e c i p i t a t e was collected by centrifugation at 10,000 X g for 20 minutes at 4°C. The supernatant was discarded and the p e l l e t was resuspended in 5 ml of guanidine thiocyanate buffer. The nucleic acids were again precipitated with 1 volume of isopropanol at -20°C for 1 hour. The p r e c i p i t a t e was collected as described above and the procedure was  repeated  two additional times. The p e l l e t was washed with 70% ethanol, dried and resuspended in s t e r i l e d i s t i l l e d water. An aliquot was removed, the amount of r a d i o a c t i v i t y and the absorbance at 260 nm and 280 nm was determined. The  30  r a t i o of the absorbances at 260/280 nm was always greater than 1.8.  b) Selection of Polv (A+l RNA Selection of poly(A+)RNA was achieved by a f f i n i t y chromatography through an oligo-dT c e l l u l o s e column as described in a previous section (page 20). After loading the specimen, the column was washed with the loading buffer until the absorbance of an undiluted fraction was less than 0.050 absorbance u n i t s . Following e l u t i o n , an aliquot of each fraction was removed, and the r a d i o a c t i v i t y present and the absorbance260 nm determined. The fractions with the highest absorbance units at 260 nm were pooled and ethanol-precipitated overnight at -20°C as previously described.  c) Hybridization of Factor II cDNA to Porcine mRNA The mRNA was resuspended in s t e r i l e d i s t i l l e d water then applied in three separate samples to a n i t r o c e l l u l o s e membrane, previously moistened in 20 X SSC, using a Biodot M i c r o f i l t r a t i o n " apparatus. Control mRNA, previously p u r i f i e d from a 20 kg p i g , was also applied to the membrane in the same manner. The samples and controls were then washed under vacuum with 1 M ammonium acetate (as described on page 26). The membrane was baked at 80°C for 2 hours. Following t h i s , the membrane was prehybridized and then hybridized as described in a previous section (page 26). The membrane was then washed and autoradiography was performed as described (page 27). The portions of the membrane containing the hybridized bands were removed and s o l u b i l i z e d as described (page 27). The amount of r a d i o a c t i v i t y present in the samples and controls was quantitated as described (page 27). The amount of r a d i o a c t i v i t y in the samples was then expressed as a percentage of the  31  control value and corrected for the recovery (by using the recovery of the internal standard). The value obtained was then expressed r e l a t i v e to the amount of DNA present in the starting material.  32  PART III - FACTOR II Prothrombin (Factor II) was activated to thrombin (Factor Ila) by the action of Ecarin, a procoagulant from the venom of the Echis carinatus snake. The thrombin, in turn, cleaved the synthetic substrate, Chromozym Th (Boehringer Mannheim, Dorval, Quebec), l i b e r a t i n g p - n i t r o a n i l i d e . The amount of p - n i t r o a n i l i d e generated was measured spectrophotometrically. A b r i e f description of the method follows.  A: Method To 0.008 mL of platelet-poor plasma and 0.192 mL of d i s t i l l e d H20, 0.600 mL of ecarin at a concentration of 0.111 mg/mL in a 0.075 M T r i s buffer, pH 8.4 was added. The mixture was incubated at room temperature f o r 10 minutes on a shaker (Eberback Corporation, Ann Arbor, Michigan) at a speed of 148 o s c i l l a t i o n s per minute with a horizontal travel of 2 inches. Following the incubation period, 0.020 mL of Chromozym Th at a concentration of 20 mM was added and mixed vigorously. Following another incubation period of 4 minutes, the reaction was stopped with the addition of 0.100 mL of a 50% acetic acid s o l u t i o n . The absorbance at 405 nm was then determined spectrophotometrically (Lambda Array 8500 UV/Visible spectrophotometer, Perkin Elmer, Oak Brook, I l l i n o i s ) . The amount of Factor II in the plasma was determined from a standard curve of s e r i a l d i l u t i o n s of normal heparinized porcine platelet-poor plasma and expressed as a percentage of each animal's pre-operative value. Each sample was run in t r i p l i c a t e .  33  B: Concentration of the Activator A study was undertaken to determine the most suitable concentration of activator to use given the conditions of the assay. Undiluted porcine platelet-poor plasma (0.008 mL) was incubated under the conditions described above with ecarin at concentrations of 0.111  mg/mL and 0.0111 mg/mL. After  the substrate had been added as described previously, the reaction was stopped with 0.100  mL of 50% acetic acid at increasing time intervals and the  absorbance at 405 nm was determined as described above.  C: Determination of Substrate Reaction Time Undiluted platelet-poor plasma and 1/10 diluted plasma was incubated with 0.111  mg/mL of ecarin for 10 minutes. Substrate was added following the  incubation period as described above and the reaction was stopped at increasing time intervals by the addition of 0.100  mL of 50% acetic a c i d . The  absorbance at 405 nm was determined as previously described.  D: The Effect of Heparin on Factor II Levels Two separate studies were conducted to ensure that the level of heparin being used in the design of the model did not interfere with the assay. These studies included: 1) an investigation of the effect of heparin on a control plasma that had been spiked with increasing amounts of a l i q u i d heparin preparation similar to that used during the operative procedure (Hepalean  R  1000 USP units/mL),  (Organon Canada Ltd., Toronto, Ontario). The plasma was assayed for Factor II and the results were compared with control plasma that had been spiked  34  with the same volume of a 0.9% NaCl, 0.9% benzyl alcohol s o l u t i o n . 2) an investigation of the effect of heparin contained in the blood c o l l e c t i o n tubes on plasma levels of Factor I I . Heparinized and c i t r a t e d plasma were collected from a normal pig by venipuncture. Serial d i l u t i o n s of the c i t r a t e d and heparinized plasma were assayed f o r prothrombin and the results were compared.  35  PART IV - INDOCYANINE GREEN CLEARANCE STUDIES  A: Indocvanine Green (ICG) Clearance Protocol To reconstitute the dye, the ICG (Hysson, Wescott & Dunning, Baltimore, Maryland) i s mixed with a 1:1 plasma to diluent mixture. The diluent i s that which i s supplied by the manufacturer and the plasma i s that of the animal being studied. The dye i s reconstituted to a f i n a l concentration of 5 mg/mL. Prior to infusion of the dye, two blood samples of 2 mL each were drawn from the animal. One was placed in a heparinized tube (Becton Dickinson, Rutherford, New Jersey) and was subsequently used as the plasma blank; the other sample was drawn into a heparinized syringe, mixed, and then used to flush the l i n e after injection of the dye. At time 0, a bolus of the dye (1 mg/kg) was infused through the venous l i n e . The l i n e was then flushed with two m i l l i l i t e r s of the animal's own blood (drawn previously), followed by 2 mL of heparinized saline (200 units/mL). Blood samples were drawn from the a r t e r i a l l i n e at 5, 10, 15, 20, 30, 40, 50, and 60 minutes. After drawing each sample the l i n e was flushed with 2 mL of heparinized s a l i n e . The plasma was separated from the c e l l s , placed in a 1.5 mL polypropylene tube, (Brinkman Instruments, Westbury, New York) and then centrifuged at 13,000 X g for 2 minutes (Model 235-B, Fisher S c i e n t i f i c Co., Ottawa, Ontario). The maximal absorbance of the plasma at 800 nm was then determined spectrophotometrically (Lambda Array 8500 UV/Visible spectrophotometer, Perkin Elmer, Oak Brook, I l l i n o i s ) . The c a l c u l a t i o n of the rate disappearance constants was as follows: a) K, i s defined as the slope of the l i n e of log of absorbance at 800  36  nm versus minutes ( up to 20 minutes) and; b) K2 i s defined as the slope of the l i n e of log of absorbance at 800 nm versus minutes, from 30 to 60 minutes.  B: Dye S t a b i l i t y Study A 0.020 mL aliquot of ICG reconstituted, as described, was added to 20 ml of a 1:1 human plasma/diluent mixture. This mixture was then aliquoted and stored at either 4°C or -70°C. Each day, the absorbance at 800 nm was determined as described above and compared to the original absorbance determined on day 0.  C: Dye Interference Study ICG was added to a control plasma so that i t s f i n a l concentration ranged from 5.0 mg/L to 20.0 mg/L. The plasma was then analyzed f o r potassium, glucose, l a c t a t e , AST, total b i l i r u b i n and Factor I I . The results were compared to an aliquot which contained no dye.  37  PART V - ROUTINE PLASMA MEASUREMENTS  A l l routine chemistries, unless otherwise indicated, were performed using the Kodak Ektachem 700 C l i n i c a l Analyzer, (Eastman Kodak, Rochester, New York) with reagents supplied by the manufacturer. A b r i e f description of each method follows.  A: Aspartate Aminotransferase In the assay for aspartate aminotransferase (AST) (EC 2.6.1.1), the amino group of aspartate i s transferred to alpha-ketoglutarate in the presence of sodium pyridoxal-5-phosphate and AST to produce oxaloacetate and glutamate. The oxaloacetate formed i s converted to malate by malate dehydrogenase with the concomitant oxidation of NADH to NAD*. The oxidation step i s monitored by reflectance spectroscopy at 340 nm (60).  