Human Donor Livers Research Articles

Hepatic nucleotide triphosphate regeneration after hypothermic reperfusion in the pig model: an in vitro P-NMR study.

The aim of this study was to assess the possibility of regenerating nucleotide triphosphates (NTP) in the pig liver following its harvest and subsequent storage on ice. This study has used a pig model that allowed human donor liver retrieval techniques and methods of storage to be utilized. In vitro phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy was used to evaluate the changes associated with phosphorus containing metabolites such as NTP, phosphomonoesters (PME), phosphodiesters (PDE), and inorganic phosphate (Po). During 4 hr storage NTP levels were reduced to undetectable levels but its regeneration was possible over a period of 2 hr of oxygenated hypothermic reperfusion. Resynthesized NTP reached values that were only 30% reduced from pre-harvest values. There was a corresponding reduction in Pi over the same period. Glycolytic intermediates, 3-phosphoglycerate and 2,3 diphosphoglycerate, both increased significantly during the period of storage and subsequently declined following hypothermic reperfusion. Cellular damage, indicated by the concentrations of glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine (GPE) was minimal during cold storage. However upon hypothermic reperfusion, concentrations of GPC and GPE reduced, indicating a degree of cellular damage caused by reperfusion. This study has shown for the first time that is possible to regenerate high energy phosphate nucleotides following a period of hypothermic reperfusion in a large, clinically related animal model. This technique warrants investigation clinically to improve the outcome of orthotopic liver transplantation. It also provides a method to study the effects of different preservation fluids and methods of storage and organ reperfusion.

Tissue pH in cold-stored human donor livers preserved in University of Wisconsin solution. A noninavasive clinical study with 31P-magnetic resonance spectroscopy.

It is not known whether the tissue acidosis that accompanies cold storage is the beginning of irreversible cell injury, ultimately leading to cell death, or whether it is a natural "protective" mechanism for cells to survive hypoxic periods. To answer this question, the tissue pH of 45 cold-stored human donor livers preserved in University of Wisconsin solution (UW) was assessed shortly before implantation using noninvasive 31P magnetic resonance spectroscopy. We conclude that tissue pH during cold storage may be partly dependent upon hepatic glycogen stores and donor age. The wide range of tissue pH values that was observed at the time of implantation does not result in significant effects on cellular damage after transplantation. This indicates that tissue pH is not a major determinant for the viability of UW solution-preserved human donor livers, as indicated by postoperative hepatocellular damage and liver synthesis function. The membrane stabilizing and buffering capacity of UW solution appears to protect liver viability against tissue acidosis. Our results also indicate that liver tissue pH can be lower than has been previously assumed in the literature without significant adverse effects on liver viability.

Venous air embolism, preservation/reperfusion injury, and the presence of intravascular air collections in human donor livers: a retrospective clinical study

Venous air embolism, preservation/reperfusion injury, and the presence of intravascular air collections in human donor livers: a retrospective clinical study

Venous air embolism, preservation/reperfusion injury, and the presence of intravascular air collection in human donor livers: a retrospective clinical study.

In human liver transplantation, air embolism is seldom encountered after graft reperfusion. Nevertheless, despite adequate flushing and clamping routines, air emboli have been reported in transesophageal echocardiography (TEE) studies performed during the reperfusion phase. We retrospectively investigated whether air in the donor liver -- as observed with pretransplant magnetic resonance imaging (MRI) -- resulted in clinical air embolism or contributed to preservation/reperfusion injury. Clinical air embolism was assessed by intraoperative hemodynamics and end-tidal CO2 monitoring. Preservation/reperfusion injury was assessed in postoperative biochemical measurements. The outcomes were compared between patients receiving livers containing significant intrahepatic air and patients receiving livers without intrahepatic air. Forty-three livers were studied, seven which had major intrahepatic air and ten of which had no evidence of air collections. Twenty-six livers showed minor amounts of air and were excluded from further study. One patient who received a liver that did not contain intrahepatic air had clinical evidence of air embolism. Clinical air embolism did not appear to be associated with the presence of significant intrahepatic air based upon pretransplant MRI. Intrahepatic air did not seem to affect the amount of preservation/reperfusion injury. Our data indicate that air bubbles in the portal and arterial branches are absorbed during reperfusion and that the majority of intrahepatic air is effectively removed by the specific flushing routines.

