Assessment of Graft Function Before Liver Transplantation

The assessment of a donor liver before transplantation has been the subject of much research, however, the transplant surgeon still has to rely on a subjective interpretation of donor data and the macro- and microscopic appearance of the liver to decide whether to use the graft (1). This decision will only be proved to have been right if the liver is transplanted and the recipient discharged safely from the hospital (2). Over the past few years the increasing demand for donor livers has not been matched by an increase in the number of organ donors. To compensate for the relative shortfall, many donor organs that were previously not considered suitable for transplantation (“marginal” grafts) are now being used in selected circumstances (3). The term marginal is used to refer to grafts from a “high-risk” group of donors based on demographic, clinical, laboratory, and histological data (4). Donor age of more than 70 years or less than 3 months, donor body weight over 100 kg, moderate or severe macrovesicular fat infiltration of the liver, abnormal liver function tests (serum aspartate aminotransferase [AST] >160 IU/L or international normalized ratio >2), serum sodium above 160 mmol/L, donor stay in intensive care of more than 5 days, significant periods of hypotension (<60 mmHg systolic for more than 30 min associated with a rise in serum AST), significant systemic infection, and patchy graft perfusion of the grafts during organ retrieval are parameters considered in our center to define marginal grafts. Donor liver biopsies have always been used to assess graft quality, however, their primary role has been to help estimate the degree of hepatic steatosis. Severe macrovesicular steatosis (more than 60% of hepatocytes) has been associated with increased incidence of primary graft dysfunction. Grafts with mild to moderate fat infiltration (up to 45%), although associated with increased early graft dysfunction (EGD) proportional to the degree of infiltration, usually function satisfactorily (5), but not all studies have shown this (6), reflecting the difficulty of accurately predicting graft viability using these morphological changes, which may have no direct functional significance (7). FUNCTIONAL GRAFT ASSESSMENT It is clear that more reliable predictors of graft function are required. Significant efforts have been made to try to assess donor grafts by evaluating different aspects of liver function, which have included the ability of the liver to synthesize proteins, metabolize drugs, secrete bile, produce high-energy phosphates, and follow the levels of markers of microvascular injury. Each of these aspects will be reviewed. SYNTHETIC FUNCTION Hepatic Protein Synthesis Protein synthesis is impaired early after hepatocyte injury and can be used as a marker of graft function. The hepatic protein synthesis rate can be determined from liver tissue obtained by biopsy and calculated by the uptake of an amino acid (L-[4.5 3H] leucine) by the protein fraction during a 10-min incubation in a test tube. It requires the availability of a liquid scintillation counter and takes approximately 45 min. Low rates of hepatic protein synthesis correlate with poor survival in animal experiments (8). However, this technique may be influenced by the nutritional status of the donor and has not been used in clinical practice. Protein synthesis can also be examined by measuring the ability of the liver to remove amino acids from the plasma per minute. Various methods based on this concept have shown good correlation with early graft function, but again they have not been used to assess human donor livers before transplantation (9, 10). Plasma Lecithin/Cholesterol Acyltransferase Activity This enzyme participates in the esterification of free cholesterol and plays a role in the transportation of cholesterol and in lipoprotein metabolism. A commercially available kit and a spectrophotometer are required to determine plasma lecithin/cholesterol acyltransferase (LCAT) activity. Reduced levels are considered to be due to impaired protein synthesis secondary to hepatic dysfunction. Plasma LCAT activity has been noted to be significantly lower in liver donors than healthy controls (11), however, donors with good graft function had significantly higher levels than those with graft dysfunction (12). The effect of blood transfusions and aggressive fluid replacement in brain-dead donors, possibly reducing plasma concentrations, may interfere with the accuracy of this test. Low levels of LCAT have also been reported in diabetics and smokers (13, 14), which may interfere with the interpretation of results in donors. DRUG METABOLISM Monoethylglycinexylidide Test Lidocaine is metabolized exclusively in the liver, resulting in the formation of monoethylglycinexylidide (MEGX). Metabolism depends on hepatic blood flow and hepatocyte function. The mitochondrial cytochrome P-450 III A4 subsystem has a central role in this function and can be damaged by acute severe hypoxia. The MEGX test is safe, rapid, and easy. It requires the administration of an intravenous bolus injection of lidocaine hydrochloride (1 mg/kg) over 2 min before organ retrieval. Samples of serum are collected immediately before and 15 min after the injection. The presence of MEGX in serum is determined by a fluorescence polarization immunoassay (which takes 20 min). It has a reported prognostic sensitivity of 73% and a specificity of 78% (15). MEGX levels lower than 50 μg/L, 15 min after lidocaine injection, correlate with early graft loss and levels greater than 90 μg/L with good early graft function (16). Other studies, however, have failed to correlate MEGX levels with graft viability and have found it to be of no predictive value (17, 18). A series of grafts with MEGX levels less than 50 μg/L were shown to be associated with good function after transplantation (17). Hemodynamic instability of the donor and the unpredictability of cytochrome P-450 III A4 enzyme activity may be related to these discrepant findings (17, 18). BILE SECRETION Bile Acids Analysis T-tube bile composition was initially used to monitor graft function after transplantation, and measurements of bile flow and bile acid concentrations followed. Canalicular bile secretion, expressed by physiological concepts such as bile acid-dependent bile flow, bile acid-independent bile flow and apparent choleretic activity, were also developed, using T-tube bile analysis (19), however, until recently there were no studies of biliary bile acid secretion in donor livers before transplantation. A new technique for hepatic bile collection in donors, without the interruption of the entero-hepatic circulation, has been developed (20), which may help to analyze parameters of bile secretion in donors and recipients as a tool of assessment of early graft function. The use of serum total bile acids levels in the assessment of posttransplantation early graft function and diagnosis of acute cellular rejection have been shown to be reliable (21, 22). However, serum total bile acids analysis has not been used in donor assessment. ENERGY METABOLISM Adenine Nucleotides and Energy Charge Adenine nucleotide concentrations have been determined from human liver biopsy specimens using high-performance liquid chromatography with an anion exchanger (23, 24). During ischemia, the level of adenosine triphosphate (ATP) decreases markedly, while the level of adenosine diphosphate (ADP) decreases more gradually. The level of adenosine monophosphate (AMP) increases concomitantly but is then degraded to uric acid (23). Therefore, the overall levels of total adenine nucleotides and individual nucleotide concentrations have been shown to decrease during graft preservation and considered as markers of early graft function. However, the ability of the mitochondria to regenerate ATP after reperfusion correlates better with graft viability than the absolute levels of adenine nucleotides (25). Hepatic energy charge is calculated by the formula: Energy charge (EC)=(ATP+0.5 ADP)/(ATP+ADP+AMP). The energy charge falls during cold preservation and recovers after liver reperfusion (25). Hepatic ATP concentrations above 2 nmol per mg protein and an energy charge above 0.3 in donor grafts have correlated with good graft function after transplantation (24). However, other studies showed that absolute pretransplantation levels of ATP, ADP, and total adenine nucleotides did not correlate with early graft function (26). Arterial Ketone Body Ratio The liver is the only organ capable of producing ketone bodies from acetyl coenzyme A derived from fatty acid oxidation. In liver mitochondria, acetoacetate undergoes reduction to 3-hydroxy-butyrate by 3-hydroxybutyrate dehydrogenase, and the equilibrium between these two compounds, expressed by the arterial ketone body ratio, reflects the hepatic mitochondrial redox state. Arterial blood ketone bodies are measured enzymatically using commercially available kits and a spectrophotometer, which takes approximately 40 min. During the anhepatic phase of liver transplantation arterial ketone body ratio levels are very low, but there is a marked increase after reperfusion if the graft is functioning and correlates well with subsequent function (27). A ratio of less than 0.7 for longer than 24 hr, in nondiabetic recipients, indicates an increased risk of early graft nonfunction or severe dysfunction (28). In human liver donors, however, the specificity of this method was only 77% when the usual cut-off point was used and some grafts below this value performed well after transplantation (29). Currently the test seems to be unsuitable for use alone in the assessment of graft function before transplantation. Low Temperature Pyridine Nucleotide Fluorometry This noninvasive method also allows monitoring of the hepatic mitochondrial redox state by measuring the fluorescent emission of nicotinamide adenine dinucleotide phosphate (reduced form) when excited by a 366-nm wavelength light. Continuous perfusion of an oxygenated solution has to be applied to the cold preserved graft, and the probe of the redoximeter is put in contact with the liver surface to perform the measurement. The calculated fluorometric parameters decrease proportionally to the duration of preservation in animal models and correlate well with other values of hepatic cell viability and graft survival rates (30). However, application of perfusion fluids to the graft may influence organ viability, and the technique is invasive. A practical application in clinical transplantation has not been demonstrated as yet. Respiratory Control Ratio The mitochondrial oxidative-phosphorylative activities can be measured polarographically using a membrane oxygen electrode