B: Glucose Glucose was measured according to the method described by Curme et al (61). Glucose i s oxidized to hydrogen peroxide by glucose oxidase (EC 1.1.3.4). Peroxidase (EC 1.11.1.7) catalyzes the oxidation of 4-aminoantipyrine and 1,7-dihydroxy-naphthalene  by hydrogen peroxide, to produce a  chromogen. The intensity of the chromogen formed i s proportional to the amount of glucose in the sample and i s monitored by reflected l i g h t at 540 nm. The chromogen system employed was f i r s t described by Trinder (62).  C: Total B i l i r u b i n Total b i l i r u b i n , including unconjugated, mono- and di-conjugated, and  38  albumin-bound delta b i l i r u b i n (63) was determined by a modification of the method described by Routh (64). The b i l i r u b i n fractions are dissociated from albumin and s o l u b i l i z e d by dyphylline and surfactant. These then were reacted with a diazonium s a l t to produce an azobilirubin chromaphore which was measured at 540 nm.  D: Potassium Potassium was determined potentiometrically using ion-selective +  electrode s l i d e s . The s l i d e consists of a K s e l e c t i v e membrane of valinomycin and a s i l v e r - s i l v e r chloride l a y e r , serving as the reference electrode. These were connected v i a a KC1 s a l t bridge. When the sample and reference f l u i d were applied to t h e i r respective layers a pair of electrochemical h a l f - c e l l s was created. The potassium a c t i v i t y was determined from the potentiometric difference measured between the two half c e l l s and related to the Nernst equation.  E: Lactate Lactate was measured using the Dupont Automatic C l i n i c a l Analyzer (Dupont, Wilmington, Delaware). In summary, lactate dehydrogenase catalyzes the oxidation of L-lactate to pyruvate with the simultaneous reduction of +  NAD to NADH. The absorbance of NADH i s d i r e c t l y proportional to the lactate concentration and i s measured spectrophotometrically at 340 nm. This method i s a modification of that described by Marbach (65).  39  PART VI - STATISTICAL ANALYSIS A l l s t a t i s t i c a l analyses were carried out using the ABSTAT s t a t i s t i c a l program (Anderson-Bell Company, USA). The differences between the means were compared using the Student t Test for paired data. Differences were considered s i g n i f i c a n t at p < 0.05, where p represents the probability f o r two-tailed t e s t s .  40  RESULTS PART I - EXPERIMENTAL DESIGN Of the 9 pigs operated upon, 7 survived the entire procedure. One animal died the f i r s t day post-operatively from uncontrollable bleeding. This was not attributed to any surgical i r r e g u l a r i t i e s , as no d i r e c t source of bleeding was i d e n t i f i e d . The death was, therefore, thought to be due to disseminated  intravascular coagulation, but this was not investigated  f u r t h e r . The other animal never regained consciousness  following 90 minutes  ischemia and 4 hours of reperfusion (Day 4 operative p r o t o c o l ) . The death was caused by aspiration of stomach contents during intubation, and not by the surgical procedure.  PART II - QUANTITATION OF FACTOR II MRNA IN PORCINE LIVER  A: Method Development for the Quantitation of Factor II mRNA in Porcine Liver 1. DEVELOPMENT OF AN INTERNAL STANDARD a) Method In synthesizing the internal standard, the separation of the unincorporated nucleotides from the incorporated nucleotides was achieved by gel f i l t r a t i o n chromatography. A representative chromatogram i s given in Figure 3. The leading peak corresponds to the nucleotides incorporated into the E. c o l i  3  tRNA and the t r a i l i n g peak i s the unincorporated H-ATP. Once  the internal standard was synthesized, i t was further p u r i f i e d by oligo-dTc e l l u l o s e chromatography. Representative p r o f i l e s of the elution of the internal standard, human and porcine poly (A+) RNA from an oligo-dT-  41  2.0  SEPHADEX—G50 COLUMN -i  0  2  4  6  8  VOLUME (mL)  Figure 3. Graph showing a representative chroiaatogram of a Sephadex-G50 gel f i l t r a t i o n column. The leading peak contains the incorporated nucleotides and the t r a i l i n g peak contains the unincorporated nucleotides.  c e l l u l o s e column i s shown in Figure 4. The elution p r o f i l e of the internal standard corresponds d i r e c t l y to that of human and porcine poly (A+)  RNA.  The recovery of the internal standard during the i s o l a t i o n of mRNA from l i v e r and following i t s p u r i f i c a t i o n by oligo-dT-cellulose chromatography i s given in Table 1. In a l l , 41 biopsies were processed. The majority of the loss of the internal standard occurred during the extraction procedure (26% recovery). The recovery of the internal standard following oligo-dTc e l l u l o s e chromatography was improved over the extraction procedure (58% recovery) but both techniques suffered from large standard deviations.  b) Elimination of the Gel f i l t r a t i o n Step It can be demonstrated in Figure 5 (see Figure 5), that following two sequential ethanol precipitations under the conditions outlined in materials and methods (page 19), a l l appreciable amounts of unincorporated nucleotides ( t r a i l i n g peak) were eliminated. Subsequent to this study, the gel f i l t r a t i o n step was discontinued and the unincorporated nucleotides were removed by two sequential ethanol p r e c i p i t a t i o n s .  c) S t a b i l i t y Study A time sequence of the recovery of the internal standard following storage at -20°C i s shown in Figure 6. The recovery of the internal standard from a Bio-Spin  R  6 column was constant for a period of 9 days after which any  absorbance 260 in the eluant was undetectable. However, there was  still  detectable amounts of r a d i o a c t i v i t y present in the eluant. The column used on Day 12 was further eluted with 4 column volumes of s t e r i l e TE b u f f e r , pH 8.0, and the resulting p r o f i l e in Figure 7 was obtained. The nucleic acids  43  Eution Profile of Oligo-dT-Cellulose Column  Volume (mL) Figure 4. Representative e l u t i o n p r o f i l e of the i n t e r n a l standard HB—Wk~ human l i v e r mRNA, - O — e — and porcine l i v e r mRNA r^fi oligo-dT-cellulose column. f  r  o  m  a  n  Table 1 Extraction E f f i c i e n c i e s of the Internal  Standard  Extraction E f f i c i e n c y (%)  Extraction e f f i c i e n c y of mRNA from l i v e r t i s s u e  26.2%+15.6%  Recovery following oligodT-cellulose chromatography  57.9%+24.4%  a  a  (41)  (41)  data presented as the mean + 1 S.D. with number of determinations shown in parenthesis  45  S e p h a d e x G—50 C o l u m n  TJ C 2  Volum*  (mL)  Figure 5. Elution profile of the internal standard following phenol extraction — • •and two sequential ethanol precipitations from a Sephadex G-50 gel f i l t r a t i o n column.+ +  Internal Standard Degradation Study  Days Figure 6. Schematic representation of the time sequence for the degradation of the internal standard. The recovery of the internal standard i s measured by the absorbance26fj '—13 fsJ * °f radioactivity present-O Q-. -  an{  t n e  a m o u n t  Elution Profile of the Internal Standard 100  80-  60-  40-  20-  Volume (mL) Figure 7. Chromatogram of the elution profile of degraded (Day 12) internal standard from a Biospin-6 column. The recovery of the internal standard i s measured by the absorbance25o M M and the amount of radioactivity that i s present • l_l . R  eluted from the column in a broad peak after one column volume. In contrast, the r a d i o a c t i v i t y never f u l l y eluted from the column and did not correspond to the p r o f i l e seen with the eluting nucleic acids.  2. cDNA PROBE STUDIES a) Removal of the Insert from Plasmid DNA A photograph of a representative agarose gel used in the separation of the insert from i t s plasmid following digestion with Hjn d III and Pst I i s given in Plate 1. The band corresponding to 370 base pairs (bp) was cut from the gel and the material was electroeluted as described in materials and methods (page 23). The DNA in this band was comprised of the insert only, i t did not contain any plasmid DNA.  b) Hybridization of the Human cDNA Probe to Porcine mRNA In the photograph of the autoradiographic f i l m pictured below, (see Plate 2) i t i s evident that human cDNA for Factor II does cross-hybridize to a s i n g l e , discrete band of porcine mRNA. The mRNA was electrophoresed under denaturing conditions, transferred to n i t r o c e l l u l o s e , and hybridized to the radioactive cDNA probe prior to autoradiography.  c) cDNA Probe Binding Saturation Studies r  Figure 8 describes the binding saturation of the cDNA probe to porcine mRNA under the conditions described. The binding i s saturated at 75 ng.  49  Plate 1. A representative photograph of an agarose gel used in the separation of the insert (Factor II cDNA) from the plasmid following digestion with Hin d III and Pst I. The band corresponding to 370 base pairs (bp) was removed from the g e l . The molecular weight marker used was phage (0X 174 digested with Hae H I .  