Autonomic nervous system and gut-derived endotoxin: involvement in activation of Kupffer cells after in situ organ manipulation.

Gentle in situ organ manipulation rapidly causes disturbances in the hepatic microcirculation, hypoxia, and activation of Kupffer cells. Because the mechanisms of Kupffer cell activation after organ manipulation remain unclear, the possible role of the autonomic nervous system and gut-derived endotoxin were assessed. To mimic what occurs with major abdominal surgery, livers from female Sprague-Dawley rats (200-230 g) underwent minimal dissection for 12 minutes and were manipulated gently or were left alone for 13 subsequent minutes. Kupffer cells were activated 2 hours after manipulation, reflected by a significant increase in intracellular calcium ([Ca2+]i) from about 90 nM in unmanipulated controls to more than 180 nM in response to lipopolysaccharide (LPS 100 ng/ml). Furthermore, Kupffer cells from manipulated rats produced about threefold more tumor necrosis factor-alpha after LPS (100 ng/ml) than did the unmanipulated controls. Moreover, O2 uptake of ex situ perfused liver was increased from about 110 micromol/g/hr in unmanipulated controls to more than 160 micromol/g/hr 2 hours after organ manipulation. Binding of pimonidazole (120 mg/kg IV), a 2-nitroimidazole hypoxia marker given 2 hours after manipulation, increased about 2.5-fold, and hepatic glycogen was depleted. Two hours after organ manipulation gut permeability to horseradish peroxidase was elevated and endotoxin in the portal venous blood was increased twofold. Microsurgical hepatic denervation, ganglionic blockade, adrenalectomy, and antibiotics to sterilize the gut before manipulation prevented activation of Kupffer cells by organ manipulation. Hexamethonium and adrenalectomy prevented increases in gut permeability caused by manipulation. Although antibiotics blunted the increase in portal venous endotoxin significantly, there was no effect on gut permeability. These data indicate for the first time that both the autonomic nervous system and gut-derived endotoxin are involved in activation of Kupffer cells after organ manipulation.

Activated Kupffer cells cause a hypermetabolic state after gentle in situ manipulation of liver in rats.

Harvesting trauma to the graft dramatically decreases survival after liver transplantation. Since activated Kupffer cells play a role in primary nonfunction, the purpose of this study was to test the hypothesis that organ manipulation activates Kupffer cells. To mimic what occurs with donor hepatectomy, livers from Sprague-Dawley rats underwent dissection with or without gentle organ manipulation in a standardized manner in situ. Perfused livers exhibited normal values for O(2) uptake (105 +/- 5 micromol. g(-1). h(-1)) measured polarigraphically; however, 2 h after organ manipulation, values increased significantly to 160 +/- 8 micromol. g(-1). h(-1) and binding of pimonidazole, a hypoxia marker, increased about threefold (P < 0.05). Moreover, Kupffer cells from manipulated livers produced three- to fourfold more tumor necrosis factor-alpha and PGE(2), whereas intracellular calcium concentration increased twofold after lipopolysaccharide compared with unmanipulated controls (P < 0.05). Gadolinium chloride and glycine prevented both activation of Kupffer cells and effects of organ manipulation. Furthermore, indomethacin given 1 h before manipulation prevented the hypermetabolic state, hypoxia, depletion of glycogen, and release of PGE(2) from Kupffer cells. These data indicate that gentle organ manipulation during surgery activates Kupffer cells, leading to metabolic changes dependent on PGE(2) from Kupffer cells, which most likely impairs liver function. Thus modulation of Kupffer cell function before organ harvest could be beneficial in human liver transplantation and surgery.

Reperfusion injury is dramatically increased by gentle liver manipulation during harvest.