Magnetic resonance venography in liver bipartition procedures using preservation solution as contrast agent.

To evaluate whether the hepatic veins can be visualized with a rapid noninvasive technique, and if so, whether the obtained images could be helpful in the preparation of split liver grafts for transplantation, six cold stored human donor livers were investigated with magnetic resonance imaging (MRI). The hepatic vein branches and their confluence were clearly visualized. Anatomic variations of the middle hepatic vein with consequences for the choice of the transection plane could be demonstrated. Furthermore, unexpected vascular abnormalities were detected. From these preliminary results it is concluded that visualization of the hepatic veins can be helpful in determining the feasibility of the bipartition procedure and the choice of the transection plane. A potential wide application of this fast and noninvasive technique is possible.

Ex vivo magnetic resonance imaging of pretransplant human donor liver: Clinical experience in 66 cases

Abstracts Magnetic resonance imaging (MRI) was performed on 66 cold-stored human donor livers. Spin echo images were obtained with a clinical whole body MRI system. Various parenchymal and vascular abnormalities were found. An unexpected finding was the abundant presence of intrahepatic air. Although the majority of parenchymal abnormalities that were found would not have precluded transplantation, the rationale of pretransplant MRI was to prevent the introduction of unidentified pathology into the recipient. Guided by the MR images, lesions in the isolated organ can be easily located for biopsy and resection. Unnecessary or inadequate therapeutic interventions after transplantation can thus be avoided. In addition, the visualization of the hepatic veins with their confluence appears to be useful in split-liver procedures.

Ex vivo magnetic resonance imaging of pretransplant human donor liver

Magnetic resonance imaging (MRI) was performed on 66 cold-stored human donor livers. Spin echo images were obtained with a clinical whole body MRI system. Various parenchymal and vascular abnormalities were found. An unexpected finding was the abundant presence of intrahepatic air. Although the majority of parenchymal abnormalities that were found would not have precluded transplantation, the rationale of pretransplant MRI was to prevent the introduction of unidentified pathology into the recipient. Guided by the MR images, lesions in the isolated organ can be easily located for biopsy and resection. Unnecessary or inadequate therapeutic interventions after transplantation can thus be avoided. In addition, the visualization of the hepatic veins with their confluence appears to be useful in split-liver procedures.

THE TISSUE HYDRATION STATE IN UW-PRESERVED HUMAN DONOR LIVERS

To determine the relation between tissue hydration state--as indicated by tissue proton magnetic resonance relaxation times--in UW-preserved human donor livers and viability parameters of the donor and early graft function, "ex vivo" magnetic resonance relaxometry was performed with a clinical MR imaging system. Relaxometric data were obtained from MR images in which signal intensities were directly proportional to T1 and T2. Forty-three subsequently transplanted livers and five discarded livers were studied. The donor serum concentrations of direct and total bilirubin had a positive correlation with T1 (P < 0.05 and P < 0.01, respectively). Sequential measurements in 7 livers demonstrated a firm time relation between the cold storage time and the length of the relaxation times. As cold storage time lengthened, T1 and T2 shortened. T1 of the donor liver showed a significant negative correlation with recipient ASAT and ALAT values on days 1, 2, and 3 after transplantation. T1 in the discarded group was significantly higher than T1 in the accepted group. T2 was not different in the two groups. It is concluded that in UW-preserved human donor livers, the tissue hydration state, as indicated by the tissue MR relaxation times, is largely independent of the clinical condition of the organ donor and the preservation procedure. An optimum tissue hydration state, in UW-preserved donors liver might have protective properties against parenchymal damage, although the clinical consequences appear to be of minor importance. The capacity of relaxometry as a discriminative instrument to accept or to discard donor livers is poor.

Passenger lymphocytes in human liver allografts and their potential role after transplantation.