50  Plate 2. An autoradiogram of porcine mRNA following formaldehyde agarose gel e l e c t r o p h o r e s i s , Northern t r a n s f e r to n i t r o c e l l u l o s e , and h y b r i d i z a t i o n to the r a d i o l a b e l e d cDNA probe f o r Factor I I . The f i l t e r was exposed, at 70°C, f o r 3 days.  51  cDNA Saturation  Figure 8. A schematic representation of the determination of the saturating amount of the human Factor II cDNA probe with constant amounts of porcine mRNA.  d) cDNA Probe Binding Linearity Studies Figure 9 describes the l i n e a r i t y i f the binding of the cDNA probe to increasing concentrations of porcine mRNA. The binding i s l i n e a r to 1.0 ug of porcine mRNA (r = .969).  3. TISSUE DNA QUANTITATION a) Correlation of Tissue DNA to Tissue Protein Content and Wet Weight A graph of the correlation between protein content and DNA l e v e l s in porcine l i v e r tissue i s given in Figure 10. It i s clear that a good c o r r e l a t i o n (r = 0.997) exists between them. In addition, a retrospective study was conducted on porcine tissue samples to correlate the wet weight and the DNA content. An acceptable good correlation (r = 0.866) i s evident (see Figure 11).  b) Buffer Interference Study The graph of the correlation between two standard curves, one prepared in the phosphate-saline buffer and the other with 0.020 mL of guanidinethiocyanate buffer added, i s shown in Figure 12. The c o r r e l a t i o n i s good (r = 0.999); however, a positive y-intercept i s noted (y = + 1.446).  B: Quantitation of Factor II mRNA in Porcine Liver The changes in Factor II mRNA levels during ischemic/reperfusion injury are shown in Figure 13. In three of the animals, Pigs 30, 31, and 36, a trend i s seen in which the mRNA rises during the ischemic period and reaches a peak at 90 minutes of reperfusion. With continued reperfusion, the Factor II mRNA  53  cDNA linearity  mRNA (ug)  p - r f b ^ h " ^ ^ ^  of  t h  .  b l n d i n g  l l n e a r l t y  o f  t h e  c  Protein v s D N A 4 Z3  -  3 -  2-B -  2 -  ^£  1J* -  Slope  -  0.023 .  Reg.Coeff. = 0.997  1 -  Y int.  OJJ -  = -0.52  0 O  20  40  TO  BO  100  120  140  1BO  Protein (ug/tub«)  Figure 10. Graph of the correlation between porcine l i v e r DNA content and protein content. Each point represents the mean of t r i p l i c a t e s .  1BO  Wet Weight vs DNA  DNA: B u f f e r Interference Study 100  - i — — —  —  100 Phosphate—satin* buffar (RFU)  Figure 12. Graph of the correlation between a standard curve with 0.020 mL of guanidine-thiocyanate buffer added to each standard in 2 mL of phosphate-saline buffer and a standard curve in phosphate-saline buffer alone.  Figure 13. Time sequence of mRNA levels for Factor II from four animals during ischemic/reperfusion injury. Each point represents the mean of duplicates. Pig 30 H I 5 (black), Pig 31 -fr fr(red), Pig 33 — + + (blue), Pig 36 AcA (green)  levels decrease to pre-operative values. In two of these Pigs, (36 and 31) the mRNA remains at pre-operative levels for the duration of the operative procedure. In Pig 30, the mRNA starts to r i s e again in comparison to i t s preoperative sample.  PART III - QUANTITATION OF FACTOR II IN PORCINE PLASMA  A: Concentration of the Activator Figure 14. depicts the results of an experiment to determine the optimal concentration of activator to use in subsequent assays. A sample consisting of 75% porcine plasma (75% standard) was activated with 0.111 mg/mL of e c a r i n . The slope of the curve using 0.111  mg/mL or  0.011  mg/mL of ecarin i s 15.8  and that of the curve using 0.0111 mg/mL i s 9.1. As the assays were not performed simultaneously, the absorbances of the 75% standard at each time interval and at the two d i f f e r i n g ecarin concentrations have been converted to % concentration, by use of a standard curve run concomitantly, to allow d i r e c t comparison between them.  B: Determination of the Substrate Reaction Time A graph of absorbance 40S (product formed) versus substrate reaction time of undiluted porcine plasma and 1/10 diluted plasma i s shown in  Figure 15.  The undiluted plasma exhibits substrate depletion at approximately the 8 minute mark whereas the curve of the 1/10 diluted plasma remains l i n e a r . At the 4 minute mark, the undiluted plasma has reached an absorbance 405 (about 1.0) which can s t i l l be determined with good accuracy on most spectrophotometers, yet s t i l l exhibits l i n e a r i t y .  59  C o n c e n t r a t i o n of A c t i v a t o r 330 —t  1  ISO -  100 -  80 -  o -]  0  j  j  4-  j  j  8  j  1  12  1  1  10  r  1  20  1  r  24  TImo (min)  Figure 14. Graphs of a 75% standard activated with 0.111 mg/mL of ecarin —•{ + and 0.011 mg/mL of ecarin gj — . Each point represents the mean of duplicates.  F a c t o r II Substrate Reaction Time 2-4  Tim* (minuta*)  Figure 15. A graph of absorbance at 405 nm versus the substrate reaction time for undiluted—B S— and 1/10 diluted H i — p o r c i n e plasma.  C: The Effect of Heparin in the Assay f o r Factor II Table 2 describes the recovery of Factor II a c t i v i t y following the addition of increasing levels of the l i q u i d heparin preparation similar to that used during the operative procedure. It i s apparent that the l i q u i d heparin did not interfere with the assay at a level of less than 2 USP units/mL. In a d d i t i o n , blood collected from a normal pig into heparinized and c i t r a t e d tubes, s e r i a l l y d i l u t e d , assayed f o r Factor II and regressed against each other, exhibited a regression slope of e s s e n t i a l l y one (Figure 16).  Once this method was established, i t was found to have an intra-assay CV of 2% (n = 10) and interassay CV of 9% (n = 9 ) .  D: Porcine Plasma Factor II Levels During Ischemic/Reperfusion  In.iury  By using the Factor II method developed in our laboratory, the results indicate (see Figure 17) that plasma levels of Factor II decreased s i g n i f i c a n t l y (p < 0.01) following 90 minutes of ischemia in r e l a t i o n to pre-operative values. These values remained low f o r the duration of the experimental  protocol (two days) and never recovered to pre-operative  levels.  PART IV - ICG CLEARANCE STUDIES A: Dve S t a b i l i t y Study After 15 days of storage at 4°C, the ICG dye (reconstituted in the manner described on page 35) deteriorated to levels which represented a difference  62  Table 2 Heparin Interference Study (II)  Hepalean" USP Units/mL  Factor II % Recovery  0.75  97%  2.0  100%  3.0  87%  4.0  87.6%  63  Heparin  Interference  Study  H s p a rinizad Plasma (Absorbancs 4 0 3 n m )  Figure 16. A graph of the correlation between a standard curve of Factor II collected i n citrated tube6 and a standard curve of Factor II collected in heparinized tubes.  Plasma Factor II Levels 120-,  c  i — i — i — i  0  i  30 60 90 0  pre—op ischemia  1  1  30  60  reperfusion  1  i  90 1  1  2  days post—op  Figure 17. A graph of porcine plasma Factor II levels during ischemic/reperfusion injury. The points indicate the mean of 7 animals +_ 1 S.E.M. * P< °-01  of greater than 2 standard deviations of the i n i t i a l absorbance values (see Figure 18). Dye which was stored at -70°C remained intact for at least 15 days.  B: Dve Interference Study Following the addition of increasing amounts of dye to porcine plasma, i t can be demonstrated that the dye did not interfere with the assays  for  +  plasma Factor I I , glucose, K , AST or lactate (Figures 19. 20. 21, 22, 23). However, with increasing concentrations of dye, there i s a positive interference with the method for total b i l i r u b i n (Figure 24).  C: ICG Clearance During Ischemic/Reperfusion  In.iury  Table 3 describes the clearance constants of indocyanine green in the model of warm hepatic ischemic/reperfusion injury in the p i g . There i s an 82% decrease between the pre-operative K, clearance slope and the clearance slope during the ischemic i n t e r v a l . This decrease i s s i g n i f i c a n t to a level of p < 0.0001. During reperfusion, both the K, and the K2 clearance slopes are decreased. These decreases are s i g n i f i c a n t to levels of p < 0.0001 and p < 0.0020 r e s p e c t i v e l y . A representative curve of the clearance of ICG from porcine plasma i s given in Figure 25. The curve suggests that ICG clears from the plasma in two phases. The f i r s t phase i s an exponential decay in which the majority of the dye i s cleared within the f i r s t twenty minutes. The second phase i s much slower and dye i s s t i l l detected in the plasma at 90 minutes.  66  1.27 1.26 r-  1.25  -~r— 10  -J—  12  H  DAYS  Figure 18. A graph of tlie s t a b i l i t y of lndocyanlne green after 15 days of storage at 4°C and -70°C. Tlie points indicate the mean of 10 readings + 2 S.D. Tlie dashed lines indicate the mean absorbance on day 0 and the solid lines are + 2 S.D.  67  DYE IHTERFERENCE: FACTOR II 0.25  0.24 -  0.23 -  0.22 U 0.2185-  0.21 -  0.2 IJ 0.19750.19 -  0.18 -  0.17  I  1 4  1  1 8  1  1 12  1  1 16  1  1 20  1  1 24  ICC (mg/L) Figure 19. A graph of the interference with ICG i n the method f o r Factor I I . The dashed l i n e i n d i c a t e s the mean of the sample containing no ICG + 2 S.D. ( s o l i d l i n e s ) . —  DYE INTERFERENCE : GLUCOSE  4.34  4.33 -  4.32 -I  \ o E E LJ V)  o o  D  _l  4.31 4.3051)— 4.3 - 4.2951 >— 4.29 -  o  4.28 -  4.27 -  4.26  4  8  1  ~r-  12  16  T" 20  —r-  24  ICG (mg/L) Figure 20. A graph of the interference with ICG in the method for glucose. The dashed line indicates the mean of the sample containing no ICG + 2 S.D. (solid lines).  