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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 British 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, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of <-?Q:T*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 significant damage to the organ and the success of transplantation is frequently dictated by the magnitude of this insult. It is for this reason that a high priority has been given to studying the pathological mechanisms underlying this type of ischemic and reperfusion injury. Ischemic/reperfusion injury to the liver significantly decreases the ab 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 is seen in the levels of a l l plasma protein clotting factors. In particular, Factor II levels decrease to 36% of their pre-operative level. 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 qualitatively altered. It is 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 injury. 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 arteries. Reperfusion was initiated 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 relative to the total DNA content. The extraction efficiencies were monitored and corrected for by means of a synthesized internal standard developed for this study. The effect of i i 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 similar to those of other investigators. Plasma AST (aspartate aminotransferase) levels rose significantly during the reperfusion phase indicating that hepatocellular damage had occured. Plasma glucose and lactate levels increased significantly during ischemia and returned to normal by 90 minutes of reperfusion. Plasma K+ levels decreased significantly 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 lactate, AST, glucose and 10, bilirubin values did not vary throughout the operative procedure. The clearance of ICG decreased significantly 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 significantly, and i t was concluded that this 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 significantly 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 i i i result of damage inflicted during the reperfusion phase, specifically the production of oxygen radicals. With continued reperfusion (two days post-operatively), the Factor II mRNA levels remained low in some of the animals studied; in others, the levels started to rise again. The plasma Factor II levels, however, remained low throughout. It is anticipated that these findings will further our understanding of the pathological mechanisms underlying ischemic/reperfusion injury. iv TABLE OF CONTENTS PAGE ABSTRACT i i LIST OF TABLES ix LIST OF FIGURES x LIST OF PLATES x i i 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 II. QUANTITATION OF FACTOR II mRNA IN PORCINE LIVER 18 A. Method Development for the Quantitation of Factor II mRNA from Liver.... 18 1. Development of an Internal Standard 18 a) method 18 b) elimination of gel f i l t r a t i o n step 20 c) stability 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 linearity studies 27 3. Tissue DNA Quantitation 28 a) method 28 b) correlation of DNA to tissue protein content 28 c) buffer interference study 29 B. Quantitation of Factor II mRNA in Porcine Liver 30 1. Isolation and Purification 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 31 III. QUANTITATION OF FACTOR II IN PORCINE PLASMA 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 Stability Study 37 C. Dye Interference Study 37 V ROUTINE PLASMA MEASUREMENTS 38 A. AST 38 B. Glucose 38 C. Total Bilirubin 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 Stability Study 62 B. Dye Interference Study 66 C. The Clearance of ICG During Ischemic/Reperfusion Injury 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 for 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 ICG CLEARANCE STUDIES 97 A. Clearance of ICG During Ischemic/Reperfusion Injury 97 B. Assay Optimization for the Detection of ICG in Porcine Plasma 99 V ROUTINE PLASMA MEASUREMENTS 101 SUMMARY 104 APPENDIX 1 106 APPENDIX II 107 REFERENCES 108 vi i i LIST OF TABLES TABLE PAGE 1 Recovery of the internal standard during the isolation and purification of mRNA from li v e r 45 2 Effect of heparin on the assay for plasma Factor II 63 3 Clearance constants of ICG during ischemic/ reperfusion injury 74 4 Porcine plasma AST and glucose levels during ischemic/reperfusion injury 77 5 Porcine plasma K+ and lactate values during ischemic/reperfusion injury 80 ix LIST OF FIGURES FIGURE PAGE 1 Schematic representation of the time sequence for specimen collection 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 4 Elution profile of the internal standard, human and porcine mRNA from an oligo-dT-cellulose column 44 5 Elution profile 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 profile of the degraded internal standard from a gel f i l t r a t i o n column 48 8 cDNA probe saturation studies . 52 9 cDNA probe linearity studies 54 10 Porcine l i v e r DNA content and protein content of the tissue 55 11 Porcine l i v e r DNA content and wet weight of the tissue 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 injury.... 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 citrated and heparinized plasma 64 17 Porcine plasma Factor II levels during ischemic/ reperfusion injury 65 x 18 The sta b i l i t y of Indocyanirie Green 67 19 Effect of ICG on the assay for Factor II in porcine plasma 68 20 Effect of ICG on the method for glucose 69 21 Effect of ICG on the method for potassium 70 22 Effect of ICG on the method for AST 71 23 Effect of ICG on the method for lactate 72 24 Effect of ICG on the method for total bilirubin 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 for Factor II to porcine mRNA following formaldehyde gel electrophoresis and Northern transfer xi i List 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 acid MOPS morpholinopropanesulfonic acid mm millimeter mM millimolar mg milligram mmol millimoles mL m i l l i l i t e r mRNA messenger ribonucleic acid ng nanogram nm nanometer xi i i p statistical probability r correlation coefficient 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 like to thank those individuals who contributed to the successful completion of this thesis, especially my friends, Mr. Allan Hustad and Mrs. Kris Gillespie. To the members of my supervisory committee for their helpful suggestions and constructive criticism, I express my gratitude. I am indebted to Dr. R.T.A. MacGillivray and the members of his laboratory, in particular, Dr. D. Clevland and Dr. D. Irwin, for supplying the Factor II cDNA probe and helping me gain proficiency with hybridization techniques. To my supervisors, Dr. David Seccombe and Dr. Charles Scudamore, for their support and encouragement throughout and for the good times we have shared, I thank you. The funding of this research was provided by the British Columbia Health Care Research Foundation. xv INTRODUCTION Liver transplantation is the only known cure for end-stage li v e r disease (1). Diseases of the liver that typically progress to end-stage include: malignant disorders such as hepatocellular carcinoma and cholangiocarcinoma, fulminant hepatic necrosis (either drug or viral induced), inborn errors of metabolism (Wilson's disease, hemachromatosis, alpha-l-antitrypsin deficiency etc.), chronic liver disease (chronic hepatitis, primary biliary cirrhosis) and conditions of the biliary tract such as bi l i a r y atresia (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, is one year of l i f e or when the symptoms are intolerable and there are no absolute contraindications for transplantation (4). As l i v e r grafting is now accepted therapy for end-stage li v e r disease and is no longer considered an experimental procedure, i t is 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 limiting factors in li v e r transplantation (and the transplantation of other organs) is donor availability (7). Between 1981 and 1982, one center reported that 26.5% of its l i v e r patients died while waiting for a transplant (8). So optimization of the limited donor pool is cruc 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 limit liver preservation to 8-10 hours1 (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 li v e r transplantation. It is for this reason that the National Institutes of Health consensus report on l i v e r transplantation in 1983 gave top priority 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 utilization of the donor pool (10). Furthermore, the traditional parameters of assessing the donor organ are notoriously unreliable predictors of ultimate graft function. It is estimated that up to 15% of hepatic grafts are lost after transplantation due to acute graft dysfunction, so called "primary non-function" (1, 11). The transplantation of a non-functional l i v e r graft is disastrous, and re-grafting is 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 cell mediated) and graft damage due to ischemic/reperfusion injury, with the majority of failures being attributed to ischemic/reperfusion injury (11). Ischemic/reperfusion injury is defined as " ... all 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, liver preservation intervals have been reported to be increased to 24 hours (13). 