to measure oxygen consumption. A water-jacketed reaction chamber is prepared at 23°C and a mitochondrial suspension added to the reaction medium with glutamate as a substrate. The respiratory control ratio (RCR) is calculated by the polarographic trace representing an index of ATP synthesis. In experimental models, RCR decreases gradually during cold preservation, reflecting general impairment of mitochondrial function. Grafts with an RCR value of more than 3.0 have been considered suitable for transplantation and correlate with graft survival (31). RCR has not been used yet in clinical transplantation, and it seems that in normal human livers the RCR values are lower and more variable than in livers from animals used in these experiments (32). Proton ATPase Activity Proton ATPase is a key enzyme in mitochondrial ATP synthesis and has the important role of maintaining the mitochondrial membranous electrical potential difference. During cold preservation in experimental liver transplantation, the proton ATPase function decreases markedly due to molecular damage. The measurement of mitochondrial proton ATPase activity can be determined spectrophotometrically (33) or using a fluorescent dye diS C3 (5) (34). This latter method is based on a change in the fluorescent intensity proportional to the potential difference between the inside and the outside of the inner mitochondrial membrane. It requires the preparation of mitochondria and analysis of the physical property of the enzyme. Animal liver grafts with proton ATPase activity above 40% were considered suitable for transplantation (32, 34), however, its value in clinical transplantation has yet not been assessed. Magnetic Resonance Spectrometry Magnetic resonance spectrometry (MRS) is a noninvasive, safe, and reproducible technique based on the application of strong magnetic fields capable of stimulating a sensitive nucleus. MRS of phosphorus 31 is most frequently used to evaluate hepatic function, and a typical spectrum shows various resonance peaks, each one corresponding to the addition of different elements. The phosphomonoester (PME) peak registers the presence of phospholipid cell membrane precursors, nucleotide monophosphate (NMP), and glucose 6-phosphate. The inorganic phosphate peak has the highest intensity in the spectrum, and this increases proportionally to the degradation of nucleotides. The phosphodiester peak (PDE) includes phospholipid cell membrane degradation products and endoplasmic reticulum. There are three nucleotide triphosphate (NTP) peaks that include adenosine, uridine, guanosine, inosine, and cytosine triphosphate. NTP peaks are very difficult to detect in human grafts because they have low intensity. During cold preservation, NTP falls to undetectable levels because it degenerates to nucleotide diphosphate and inorganic phosphate and in turn to NMP in a steady loss of energy substrate. High levels of NMP products increase the PME peak (35). At reperfusion, the rephosphorylation of NMP restores the NTP levels, expressed by the PME/inorganic phosphate ratio, which has been considered as a better indicator of graft viability than individual static NTP measurement (36). Much attention has also focused on the PDE peak, which when elevated, represents membrane cell damage with increased phospholipid breakdown products (35). The accuracy of using the PME and PDE peaks has been questioned because they may vary with the donor nutritional status. To date, MRS of phosphorus 31 has not been considered sufficiently reliable to use in the prediction of graft dysfunction in human livers (37). MARKERS OF MICROVASCULAR INJURY Sinusoidal endothelial cells are considered to be the most important target of cold preservation giving rise to the postreperfusion microvascular injury. A variety of substances that appear in the graft caval effluent (GCE) after flushing of the liver and the degree of platelet adherence in donor livers have been suggested as markers of this microvascular injury. Hyaluronic Acid Hyaluronic acid (HA) is produced by mesenchymal cells throughout the body, particularly within connective tissue. It is rapidly cleared from the circulation by a specific high-affinity receptor in the hepatic vascular endothelium. Since HA uptake by endothelial cells is significantly greater than its production, a high level of HA in the GCE after perfusion may reflect impaired uptake by the injured microvasculature (38). The GCE can be collected after flushing of the liver through the portal vein on the back table or at the time of implantation. HA levels are analyzed by a sensitive radiometric assay (3-hr test). A significant correlation has been demonstrated between HA levels and graft viability. A cut-off level of 400 μg/L has been proposed to differentiate good or “bad” livers (38, 39). Other groups, however, have failed to demonstrate that the use of HA in the GCE is of value as a predictor of graft outcome (40). Creatine Kinase-BB Significant creatine kinase activity has been found in mouse liver cell cultures. Brain-type creatine kinase isoenzyme was present exclusively in endothelial and Kupffer cells, muscle-type creatine kinase isoenzyme was present in Ito cells, and mitochondrial CK in hepatocytes. Because of its endothelial origin, brain-type creatine kinase isoenzyme has been considered as a marker of liver sinusoidal damage after ischemia-reperfusion