Rare cases of graft-versus-host disease after liver transplantation indicate that donor lymphocytes may be transferred to the recipient by human liver grafts. In this study, we have analyzed the number and subpopulations of donor lymphocytes transferred by liver grafts in order to evaluate the potential relevance of these cells after transplantation. Therefore, mononuclear cells were isolated from the tissue of perfused human donor livers and from the associated lymph nodes. The number of lymphocytes, their location, and surface marker expression were determined by immunostaining. The majority of lymphocytes transferred by the grafts were found within the liver tissue (5.3 +/- 2.9 x 10(9) cells). These lymphocytes are mainly T and NK cells, predominantly CD8+, are partially activated (28% HLA-DR+), and show strong adhesion molecule expression (88% LFA-1(3+)). In addition, 20-500 x 10(6) of resting lymphocytes, predominantly T and B cells, are transmitted by lymph nodes. These findings demonstrate that considerable numbers of donor lymphocytes of distinct phenotype are regularly transmitted to the recipient by human liver grafts and may be of functional relevance after transplantation.

Tissue hydration state in UW preserved human donor liver

Tissue hydration state in UW preserved human donor liver

Prediction of the outcome of transplantation in man by platelet adherence in donor liver allografts. Evidence of the importance of prepreservation injury.

We examined platelet adhesion in thirty human donor livers to determine if the degree of platelet adhesion could predict outcome of transplantation. Wedge liver biopsies were taken at the start of the donor operation (biopsy 1) and 1 hr after reperfusion in the recipient (biopsy 2). Biopsies were stained with a monoclonal antibody against platelet glycoprotein Ib and graded for platelet adhesion. Hematoxylin and eosin-stained sections were examined for polymorphonuclear leukocyte adhesion and necrosis. Platelet adhesion was much more frequent and extensive than expected in biopsy 1. Nine of 30 biopsies showed moderate or high-grade platelet adhesion. Thus in this study endothelial cell damage was present in about one-third of donors before the donor operation. The injury was not detectable by routine microscopic or clinical examination or biochemical tests. The degree of platelet adhesion in biopsy 1 predicted development of PMN adhesion and necrosis in biopsy 2 and postoperative transaminase concentrations and prothrombin times in recipients. During preservation and implantation some livers converted from low to either moderate or high grades of platelet adhesion. The grade of platelet adhesion in biopsy 2 predicted postoperative outcome as measured by transaminase and PT levels. Patients whose platelet grade converted to a higher level during preservation and implantation did not do as well as patients who remained at a low adhesion grade. These findings strongly suggest that the degree of platelet adhesion is an important determinant in assessing outcome and may provide a means of measuring the status of liver allografts prior to transplantation.

Measuring impedance for evaluating ischemia damage to the human liver in preparation for transplantation

In 22 human donor livers the measurement of the non-invasive bioelectrical impedance was performed prospectively to evaluate the degree of tissue damage sustained during cold ischemia. The results of the measurement were correlated with liver function, the method of organ preservation and the period of ischemia. The impedance was measured in vivo as 620 ohm (at 192 Hz), the phase angle as -7.4 degrees (at 5 kHz). The results were compared with the data obtained from 72 patients who underwent elective laparotomies. The 22 donor livers were studied further during ischemia. The method was found to be a reliable way of detecting severe damage to the hepatocytes during the cold ischemia.

Secretion of 72 kDa type IV collagenase/gelatinase by cultured human lipocytes. Analysis of gene expression, protein synthesis and proteinase activity.