DYE INTERFERENCE :K+  4.7  4.65 4.6141Y 4.6 -  4.55 -  \  0 E E o  4.5  •  —  + 4.45 -  4.4 4.386* 4.35 -  4.3  1 0.00  4.00  8.00  12.00  I 16.00  1 20.00  1  I 24.00  ICG (mg/L)  Figure 21 A graph of the interference with ICG in the method for K+. The dashed line indicates the mean of the sample containing no ICG + 2 S.D. (solid lines). ~  DYE INTERFERENCE : AST  1G.56IJ16 -  6 5.35 IH  i  1  8  12  16  20  24  ICG (mg/L) Figure 22. A graph of the interference with ICG i n the method for AST. The dashed line indicates the mean of the sample containing no ICG + 2 S.D. (solid lines). ~  DYE INTERFERENCE : LACTATE  1.8  l  - 111.6927  1.6 -  1.5  b-  1.4 -  1.3 J) 1.308-  1.2 -  1.1 -  1  I 4  I  I 8  I  I 12  I  1 16  —I  1 20  1  [ 24  ICG (mg/L) Figure 23. A graph of the interference with ICG i n the method f o r l a c t a t e . The dashed l i n e indicates the mean of the sample containing no ICG + 2 S.D. (solid l i n e s ) . ~~  DYE INTERFERENCE : TOTAL BILIRUBIN  14 13 12 11 -  -I \  0 E 3  v •  2 m  10 97.6?6 7 H  D tr  _j m _j  o  54.3241V 3 -  210  T 4  T 8  I  12  16  I  20  I  24  ICG (mg/L) Figure 24. A graph of the interference with ICG i n the method f o r t o t a l b i l i r u b i n . The dashed l i n e indicates the mean of the sample containing no ICG + 2 S.D. ( s o l i d l i n e s ) .  Table 3 Mean ICG Clearance Constants from Porcine Plasma  Time  n = 7  K,  K  2  pre-op  .0419  ischemia  .008 •  reperfusion  .009 •  .0046T  1 day post-op  .0414  .0123  2 days post-op  .0439  .0103  - p<0.0001  .0112  Tp<0.0020 from pre-operative values  74  ICG C l e a r a n c e 7 -i  Curve  —  6H  5H  4H  3H 2  i H  •  0  •  i  i  i  20  40  i  1 60  Tim* (minutes)  Figure 25. A representative  curve of the clearance of ICG from porcine pla  PART V - ROUTINE PLASMA MEASUREMENTS  Aspartate aminotransferase (AST) a c t i v i t y i n plasma increased s i g n i f i c a n t l y (p < 0.05) from pre-operative values at 5, 15, and 30 minutes of reperfusion (Table 4 and Figure 26). The plasma glucose increased s i g n i f i c a n t l y from pre-operative values at: 30 minutes of ischemia and 15, 30, and 60 minutes of reperfusion (p < 0.05) (Table 4 and Figure 27). At one and two days post-operatively, the glucose levels returned to pre-operative +  values. Plasma K levels decreased s i g n i f i c a n t l y from pre-operative values at: 15 (p < 0.005), 30 (p < 0.02), and 60 (p < 0.05) minutes of reperfusion (Table 5 and Figure 28). By the end of the reperfusion phase, and at one and +  two days post-operatively, K levels had returned to pre-operative values. The plasma lactate increased s i g n i f i c a n t l y from pre-operative values at 30, 60 (p < 0.002), and 90 minutes of ischemia, and 5, 15, 30, and 60 minutes of reperfusion (p < 0.02) (Table 5 and Figure 29). In contrast to the other routine chemistries performed, b i l i r u b i n values did not change throughout the operative procedure and remained at a mean of 5.3 umol/L (results not shown).  76  Table 4 Porcine Plasma AST and Glucose Values following 90 Minutes of Ischemia and 2 Days of Reperfusion  Time  AST (U/L)  Pre-op  62±8  4.9±.025  45+5 45+5 50±9  8.2+1.377.0+0.98 6.0±0.67 10.0+1.829.6+1.70 • 9.2+1.61 • 8.1+1.18 • 7.6±1.14  Ischemia  30 min. 60 min. 90 min.  Reperfusion  5 min. 15 min. 30 min. 60 min. 90 min.  113+20130+31163+48217+81 241+105  1 Day 2 Days  482+259 174+85  n = 7 ip < 0.05  Glucose (mmol/L)  5.0+0.26 4.8±0.32  x ± 1 S.E.M. from the pre-operative values  77  Plasma AST Levels 8OO-1 700600-  Oh  1  pre—op  i  1  1  0  30  60  ischemia  i  1  1  90 0  1  30  60  reperfusion  1  i  90 1  1  2  days post—op  F i g u r e 2 6 . Time sequence o f plasma AST l e v e l s f o l l o w i n g I s c h e m i c / r e p e r f u s i o n I n j u r y i n p o r c i n e l i v e r . Each p o i n t r e p r e s e n t s mean o f 7 a n i m a l s +_ 1 S . E . M . f p < 0 . 0 5  the  Plasma Glucose Levels 15-,  E E io  o o  5-  X  O  i  30  pre—op  —i  60  -ischemia  1  r  90 0  30  60  reperfusion  -~i r 90 1  days post-op  Figure 27. Time sequence of plasma glucose levels following ischemic/reperfusion injury in porcine l i v e r . Each point represents the mean of 7 animals + 1 S.E.M. f p < 0.05  Table 5 +  Porcine Plasma K and Lactate Values following 90 Minutes of Ischemia and 2 Days of Reperfusion  Time  10 (mmol/L)  Pre-op  Lactate (mmol/L)  3.58+.08  1.7±0.45  Ischemia  30 min. 60 min. 90 min.  3.27+0.13 3.33+0.14 3.41±0.14  3.5+0.64 T 3.6+0.52T 4.0±0.62T  Reperfusion  5 min. 15 min. 30 min. 60 min. 90 min.  3.41+0.18 2.80+0.182.98+0.183.20+0.153.65+0.12  4.8+1.1 T 3.8+0.85 T 3.4+0.72 T 2.7+0.53 T 2.2±0.54  3.62+0.05 3.57±0.08  1.3+0.49 1.8+0.48  1 Day 2 Days  n =7  x ± 1S.E.M. TP<0.02  -p<0.05  from the pre-operative values  80  Plasma Potassium Levels 5n  o £  E - 2H 00  to o O  CL  1-  30 pre~op  i  60  ischemia  90 0  30  ~T~  60  reperfusion  ~i r 90 1  1  2  days post—op  Figure 28. Time sequence of plasma K* levels following ischemic/reperfusion injury in porcine l i v e r . Each point represents the mean of 7 animals + 1 S.E.M. TP < 0.05  P l a s m a Lactate Levels 6-1  u-t  1  pre-op  i  1  1  0  30  60  ischemia  1  i  90 0  1  1  1  i  1  30  60  90  1  2  reperfusion  days post—op Figure 29. Time sequence of plasma l a c t a t e l e v e l s following ischemic/reperfusion injury i n porcine l i v e r . Each point represents mean of 7 animals + 1 S.E.M. T P < 0.02  DISCUSSION  PART I - EXPERIMENTAL DESIGN The specimen c o l l e c t i o n time sequence used in the model of warm hepatic ischemic/reperfusion injury made possible the study of the injury process at s p e c i f i c intervals of the operative procedure. The design of the time sequence was such that the test values obtained during ischemia  and  reperfusion could be related d i r e c t l y to each animal's pre-operative values. In t h i s respect, each animal served as i t s own  control.  The survival rate of pigs subjected to various ischemic periods i s largely dictated by the surgical procedure used. In 1974, Battersby et al achieved a good survival rate following 30 minutes of warm hepatic  ischemia  in pigs (66). In t h e i r model, splenic decompression was effected by means of a wide bore catheter inserted into the splenic vein and advanced to the junction with the superior mesenteric vein. This was connected to a similar catheter in the right external jugular vein (66). Harris et al achieved an 80% survival rate following 180 minutes of warm ischemia (67). To achieve ischemia, t h e i r surgical model employed a porto-jugular shunt, and clamping of the hepatic and gastroduodenal arteries and other c o l l a t e r a l s . Kahn et al achieved a 6 hour ischemic interval with 100% survival rate (68). To induce ischemia, however, a side-to-side portocaval shunt was used and only the hepatic artery was clamped (68). The surgical model we employed was similar to that described by Harris et a l and our survival rate after 90 minutes of ischemia (and 2 days of reperfusion) was 78% (n=9)  (67).  83  PART II - THE QUANTITATION OF FACTOR II MRNA IN PORCINE LIVER A: Method Development for the Quantitation of Factor II mRNA in Porcine Liver The detection of mRNA in biological samples i s , for the most part, a r e l a t i v e l y q u a l i t a t i v e procedure. For the purposes of this project a quantitative procedure for the detection of Factor II mRNA was required. To meet t h i s requirement, preliminary investigations were performed on the existing q u a l i t a t i v e methods. We then modified the q u a l i t a t i v e conventional methodologies and added the steps necessary to produce a quantitative method. The modifications consisted of: 1. the development of an internal standard; 2. cDNA probe studies, and; 3. the quantitation of tissue DNA.  1. DEVELOPMENT OF THE INTERNAL STANDARD Gene expression following chemical or hormonal manipulation i s frequently monitored by quantitating mRNA levels coding for the s p e c i f i c protein of interest (69,70). This can either be achieved by in v i t r o t r a n s l a t i o n systems or, more frequently, by direct hybridization of the mRNA to i t s corresponding cDNA (or RNA) probe. The assumption made in these assay systems i s that recovery of the mRNA from the starting material i s 100% (69,71). However, due to the large amount of sample manipulation during the extraction and p u r i f i c a t i o n of the mRNA from tissue p r i o r to being quantitated, this assumption has not been substantiated. A few authors have attempted to address this concern (72,73). Toscani et al have normalized  84  multiple RNA samples with the use of an "externally added standard" (73). Their externally added standard consisted of a synthesized RNA control previously cloned in t h e i r laboratory (73). Other investigators have circumvented the issue of recovery by expressing the amount of mRNA detected r e l a t i v e to the amount of total RNA or to the amount of other species of mRNA in the sample (70,72). A l l of these techniques have proved to be s a t i s f a c t o r y . However, the amount of mRNA detected cannot be related back to the o r i g i n a l amount of starting material, either tissues or c e l l suspensions. In addition, these methods have several problems. One problem in expressing the amount