Kupffer cell-dependent injury in livers gently manipulated during harvest develops upon reperfusion. The purpose of this study was to characterize this injury and to detect underlying mechanisms. Livers from female Sprague-Dawley rats were harvested for transplantation within 25 min. Minimal dissection was performed during the first 12 min, including freeing the liver from ligaments. After this, for further 13 min, livers were either left alone or manipulated gently. The livers were then cold-stored for 24 h in University of Wisconsin (UW) solution and perfused with oxygen-saturated Krebs-Henseleit buffer at 37 degrees C. Trypan blue in the buffer was used to index microcirculation. Cell damage was assessed with histology. Initial dissection during harvest and cold storage had minimal effects on sinusoidal lining cells; in contrast, the subsequent gentle organ manipulation dramatically increased cell death 6.5-fold, while the time for complete trypan blue distribution increased 2.3-fold (P < 0.05). Manipulation increased proteolysis 2-fold (P < 0.05). At harvest, manipulation increased portal venous pressure significantly by 68%. Treatment of donors with gadolinium chloride, a selective Kupffer cell toxicant, or with dietary glycine, an inhibitor of Kupffer cell activation, prevented effects of organ manipulation on all parameters studied. These findings demonstrate Kupffer cell-dependent reperfusion injury of sinusoidal lining cells caused by manipulation of the liver during its recovery. The mechanisms are those of proteolysis and impaired hepatic microcirculation.

Gene delivery of Cu/Zn-superoxide dismutase improves graft function after transplantation of fatty livers in the rat.

Oxygen-derived free radicals play a central role in reperfusion injury after organ transplantation, and fatty livers are particularly susceptible. Endogenous radical scavengers such as superoxide dismutase (SOD) degrade these radicals; however, SOD is destroyed rapidly when given exogenously. Therefore, an adenoviral vector encoding the Cu/Zn-SOD gene (Ad.SOD1) was used here to test the hypothesis that organ injury would be reduced and survival increased in a rat model of transplantation of fatty livers. Donors received chow diet (untreated), high-fat diet, or ethanol-containing high-fat diet. Some of the ethanol-fed donors were infected either with the gene lacZ encoding bacterial beta-galactosidase (Ad.lacZ), or Ad.SOD1. After liver transplantation, SOD activity and protein expression in liver, survival, histopathology, release of transaminases, free radical adducts in bile, and activation of NF-kappaB, IkappaB kinase (IKK), Jun-N-terminal kinase (JNK), and TNFalpha were evaluated. Ad.SOD1 treatment increased survival dramatically, blunted transaminase release, and reduced necrosis and apoptosis significantly. Free radical adducts were increased two-fold in the ethanol group compared with untreated controls. Ad. SOD1 blunted this increase and reduced the activation of NF-kappaB. However, release of TNFalpha was not affected. Ad.SOD1 also blunted JNK activity after transplantation. This study shows that gene therapy with Ad.SOD1 protects marginal livers from failure after transplantation because of decreased oxygen radical production. Genetic modification of fatty livers using viral vectors represents a new approach to protect marginal grafts against primary nonfunction.

Delivery of Cu/Zn-superoxide dismutase genes with a viral vector minimizes liver injury and improves survival after liver transplantation in the rat.

BACKGROUND: Oxygen-derived free radicals play a central role in pathomechanisms of reperfusion injury after organ transplantation. Endogenous radical scavenger systems such as superoxide dismutase (SOD) degrade toxic radicals; however, SOD is degraded rapidly when given exogenously. Therefore, the hypothesis that treatment of the donor liver with an adenoviral vector encoding the Cu/Zn-SOD gene (Ad-SOD1) would lead to permanent gene expression and therefore protect the organ against injury and increase survival in a rat model of liver transplantation was tested. METHODS: Some donors were infected with Ad-SOD1, whereas untreated grafts and livers infected with the indicator gene lacZ encoding bacterial beta-galactosidase (Ad-lacZ) served as controls. After orthotopic liver transplantation, survival, serum transaminases, and histopathology were evaluated. RESULTS: Approximately 80% of hepatocytes expressed beta-galactosidase 72 hr after injection of Ad-lacZ. Moreover, SOD1 gene expression and activity were increased 3- and 10-fold in the Ad-SOD1 group, respectively. After transplantation, 20-25% of rats treated with Ad-lacZ survived. In contrast, all SOD1-treated animals survived. Transaminases measured 8 hr after transplantation in Ad-SOD1 rats were only 40% of those in controls, which increased 40-fold above normal values. Approximately 20% of hepatocytes in untreated and Ad-lacZ-infected organs were necrotic 8 hr after reperfusion, whereas necrosis was nearly undetectable in grafts from rats treated with Ad-SOD1. CONCLUSIONS: This study provides clear evidence for the first time that gene therapy with Ad-SOD1 increases survival and decreases hepatic injury after liver transplantation. Genetic modification of the liver represents a future approach to protect organs against injury where oxygen-derived free radicals are involved.

Glycine inhibits growth of T lymphocytes by an IL-2-independent mechanism.