2 reperfusion" (12). But as yet, the pathological mechanisms underlying ischemic/reperfusion injury are poorly understood. It is only when these mechanisms are more full y understood can the amount of damage an organ has sustained be assessed prior to transplantation. Further benefits from studying ischemic/reperfusion injury is 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 is defined as the cessation of blood flow. This diminishes substrate supply and results in tissue anoxia. Whether or not the anoxic damage is reversible depends upon the temperature, tissue type, duration of ischemia, availability of stored substrate and other unknown factors (14). In the kidney and l i v e r , cell death from ischemia occurs 1-2 hours after injury at 37°C, whereas, in bronchial epithelium and pancreas irreversible damage occurs later (3 hours) (14). Although the reasons for these different rates are unknown, a l l cells progress through the same series of biochemical events that result in cell death (14). This biochemical progression corresponds to distinct morphological stages which have been identified by Trump et al (14). Mitochondria depend on a continuous supply of oxygen and substrate to support the production of ATP. Ischemia disrupts this process, resulting in a decreased energy charge2 in the cell (15). The levels of ADP and i t s 2 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 activities which results in increasing the rates of glycogenolysis and anaerobic glycolysis (16). But, because of the anaerobic state of the organ, glycolysis is inefficient. Consequently, glycogen stores are quickly depleted, lactate accumulates, and the intracellular pH f a l l s , causing clumping in nuclear chromatin (14). As intracellular lactate levels increase, phosphofructokinase and hexokinase activities decrease, further impairing ATP production (15). With the continuing deterioration of ATP levels, ATP-dependant ion pump activity decreases resulting in the leakage of K+ and Mg++ from the cell and the diffusion of Na+ and Ca++ into the c e l l . Due to these changes in ion homeostasis, the cellular volume expands with water and ultrastructurally this results in the appearance of small "blebs" on the cell membrane. Concurrently, the nuclear envelope and the cisternae of the rough endoplasmic reticulum dilate, 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 cell attempts to enhance substrate supply for ATP generation further depleting the glycogen and accelerating gluconeogenesis (15). However, intracellular AMP levels are sufficient to inhibit phosphoenolpyruvate carboxykinase, an essential enzyme for gluconeogenesis. In addition, the activities 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 fatty acids normally undergo an ATP-dependant activation process generating acyl-coenzyme A. The acyl-coenzyme A is then esterified to L-carnitine by carnitine palmityltransferase and transported into the mitochondria where they undergo beta-oxidation (16,17). However, during ischemia, the availability of ATP becomes the rate limiting factor for the oxidation of free fatty acids. With anaerobic metabolism and intracellular lactate accumulation, the carnitine esterification reaction is inhibited, letting free fatty acids and acyl-coenzyme A moieties accumulate within the c e l l . Acyl-coenzyme A, a known inhibitor of adenine nucleotide translocase activity, impairs effective ADP/ATP mitochondrial exchange (17). This has a direct uncoupling effect on oxidative phosphorylation (18). By virtue of these effects, intracellular acyl-coenzyme A accumulation leads to a further reduction in cellular ATP content which, in part, is manifested by margination of the nuclear chromatin and "high amplitude swelling" of the mitochondria (14). All of these ischemia-induced changes are reversible (14). However, i f ischemia continues, an undefined " point of no return" is reached in which further damage to membranes and mitochondria becomes irreversible (14,18). Studies directed at elucidating the intracellular pathological mechanisms underlying ischemic injury have more recently focused on the role played by calcium. Evidence indicates that mitochondria lose their 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 is lost, calcium accumulates within the cytosol, activating membrane-bound lipases 5 (phospholipase, lysophospholipase) which bring about degradation of the membrane (14,17,18). This increase in cytosolic calcium is thought to have other damaging effects. It may interfere directly with mitochondrial functions by unknown mechanisms and through the activation of normal calcium-dependent processes. Such processes include the inhibition of phospholipid synthesis, regulation of hormonal activity, and binding of prostaglandins to the cell membrane (14,18). Overall, calcium-initiated membrane damage results in cytosolic contents leaking into the extracellular space. This signals cell 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 re-introduction of blood flow to the area may i t s e l f be deleterious. The reperfusion with oxygenated blood removes degradation by-products such as adenosine, hypoxanthine, free fatty acids and lactate. This removal of potential energy substrates can further retard ATP synthesis and membrane repair. Furthermore, reperfusion delivers calcium and oxygen to cells already compromised in their a b i l i t y to handle these substances. As oxygen is reintroduced into the system, i t is converted to partially reduced forms of dioxygen or oxygen radicals (superoxide, hydrogen peroxide, hydroxyl radicals) (19,20,21). These free radicals are extremely reactive (22). They damage tissue by combining and reacting with all major classes of macromolecules: polysaccharides, l i p i d s , nucleic acids and proteins. Oxygen radicals have been shown to fragment a major polysaccharide, hyaluronic acid (22). Lipid peroxidation is initiated by free radicals reacting with polyunsaturated fatty 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 directly by causing polymerization and peptide bond cleavage (20,22). Indirect damage to proteins by limited free-radical oxidation has been shown to increase their susceptibility 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 via base substitution, induce DNA cross-linkage, polymerize protein and decrease the synthesis of RNA, DNA and proteins in in vitro systems (23). The evidence that supports the presence of free radicals is both direct and indirect. Direct evidence is given by the actual detection of free radicals by electron spin resonance spectroscopy (24). Their existence is 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). Clearly, reperfusion-induced free radical production potentiates the ischemic insult. What is the origin, 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 cells and other c e l l s , is the major source of free radicals. Under normal physiological processes, the enzyme exists in the dehydrogenase form. Yet during ischemia, xanthine dehydrogenase is converted to xanthine oxidase via the action of a calcium-dependant protease which is thought to be activated due to the accumulation of intracellular calcium (28). Another consequence of the ischemic injury is the accumulation of hypoxanthine, a degradation product of ATP and the 7 natural substrate for xanthine oxidase. Once the organ is reperfused, oxygen is reintroduced into the tissues where i t reacts with the hypoxanthine and xanthine oxidase to produce oxygen free radicals. In addition to i t s effect on the direct production of oxygen radicals, xanthine oxidase has also been shown to augment the production of free radicals i n d i r e c t l y . Xanthine oxidase has the a b i l i t y to mobilize iron from it s storage protein, f e r r i t i n , by both 02~-dependant and -independent pathways (29). The role of iron and other transition metals as catalysts in the production of free radicals y_j_a the modified Haber-Weiss reaction is well established3 (30). Roy and McCord have studied the kinetics 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 is quite slow in comparison to the conversion in the intestine and heart (31). However, in the normothermic ischemic l i v e r , near-complete conversion from the dehydrogenase form to the oxidase form is achieved in 10 minutes with a concommitant four-fold increase in the oxidase activity (31). Next to the small intestine, the l i v e r is the richest source of xanthine dehydrogenase (31). It is also the storage organ for the body's trace elements and is the site of f e r r i t i n synthesis (32). In view of these facts, one might conclude that i t s potential for free radical induced damage is high. The l i v e r is also highly susceptible to ischemic injury due to i t s 02"+ Fe3 Fe2+ + 0, Fe2+ + H202 Fe3+ + 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 arteriole and portal venule into the sinusoids then drains into the central vein. This structural organization gives rise to gradients in substrate and oxygen concentrations. As a result, the hepatocytes in the acinus are divided into three different zones of metabolic activity, those cells in zone three (perivenous hepatocytes) being most susceptible to anoxic conditions (32). Most metabolic activities 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 is an integral part of liver function, i t has been used extensively as a marker of ischemic liver damage (33,34,35). Relatively mild conditions of l i v e r ischemia (hepatic artery ligation) have been shown to decrease protein synthesis in vitro and in vivo by 60% and 80%, respectively, as judged by the incorporation of radiolabeled amino acids (35). In addition, protein synthesis has been shown to decrease progressively as the ischemic interval continues and the abil i t y of the l i v e r to regain i t s protein synthesizing capability is 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 abi l i t y of protein synthesis recovered, the production of albumin was decreased in relation to its control value and the investigators found that this decrease in albumin production was a direct result of depressed relative amounts of translatable albumin mRNA (37). These changes were found to occur during reperfusion, 16 hours after 9 termination of the ischemic event. In that both ischemia-induced injury and free radical-induced injury, (analogous to that which is produced during reperfusion), have been demonstrated to cause DNA strand breaks in vitro and in vivo, 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 specific aim of this thesis, 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 liver damage for several reasons. Prothrombin is 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 their existing l i v e r disease. Furthermore, a decrease in a l l coagulation factors, in comparison to pre-operative values, is noted during the transplant procedure (40). In addition, once the organ has been transplanted and the recipients' blood allowed to reperfuse the graft, a coagulopathy may be initiated by factor(s) released from the damaged graft (41). For these reasons, an immediately functioning graft is essential to help replace depleted coagulation factors as uncontrollable bleeding is a significant factor in peri-operative mortality (42). The biological h a l f - l i f e of Factor II (96-100 hours) is such that under normal circumstances its turnover is slow in comparison to other coagulation factors (43). 10 Factor II mRNA was quantitated by using its human cDNA probe previously cloned and supplied by the laboratory of Dr. R.T.A. MacGillivray (44). The established methods for the detection of mRNA in tissue using their DNA (or RNA) probes, are qualitative procedures and for the purpose of this thesis i t was necessary to make these methods quantitative. The objectives of this thesis were to: 1. establish a method for the accurate quantitation of Factor II mRNA using a human cDNA probe; 2. making use of this method, quantitate Factor II mRNA in liv e r biopsies from the established model of warm hepatic ischemic/reperfusion injury in the pig. 3. develop and characterize a method to quantitate porcine Factor II levels in porcine plasma; 4. making use of this method, analyze plasma specimens obtained from the established model of warm hepatic ischemic/reperfusion injury in the pig. 