The matrix metalloproteinases play an important role in matrix degradation, but there is limited information about this family of enzymes in either normal or diseased human liver. In this study, we have examined the synthesis of a 72 kDa type IV collagenase/gelatinase by human hepatic lipocytes in primary culture. Hepatic lipocytes were isolated from wedges of normal human donor liver by Pronase/collagenase perfusion, purified by density-gradient centrifugation, and established in primary culture on uncoated plastic. By Northern-blot analysis, the total RNA extracted from cultured human lipocytes was found to contain 3.4 kb mRNA for the 72 kDa type IV collagenase/gelatinase. Low levels of expression of this mRNA were observed in freshly isolated lipocytes but expression increased with the duration of lipocyte culture. Using anti-human 72 kDa type IV collagenase/gelatinase IgG, synthesized enzyme was immunolocalized to monensin-treated human lipocyte cultures. De novo synthesis and secretion of 72 kDa type IV collagenase/gelatinase were confirmed by immunoprecipitation of radiolabelled enzyme from medium obtained from [35S]methionine-treated cells. Activity of the secreted enzyme was demonstrated by gelatin-zymography and by degradation of soluble, radiolabelled [14C]gelatin. The enzyme was released both in active and latent pro-enzyme forms and its inhibition profile was that of a metalloproteinase. These studies indicate that cultured human hepatic lipocytes express the gene for the 72 kDa type IV collagenase/gelatinase, and secrete this enzyme, particularly in prolonged primary culture. As this enzyme exhibits degradative activity against basement membrane collagen, its release by activated hepatic lipocytes in the space of Disse could lead to disruption of the normal subendothelial liver matrix. It is suggested that this enzyme may have an important role in human liver injury and fibrosis.

Rapid donor liver nutritional enhancement in a large animal model

Organ donors are typically subject to acute hyponutrition that might affect postpreservation liver function. Livers from nutritionally supplemented rats function better after preservation than livers from fasted rats. We have developed a method to glycogenate the liver of large animals in the temporal context of a human donor liver operation and have studied the fate of glycogen stores during preservation. Starved anesthetized pigs were infused with a hexose solution (glucose, fructose or galactose) by way of the superior mesenteric vein for 3 hr. Regular porcine insulin was infused to maintain a hyperglycemic hyperinsulinemic arterial glucose clamp at 12 to 16 mmol/L. Liver biopsy specimens and blood samples were taken before infusion and hourly. At 3 hr the liver was excised, stored for 24 hr at 1°C in University of Wisconsin solution and biopsied. It was then placed at 20° C for 1 hr to simulate the reimplantation stage of transplantation. Glycogen and nucleotide levels were measured, and results were corrected for starch in the University of Wisconsin solution. A 20% glucose infusion produced rapid hepatic glycogenation without side effects. Greater glycogenation was obtained with 20% fructose but at the cost of lactic acidosis and a fall in pH. A combination of 15% glucose and 5% fructose produced intermediary glycogenation without significant side effects. Galactose (20%) was less efficient than glucose alone. The addition of alanine and glutamine (20 mmol/L) did not significantly improve glycogenation. Metabolism of glycogen at 1° C did occur. Glycogen content fell 0.15% ± 0.05% dry weight liver per hour during cold preservation and 5.49% ± 2.15% per hour during ischemic rewarming. The rate of glycogen depletion during rewarming was dependent on the amount of glycogen available at the start of the period. Phosphorylated nucleotide levels were maintained at significantly higher levels in glycogenated livers. Periodic acid–Schiff staining showed a clear gradient of glycogenation maximal in zone 1 and minimal in zone 3. Rapid hepatic glycogenation is possible and beneficial in terms of maintaining adenine nucleotide levels in a large animal model. (HEPATOLOGY 1992;16:1271–1279.)

Rapid donor liver nutritional enhancement in a large animal model

Organ donors are typically subject to acute hyponutrition that might affect postpreservation liver function. Livers from nutritionally supplemented rats function better after preservation than livers from fasted rats. We have developed a method to glycogenate the liver of large animals in the temporal context of a human donor liver operation and have studied the fate of glycogen stores during preservation. Starved anesthetized pigs were infused with a hexose solution (glucose, fructose or galactose) by way of the superior mesenteric vein for 3 hr. Regular porcine insulin was infused to maintain a hyperglycemic hyperinsulinemic arterial glucose clamp at 12 to 16 mmol/L. Liver biopsy specimens and blood samples were taken before infusion and hourly. At 3 hr the liver was excised, stored for 24 hr at 1 degrees C in University of Wisconsin solution and biopsied. It was then placed at 20 degrees C for 1 hr to simulate the reimplantation stage of transplantation. Glycogen and nucleotide levels were measured, and results were corrected for starch in the University of Wisconsin solution. A 20% glucose infusion produced rapid hepatic glycogenation without side effects. Greater glycogenation was obtained with 20% fructose but at the cost of lactic acidosis and a fall in pH. A combination of 15% glucose and 5% fructose produced intermediary glycogenation without significant side effects. Galactose (20%) was less efficient than glucose alone. The addition of alanine and glutamine (20 mmol/L) did not significantly improve glycogenation. Metabolism of glycogen at 1 degree C did occur. Glycogen content fell 0.15% +/- 0.05% dry weight liver per hour during cold preservation and 5.49% +/- 2.15% per hour during ischemic rewarming.(ABSTRACT TRUNCATED AT 250 WORDS)