Previously, it was shown that glycine prevented increases in intracellular calcium ([Ca2+]i) in Kupffer cells. Since Kupffer cells and T lymphocytes are derived from the same pluripotent stem cell, it was hypothesized that glycine would prevent increases in [Ca2+]i in lymphocytes and inhibit cell proliferation. Lymphocyte proliferation was measured in one-way MLC with spleen cells from DA and Lewis rats and in enriched T lymphocyte preparations stimulated by immobilized anti-CD3 Ab. Glycine caused a dose-dependent decrease in cell proliferation to about 40% of control. Con A caused a dose-dependent increase in [Ca2+]i in Jurkat cells which was blunted maximally with 0.6 mM glycine. The effect of glycine was dependent on extracellular chloride and reversed by strychnine, an antagonist of the glycine-gated chloride channel. Similar results were obtained with rat T lymphocytes stimulated by anti-CD3 Ab. Surprisingly, glycine had no effect on IL-2 production in the mixed lymphocyte culture; therefore, the effect of glycine on IL-2-dependent proliferation was tested. Glycine and rapamycin caused dose-dependent decreases in IL-2-stimulated growth of Ctll-2 cells to about 60% and 40%, respectively, of control. Moreover, glycine also inhibited the IL-2-stimulated growth of rat splenic lymphocytes. It is concluded that glycine blunts proliferation in an IL-2-independent manner. This is consistent with the hypothesis that glycine activates a glycine-gated chloride channel and hyperpolarizes the cell membrane-blunting increases in [Ca2+]i that are required for transcription of factors necessary for cell proliferation.

Inhibition of chronic rejection of aortic allografts by dietary glycine.

BACKGROUND: Chronic rejection is influenced by a variety of risk factors, including histoincompatibility and ischemia. Glycine, a cytoprotective agent, has been shown to protect against ischemia-reperfusion injury in the liver, inactivate hepatic resident macrophages, minimize cyclosporin A-induced nephrotoxicity, and exhibit immunosuppressive properties in vitro. The aim of this study was to investigate whether dietary glycine could reduce development of chronic rejection. METHODS: Lewis recipients of Fisher-344 abdominal aortic allografts received diets that contained either 5% glycine plus 15% casein or 20% casein as control for 10 weeks. Vascular lesions of aortic isografts and allografts were evaluated quantitatively with image analysis and cell counting. RESULTS: No significant vascular changes were observed in isografts (mean medial areas of 3.3 +/- 0.3x0(5) microm2). However, dramatic intimal thickening (neointimal area 2.1+/-0.3) and medial thinning (1.5+/-0.3) were observed in allografts from rats fed control diet. In contrast, glycine significantly reduced the neointima by 45% (1.2+/-0.3) and protected the media (3.5+/-0.2). This led to intima to media area ratios almost twice as large in the control group as in glycine-fed rats (2.2+/-0.4 vs. 1.1+/-0.3, P<0.05). Moreover, infiltrating leukocytes, especially macrophages, were reduced significantly in the adventitia by glycine. In addition, glycine inhibited proliferation and migration of rat aortic smooth muscle cells in culture by 45 and 60%, respectively. CONCLUSION: These results indicate that dietary glycine minimizes histopathological changes of chronic rejection by reducing the immune response and, in part, by minimizing proliferation and migration of smooth muscle cells.

Gentle organ manipulation during harvest as a key determinant of survival of fatty livers after transplantation in the rat.

Both in situ organ manipulation during harvest and steatosis reduce survival after liver transplantation via mechanisms involving Kupffer cells; thus, their effect on survival was compared here. Moderate steatosis was induced by a single dose of ethanol to Lewis rats, while long-term administration of ethanol yielded severe steatosis in donor animals. After minimal dissection during the first 12 min, livers were either manipulated gently or left alone for 13 min subsequently. Orthotopic liver transplantation was performed after 1 h of cold storage in UW solution. Ethanol increased hepatic lipid content to a level of moderate or severe steatosis that reduced survival after transplantation from 100% to approximately 70% (P < 0.05). However, gentle manipulation decreased survival to approximately 30% (P < 0.05) in livers from normal, saline-treated rats and in livers from rats fed a high-fat control diet. Moreover, after short- or long-term ethanol administration, manipulation of fatty livers decreased survival from 70% to approximately 13% (P < 0.05). Further, manipulation elevated serum transaminases, total bilirubin, and necrosis significantly about 2- to 20-fold in fatty grafts after transplantation. At the end of harvest, trypan blue distribution time and hypoxia assessed from 2-nitroimidazole binding were elevated significantly about two- to fourfold by manipulation of fatty grafts. Gadolinium chloride, a Kupffer cell toxicant, blocked the detrimental effects of manipulation. These data demonstrate for the first time that, while steatosis is detrimental for survival, organ manipulation plays a much greater role than fat in mechanisms of primary nonfunction.