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 its hepatic architecture and vasculature is similar to that of man (45). The size of the animal facilitated sample collection once the chronic blood lines 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 ice. The plasma was separated from the cells by centrifugation at 3,000 X g (Silencer H-103NA, VWR S c i e n t i f i c , San Francisco, California), frozen within 20 minutes of collection, and stored at -70°C until analyzed. Liver biopsies were processed immediately. The tissue was rinsed with sterile 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 light microscopy. The remaining segment, (1 gram) was frozen in liquid nitrogen and used to quantitate tissue levels of Factor II mRNA. A graphic representation of the time sequences for specimen collection (blood and tissue) is given below (Figure 1). A more in-depth description is given in Appendix I. 12 Model pre-op ischemia •reperfusion- 1 day 2 days post-op post-op A A 0 90. • i t A B 90 180 D 240 f •ih A G Figure 1 . Schematic representation of the Lime sequence for specimen c o l l e c t i o n i n the model of warm hepatic ischemic/reperfusion i n j u r y . Liver biopsies and peripheral blood specimens for routine chemistries are taken at times indicated as B through G . ICG clearances? are performed at A , B, F , and G . Blood for pre-operative routine chemistries are drawn at 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 injection of ketamine (11 mg/kg) given in the soft tissues of the neck. The animal was anesthetized by mask using isoflurane to s u f f i c i e n t depth to enable endotracheal intubation. After intubation, the animal was ventilated with a mixture of isoflurane (1-3%) 13 and 100% oxygen. A gastric 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 table. 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 incision was made parallel and lateral to the sternocleidomastoid muscle and the right carotid artery and internal jugular vein were exposed by careful dissection. An arterial line (poly-vinyl chloride, 1.69 mm internal diameter X 3.07 mm outer diameter) was placed into the right carotid artery with the distal artery being ligated. A venous line was placed into the right internal jugular vein and advanced to the right side of the heart. Following placement, both lines were tunnelled subcutaneously to the interscapular midline and exteriorized. The arterial line was used to monitor arterial blood pressure and blood gases. Both lines were used for acute and chronic blood sampling. The lines were kept patent on a chronic basis with heparinized saline (200 units/mL). Following line placement, the neck was closed with 2-0 polyglycolic acid sutures. The abdomen was opened through a midline incision from the xyphoid to the midhypogastrium. Adequate exposure was obtained by means of a Balfour self-retaining retractor secured with towel c l i p s . The li v e r was mobilized and the portal structures were isolated. The hepatic artery was dissected throughout its entire length beginning at the aorta below the takeoff of the splenic artery. Minor arterial branches were cauterized and the gastroduodenal and l e f t gastric arteries were identified. At this point, 14 Doppler flow probe cuffs of the appropriate size were placed around the hepatic artery and the portal vein. The peritoneum and fascial layers were closed with continuous 0-polyglycolic acid sutures and the skin with 2-0 polyglycolic acid sutures. The anesthetic agents were discontinued and the animal was fed following recovery. Food was subsequently withdrawn for an overnight fast. b) Day 2 Following an overnight fast, a blood sample was drawn from the carotid artery catheter and processed for Factor II and routine chemistries (AST, glucose, total b i l i r u b i n , lactate and K+). Subsequently, an indocyanine green clearance study was performed as per protocol. Following the clearance study, the animal was fed. c) Day 3 A 24 hour fast is begun. d) Day 4 Following the 24 hour fast, the animal was anesthetized and prepared for surgery as previously outlined (Day 1). The arterial line was connected to a high pressure sampling port which allowed for blood pressure monitoring as well as arterial blood sampling. The abdomen was opened as previously described (Day 1). A 16 gauge angiocath was introduced through a venotomy in the infrahepatic inferior 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 right external jugular vein. A Bardec cannula with a s i l i c o n connector was placed into the portal vein on the intestinal side with the distal end clamped. Blood was allowed to f i l l the shunt displacing any a i r . The shunt was then clamped and introduced into the right external jugular vein. Subsequently, the clamps were removed. The celiac artery was clamped with a pediatric Statinsky clamp and the l e f t gastric artery was clamped with a Dietrick clamp. The gastroduodenal artery was tied d i s t a l l y with a s i l k ligature and the artery was p a r t i a l l y divided leaving the proximal branch untied. Bleeding was noted to be present or absent. If absent, the hepatic ci r c u l a t i o n had been adequately isolated. The hepatic artery 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 ischemia, the portal vein was b r i e f l y clamped and the porto-jugular shunt removed and the integrity of the vessel re-established. Subsequently, the clamps were removed from the portal vein, hepatic artery and i t s c o l l a t e r a l s - thus marking the start of the reperfusion phase. JV Figure ?.. Schematic representation of the surgical procedure. Ischemia is achieved by diverting blood, through an external shunt (ST), from the portal vein (PV) to the external jugular vein (JV) and by clamping the hepatic artery (HA) and the gastroduodenal artery (GD) (42). ST 16 All blood samples taken during the reperfusion phase were taken from the hepatic vein angiocath following a 30 second vena caval clamp to collect 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 vein. The lines 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 fast, a blood sample was drawn from the arterial line and processed for Factor II and routine chemistries. Subsequent to th i s , an indocyanine green clearance study was conducted as per protocol. 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 is followed. The animal was then euthanized. 17 PART II - QUANTITATION OF FACTOR II MRNA IN PORCINE LIVER A. Method Development for the Quantitation of mRNA in Porcine Liver 1. DEVELOPMENT OF AN INTERNAL STANDARD a) Method The internal standard consisted of E. coli tRNA that had been polyadenylated and radiolabeled using poly (A) polymerase according to the method of Sippell (47). Briefly, the procedure was as follows. E. coli tRNA (30 ug), (Boehringer Mannheim, Montreal, Quebec) was incubated at 37°C in a buffer (0.100 mL total volume) of 50 mM Tris-HCl4, pH 7.9, 10 mM MgCl2, 2.5 mM MnCl2, 250 mM NaCl, 50 ug bovine serum albumin, 9.43 X 10"g mmoles (2,5',8-H3)-ATP (specific activity approximately 50-60 Ci/mmole), (Amersham, Oakville, 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) Purification of the Nucleic Acids The polyadenylated RNA and unincorporated nucleotides were purified 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, all chemical were purchased from Sigma Chemical Company, St. Louis, Missouri. 18 of this organic mixture was added to the reaction buffer, mixed vigorously and centrifuged at 14,000 X g for 30 seconds (Model 235-B, Fisher Scientific Co., Ottawa, Ontario). The aqueous (upper) phase was removed and transferred to a st e r i l e tube. The organic phase was "back extracted" twice with an equal volume of sterile 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 Filtration 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 precipitation with ethanol as described below. i i i ) Concentration of the Nucleic Acids by Ethanol Precipitation Polyadenylated RNA was concentrated by the addition of sodium acetate, pH 5.0, to a final concentration of 0.3 M, and 2 volumes of 99% ethanol. The sample was placed at either -20°C overnight or at -70°C for 1 hour. Subsequently, the precipitate was collected by centrifugation at 14,000 X g for 10 minutes. The pellet was dried and resuspended in sterile d i s t i l l e d 19 water (50). iv) Selection of Poly (A+) RNA Polyadenylated RNA was purified 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 ice. Sodium acetate, pH 7.0, and SDS were added to a final concentration of 0.4 M and 0.1%, respectively. The sample was then applied five 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 this 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 radioactivity were pooled, ethanol-precipitated overnight, resuspended in sterile d i s t i l l e d water, and stored at -70°C until needed. b) Elimination of the Gel Filtration 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 radiolabelling E. coli tRNA and phenol:chloroform:isoamyl alcohol extractions were performed as described previously. Following this, 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) Stabilitv Study The internal standard was synthesized and purified as described above. The absorbance260 and the amount of radioactivity present was determined (Lambda 3B UV/VIS Spectrophotometer, Perkin Elmer Co., Norwalk, Connecticut; Beckman LS-9000 Liquid Scintillation Counter, Beckman Instruments, Fullerton, California). The sample was then aliquoted and stored at -20°C. At specific time intervals, samples were thawed and then applied to a Bio-Spin 6 Column" (Bio-rad, Richmond, California). The absorbance260 and the radioactivity 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 sterile TE buffer, pH 8.0. The radioactivity 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 Purification of the Plasmid After harvesting the E. coli containing the plasmid, the bacterial pellet 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 buffer). This mixture was centrifuged at 3,000 X g for ten minutes. The supernatant was discarded and the pellet 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 solution, pH 4.8, was added and the mixture was placed on ice for 10 minutes. The mixture was centrifuged at 10,000 X g (Beckman J2-21, Beckman Instruments, Fullerton, California) for 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 precipitate was collected by centrifugation at 12,000 X g for 30 minutes at room temperature. The DNA pellet was washed with 70% ethanol, dried briefly in a vacuum dessicator and resuspended in 11 mL of TE buffer. To this suspension, 11 grams of CsCl and 0.006 grams of ethidium bromide was added. This mixture was protected from light 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 for 10 minutes. The supernatant was centrifuged at 140,000 X g for 42 hours at 20 °C. The lower band, (visualized by ultraviolet light) consisting of closed, circular plasmid DNA was collected through an 18 gauge needle into a st e r i l e tube. The ethidium bromide was removed by extraction (4 times) with isoamyl alcohol. The aqueous phase (containing plasmid DNA) was diluted with 2 volumes of sterile 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 for 30 minutes at 4°C (52). The pellet was dried, resuspended in sterile d i s t i l l e d H20 and the absorbance at 260 nm. was determined. (A map of the plasmid is 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. Briefly, ultrapure agarose (1%) (Bethesda Research Laboratories, Gaithersberg, Maryland) was melted in a 0.04 M Tris-acetate, 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 sterile d i s t i l l e d H20 and applied to the gel. 