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Hepatic energy booster effect and lipo-modulatory effect on postreperfusional endotoxemia by intraoperative lipid infusion in porcine liver transplantation
Hepatology Research 17 (2000) 72 – 82 www.elsevier.com/locate/ihepcom
Short communication
Hepatic energy booster effect and lipo-modulatory effect on postreperfusional endotoxemia by intraoperative lipid infusion in porcine liver transplantation Takahito Yagi *, Takashi Ishikawa, Masahiro Oishi, Hiroaki Matsuda, Akira Endo, Hiroyoshi Matsukawa, Atsunori Nakao, Yutaka Okada, Hiroshi Sadamori, Masaru Inagaki, Norihisa Takakura, Noriaki Tanaka First Department of Surgery, Okayama Uni6ersity Medical School, 2 -5 -1 Shikata-Cyo, Okayama City, Okayama 700 -8558, Japan Received 21 April 1999; received in revised form 8 July 1999; accepted 16 July 1999
Abstract We investigated whether intraoperative infusion of fat emulsion could suppress postoperative endotoxemia and improve energy status in the transplanted liver graft using a swine orthotopic liver transplantation (OLT) model. Intraoperative free fatty acid (FFA) concentration, hepatic FFA clearance, serum hyaluronic acid levels, ATP content and hepatic energy charge (HEC) in liver grafts, postreperfusional endotoxin levels and recipient outcome were compared between a fat emulsion-treated group (LCT-treated group, n= 6) and a saline infused-group (control group, n= 7). Independent of massive surgical stress and administration of heparin, FFA concentration was significantly elevated in the LCT-treated group (P= 0.003). Since FFA clearance, ATP contents and HEC were also increased in the LCT-treated group (P =0.024, 0.006, 0.005, respectively), intraoperative LCT-treatment was shown to increase FFA concentration and improve energy status of liver grafts. Because
* Corresponding author. Tel.: +81-86-235-7257; fax: +81-86-221-8775. E-mail address: [email protected] (T. Yagi) 1386-6346/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 3 8 6 - 6 3 4 6 ( 9 9 ) 0 0 0 5 6 - X
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there were no significant differences in postoperative hyaluronic acid levels, infused fat emulsion (0.1 g/kg per hour) prevented endothelial cell injury. Significant improvement of postoperative endotoxemia was obtained at 1 and 2 h after reperfusion (P =0.01 and 0.003, respectively). Energy booster effect and antiseptic effect led to prolonged survival of recipients in the LCT-treated group (28.5 913.3 days, P= 0.013 vs. control). We concluded that the hepatic energy booster effect and the lipo-modulatory effect on postreperfusional endotoxemia caused by intraoperative infusion of fat emulsion may be of great use in human liver transplantation. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Liver transplantation; Lipid emulsion; ATP; Energy charge; Endotoxin; Free fatty acid; Clearance
1. Introduction We previously reported that reperfused liver grafts require excessive FFA as an energy source when the graft is exposed to mild–moderate reperfusion injury [1,2]. Synthetic lipid emulsion, which is an extrinsic source of FFA, was recently reported by Read et al. to have a potent antiendotoxic property [3]. The accepted concept that lipid emulsion as a foreign body impairs non-perenchymal cell functions and leads to irreversible hepatic injury, is once again debatable. Liver transplantation recipients are inevitably exposed to iatrogenic ‘spill over endotoxemia’ because they have complete porta-caval shunts during the anhepatic-phase. Since a plasma endotoxin value of \100 pg/ml in the anhepatic phase was reported to be a strong risk factor for postoperative graft death in human liver transplantation in the Pittsburgh series [4], suppression of intraoperative endotoxemia was supposed to provide some favorable effects on the outcome of liver transplantation recipients. Therefore, we investigated whether intraoperative administration of synthetic lipid emulsion could prevent ‘spill over endotoxemia’ during transplantation surgery and improve the energy status in the graft using a pig OLT model.