Dietary glycine and renal denervation prevents cyclosporin A-induced hydroxyl radical production in rat kidney.

Cyclosporin A (CsA) nephrotoxicity is associated with renal hypoxia and increases in free radicals in the urine. This study was designed to elucidate the mechanism of radical production caused by CsA. Pretreatment of rats with CsA (25 mg/kg, i.g.) for 5 days decreased glomerular filtration rates by 65%, an effect largely prevented by both dietary glycine (5%) or renal denervation. CsA dissolved in olive oil produced a 6-line alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (4-POBN)/free radical signal in the urine, which partitioned predominantly into the aqueous phase after chloroform extraction (i.e., it is water soluble). Dimethyl sulfoxide (DMSO) is attacked by the hydroxyl radical to produce a methyl radical; administration of CsA with [(12)C]DMSO produced two radical species in urine, one with hyperfine coupling constants similar to the 4-POBN/methyl radical adduct found in aqueous solution. CsA given with [(13)C]DMSO produced a 12-line spectrum, confirming the formation of hydroxyl radicals. The methyl radical produced by the hydroxyl radical represented 62% of radicals detected in urine but only 15% in bile. Therefore, hydroxyl radicals are produced largely in the kidney. Free radicals in urine were increased about 5-fold by CsA, an effect completely blocked by the inhibitory neurotransmitter, glycine, or by renal denervation. CsA infusion for 30 min increased efferent renal nerve activity 2-fold, and dietary glycine (5%) totally blocked this phenomenon. Taken together, these data are consistent with the hypothesis that CsA increases hydroxyl radical formation by increasing renal nerve activity resulting in vasoconstriction and hypoxia-reoxygenation. Glycine blunts the effect of CsA on the renal nerve, which explains, in part, prevention of nephrotoxicity.

Amino acids in rinse effluents as a predictor of graft function after transplantation of fatty livers in rats.

There are too few reliable markers by which one can predict future function of a liver before implantation. Consequently, the purpose of this study was to test the hypothesis that amino acids in rinse-effluents could predict transplant outcome in marginal fatty livers from rats. Amino acids were measured in the rinse effluent from the livers immediately after harvest and graft preparation or cold storage. Amino acids in the effluent were twice as high in ethanol-treated animals compared to those in nonfatty controls. Ethanol-treated fatty livers survived for no longer than 7 days after transplantation while 83% of nonfatty controls survived (P < 0.05). In subsequent studies, the cold-storage time was decreased to 6 h to determine whether failing fatty livers released more amino acid than grafts that would function normally. There was a significant increase in amino acids in the effluent of fatty grafts compared to controls. Moreover, the sum of the four selected amino acids (alanine, valine, histidine, leucine) was lower than 23 nmol/g liver in functional livers, whereas failing grafts had totals significantly higher than 25 nmol/g liver. The sum of the four amino acids correlated well with 24 h post-transplant serum AST levels (r = 0.78, P < 0.0001). So we can conclude that amino acid release can serve as a useful marker of graft viability and reliably predicts survival.

Reperfusion injury in livers due to gentle in situ organ manipulation during harvest involves hypoxia and free radicals.