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 ultraviolet 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 dialysis tubing. The tubing was f i l l e d , with the least volume possible, 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 slice by the application of 0.02 amps of current for 10 minutes. The polarity of the current was then reversed for 10 seconds to release the DNA from the wall of the dialysis tubing. The buffer surrounding the gel slice in the dialysis tubing was removed and the DNA was purified 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, dried, resuspended in sterile 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 oligo-labelled using the Klenow fragment of DNA Polymerase I. A brief 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 to a mixture which contained 20 ug BSA, 50 uCi 32P-dATP, (specific activity was approximately 3000 Ci/mmole) (New England Nuclear, Lachine, Quebec) 2.5 units of enzyme (Pharmacia, Dorval, Quebec) 0.01 mL of Klenow labelling buffer (described below) and sufficient sterile d i s t i l l e d water for a final volume of 0.05 mL. The Klenow labelling buffer was a combination of three stock solutions (A,B,C) that were mixed in a ratio 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 buffer, 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 is essentially 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 buffer, 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 final concentrations, respectively. 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 microliters of a sterile loading buffer (50% glycerol, 1 mM EDTA. 0.4% bromophenol blue, 0.4% xylene cyanol) was added and the samples were loaded onto the gel. 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 Tr i s , pH 7.5, for 45 minutes. Before transfer, 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 nitrocellulose membrane (Bio-rad, Richmond, California), cut slightly smaller than the gel and wettened in 10 X SSC, was placed on top of the gel. F i l t e r papers, slightly smaller than the nitrocellulose, were placed on top of the nitrocellulose. An 8 cm stack of paper towels, cut slightly 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 transfer, the nitrocellulose membrane was air-dried then baked for 4 hours at 80°C (55). i i - Hybridization of cDNA to mRNA The nitrocellulose membrane was placed in heat-seal able plastic bags (Dazey Appliances, Industrial Airport, 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 is 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 Airport, 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 nitrocellulose f i l t e r s , moistened in 20 X SSC, using a Bio-DotR microfiltration apparatus, (Biorad, Richmond, California 94804) then washed under vacuum with 1 M ammonium acetate. The membrane was baked at 80°C for 2 hours. These f i l t e r s were then hybridized with different 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 for hybridization is 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 liquid 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 vials were incubated at 55°C for 3 hours (56). Following this, 10 mL of scintillant (Biofluor, 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 radioactivity 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 for Factor II. 27 3. TISSUE DNA QUANTITATION a) Method DNA was determined by a fluorometric method described by Labarca et al (57). In summary, the method is as follows. Approximately 1 gram of porcine liver 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 citrate, pH 7.0, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol). The homogenate was then sonicated (Sonifier Cell Disruptor 350, Branson Ultrasonics, Scarboro, Ontario) for 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 for mRNA extraction. From this aliquot, 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 for 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., Silver Springs, Maryland). The DNA was quantitated from a standard curve constructed from calf thymus DNA in phosphate-saline buffer. By using this method, i t was found that the amount of DNA per gram of wet liver tissue was close to that reported in the 1 iterature (57). b) Correlation of DNA to Tissue Protein Content Because this assay was to serve as a reflection of the amount of tissue that was originally available, a study was undertaken to determine i f the 28 amount of DNA correlated with the protein content of the tissue. 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 is 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 tartrate was added and mixed. One hundred microliters 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 for 30 minutes at room temperature. The absorbance at 500 nm was then determined spectrophotometrically. This method is 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 this buffer is not the same as that described in the original DNA method, a study was conducted to ensure that this 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 Purification 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 is as follows. The tissue was homogenized in 15 mL of guanidine thiocyanate buffer (4.0 M guanidine thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol). 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 final mixture was placed on ice for 10 minutes then centrifuged at 10,000 X g for 20 minutes at 4°C. Following centrifugation, the aqueous phase was removed and re-extracted as described above. The aqueous phase was then placed in a sterile tube, mixed with 10 ml of isopropanol and placed at -20°C for 1 hour to precipitate the nucleic acids. The precipitate was collected by centrifugation at 10,000 X g for 20 minutes at 4°C. The supernatant was discarded and the pellet 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 precipitate was collected as described above and the procedure was repeated two additional times. The pellet was washed with 70% ethanol, dried and resuspended in sterile d i s t i l l e d water. An aliquot was removed, the amount of radioactivity and the absorbance at 260 nm and 280 nm was determined. The 30 ratio 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 affinity chromatography through an oligo-dT cellulose 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 units. Following elution, an aliquot of each fraction was removed, and the radioactivity 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 sterile d i s t i l l e d water then applied in three separate samples to a nitrocellulose membrane, previously moistened in 20 X SSC, using a Biodot Microfiltration" apparatus. Control mRNA, previously purified from a 20 kg pig, 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 this, 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 solubilized as described (page 27). The amount of radioactivity present in the samples and controls was quantitated as described (page 27). The amount of radioactivity 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 relative 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), liberating p-nitroanilide. The amount of p-nitroanilide generated was measured spectrophotometrically. A brief 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 Tris buffer, pH 8.4 was added. The mixture was incubated at room temperature for 10 minutes on a shaker (Eberback Corporation, Ann Arbor, Michigan) at a speed of 148 oscillations 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 solution. 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 serial dilutions 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 acid. 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 liquid heparin preparation similar to that used during the operative procedure (HepaleanR 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 solution. 2) an investigation of the effect of heparin contained in the blood collection tubes on plasma levels of Factor II. Heparinized and citrated plasma were collected from a normal pig by venipuncture. Serial dilutions of the citrated and heparinized plasma were assayed for 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) is mixed with a 1:1 plasma to diluent mixture. The diluent is that which is supplied by the manufacturer and the plasma is that of the animal being studied. The dye is reconstituted to a final 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 line after injection of the dye. At time 0, a bolus of the dye (1 mg/kg) was infused through the venous li n e . The line 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 arterial line at 5, 10, 15, 20, 30, 40, 50, and 60 minutes. After drawing each sample the line was flushed with 2 mL of heparinized saline. 