of mRNA detected to the amount of total RNA i s that, with this approach, one of the most common methods used to quantitate the amount of RNA, the measurement of the absorbance at 260 nm, requires RNA concentrations of > 1 ug/mL. Furthermore, i f one i s measuring the total amount of RNA by the absorbance 260 , the type of nucleic acids present in the sample w i l l depend on the tissue homogenization method used as the Polytron tissue homogenizer i s f i t t e d with an ultrasonic probe. This results in the disruption of nuclei and the release of DNA into the surrounding buffer. To express the amount of mRNA r e l a t i v e to other species of mRNA in the sample, the membrane must f i r s t be subjected to harsh washing procedures to remove the f i r s t probe and then the sample must be reprobed to detect the other species (72). This technique prolongs the procedure and may r e s u l t in loss of the sample. The technique we have developed was devised to circumvent these problems. Our method involved the development of a synthesized internal standard. An internal standard i s a chemical compound added in known amounts to a sample and carried through a l l steps of the analytical procedure. In that  85  the internal standard i s similar chemically and s t r u c t u r a l l y to the analyte of i n t e r e s t , i t s recovery w i l l be comparable to that of the analyte and, therefore, i t can be used to monitor analyte extraction e f f i c i e n c i e s . In order to accurately quantitate l i v e r mRNA l e v e l s , an internal standard was developed in our laboratory and was used to normalize mRNA recovery for the d i f f e r i n g e f f i c i e n c i e s in extracting and purifying i t from l i v e r . The synthesized internal standard behaved both chemically and physically l i k e eukaryotic mRNA in our test system, i e : a)  i t was soluble in the aqueous phase during phenol/ chloroform extraction;  b)  i t precipitated with ethanol and isopropanol;  c)  i t bound to an oligo-d(T)-cellulose column and eluted from  the column in the same fractions as eukaryotic mRNA. In that cDNA probes are frequently labelled with  32  P and the internal  standard i s t r i t i a t e d , i t can be readily d i f f e r e n t i a t e d from a cDNA probe by employing a dual-label counting technique (74). This technique i s less complicated than reprobing the sample with a d i f f e r e n t cDNA probe. Due to the inherent s p e c i f i c i t y of cDNA probes, there was no cross-hybridization of the probe with the internal standard to create any interference with the detection of the mRNA species. The method we have employed to synthesize the internal standard i s faster and simpler than cloning an RNA control as described by Toscani et al (73). It u t i l i z e s equipment found in most basic research l a b o r a t o r i e s . In addition, the recovery of the mRNA could be monitored at a l l stages of the analytical procedure and not only at the f i n a l hybridization step (72,73). This internal standard could be u t i l i z e d in both the "dot-blot" (70) or  86  "cytodot" (75) hybridization assays. In the Northern blot assay (76) i t i s unknown whether the internal standard could correct for the completeness of transfer of the mRNA species as this has not as yet been studied. One additional benefit in the use of the internal standard may be the enhanced recovery of mRNA. Gautron et al have reported that the addition of E. c o l i RNA to a sample augments the recovery of mRNA as i t acts as a c a r r i e r during the p r e c i p i t a t i o n of the nucleic acids with ethanol  (77).  The s t a b i l i t y of the internal standard was investigated by use of gel f i l t r a t i o n columns which have the a b i l i t y to exclude nucleotides of greater than 6 base pairs in length. There are four possible mechanisms by which the internal standard could degrade: loss of the radioactive adenines, loss of the radioactive label ( t r i t i u m ) , random degradation of the entire poly (A) labelled nucleotide and a combination of these mechanisms. By using these columns the most probable mechanism could be determined. A time-sequence of the s t a b i l i t y of the internal standard revealed that i t degrades to unacceptable levels (46% recovery of DPM from the gel f i l t r a t i o n column) at 12 days of storage at -20°C. Although the recovery of the internal standard in the eluant, as measured by absorbance 260 and DPM in the degradation study (Figure 6). does not appear to be in agreement, i t must be noted that the detection of r a d i o a c t i v i t y i s much more sensitive than an absorbance reading. Further washing of the column ( 12 day column) resulted in the elution of 75% of the i n i t i a l nucleic acids applied but no appreciable amounts of r a d i o a c t i v i t y , t h i s suggests that there was inclusion of the nucleic acids by the g e l . This could be a result of the loss of the individual adenine nucleotides from the t a i l or from endonuclease or exonuclease a c t i v i t y on the entire length of the poly (A) t a i l e d nucleic  87  a c i d . Of interest i s the loss of the radioactive label from the nucleic acids. In that the recovery of the nucleic acids subsequent to washing the column i s almost complete, i t can be concluded that the t r i t i u m label has been l o s t from the adenine. In addition, the evidence indicates that i t has actually bound to the column. 3  The l a b i l i t y of t r i t i u m i s well established (78,79). H -labelled compounds undergo degradation by a free exchange of the t r i t i u m label with hydrogen in the aqueous solutions in which they are contained. This loss i s accelerated at higher temperatures  (78,79). Other forms of radioactive ATP  that may have been used to overcome this problem with t r i t i u m in the 35  synthesis of the internal standard include: S,  14  C, and  125  I . A l l of these  isotopes are beta-emitters and t h e i r quantitation i s achieved by l i q u i d 3S  s c i n t i l l a t i o n counting (78). Both S and  14  C-labelled ATP are available  commercially; however, these isotopes share overlapping energy peaks with and, therefore, would interfere with the simultaneous detection of the labelled cDNA probe.  12S  32  P  32  P-  I has two energy peaks. One peak shares an overlapping  32  energy window with P and the other peak has a d i s t i n c t window (79). A duallabel counting technique could be employed using the isotopes  125  32  I and P i f  the counts were corrected for the overlapping energy peaks of the two isotopes (74). Unfortunately, to our knowledge, the only commercially 12S  available nucleotide that i s labelled with radioactive iodine i s 5- iodo2'-deoxycytidine-5'-triphosphate. Due to the nature of the internal standard ( E . c o l i tRNA), i t i s highly unlikely that i t would be endogenous in any mammalian tissues investigated and can therefore be widely used. The use of the cloned "externally added standard" (73), however, i s limited to tissues and c e l l s which do not  88  contain endogenous mRNA that w i l l cross-hybridize to the probe f o r the cloned external standard. Additional studies, therefore, must be carried out to investigate t h i s . The synthesized internal standard was used to monitor the extraction and p u r i f i c a t i o n e f f i c i e n c i e s of mRNA from l i v e r t i s s u e . The r e s u l t s (Table 1) indicate a low recovery (26.2%) in the i s o l a t i o n of mRNA from tissue but a good recovery following oligo-dT-cellulose chromatography (57.9%). Both methods, however, show a large standard deviation in the recovery of the internal standard at the various steps. This validates the use of an internal standard i f one i s attempting to quantitate mRNA.  2. cDNA PROBE STUDIES Once i t was determined that the human cDNA probe for Factor II did crosshybridize to porcine Factor II mRNA, the binding saturation and l i n e a r i t y of the cDNA probe was established. Saturation was achieved at 75 ng of probe when the amount spotted per f i l t e r did not exceed 2 ug of mRNA. This saturating concentration of probe was then used to determine the l i n e a r i t y of the probe. Linearity was seen from the ranges of 0.1 ug to 1.0 ug of mRNA. Papavasiliou et al achieved l i n e a r i t y with t h e i r cDNA probes to alpha and beta l u t e i n i z i n g hormone to 3 and 8 ug of RNA, r e s p e c t i v e l y , using saturating concentrations of each probe that they had previously determined (80). Schwarzenberg et al achieved l i n e a r i t y with t h e i r genomic probe f o r alpha-l-antitrypsin to 1.0 ug of RNA (81). Saturation studies of t h i s probe were not reported.  89  3. TISSUE DNA QUANTITATION The quantitation of tissue DNA was  introduced as a means of determining  the o r i g i n a l amount of tissue used in the extraction and p u r i f i c a t i o n of mRNA. We f e l t that t h i s would be more accurate than expressing the mRNA r e l a t i v e to protein content or wet weight. Protein content i s t r a d i t i o n a l l y measured by the method according to Lowry et al (58). This method i s subject to n o n - s p e c i f i c i t y due to many i n t e r f e r i n g substances such as b i l e acids and SDS  (82). Both of these substances would be present in the samples studied.  