Kupffer cell-dependent injury in livers gently manipulated during harvest develops upon transplantation; however, underlying mechanisms remain unknown. Thus, the purpose of this study was to identify factors involved in mechanisms of injury. Livers from female Sprague-Dawley rats (200-230 g) were cold stored for 24 h in University of Wisconsin solution. Subsequently, livers were perfused at 37 degrees C with oxygen-saturated Krebs-Henseleit buffer containing fluorescein-dextran to assess microcirculation. Cell death was assessed by uptake of trypan blue, a vital dye. Minimal dissection during harvest had no effects on sinusoidal lining cells; however, gentle organ manipulation dramatically increased trypan blue uptake about 5-fold (p <.05). In contrast, perfusion with N2-saturated buffer after cold storage totally prevented cell death due to manipulation. At harvest, portal venous pressure was increased significantly by 70% due to manipulation. Furthermore, vascular space and microcirculation were decreased by more than 50% (p <.05), reflecting the rate of entry and exit of fluorescein-dextran. Pimonidazole, a 2-nitroimidazole marker, was given to rats before harvest to detect hypoxia in liver. Pimonidazole adduct binding was increased significantly about 2-fold by manipulation. To detect free radical adducts by electron spin resonance (ESR) spectroscopy in bile, C-phenyl-N-tert-butylnitrone was given as spin trapping reagent to the donor before operation. Free radical formation was increased about 3-fold by organ manipulation (p <.05). Donors given gadolinium chloride, a selective Kupffer cell toxicant, or dietary glycine, which prevents activation of Kupffer cells, significantly blunted microcirculatory disturbances, hypoxia, and death of endothelial lining cells. These data indicate for the first time that gentle organ manipulation during harvest causes oxygen-dependent reperfusion injury to endothelial lining cells via mechanisms involving hepatic microcirculation, hypoxia, and Kupffer cells.

Graft survival is improved by hepatic denervation before organ harvesting.

BACKGROUND: In a recent study, disturbances of hepatic microcirculation at harvesting caused by in situ organ manipulation dramatically reduced survival after a liver transplant. Because hepatic innervation is involved in the regulation of liver hemodynamics, the effect of denervation before harvesting was assessed here. METHODS: The livers were harvested from female Lewis rats (200-230 g) within 25 min. Briefly, after minimal dissection during the first 12 min, the livers were either manipulated gently or left alone for 13 min. Subsequently, an orthotopic liver transplant was performed after 1 hr of storage in cold UW solution. Some donors livers underwent microsurgical denervation before harvesting or rats were given hexamethonium (10 mg/kg, i.v.), a ganglionic blocking agent. RESULTS: In the nonmanipulated group, survival was 100% after the transplant; however, gentle manipulation decreased survival by about 50%. Furthermore, manipulation elevated serum transaminases and bilirubin 6- to 8-fold 8 hr after the transplant and caused necrosis of about 25% of hepatocytes. After organ harvesting, the rate of entry and exit of fluorescein dextran, a dye confined to the vascular space, was decreased 2- to 4-fold, and the maximal increase of surface fluorescence was blunted about 2-fold. Pimonidazole binding, which reflects tissue hypoxia, was increased 2-fold by manipulation. Denervation of the liver before organ harvesting or treatment with hexamethonium prevented the effects of organ manipulation on all parameters studied. CONCLUSION: These data indicate for the first time that hepatic denervation before organ harvesting prevents detrimental effects of brief, gentle manipulation of the liver during harvesting on survival after the transplant. This is consistent with the hypothesis that organ manipulation disturbs the hepatic microcirculation and causes hypoxia at harvesting using mechanisms dependent on innervation.

Dietary glycine inhibits the growth of B16 melanoma tumors in mice.

Dietary glycine inhibited hepatocyte proliferation in response to the carcinogen WY-14,643. Since increased cell replication is associated with hepatic cancer caused by WY-14,643, glycine may have anti-cancer properties. Therefore, these experiments were designed to test the hypothesis that dietary glycine would inhibit the growth of tumors arising from B16 melanoma cells implanted subcutaneously in mice. C57BL/6 mice were fed diet supplemented with 5% glycine and 15% casein or control diet (20% casein) for 3 days prior to subcutaneous implantation of B16 tumor cells. Tumor volume was estimated from tumor diameter for 14 days. Tumors were excised, weighed and sectioned for histology post-mortem. B16 cells and endothelial cells were cultured in vitro to assess effects of glycine on cell growth. Statistical tests were two-sided and a P-value of 0.05 was defined as a significant difference between groups. Weight gain did not differ between mice fed control and glycine-containing diets. B16 tumors grew rapidly in mice fed control diet; however, in mice fed glycine diet, tumor size was 50-75% less. At the time of death, tumors from glycine-fed mice weighed nearly 65% less than tumors from mice fed control diet (P < 0.05). Glycine (0.01-10 mM) did not effect growth rates of B16 cells in vitro. Moreover, tumor volume and mitotic index of B16 tumors in vivo did not differ 2 days after implantation when tumors were small enough to be independent of vascularization. After 14 days, tumors from mice fed dietary glycine had 70% fewer arteries (P < 0.05). Furthermore, glycine (0.01-10 mM) inhibited the growth of endothelial cells in vitro in a dose-dependent manner (P < 0.05; IC50 = 0.05 mM). These data support the hypothesis that dietary glycine prevents tumor growth in vivo by inhibiting angiogenesis through mechanisms involving inhibition of endothelial cell proliferation.