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 Scientific 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 calculation of the rate disappearance constants was as follows: a) K, is defined as the slope of the line of log of absorbance at 800 36 nm versus minutes ( up to 20 minutes) and; b) K2 is defined as the slope of the line of log of absorbance at 800 nm versus minutes, from 30 to 60 minutes. B: Dye Stability 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 its final concentration ranged from 5.0 mg/L to 20.0 mg/L. The plasma was then analyzed for potassium, glucose, lactate, AST, total bilirubin and Factor II. The results were compared to an aliquot which contained no dye. 37 PART V - ROUTINE PLASMA MEASUREMENTS All routine chemistries, unless otherwise indicated, were performed using the Kodak Ektachem 700 Clinical Analyzer, (Eastman Kodak, Rochester, New York) with reagents supplied by the manufacturer. A brief 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 is transferred to alpha-ketoglutarate in the presence of sodium pyridoxal-5-phosphate and AST to produce oxaloacetate and glutamate. The oxaloacetate formed is converted to malate by malate dehydrogenase with the concomitant oxidation of NADH to NAD*. The oxidation step is monitored by reflectance spectroscopy at 340 nm (60). B: Glucose Glucose was measured according to the method described by Curme et al (61). Glucose is oxidized to hydrogen peroxide by glucose oxidase (EC 1.1.3.4). Peroxidase (EC 1.11.1.7) catalyzes the oxidation of 4-amino-antipyrine and 1,7-dihydroxy-naphthalene by hydrogen peroxide, to produce a chromogen. The intensity of the chromogen formed is proportional to the amount of glucose in the sample and is monitored by reflected light at 540 nm. The chromogen system employed was f i r s t described by Trinder (62). C: Total Bilirubin Total b i l i r u b i n , including unconjugated, mono- and di-conjugated, and 38 albumin-bound delta bilirubin (63) was determined by a modification of the method described by Routh (64). The bilirubin fractions are dissociated from albumin and solubilized by dyphylline and surfactant. These then were reacted with a diazonium salt to produce an azobilirubin chromaphore which was measured at 540 nm. D: Potassium Potassium was determined potentiometrically using ion-selective electrode slides. The slide consists of a K+ selective membrane of valinomycin and a silver-silver chloride layer, serving as the reference electrode. These were connected via a KC1 salt bridge. When the sample and reference f l u i d were applied to their respective layers a pair of electrochemical half-cells was created. The potassium activity was determined from the potentiometric difference measured between the two half cells and related to the Nernst equation. E: Lactate Lactate was measured using the Dupont Automatic Clinical 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 is directly proportional to the lactate concentration and is measured spectrophotometrically at 340 nm. This method is a modification of that described by Marbach (65). 39 PART VI - STATISTICAL ANALYSIS All statistical analyses were carried out using the ABSTAT statistical program (Anderson-Bell Company, USA). The differences between the means were compared using the Student t Test for paired data. Differences were considered significant at p < 0.05, where p represents the probability for two-tailed tests. 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 irregularities, as no direct source of bleeding was identified. The death was, therefore, thought to be due to disseminated intravascular coagulation, but this was not investigated further. The other animal never regained consciousness following 90 minutes ischemia and 4 hours of reperfusion (Day 4 operative protocol). 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 is given in Figure 3. The leading peak corresponds to the nucleotides incorporated into the E. coli tRNA and the tra i l i n g peak is the unincorporated 3H-ATP. Once the internal standard was synthesized, i t was further purified by oligo-dT-cellulose chromatography. Representative profiles of the elution of the internal standard, human and porcine poly (A+) RNA from an oligo-dT-41 SEPHADEX—G50 COLUMN 2.0 - i 0 2 4 6 8 VOLUME (mL) Figure 3. Graph showing a representative chroiaatogram of a Sephadex-G50 gel filtration column. The leading peak contains the incorporated nucleotides and the trailing peak contains the unincorporated nucleotides. cellulose column is shown in Figure 4. The elution profile of the internal standard corresponds directly to that of human and porcine poly (A+) RNA. The recovery of the internal standard during the isolation of mRNA from liv e r and following i t s purification by oligo-dT-cellulose chromatography is 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-dT-cellulose 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), all appreciable amounts of unincorporated nucleotides (trailing 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 precipitations. c) Stability Study A time sequence of the recovery of the internal standard following storage at -20°C is shown in Figure 6. The recovery of the internal standard from a Bio-SpinR 6 column was constant for a period of 9 days after which any absorbance260 in the eluant was undetectable. However, there was s t i l l detectable amounts of radioactivity present in the eluant. The column used on Day 12 was further eluted with 4 column volumes of sterile TE buffer, pH 8.0, and the resulting profile in Figure 7 was obtained. The nucleic acids 43 Eution Profile of Oligo-dT-Cellulose Column Volume (mL) Figure 4. Representative elution profile of the internal standard HB—Wk~ human liv e r mRNA, - O — e — and porcine l i v e r mRNA r^fi f r o m a n oligo-dT-cellulose column. Table 1 Extraction Efficiencies of the Internal Standard Extraction Efficiency (%) a Extraction efficiency of mRNA from l i v e r tissue 26.2%+15.6% (41) Recovery following oligo-dT-cellulose chromatography 57.9%+24.4% (41) a data presented as the mean + 1 S.D. with number of determinations shown in parenthesis 45 Sephadex 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 filtration 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 is measured by the absorbance26fj '—13 fsJ- a n {* t n e a m o u n t °f radioactivity present-O Q-. 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-6R column. The recovery of the internal standard is measured by the absorbance25o M M - and the amount of radioactivity that is present • l_l . eluted from the column in a broad peak after one column volume. In contrast, the radioactivity never ful l y eluted from the column and did not correspond to the profile 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 is 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 film pictured below, (see Plate 2) i t is evident that human cDNA for Factor II does cross-hybridize to a single, discrete band of porcine mRNA. The mRNA was electrophoresed under denaturing conditions, transferred to nitrocellulose, 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 is 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 gel. 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 electrophoresis, Northern transfer to nitrocellulose, and hybridization to the radiolabeled cDNA probe for Factor II. The f i l t e r was exposed, at -70°C, for 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 linearity i f the binding of the cDNA probe to increasing concentrations of porcine mRNA. The binding is linear 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 levels in porcine l i v e r tissue is given in Figure 10. It is clear that a good correlation (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) is 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 guanidine-thiocyanate buffer added, is shown in Figure 12. The correlation is good (r = 0.999); however, a positive y-intercept is 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 is 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 vs DNA 4 -Z3 -3 -2-B -2 -1J* - ^£ Slope - 0.023 . 1 - Reg.Coeff. = 0.997 OJJ -0 -Y int. = -0 .52 O 20 40 TO BO 100 120 140 1BO 1BO Protein (ug/tub«) Figure 10. Graph of the correlation between porcine liver DNA content and protein content. Each point represents the mean of triplicates. Wet Weight vs DNA DNA: Buffer 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 HI 5 (black), Pig 31 -fr fr- (red), Pig 33 — + + (blue), Pig 36 Ac- A (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 rise again in comparison to i t s pre-operative 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 or 0.011 mg/mL of ecarin. The slope of the curve using 0.111 mg/mL of ecarin is 15.8 and that of the curve using 0.0111 mg/mL is 9.1. As the assays were not performed simultaneously, the absorbances of the 75% standard at each time interval and at the two differing ecarin concentrations have been converted to % concentration, by use of a standard curve run concomitantly, to allow direct comparison between them. B: Determination of the Substrate Reaction Time A graph of absorbance40S (product formed) versus substrate reaction time of undiluted porcine plasma and 1/10 diluted plasma is 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 linear. At the 4 minute mark, the undiluted plasma has reached an absorbance405 (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 linearity. 59 Concentration of Activator 330 —t 1  ISO -100 -80 -o -] j j j j j 1 1 1 r 1 1 r 0 4- 8 12 10 20 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. Factor 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 for Factor II Table 2 describes the recovery of Factor II activity following the addition of increasing levels of the liquid heparin preparation similar to that used during the operative procedure. It is apparent that the liquid heparin did not interfere with the assay at a level of less than 2 USP units/mL. In addition, blood collected from a normal pig into heparinized and citrated tubes, serially diluted, assayed for Factor II and regressed against each other, exhibited a regression slope of essentially 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 significantly (p < 0.01) following 90 minutes of ischemia in relation to pre-operative values. These values remained low for the duration of the experimental protocol (two days) and never recovered to pre-operative levels. PART IV - ICG CLEARANCE STUDIES A: Dve Stability 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 Heparin Hepalean" USP Units/mL 0.75 2.0 3.0 4.0 Table 2 Interference Study (II) Factor II % Recovery 97% 100% 87% 87.6% 63 H e p a r i n Inter ference S t u d y H s p a rinizad Plasma (Absorbancs 403 nm) Figure 16. A graph of the correlation between a standard curve of Factor II collected in citrated tube6 and a standard curve of Factor II collected in heparinized tubes. Plasma Factor II Levels 120 - , c i — i — i — i i 1 1 1 i 1 0 3 0 6 0 9 0 0 3 0 6 0 9 0 1 2 pre—op ischemia reperfusion 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 < ° - 0 1 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 II, glucose, K+, AST or lactate (Figures 19. 20. 