In t h i s instance, wet weight is inappropriate as the tissue i s subject to varying amounts of edema due to the injury i n f l i c t e d by ischemia and  the  subsequent reperfusion. Preliminary studies done in our laboratory on normal porcine l i v e r tissue established a good correlation between tissue DNA,  protein content and wet  weight. There was a s l i g h t interference from the guanidine-thiocyanate buffer as witnessed by the positive Y intercept in Figure 14. Subsequent to t h i s study, 0.020 mL of guanidine-thiocyanate buffer was  added to each  standard. This assay proved to be simple to perform and i t s introduction was e f f e c t i v e as a more accurate means of assessing the original amount of tissue used. A quantitative method for the detection of Factor II mRNA l e v e l s in porcine l i v e r was established. This was achieved by the use of a cDNA probe for Factor II obtained from the laboratory of Dr. R.T.A. MacGillivray. The amount of radioactive probe bound to i t s corresponding mRNA in each sample was expressed as a percentage of the amount of radioactive probe bound to control mRNA that had been applied to the same f i l t e r . In this respect, the assay was controlled for variations in the pre-hybridization, hybridization  90  and, more importantly, the s p e c i f i c a c t i v i t y of the probe used from f i l t e r to f i l t e r . The sample's percent of control value was then corrected for the extraction e f f i c i e n c y during the i s o l a t i o n and p u r i f i c a t i o n of mRNA by the use of a synthesized internal standard that was developed. This r e s u l t was then expressed r e l a t i v e to the amount of DNA in the starting t i s s u e . This procedure was found to have an inter-assay c o e f f i c i e n t of variation of 32%. This i s similar to that reported by Papavasiliou et al who achieved an inter-assay CV of 28% (80). In future, this CV could be decreased to a more acceptable level by the addition of the internal standard to the control mRNA to correct for percent binding to the n i t r o c e l l u l o s e membrane during the f i l t r a t i o n step.  B: Levels of Factor II mRNA in Liver and Factor II in Plasma Following Ischemic/Reperfusion  In.iurv  Uncontrollable bleeding i s one of the major causes of peri-operative mortality in l i v e r transplant recipients (83). Although the recipient i s already in a compromised hemostatic state prior to the transplant, a reperfusion coagulopathy or disseminated  intravascular coagulation i s one of  the usual p r e c i p i t a t i n g events that results in mortality (84).  Reperfusion  coagulopathy has been reported to be caused by the release of tissue thromboplastin from the stored, damaged g r a f t , or from the release of sequestered heparin from the graft (83,84). In addition, even though a coagulopathy i s not evident in the l i v e r transplant r e c i p i e n t , a decrease in a l l plasma protein c l o t t i n g factors from those of pre-operative levels i s seen (40). S p e c i f i c a l l y , Factor II has been shown to decrease to 78% of i t s pre-operative value on the induction of anesthesia. It then further drops to  91  36% of i t s pre-operative level after 70 minutes of reperfusion (40). This decrease in Factor II levels may be due, in part, to the decreased a b i l i t y of the graft to synthesize protein, as i t has been subjected to a period of both warm and cold ischemia followed by reperfusion. The phenomenon described above i s duplicated in the model of warm hepatic ischemic reperfusion injury in the porcine l i v e r investigated in t h i s t h e s i s . The level of plasma Factor II decreased at 30 minutes of ischemia to approximately 58% of i t s pre-operative value. (Levels of plasma Factor II were not investigated immediately after the induction of the anesthesia). This decrease was not due to any u t i l i z a t i o n of the factor as there was no apparent bleeding and the animal was f u l l y heparinized. One possible explanation i s the sequestration of the factor in organs such as the spleen or l i v e r . Although an adequate explanation of i t s loss cannot be made, the lowered levels of Factor II may have acted as a stimulus for i t s own renewal, as seen by a trend of r i s i n g levels of mRNA which s p e c i f i c a l l y codes for Factor I I . This stimulus may have been provided in the fashion of a recently described humoral f a c t o r , coaguloprotein I I , which has been shown to activate the production of Factor II (85). The trend of increasing levels of Factor II mRNA, seen during ischemia and at the e a r l i e r part of reperfusion, do not r e s u l t in the production of any s i g n i f i c a n t increase in the amount of Factor II in the plasma. It may be that the time frame (approximately 90 minutes) i s too short to allow for the production, post-translational modifications and release of the protein into the systemic c i r c u l a t i o n . On further reperfusion, a further trend i s noted in which the l e v e l s of Factor II mRNA decrease. This could be as a result of the production of toxic oxygen radicals which have been shown to be produced during reperfusion. In  92  that oxygen radicals have been shown to cause DNA strand breaks in v i t r o and in v i v o , i t i s conceivable that they might act in a similar manner on mRNA. Although mRNA strand breakage may be e f f e c t e d , i f the oxy-radicals have the a b i l i t y to damage the poly (A+) t a i l , this damage alone would not only affect the s t a b i l i t y of the mRNA, but also i t s involvement in the actual role of protein synthesis (86,87). Although maximal oxygen radical production would be at the i n i t i a t i o n of reperfusion, radical scavengers and antioxidants endogenous to the l i v e r must f i r s t be depleted before s i g n i f i c a n t injury occurs (88,89). As the l i v e r contains more copper/zinc superoxide dismutase and glutathione than any other t i s s u e , and i t i s one of the richest sources of catalase and alpha-tocopherol, i t i s not surprising that the levels of Factor II mRNA do not start to decline until after 90 minutes of reperfusion.  Cairo et a l . in t h e i r model of hepatic ischemia in r a t s , found that at 16 hours of reperfusion a decrease in albumin synthesis accompanied by an unchanged total protein synthesis was evident. The decrease in albumin synthesis was seen as a d i r e c t result of lowered l e v e l s of translatable albumin mRNA (37). They hypothesized that because albumin i s a protein which functions outside of the c e l l , protein synthesizing energies were directed at proteins which function inside the c e l l probably to repair the damage i n f l i c t e d during the ischemic i n t e r v a l . Although prothrombin i s also a protein which functions outside of the c e l l , lowered plasma l e v e l s acted as a stimulus to produce increased amounts of i t s mRNA. During reperfusion, however, this increased Factor II mRNA was o b l i t e r a t e d , perhaps as a result of damage i n f l i c t e d during reperfusion such as by toxic oxygen r a d i c a l s . At one and two days of reperfusion, the levels of Factor II mRNA remained  93  at control biopsy levels and the plasma Factor II also remained low. In contrast, the level of Factor II mRNA in Pig 30 started to r i s e again at one and two days of reperfusion. This could be a result of the continued  stimulus  from the s t i l l - d e p r e s s e d levels of plasma Factor I I . It i s interesting to note that with this p a r t i c u l a r animal, i t s plasma Factor II level increased to 91% of i t s pre-operative value at two days of reperfusion. Of the seven pigs that survived the operative procedure, only four had Factor II mRNA levels that could be quantitated. Factor II mRNA could be detected in a l l animals, but not quantitated according to the procedure outlined in Materials and Methods. This was attributed to poor recovery following the extraction of the nucleic acids and the p u r i f i c a t i o n of mRNA from the l i v e r biopsy.  The advent of recombinant DNA technology and the a v a i l a b i l i t y of pure probes for RNA products of certain genes has opened new avenues for medical research. Presently, recombinant DNA technology has had an impact on many f i e l d s of c l i n i c a l research including cardiovascular diseases, diabetes, hematology and endocrinology  (90,91,72,92). The technology i s used not only  to study the disease mechanisms but to elucidate the normal physiological processes. This thesis used recombinant DNA technology to study one of the underlying mechanisms of warm hepatic ischemic/reperfusion i n j u r y . The results indicate that recombinant DNA technology can be successfully applied to the f i e l d of c l i n i c a l transplantation research. The adaption of recombinant DNA technology to routine c l i n i c a l use i s hindered only by the lengthy and involved nature of the present technology. This i s p a r t i c u l a r l y  94  relevant in the case of c l i n i c a l transplantation where the amount of time required and the amount of sample needed to perform these techniques prohibit t h e i r use. In time, this problem w i l l be solved.  