Presence of large amounts of activated lymphocytes in the parenchyma of human donor livers

Presence of large amounts of activated lymphocytes in the parenchyma of human donor livers

Viability testing of transplantation donor liver by 1H NMR relaxometry

We present preliminary results of water proton relaxation time measurements on perfused, cold-stored rat livers as a basis of a simple method for testing the viability of human donor livers in liver transplantation surgery. Proton T1 and T2 data correlated strongly (p less than 0.01) with bile flow measurements, indicating that relaxation times reflect organ viability and therefore might be used for rapid testing of donor livers shortly before transplantation.

In vitro characterization of the human cytochrome P-450 involved in polymorphic oxidation of propafenone.

Propafenone is a new class 1 antiarrhythmic agent. The drug is extensively metabolized. 5-Hydroxylation and N-dealkylation constitute major metabolic pathways. Recently it has been demonstrated that the in vivo metabolism of propafenone is controlled by the debrisoquin/sparteine polymorphism. To elucidate which of the above metabolic reactions is catalyzed by cytochrome P-450db1, the formation of 5-hydroxypropafenone and N-desalkylpropafenone was studied in the microsomal fraction of four human kidney donor livers previously characterized with regard to their ability to hydroxylate the beta-adrenergic antagonist bufuralol. The l'hydroxylation of bufuralol is catalyzed by the P-450db1 responsible for polymorphic debrisoquin/sparteine oxidation. The formation of 5-hydroxypropafenone but not N-desalkylpropafenone was closely related to bufuralol l'hydroxylation. Incubation with LKM1 antibodies, which selectively recognize P-450db1, inhibited 5-hydroxypropafenone formation completely whereas N-dealkylation was unimpaired. Propafenone was a strong competitive inhibitor of bufuralol l'hydroxylation. Thus it can be concluded that 5-hydroxypropafenone is formed by the cytochrome P-450 isozyme involved in polymorphic bufuralol oxidation.

Decreased loss of liver adenosine triphosphate during hypothermic preservation in rats pretreated with glucose: Implications for organ donor management

Recent studies of human donor livers indicate an association between ex vivo hepatocellular adenosine triphosphate and posttransplant graft function. To test the hypothesis that prior glucose loading of donor liver would optimize its adenosine triphosphate production and adenylate energy charge during ex vivo organ preservation, adult male rats were randomized to receive either intravenous dextrose or saline for 44 h. After this infusion, a liver lobe was exposed and freeze-clamped (time 0). The remaining liver was quickly flushed, excised, and stored in Collins' II solution at 2 °C for 8 h. Additional lobes were freeze-clamped at 1, 4, and 8 h. Liver adenosine triphosphate, total nucleoside triphosphates, and energy charge losses were significantly reduced in the dextrose-treated rats in comparison with saline-treated rats during the first 4 h of preservation. Although the livers from rats receiving intravenous dextrose were able to generate lactate, their glycogen stores were not utilized appreciably, suggesting that exogenous glucose served as a substrate for anaerobic glycolysis. Unesterified choline levels of the fasted rat livers were significantly higher than those from the rats receiving intravenous dextrose by the first hour, indicative of increased membrane breakdown. These results indicate that prior infusion of glucose enhances the capacity of the ex vivo liver, presumably through the induction and stabilization of key glycolytic enzymes, to anaerobically generate adenosine triphosphate. Administration of glucose to liver donors before organ procurement may improve posttransplant graft function by reducing the loss of hepatocellular energy, retarding membrane damage, and fostering glycogen storage for use in the early postoperative period.