21, 22, 23). However, with increasing concentrations of dye, there is a positive interference with the method for total bilirubin (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 pig. There is an 82% decrease between the pre-operative K, clearance slope and the clearance slope during the ischemic interval. This decrease is significant to a level of p < 0.0001. During reperfusion, both the K, and the K2 clearance slopes are decreased. These decreases are significant to levels of p < 0.0001 and p < 0.0020 respectively. A representative curve of the clearance of ICG from porcine plasma is given in Figure 25. The curve suggests that ICG clears from the plasma in two phases. The f i r s t phase is an exponential decay in which the majority of the dye is cleared within the f i r s t twenty minutes. The second phase is much slower and dye is s t i l l detected in the plasma at 90 minutes. 66 1.27 -1.26 r-1.25 DAYS -~r— 10 12 -J— H Figure 18. A graph of tlie stability 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 0.25 DYE IHTERFERENCE: FACTOR II 0.24 -0.23 -0.22 -U 0.2185-0.21 -0.2 -0.19 -0.18 -0.17 IJ 0.1975-I 1 1 1 1 1 1 1 1 1 1 1 4 8 12 16 20 2 4 ICC (mg/L) Figure 19. A graph of the interference with ICG i n the method for Factor II. The dashed line indicates the mean of the sample containing no ICG + 2 S.D. (solid l i n e s ) . — 4.34 DYE INTERFERENCE : GLUCOSE -I \ o E E LJ V) o o D _l o 4.33 -4.32 -4.31 -4.3051)— 4.3 - -4.2951 >— 4.29 -4.28 -4.27 -4.26 4 8 1 12 ICG (mg/L) ~r-16 T " 20 —r-24 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). 4.7 DYE INTERFERENCE :K+ o \ 0 E E + 4.65 -4.6141Y 4.6 -4.55 -4.4 4 .386* 4.35 -4.3 4.5 • — 4.45 -0.00 4.00 8.00 1 12.00 ICG (mg/L) I 16.00 1 20.00 1 I 24.00 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.56IJ-16 -6 -5.35 IH i 8 1 12 ICG (mg/L) 16 20 24 Figure 22. A graph of the interference with ICG in 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- 7 111.692-1.6 -1.5 b -1.4 -1.3 J) 1.308-1.2 -1.1 -1 I I I I I I 1 —I 1 1 [ 4 8 12 16 20 24 ICG (mg/L) Figure 23. A graph of the interference with ICG in the method for lactate. The dashed line indicates the mean of the sample containing no ICG + 2 S.D. (solid lines). ~~ -I \ 0 E 3 v • 2 m D tr _j m _ j o 14 DYE INTERFERENCE : TOTAL BILIRUBIN 13 -12 -11 -10 -9 -7.6?6 7 H 5 -4.3241V 3 -2 -1 -0 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 for tota l b i l i r u b i n . The dashed line indicates the mean of the sample containing no ICG + 2 S.D. (solid l i n e s ) . Table 3 Mean ICG Clearance Constants from Porcine Plasma Time K, K 2 pre-op .0419 .0112 ischemia .008 • reperfusion .009 • .0046T 1 day post-op .0414 .0123 2 days post-op .0439 .0103 n = 7 - p<0.0001 Tp<0.0020 from pre-operative values 74 ICG C l e a r a n c e Curve 7 -i — 6 H 5 H 4H 3H 2 i H • • i i i i 1 0 20 40 60 Tim* (minutes) Figure 25. A representative curve of the clearance of ICG from porcine pla PART V - ROUTINE PLASMA MEASUREMENTS Aspartate aminotransferase (AST) activity in plasma increased significantly (p < 0.05) from pre-operative values at 5, 15, and 30 minutes of reperfusion (Table 4 and Figure 26). The plasma glucose increased significantly 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 significantly 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 significantly 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, bilirubin 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) Glucose (mmol/L) Pre-op 62±8 4.9±.025 Ischemia 30 min. 45+5 8.2+1.37-60 min. 45+5 7.0+0.98 90 min. 50±9 6.0±0.67 Reperfusion 5 min. 113+20- 10.0+1.82-15 min. 130+31- 9.6+1.70 • 30 min. 163+48- 9.2+1.61 • 60 min. 217+81 8.1+1.18 • 90 min. 241+105 7.6±1.14 1 Day 482+259 5.0+0.26 2 Days 174+85 4.8±0.32 n = 7 x ± 1 S.E.M. ip < 0.05 from the pre-operative values 77 Plasma AST Levels 8OO-1 7 0 0 -6 0 0 -O h 1 i 1 1 1 i 1 1 1 i 1 0 3 0 60 90 0 3 0 60 9 0 1 2 pre—op ischemia reperfusion days post—op F i g u r e 26. Time sequence of 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 the mean o f 7 animals +_ 1 S . E . M . f p < 0 .05 Plasma Glucose Levels 15- , E E i o o o O 5- X i 30 —i 1 r 60 90 0 3 0 6 0 -~i r 9 0 1 pre—op -ischemia reperfusion days post-op Figure 27. Time sequence of plasma glucose levels following ischemic/reperfusion injury in porcine l iver. 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) Lactate (mmol/L) Pre-op 3.58+.08 1.7±0.45 Ischemia 30 min. 3.27+0.13 3.5+0.64 T 60 min. 3.33+0.14 3.6+0.52T 90 min. 3.41±0.14 4.0±0.62T Reperfusion 5 min. 3.41+0.18 4.8+1.1 T 15 min. 2.80+0.18- 3.8+0.85 T 30 min. 2.98+0.18- 3.4+0.72 T 60 min. 3.20+0.15- 2.7+0.53 T 90 min. 3.65+0.12 2.2±0.54 1 Day 3.62+0.05 1.3+0.49 2 Days 3.57±0.08 1.8+0.48 n = 7 x ± 1 S.E.M. TP<0.02 -p<0.05 from the pre-operative values 80 Plasma Potassium Levels o £ E O CL 5n - 2H 00 to o 1-30 i 60 90 0 30 ~T~ 60 ~ i r 90 1 1 2 pre~op ischemia reperfusion days post—op Figure 28. Time sequence of plasma K* levels following ischemic/reperfusion injury in porcine l iver. Each point represents the mean of 7 animals + 1 S.E.M. TP < 0.05 Plasma Lactate Levels 6-1 u-t 1 i 1 1 1 i 1 1 1 i 1 0 30 60 90 0 30 60 90 1 2 pre-op ischemia reperfusion days post—op Figure 29. Time sequence of plasma lactate levels following ischemic/reperfusion injury in 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 collection time sequence used in the model of warm hepatic ischemic/reperfusion injury made possible the study of the injury process at specific 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 directly to each animal's pre-operative values. In this respect, each animal served as its own control. The survival rate of pigs subjected to various ischemic periods is 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 their 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, their surgical model employed a porto-jugular shunt, and clamping of the hepatic and gastroduodenal arteries and other collaterals. 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 relatively qualitative procedure. For the purposes of this project a quantitative procedure for the detection of Factor II mRNA was required. To meet this requirement, preliminary investigations were performed on the existing qualitative methods. We then modified the qualitative 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 is frequently monitored by quantitating mRNA levels coding for the specific protein of interest (69,70). This can either be achieved by in vitro translation systems or, more frequently, by direct hybridization of the mRNA to it s corresponding cDNA (or RNA) probe. The assumption made in these assay systems is that recovery of the mRNA from the starting material is 100% (69,71). However, due to the large amount of sample manipulation during the extraction and purification of the mRNA from tissue prior 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 their laboratory (73). Other investigators have circumvented the issue of recovery by expressing the amount of mRNA detected relative to the amount of total RNA or to the amount of other species of mRNA in the sample (70,72). All of these techniques have proved to be satisfactory. However, the amount of mRNA detected cannot be related back to the original amount of starting material, either tissues or cell suspensions. In addition, these methods have several problems. One problem in expressing the amount of mRNA detected to the amount of total RNA is 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 is measuring the total amount of RNA by the absorbance260, the type of nucleic acids present in the sample will depend on the tissue homogenization method used as the Polytron tissue homogenizer is fitted 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 relative 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 result 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 is a chemical compound added in known amounts to a sample and carried through all steps of the analytical procedure. In that 85 the internal standard is similar chemically and structurally to the analyte of interest, i t s recovery will be comparable to that of the analyte and, therefore, i t can be used to monitor analyte extraction efficiencies. In order to accurately quantitate liver mRNA levels, an internal standard was developed in our laboratory and was used to normalize mRNA recovery for the differing efficiencies in extracting and purifying i t from l i v e r . The synthesized internal standard behaved both chemically and physically like eukaryotic mRNA in our test system, ie: 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 32P and the internal standard is t r i t i a t e d , i t can be readily differentiated from a cDNA probe by employing a dual-label counting technique (74). This technique is less complicated than reprobing the sample with a different cDNA probe. Due to the inherent specificity 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 is faster and simpler than cloning an RNA control as described by Toscani et al (73). It uti l i z e s equipment found in most basic research laboratories. In addition, the recovery of the mRNA could be monitored at all stages of the analytical procedure and not only at the final hybridization step (72,73). This internal standard could be utilized in both the "dot-blot" (70) or 86 "cytodot" (75) hybridization assays. In the Northern blot assay (76) i t is 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. coli RNA to a sample augments the recovery of mRNA as i t acts as a carrier during the precipitation 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 (tritium), 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 stability 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 absorbance260 and DPM in the degradation study (Figure 6). does not appear to be in agreement, i t must be noted that the detection of radioactivity is 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 radioactivity, this suggests that there was inclusion of the nucleic acids by the gel. This could be a result of the loss of the individual adenine nucleotides from the t a i l or from endonuclease or exonuclease activity on the entire length of the poly (A) tailed nucleic 87 acid. Of interest is the loss of the radioactive label from the nucleic acids. In that the recovery of the nucleic acids subsequent to washing the column is almost complete, i t can be concluded that the tritium label has been lost from the adenine. In addition, the evidence indicates that i t has actually bound to the column. The l a b i l i t y of tritium is well established (78,79). 