Intravenous glycine improves survival in rat liver transplantation.

In situ manipulation by touching, retracting, and moving liver lobes gently during harvest dramatically reduces survival after transplantation (P. Schemmer, R. Schoonhoven, J. A. Swenberg, H. Bunzendahl, and R. G. Thurman. Transplantation 65: 1015-1020, 1998). The development of harvest-dependent graft injury upon reperfusion can be prevented with GdCl3, a rare earth metal and Kupffer cell toxicant, but it cannot be used in clinical liver transplantation because of its potential toxicity. Thus the effect of glycine, which prevents activation of Kupffer cells, was assessed here. Minimal dissection of the liver for 12 min plus 13 min without manipulation had no effect on survival (100%). However, gentle manipulation decreased survival to 46% in the control group. Furthermore, serum transaminases and liver necrosis were elevated 4- to 12-fold 8 h after transplantation. After organ harvest, the rate of entry and exit of fluorescein dextran, a dye confined to the vascular space, was decreased about twofold, indicating disturbances in the hepatic microcirculation. Pimonidazole binding, which detects hypoxia, increased about twofold after organ manipulation, and Kupffer cells isolated from manipulated livers produced threefold more tumor necrosis factor-alpha after lipopolysaccharide than controls. Glycine given intravenously to the donor increased the serum glycine concentration about sevenfold and largely prevented the effect of gentle organ manipulation on all parameters studied. These data indicate for the first time that pretreatment of donors with intravenous glycine minimizes reperfusion injury due to organ manipulation during harvest and after liver transplantation.

Gentle in situ liver manipulation during organ harvest decreases survival after rat liver transplantation: role of Kupffer cells.

BACKGROUND: The etiology of primary graft nonfunction and dysfunction is unknown but most likely involves Kupffer cell-dependent reperfusion injury. However, the donor operation and surgical technique may also have an effect on the outcome after transplantation. Because liver manipulation during harvest cannot be prevented completely with standard procedures, its effect on survival was assessed here. METHODS: Donor livers were harvested from female Sprague-Dawley rats (200-230 g). Briefly, after minimal dissection during the first 12 min, livers were either manipulated gently or left alone for 13 subsequent minutes. At 25 min, all livers were perfused with cold University of Wisconsin solution via the portal vein, and transplantation was performed after cold storage (1 hr). In some rats, Kupffer cells were destroyed with gadolinium chloride or inactivated with dietary glycine before harvest. Survival, proteolytic activity in the rinse effluent, serum transaminases, trypan blue distribution to index microcirculation, and histology were compared. RESULTS: In the nonmanipulated group, survival was 100% after transplantation; however, gentle manipulation decreased survival by 70%. Further, manipulation elevated transaminases fivefold and caused about 200% necrosis. At harvest, proteolytic activity and the time for trypan blue to distribute homogeneously were elevated three- to eightfold by manipulation. Gadolinium chloride and glycine prevented the effects of manipulation on all parameters studied. CONCLUSION: These data indicate for the first time that brief, gentle manipulation of the donor liver has a marked detrimental effect on survival by priming or activating Kupffer cells. This may represent an important early event in pathogenesis, because Kupffer cells play an important role in primary graft nonfunction.

Kupffer cell-dependent reperfusion injury in liver transplantation: new clinically relevant use of glycine.

Kupffer cell-dependent reperfusion injury occurs to the liver following transplantation, most often in fatty livers which fail most frequently due to primary nonfunction. Failure was largely blocked with Carolina rinse solution, which contains glycine, and prevents the activation of Kupffer cells. Furthermore, gentle in situ organ manipulation, which cannot be prevented using standard harvesting techniques, has a detrimental effect on survival. These effects were also prevented by glycine. Since proteolytic activity is increased in both fatty and manipulated livers, amino acids were measured in rinse effluents collected at harvest. A combination of four amino acids correlated with graft function. It is concluded that glycine could be beneficial in clinical liver transplantation to prevent reperfusion injury, and that amino acids measured at harvest may predict graft function.