injury in an experimental models (41). It is also considered a marker of brain damage after head injury but its value as an index of liver graft viability has not been confirmed when studied in the GCE of human liver grafts before (42) and during transplantation (40). Endothelin-1 Endothelin-1 (ET-1) is produced and released by endothelial cells, has a potent vasoconstrictor effect, and has a basal secretion into plasma under physiological conditions. Several stimuli (interleukin-1, thrombin, angiotensin II, and oxidized low-density lipoproteins) and clinical conditions (hypoxia, increased fluid flow rates, acute renal failure, hemodialysis, myocardial ischemia, liver cirrhosis, subarachnoid hemorrhage, endotoxic shock, Raynaud‘s phenomenon, and surgical stress) may increase plasma levels. can be measured by High GCE levels have been reported in with plasma levels at liver transplantation but did not correlation with graft or outcome it is not a sensitive marker of reperfusion injury because there is no of and its secretion from endothelial cells depends on has been used to try to assess donor liver function by measuring its GCE levels on the back however, its clinical value has not been demonstrated (38). This is an endothelial cell membrane protein that as a for and has an important role in the protein It is released into the blood in in to endothelial damage. There is a commercially available assay based on an enzyme immunoassay High levels in the GCE before transplantation and in the hepatic vein blood immediately after liver reperfusion correlate well with and primary However, this correlation has not been confirmed by and clinical is This is produced by vascular and has a in It is a key enzyme in of and is released in to a number of stimuli and was measured in the GCE of grafts during the back and of the levels was found in grafts with cold levels were also measured in the GCE of human grafts during liver implantation. were increased during the anhepatic phase and immediately after reperfusion, however, this may not be to by the donor liver but due to a of hepatic The degree of platelet to graft has been considered to be a good index in predicting outcome after liver transplantation. biopsy specimens were from donor livers and with a platelet and for platelet correlated with one of the donors studied had platelet before organ that platelet was associated with the to brain however, the predictive value of this method was not shown in this MARKERS OF GRAFT FUNCTION Other in are cellular degradation products of ATP and if they during cold preservation, can be measured in the GCE after perfusion in animal such as adenosine, inosine, and uric acid can be in the GCE of the liver by high-performance liquid chromatography using a exchanger and with the total loss of cellular adenine nucleotides during cold However, in a clinical the levels of obtained from GCE during liver did not correlate with early function dehydrogenase, hepatocyte vascular endothelial and have also been analyzed in the caval effluent of human grafts on the back table and before reperfusion can be considered as of the injury as markers of graft damage. A variety of methods have been used to detect these with higher levels found in with with those with good graft function, however, their predictive value has not been of is a new marker with the potential to evaluate graft function before transplantation. It is an abnormal which is undetectable in healthy but in serum of in are a and in with can be measured in from serum by using a specific It is in donors have had a hospital using it has been found that donor is associated with an increased risk of graft dysfunction. The effect of donor on levels and graft dysfunction has not been assessment of the liver has an important of for many transplant The test all donor livers that have the potential to or their function and those grafts that will not of the liver can now be using a variety of however, it is difficult to assess the value of the methods because no studies have been MEGX arterial ketone body ratio, MRS of phosphorus and acid in GCE have been studied and initially to be but studies have questioned their and value in liver grafts. Other tests such as plasma LCAT activity, in in and platelet adherence have and clinical studies to be performed to evaluate their predictive tests in the experimental proton ATPase activity seems to be the most for clinical Donor biliary bile acid analysis in donors has the potential of the function of the liver to assess the graft before and new have been developed to the of bile during organ retrieval new of include the assessment of in donor the by molecular of bile acid and the application of proton magnetic resonance to hepatic bile from potential donor grafts. of graft function before liver of the methods to be better than liver function tests and liver graft appearance in whether to use a graft in clinical practice. However, donor are not reliable and may to livers that function and if a poor graft is to the of a The organ donor and the increasing to use more marginal donor livers the of being to accurately significant graft dysfunction or nonfunction and to early of graft dysfunction to the number of grafts. This in turn has the potential to and after liver transplantation efforts and are in the for more reliable methods of graft assessment before liver transplantation.