3H -labelled compounds undergo degradation by a free exchange of the tritium label with hydrogen in the aqueous solutions in which they are contained. This loss is accelerated at higher temperatures (78,79). Other forms of radioactive ATP that may have been used to overcome this problem with tritium in the synthesis of the internal standard include: 35S, 14C, and 1 2 5I . All of these isotopes are beta-emitters and their quantitation is achieved by liquid s c i n t i l l a t i o n counting (78). Both 3SS and 1 4C-labelled ATP are available commercially; however, these isotopes share overlapping energy peaks with 32P and, therefore, would interfere with the simultaneous detection of the 32P-labelled cDNA probe. 12SI has two energy peaks. One peak shares an overlapping energy window with 32P and the other peak has a distinct window (79). A dual-label counting technique could be employed using the isotopes 125I and 32P i f the counts were corrected for the overlapping energy peaks of the two isotopes (74). Unfortunately, to our knowledge, the only commercially available nucleotide that is labelled with radioactive iodine is 5-12Siodo-2'-deoxycytidine-5'-triphosphate. Due to the nature of the internal standard (E . coli tRNA), i t is 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, is limited to tissues and cells which do not 88 contain endogenous mRNA that will cross-hybridize to the probe for 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 purification efficiencies of mRNA from liver tissue. The results (Table 1) indicate a low recovery (26.2%) in the isolation 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 is attempting to quantitate mRNA. 2. cDNA PROBE STUDIES Once i t was determined that the human cDNA probe for Factor II did cross-hybridize to porcine Factor II mRNA, the binding saturation and linearity 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 linearity of the probe. Linearity was seen from the ranges of 0.1 ug to 1.0 ug of mRNA. Papavasiliou et al achieved linearity with their cDNA probes to alpha and beta luteinizing hormone to 3 and 8 ug of RNA, respectively, using saturating concentrations of each probe that they had previously determined (80). Schwarzenberg et al achieved linearity with their genomic probe for alpha-l-antitrypsin to 1.0 ug of RNA (81). Saturation studies of this probe were not reported. 89 3. TISSUE DNA QUANTITATION The quantitation of tissue DNA was introduced as a means of determining the original amount of tissue used in the extraction and purification of mRNA. We f e l t that this would be more accurate than expressing the mRNA relative to protein content or wet weight. Protein content is traditionally measured by the method according to Lowry et al (58). This method is subject to non-specificity due to many interfering substances such as bile acids and SDS (82). Both of these substances would be present in the samples studied. In this instance, wet weight is inappropriate as the tissue is subject to varying amounts of edema due to the injury inflicted 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 slight interference from the guanidine-thiocyanate buffer as witnessed by the positive Y intercept in Figure 14. Subsequent to this study, 0.020 mL of guanidine-thiocyanate buffer was added to each standard. This assay proved to be simple to perform and its introduction was effective as a more accurate means of assessing the original amount of tissue used. A quantitative method for the detection of Factor II mRNA levels 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 its 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 specific activity 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 efficiency during the isolation and purification of mRNA by the use of a synthesized internal standard that was developed. This result was then expressed relative to the amount of DNA in the starting tissue. This procedure was found to have an inter-assay coefficient of variation of 32%. This is 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 nitrocellulose 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 is one of the major causes of peri-operative mortality in li v e r transplant recipients (83). Although the recipient is already in a compromised hemostatic state prior to the transplant, a reperfusion coagulopathy or disseminated intravascular coagulation is one of the usual precipitating events that results in mortality (84). Reperfusion coagulopathy has been reported to be caused by the release of tissue thromboplastin from the stored, damaged graft, or from the release of sequestered heparin from the graft (83,84). In addition, even though a coagulopathy is not evident in the liver transplant recipient, a decrease in all plasma protein clotting factors from those of pre-operative levels is seen (40). Specifically, Factor II has been shown to decrease to 78% of its pre-operative value on the induction of anesthesia. It then further drops to 91 36% of it s pre-operative level after 70 minutes of reperfusion (40). This decrease in Factor II levels may be due, in part, to the decreased ab 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 is duplicated in the model of warm hepatic ischemic reperfusion injury in the porcine liver investigated in this thesis. The level of plasma Factor II decreased at 30 minutes of ischemia to approximately 58% of its 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 utilization of the factor as there was no apparent bleeding and the animal was full y heparinized. One possible explanation is the sequestration of the factor in organs such as the spleen or l i v e r . Although an adequate explanation of its 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 rising levels of mRNA which specifically codes for Factor II. This stimulus may have been provided in the fashion of a recently described humoral factor, coaguloprotein II, 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 earlier part of reperfusion, do not result in the production of any significant increase in the amount of Factor II in the plasma. It may be that the time frame (approximately 90 minutes) is too short to allow for the production, post-translational modifications and release of the protein into the systemic circulation. On further reperfusion, a further trend is noted in which the levels 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 vitro and in vivo, i t is conceivable that they might act in a similar manner on mRNA. Although mRNA strand breakage may be effected, i f the oxy-radicals have the ab i l i t y to damage the poly (A+) t a i l , this damage alone would not only affect the st a b i l i t y of the mRNA, but also its involvement in the actual role of protein synthesis (86,87). Although maximal oxygen radical production would be at the ini 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 significant injury occurs (88,89). As the l i v e r contains more copper/zinc superoxide dismutase and glutathione than any other tissue, and i t is one of the richest sources of catalase and alpha-tocopherol, i t is 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 their model of hepatic ischemia in rats, 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 direct result of lowered levels of translatable albumin mRNA (37). They hypothesized that because albumin is a protein which functions outside of the c e l l , protein synthesizing energies were directed at proteins which function inside the cell probably to repair the damage infli c t e d during the ischemic interval. Although prothrombin is also a protein which functions outside of the c e l l , lowered plasma levels acted as a stimulus to produce increased amounts of its mRNA. During reperfusion, however, this increased Factor II mRNA was obliterated, perhaps as a result of damage infl i c t e d during reperfusion such as by toxic oxygen radicals. 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 rise again at one and two days of reperfusion. This could be a result of the continued stimulus from the still-depressed levels of plasma Factor II. It is interesting to note that with this particular animal, its plasma Factor II level increased to 91% of it 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 purification of mRNA from the l i v e r biopsy. The advent of recombinant DNA technology and the availability 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 fields of c l i n i c a l research including cardiovascular diseases, diabetes, hematology and endocrinology (90,91,72,92). The technology is 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 injury. The results indicate that recombinant DNA technology can be successfully applied to the f i e l d of cl 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 is hindered only by the lengthy and involved nature of the present technology. This is particularly 94 relevant in the case of cli n i c a l transplantation where the amount of time required and the amount of sample needed to perform these techniques prohibit their use. In time, this problem will be solved. PART III - QUANTITATION OF PLASMA FACTOR II IN PORCINE PLASMA Methods for the determination of Factor II in plasma include clotting assays, enzymatic assays and ELISA (enzyme-linked immunosorbant assay) techniques (93,94,95). Because Factor II is a zymogen, we chose to quantitate i t using an enzymatic assay. With the recent development of synthetic chromogenic peptide substrates, assays for individual coagulation factors have been devised that do not suffer from the same degree of non-specificity that clotting assays do. The two synthetic substrates available for the determination of prothrombin are S-22385 (Kabi Vitrum Ltd., Stockholm, Sweden) and Chromozym Th6. 