PART III - QUANTITATION OF PLASMA FACTOR II IN PORCINE PLASMA  Methods f o r the determination of Factor II in plasma include c l o t t i n g assays, enzymatic assays and ELISA (enzyme-linked immunosorbant assay) techniques (93,94,95). Because Factor II i s a zymogen, we chose to quantitate i t using an enzymatic assay. With the recent development of synthetic chromogenic peptide substrates, assays f o r individual coagulation factors have been devised that do not suffer from the same degree of nons p e c i f i c i t y that c l o t t i n g assays do. The two synthetic substrates available f o r the determination of 5  prothrombin are S-2238 (Kabi Vitrum Ltd., Stockholm, Sweden) and Chromozym 6  Th . Both are substrates for thrombin rather than prothrombin; therefore, the prothrombin must f i r s t be activated to thrombin. Under normal physiological ++  processes, prothrombin i s activated by the Xa, Ca , P l a t e l e t Factor 3, Va, VIII a complex. If the prothrombin molecule has not been gamma-carboxylated via the action of a vitamin K-dependant carboxylase, i t cannot bind to the activating complex and, therefore, cannot be converted to thrombin. Because  5  HD-phenylalanyl-L-pipecolyl-L-arginine-pnitroanilide-HCl 6  tosyl-L-glycyl-L-prolyl-L-arginyl-p-nitroanilide-  HCl 95  t h i s assay was used to correlate plasma Factor II levels and tissue mRNA l e v e l s that code for Factor I I , r e f l e c t i n g the protein synthesizing a b i l i t y of the organ, we wanted i t to be independent of c e l l u l a r processes that are post-translational modifications. To make this assay independent of the Vitamin K status and gamma-carboxylating processes, a non-physiological a c t i v a t o r , Ecarin, was used. Ecarin i s one procoagulant found in the venom of the Echis carinatus snake (96). Its action i s described to be independent of the calcium ion concentration, p l a t e l e t factor 3 l e v e l s , and therapeutic levels of heparin (97). In addition, the ecarin-thrombin complex i s not inhibited by antithrombin and has a higher a c t i v i t y in cleaving synthetic substrates than does the physiological thrombin (97). Other nonphysiological activators that have been used include staphylocoagulase  and  Taipan Snake Venom (TSV). TSV activation is dependant upon Ca~ concentration (98). Although ecarin was the activator of choice, i t has been reported to activate the chromogenic substrate S-2238 d i r e c t l y (98). For t h i s reason, Chromozym Th was used. Other investigators have used this combination of activator and substrate (98). However, t h e i r buffer system included NaCl, Tris-HCl and imidazole. We found t h a t , in such a buffer system, the substrate p r e c i p i t a t e d . When the ionic strength was reduced, by using 0.075 M T r i s buffer, pH 8.4, t h i s p r e c i p i t a t i o n was prevented. In addition, the pH of the buffer was  increased  to 8.4, as t h i s i s the reported pH maxima for Ecarin (99). Since imidazole is used primarily to keep calcium ions in solution, i t s elimination had no detrimental e f f e c t s on the assay (100). In order to optimize this assay, i t was necessary to investigate the  96  concentration of the a c t i v a t o r , the reaction time with the substrate and the e f f e c t of heparin. A higher concentration of activator was used (0.111 mg/mL) because i t contributed to a more sensitive standard curve, as witnessed by the steeper slope. An even higher concentration of substrate might have resulted in more s e n s i t i v i t y , but this was not investigated due to the cost of the substrate. The endpoint chosen for the reaction time with the substrate (4 minutes) resulted in maximal product formation while s t i l l retaining a l i n e a r relationship between product formation and time. The material of choice for assay of coagulation proteins i s c i t r a t e d plasma, as the heparin in heparinized plasma interferes with most other assay techniques. Although the action of the activator i s reported to be independent of therapeutic levels of heparin, studies were conducted to ensure that the heparin used in the operative procedure as well as the heparin in the blood c o l l e c t i o n tubes did not i n t e r f e r e . We confirmed the findings of other investigators that therapeutic levels of heparin R  (Hepalean ) did not interfere with the assay. Although other investigators have found that levels up to 10 units did not i n t e r f e r e , t h e i r unit value  was  not defined (99).  PART IV - INDOCYANINE GREEN CLEARANCE STUDIES A: The Clearance of ICG During Ischemic/Reperfusion In.iury Indocyanine green i s a non-toxic, tricarbocyanine dye (101). Once infused into the c i r c u l a t i o n , i t i s cleared exclusively by the hepatocytes and excreted into the b i l e in an unconjugated form (102). The clearance of ICG from the peripheral c i r c u l a t i o n has been described as a single exponential decay (102). We, and others, have found that i f the sampling interval i s  97  extended past the recommended 20 minutes, a second exponential decay i s evident (103). In that there i s no extra-hepatic uptake nor enterohepatic r e c i r c u l a t i o n of the dye and that i t i s cleared rapidly from the c i r c u l a t i o n (faster than BSP), ICG has been used to monitor both hepatic function and hepatic blood flow (104,105). The clearance of ICG was included in this study as a measure of hepatic function. Rather than calculate the clearance r a t e , we used the rate disappearance constant (defined as the slope of the log of absorbance versus time) as an index of l i v e r function as i t is considered more r e l i a b l e (106). This interpretation of l i v e r function assumes constant l i v e r blood flow. In this model this assumption cannot be made. To overcome t h i s d i f f i c u l t y , an attempt was made to monitor and correct a l l the ICG clearances for hepatic blood flow by the use of indwelling Doppler flow probes. Due to the high rate of f a i l u r e of these probes, especially the probe around the portal vein, this correction could not be accomplished. This made the data d i f f i c u l t to interpret. As expected, during the ischemic phase there was an 82% reduction (over a 90 minute time interval) in the clearance of ICG in comparison to the preoperative values. This observation i s indicative of the blood flow status rather than hepatocellular dysfunction. The residual uptake of the ICG could have occurred from back flow into the l i v e r from the hepatic v e i n , or from very small c o l l a t e r a l s to the l i v e r that could not be i d e n t i f i e d and clamped. Whatever the mechanism, the clearance of ICG during the ischemic period remained low and constant. During reperfusion, the rate disappearance constants (K, and K2) remained blunted and this reduction proved to be s i g n i f i c a n t . The decrease in the rate disappearance constants may be  98  explained by the hepatocellular damage i n f l i c t e d during ischemia and reperfusion or from the "no reflow phenomenon" that has been described in other models of ischemia/reperfusion injury (107,108). The "no reflow phenomenon" describes the i n a b i l i t y of the blood to perfuse an organ after a period of ischemia due to swelling of the vascular endothelium which impedes blood flow on reperfusion (107,108). In interpreting these data, differences in blood flow to the organ cannot be ruled out. At one and two days postoperatively, the rate disappearance constants returned to normal. The normalization of the rate disappearance constants may be explained by the l i v e r ' s large reserve capacity thereby making this test i n s e n s i t i v e to ischemic/reperfusion injury or to the fact that the injury i n f l i c t e d was not severe enough to allow for differences at one and two days post-operatively.  B: Assay Optimization f o r the Determination of ICG in Porcine Plasma The clearance of ICG was measured spectrophotometrically. Other methods, including fluorometric and HPLC assays have been described (109,110). The advantage these assays offer over the spectrophotometric method i s s e n s i t i v i t y . Due to the dose of ICG used (1 mg/kg), s e n s i t i v i t y was not an issue. Once reconstituted in an aqueous s o l u t i o n , indocyanine green degrades rapidly (102). However, after the dye has been bound to protein i t remains r e l a t i v e l y stable (104). In order to increase i t s s t a b i l i t y , we devised an alternative method of reconstituting the dye by taking advantage of this f a c t . We found that by reconstituting the dye in a 1:1 plasma to diluent mixture, the dye i s stable for at least 10 days. This time frame i s more than adequate f o r the performance and measurement of the ICG in the model used.  99  Other investigators have reconstituted the dye with a solution of 5% s t e r i l e human albumin. However, the extent to which the dye was  s t a b i l i z e d was  never  reported (111). Since many of the methods used in this study depend on spectrophotometric analysis of t h e i r reaction endpoint, an investigation was conducted to determine i f the ICG in the animal's plasma would interfere with any of these methods. It was determined that there was  a p o s i t i v e , dose-dependant  interference with the method used for total b i l i r u b i n due to the presence of indocyanine green. In the method for the determination of plasma b i l i r u b i n , a surfactant (Triton X-100) is used to dissociate b i l i r u b i n from albumin (112). It might be expected that t h i s surfactant could also dissociate the ICG from the albumin. The ICG, therefore, i s free to enter the spreading and reaction layer of the f i l m cassette. In t h i s assay, a bichromatic reflectance reading i s performed at 540 and 420 nm, the l a t t e r wavelength being used to correct for any spectral interference. The p o s i t i v e interference from the ICG may  or may  not be solely attributed to spectral  interference. ICG has a higher absorbtivity at 420 nm than i t does at 540 when i t i s protein bound. It may  nm  be that the ICG is actually reacting with  the diazonium s a l t in the reagent layer and producing a colored azo compound that r e f l e c t s strongly at 540 nm. However, i t may  also be that the spectral  c h a r a c t e r i s t i c s of unbound ICG do contribute to the positive interference under the conditions of the assay. To our knowledge, the observation of positive interference of ICG with the total b i l i r u b i n method has not been reported previously. As a result of t h i s study, only specimens which did not contain dye were analyzed for total b i l i r u b i n .  100  PART V - ROUTINE PLASMA MEASUREMENTS During the reperfusion phase, the AST levels increased s i g n i f i c a n t l y . These changes were also noted by Harris et al after 60 and 180 minutes of ischemia and by Battersby et al after 30 minutes of ischemia (66,67). The increase in AST seen during reperfusion i s probably not due to injury caused during reperfusion; rather, enzymes which are released from hepatocytes during the ischemic insult are being washed out of the l i v e r into the peripheral c i r c u l a t i o n on reperfusion. Both Battersby et al and ourselves reported a s i g n i f i c a n t r i s e in the peripheral glucose level during the ischemic phase (66). They hypothesized that enzymes in the hepatocytes responsible f o r the uptake of glucose from the peripheral c i r c u l a t i o n had been damaged to a s i g n i f i c a n t enough degree during ischemia so that glucose uptake was impaired (66). We believe that t h i s increase in glucose during ischemia can best be explained by the fact that the l i v e r , in i t s e l f , u t i l i z e s a large proportion of blood glucose f o r i t s own metabolic a c t i v i t i e s . During ischemia, however, c i r c u l a t i o n to the l i v e r i s e f f e c t i v e l y cut o f f and, as a r e s u l t , the peripheral glucose levels r i s e . We confirmed the findings of Battersby et al of high glucose levels from the hepatic venous effluent during reperfusion (66). Battersby et al hypothesized that this release of glucose from the l i v e r was a further indication of damage to hepatocellular g l y c o l y t i c enzymes resulting in impaired glucose homeostasis. However, i t may be that the l i v e r i s reacting normally to the stress of the operative procedure and i s releasing glucose for the u t i l i z a t i o n in the peripheral  tissue.  Harris et al and Battersby et al found a s i g n i f i c a n t increase in the level  101  of 10 in the peripheral c i r c u l a t i o n and the venous effluent from the l i v e r on reperfusion of the organ (66,67). This increase was rapid and t r a n s i e n t . In contrast to both of these investigators, we found a rapid and transient decrease in the l e v e l s of 10 on reperfusion of the l i v e r . As both Harris et a l and Battersby et al were investigating l i v e r survival a f t e r extended periods of ischemia, the l i v e r injury was more severe and resulted in death of hepatocytes and leakage of 10 from the l i v e r c e l l s . We found s i g n i f i c a n t l a c t i c acidosis during the ischemic period which continued into the reperfusion phase indicating that a pronounced degree of anaerobic metabolism had occurred. The return of the lactate to normal levels following 90 minutes of reperfusion indicated a rapid recovery of the hepatocytes (with respect to lactate metabolism) when blood flow and oxygen were restored. These findings agree with those of Harris et al (67). We confirmed the observations of Harris et al who  found no s i g n i f i c a n t  changes in b i l i r u b i n values (67). This can best be explained by the  liver's  large reserve capacity for the uptake, conjugation and release of b i l i r u b i n .  Ischemic/reperfusion  injury to an organ during donor harvesting, storage  and transplantation i s i n e v i t a b l e . However, assessing the amount of injury an organ has sustained p r i o r to transplantation i s crucial to a successful outcome. Currently, the analysis of routine biochemical parameters of l i v e r function in the donor before harvesting the organ have proved to be incapable of predicting whether or not the organ w i l l function once transplanted. Therefore, the transplantation of a non-functioning  102  graft i s a  recurring r e a l i t y and in the case of hepatic transplantation, may prove to be f a t a l . Before the effects of ischemic/reperfusion injury can be assessed  and  controlled t h e i r pathological mechanisms must be further investigated. We chose to study the effects of warm hepatic ischemic/reperfusion injury on the protein synthesizing a b i l i t y of the porcine l i v e r , in p a r t i c u l a r , the l e v e l s of Factor II mRNA. These Factor II mRNA levels were compared to other indicators of hepatocellular damage that have been used to assess ischemic/reperfusion i n j u r y . Our studies indicate that Factor II mRNA may be more suseptible to the effects of reperfusion rather than ischemia given the changes seen during each p a r t i c u l a r time period. Although t h i s may be the case one cannot be certain that the effects seen are not due to a prolonged ischemic event as a result of swollen endothelium r e s t r i c t i n g or stoppnig the blood flow through the l i v e r when reperfusion was thought to be i n i t i a t e d . In addition, the lower levels of Factor II mRNA seen on reperfusion may be as a result of damage that was i n i t i a t e d during ischemia rather than reperfusion. Although the actual time course of the injury process i s not perfectly c l e a r , Factor II mRNA levels have proven to be more sensitive to the e f f e c t s of ischemic/reperfusion injury than the other routine biochemical parameters of hepatocellular dysfunction.  103  SUMMARY  1. A quantitative method for the detection of Factor II mRNA was established and t h i s included the development of an internal  standard,  saturation and l i n e a r i t y binding studies on the cDNA probe and the introduction of the quantitation of tissue DNA l e v e l s .  2. By using the method established, Factor II mRNA levels were quantitated in porcine l i v e r following 90 minutes of ischemia and 2 days of reperfusion. During the ischemic period, the Factor II mRNA l e v e l s increased and t h i s trend was exhibited in four animals. The increase i n the l e v e l s of Factor II mRNA may have been in response to decreased levels of Factor II in the plasma. With continued reperfusion, the levels of Factor II mRNA decreased and returned to control biopsy values. This decrease may be due to the effects of oxygen radicals that are known to be produced during reperfusion.  3. A quantitative method for the detection of Factor II in plasma was developed and used to assess the affect of ischemia and reperfusion on plasma l e v e l s of Factor I I . Factor II plasma levels decreased at the s t a r t of ischemia and t h i s decrease was found to be s i g n i f i c a n t .  4. Other established parameters of hepatocellular damage studied included +  routine plasma measurements (AST, l a c t a t e , glucose and K ) and indocyanine green clearances. The changes in routine plasma measurements were found to be similar to those of other investigators. The r i s i n g levels of AST during  104  reperfusion indicated that hepatocellular damage had occurred, thereby validating the model of ischemic/reperfusion i n j u r y . The clearance of indocyanine green was s i g n i f i c a n t l y decreased during ischemia and reperfusion. The decreased clearance during ischemia was attributed to a decreased blood flow. The decreased clearance of ICG was thought to be due to some degree of hepatocellular injury although the differences in blood flow cannot be ruled out. At one and two days of reperfusion the clearance of ICG was normal.  105  APPENDIX I TISSUE SAMPLING PROTOCOL  PHASE  TIME  HV  RA  CA  Baseline (pre-op)  0  -  ICG  X*  Control  0  -  -  X*  Ischemia (90 min)  Reperfusion  0 30 60 90  X  ICG -  5 10 15 20 30 40 50 60 90  # * # * # * * # #  -  # # #  +  -  -  -  +  Reperfusion (4 hours)  -  -  -  +  Reperfusion (24 hours)  -  ICG  -  +  Reperfusion (48 hours)  -  ICG  -  +  HV - hepatic vein RA - internal jugular vein catheter tunnelled to the right side of the heart (right atrium) CA - carotid artery H - histology (routine l i g h t and electron) and cDNA/mRNA hybridization studies + # - blood drawn f o r AST, K , l a c t a t e , glucose, total b i l i r u b i n , ICG and Factor II * - blood drawn f o r ICG only  106  APPENDIX II MAP OF PLASMID PKK2233 AND INSERT  a XI  O o  II  CM"  m-  E u  I  e CM  m ' 3 >  e a  a.  H OS  w SB  ID CM  3  in  in  00.  107  S  REFERENCES  1. 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