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 is activated by the Xa, Ca++, Platelet Factor 3, Va, VIIIa 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-p-nitroanilide-HCl 6 tosyl-L-glycyl-L-prolyl-L-arginyl-p-nitroanilide-HCl 95 this assay was used to correlate plasma Factor II levels and tissue mRNA levels that code for Factor II, reflecting the protein synthesizing abil i t y of the organ, we wanted i t to be independent of cellular processes that are post-translational modifications. To make this assay independent of the Vitamin K status and gamma-carboxylating processes, a non-physiological activator, Ecarin, was used. Ecarin is one procoagulant found in the venom of the Echis carinatus snake (96). Its action is described to be independent of the calcium ion concentration, platelet factor 3 levels, and therapeutic levels of heparin (97). In addition, the ecarin-thrombin complex is not inhibited by antithrombin and has a higher activity in cleaving synthetic substrates than does the physiological thrombin (97). Other non-physiological 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 directly (98). For this reason, Chromozym Th was used. Other investigators have used this combination of activator and substrate (98). However, their buffer system included NaCl, Tris-HCl and imidazole. We found that, in such a buffer system, the substrate precipitated. When the ionic strength was reduced, by using 0.075 M Tris buffer, pH 8.4, this precipitation was prevented. In addition, the pH of the buffer was increased to 8.4, as this is the reported pH maxima for Ecarin (99). Since imidazole is used primarily to keep calcium ions in solution, its elimination had no detrimental effects on the assay (100). In order to optimize this assay, i t was necessary to investigate the 96 concentration of the activator, the reaction time with the substrate and the effect 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 sensitivity, 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 linear relationship between product formation and time. The material of choice for assay of coagulation proteins is citrated plasma, as the heparin in heparinized plasma interferes with most other assay techniques. Although the action of the activator is 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 collection tubes did not interfere. We confirmed the findings of other investigators that therapeutic levels of heparin (HepaleanR) did not interfere with the assay. Although other investigators have found that levels up to 10 units did not interfere, their unit value was not defined (99). PART IV - INDOCYANINE GREEN CLEARANCE STUDIES A: The Clearance of ICG During Ischemic/Reperfusion In.iury Indocyanine green is a non-toxic, tricarbocyanine dye (101). Once infused into the circulation, i t is cleared exclusively by the hepatocytes and excreted into the bile in an unconjugated form (102). The clearance of ICG from the peripheral circulation has been described as a single exponential decay (102). We, and others, have found that i f the sampling interval is 97 extended past the recommended 20 minutes, a second exponential decay is evident (103). In that there is no extra-hepatic uptake nor enterohepatic recirculation of the dye and that i t is cleared rapidly from the circulation (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 rate, we used the rate disappearance constant (defined as the slope of the log of absorbance versus time) as an index of liver function as i t is considered more reliable (106). This interpretation of liver function assumes constant l i v e r blood flow. In this model this assumption cannot be made. To overcome this d i f f i c u l t y , an attempt was made to monitor and correct all the ICG clearances for hepatic blood flow by the use of indwelling Doppler flow probes. Due to the high rate of failure 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 pre-operative values. This observation is indicative of the blood flow status rather than hepatocellular dysfunction. The residual uptake of the ICG could have occurred from back flow into the liv e r from the hepatic vein, or from very small collaterals to the liv e r that could not be identified 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 significant. The decrease in the rate disappearance constants may be 98 explained by the hepatocellular damage inflicted 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 inability 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 post-operatively, the rate disappearance constants returned to normal. The normalization of the rate disappearance constants may be explained by the liver's large reserve capacity thereby making this test insensitive to ischemic/reperfusion injury or to the fact that the injury inflicted was not severe enough to allow for differences at one and two days post-operatively. B: Assay Optimization for 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 is sensitivity. Due to the dose of ICG used (1 mg/kg), sensitivity was not an issue. Once reconstituted in an aqueous solution, indocyanine green degrades rapidly (102). However, after the dye has been bound to protein i t remains relatively stable (104). In order to increase its s t a b i l i t y , we devised an alternative method of reconstituting the dye by taking advantage of this fact. We found that by reconstituting the dye in a 1:1 plasma to diluent mixture, the dye is stable for at least 10 days. This time frame is more than adequate for the performance and measurement of the ICG in the model used. 99 Other investigators have reconstituted the dye with a solution of 5% sterile human albumin. However, the extent to which the dye was stabilized was never reported (111). Since many of the methods used in this study depend on spectrophotometric analysis of their 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 positive, dose-dependant interference with the method used for total bilirubin due to the presence of indocyanine green. In the method for the determination of plasma bi l i r u b i n , a surfactant (Triton X-100) is used to dissociate bilirubin from albumin (112). It might be expected that this surfactant could also dissociate the ICG from the albumin. The ICG, therefore, is free to enter the spreading and reaction layer of the film cassette. In this assay, a bichromatic reflectance reading is performed at 540 and 420 nm, the latter wavelength being used to correct for any spectral interference. The positive 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 nm when i t is protein bound. It may be that the ICG is actually reacting with the diazonium salt in the reagent layer and producing a colored azo compound that reflects strongly at 540 nm. However, i t may also be that the spectral characteristics 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 bilirubin method has not been reported previously. As a result of this 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 significantly. 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 is 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 circulation on reperfusion. Both Battersby et al and ourselves reported a significant rise in the peripheral glucose level during the ischemic phase (66). They hypothesized that enzymes in the hepatocytes responsible for the uptake of glucose from the peripheral circulation had been damaged to a significant enough degree during ischemia so that glucose uptake was impaired (66). We believe that this 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 , utilizes a large proportion of blood glucose for its own metabolic a c t i v i t i e s . During ischemia, however, circulation to the liv e r is effectively cut off and, as a result, 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 liver was a further indication of damage to hepatocellular glycolytic enzymes resulting in impaired glucose homeostasis. However, i t may be that the li v e r is reacting normally to the stress of the operative procedure and is releasing glucose for the utilization in the peripheral tissue. Harris et al and Battersby et al found a significant increase in the level 101 of 10 in the peripheral circulation and the venous effluent from the l i v e r on reperfusion of the organ (66,67). This increase was rapid and transient. In contrast to both of these investigators, we found a rapid and transient decrease in the levels of 10 on reperfusion of the l i v e r . As both Harris et a l and Battersby et al were investigating liver survival after 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 liver c e l l s . We found significant lactic 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 significant changes in bilirubin 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 is inevitable. However, assessing the amount of injury an organ has sustained prior to transplantation is 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 will function once transplanted. Therefore, the transplantation of a non-functioning graft is a 102 recurring reality 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 their pathological mechanisms must be further investigated. We chose to study the effects of warm hepatic ischemic/reperfusion injury on the protein synthesizing abil i t y of the porcine l i v e r , in particular, the levels 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 injury. 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 particular time period. Although this 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 restricting or stoppnig the blood flow through the liver when reperfusion was thought to be initi 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 initiated during ischemia rather than reperfusion. Although the actual time course of the injury process is not perfectly clear, Factor II mRNA levels have proven to be more sensitive to the effects 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 this included the development of an internal standard, saturation and linearity binding studies on the cDNA probe and the introduction of the quantitation of tissue DNA levels. 2. By using the method established, Factor II mRNA levels were quantitated in porcine liver following 90 minutes of ischemia and 2 days of reperfusion. During the ischemic period, the Factor II mRNA levels increased and this trend was exhibited in four animals. The increase in the levels 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 levels of Factor II. Factor II plasma levels decreased at the start of ischemia and this decrease was found to be significant. 4. Other established parameters of hepatocellular damage studied included routine plasma measurements (AST, lactate, glucose and K+) and indocyanine green clearances. The changes in routine plasma measurements were found to be similar to those of other investigators. The rising levels of AST during 104 reperfusion indicated that hepatocellular damage had occurred, thereby validating the model of ischemic/reperfusion injury. The clearance of indocyanine green was significantly 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 0 - ICG X* (pre-op) Control 0 - - X* Ischemia 0 - ICG (90 min) 30 - - # 60 - - # 90 X - # + Reperfusion 5 # - - -10 * - -15 # - -20 * - -30 # - -40 * - -50 * -60 # - -90 # - - + Reperfusion - - - + (4 hours) Reperfusion - ICG - + (24 hours) Reperfusion - ICG - + (48 hours) HV - hepatic vein RA - internal jugular vein catheter tunnelled to the right side of the heart (right atrium) CA - carotid artery H - histology (routine light and electron) and cDNA/mRNA hybridization studies # - blood drawn for AST, K+, lactate, glucose, total b i l i r u b i n , ICG and Factor II * - blood drawn for ICG only 106 APPENDIX II MAP OF PLASMID PKK2233 AND INSERT a X I O o CM" m- I e CM in 0 0 . II E u m ' 3 > a. e a ID CM H OS w SB 3 S i n 107 REFERENCES 1. Gordon, R.D., Sharo, B.W., Iwatsuki, S., Esquivel, CO., Starzl, T.E. 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