2. Materials and methods
2.1. Transplantation surgery and protocol Thirteen pairs of female large-white pigs, aged 4–6 months and weighing 20–30 kg, were used as donors and recipients. After 9 h of fasting for both donors and recipients, OLT was performed. After irrigation with cold (4°C) Euro-Collins’ solution (EC), retrieved grafts were cold-preserved in EC for 4 h. The recipients were divided into two groups according to intraoperative lipid emulsion treatment. In group 1 (control group, n = 7), saline was administered at 0.5 ml/kg per hour from 30 min prior to reperfusion up to 60 min after reperfusion. In group 2 (LCT-treated group, n =6), an emulsion of long-chain triglycerides (20% LCTs; Intralipid®; Otsuka Pharmaceutical, Tokyo, Japan) was administered instead of saline (0.5 ml/kg per hour). In both groups, intraoperative blood glucose level was
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monitored every 45 min and maintained at 150–200 mg/dl by continuous infusion of 10% glucose solution. In order to keep FFA induction from the peripheral adipose tissues, insulin was not used during collection of blood samples in this study. None of the recipients manifested life-threatening hyperglycemia during transplant surgery. Before induction of veno-venous bypass using the centrifugal pump in the anhepatic phase, recipients were given 100 U/kg of heparin sodium as an anticoagulant for use in non-heparin-coated bypass circuit. Extracorporeal circulation was maintained at a flow rate of 25 ml/min per kilogram. No immunosuppressive reagents, except for bolus steroids and heparin antagonists, were used in the present study. Cephazolin sodium (20 mg/kg) was administered up to postoperative day 2 for prophylaxis of postoperative infection.
2.2. E6aluation Serial serum FFA levels were measured and compared in the two groups. Free fatty acid levels in the portal vein (PFFA, mEq/l) and in the hepatic vein (VFFA, mEq/l) were also measured in order to calculate hepatic FFA clearance (CFFA), as shown in Fig. 1, which reflected hepatic FFA consumption as an energy source. CFFA was defined as CFFA = (100− Hct)/100 × PBF× (PFFA − VFFA), where PBF is the portal blood flow (ml/min). PBF was measured using ultrasound transit time flowprobes and a flowmeter (Transonic Systems, Ithaca, NY). Samples from the hepatic and portal veins were obtained via a liver-wedged infantile Swan-Gantz catheter and a cannula into the ileo-cecal vein. The serum asparate aminotransferase (AST) and hyarulonic acid (HA) levels were determined as functional indices of hepatocyte (HC) and sinusoidal endothelial cell (SEC) damage, respectively.
Fig. 1. Serum FFA levels in both groups. The open triangle connected to a broken line and the chain line connecting closed squares demonstrate the LCT-treated group and the control group, respectively. The dark hatched area indicates the duration of LCT-administration (30 min before to 60 min after reperfusion). The black arrow indicates intravenous, bolus injection of 100 U/kg heparin sodium in both groups. Heparin-independent, significant elevation of serum FFA levels was observed in the LCT-treated group (P= 0.003, by repeated ANOVA). aRPF, after reperfusion
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Serial biopsy specimens \ 1 cm3 were obtained from the graft before organ harvesting, during cold preservation and 1 h after reperfusion. The concentrations of adenin nucleotides in the liver graft were determined in perchloric acidextracted tissues. They were separated from other nucleotides by high-performance liquid chromatography and the concentrations were determined by comparison of absorbance with a standard. HEC was calculated as (ATP+ ADP/2)/AMP +ADP + ATP. Plasma endotoxin assay was performed by the Endospecy method using Endotoxin test® kit (Wako Pure Chemical Industries, Osaka, Japan). Autopsy and postmortem blood sampling from the right atrium were immediately commenced after death of the recipients. All atrial blood samples and surgically diagnosed abscess formation and/or inflammatory tissues were cultured for diagnosis of septic complication. All liver grafts were removed and histologically investigated in order to detect the presence of acute rejection. The individual which had an optimal body weight loss \ 20% without rejection and proof of septic events was diagnosed as having emerciation.
2.3. Statistical analysis Values are presented as means9 S.D. The significance of differences was tested using repeated ANOVA and Student’s t-test. Findings of PB 0.05 indicated significant difference.
3. Results
3.1. FFA le6els in peripheral blood The mean preanhepatic FFA level was 0.4579 0.28 mEq/l. In the anhepatic phase, serum FFA levels in the control group did not increase despite a 100 U/kg heparin challenge, whereas in the LCT-treated group, FFA levels significantly increased (0.2790.13 vs. 1.45 9 0.38 mEq/l, Fig. 1). Significant elevation of FFA levels in the LCT treated group lasted for up to 60 min after reperfusion (0.4190.13 vs. 1.019 0.16 mEq/l, P=0.003).
3.2. CFFA (li6er FFA clearance) FFA clearance of the liver graft was significantly increased in the LCT-treated group soon after reperfusion (up to 2 h, P= 0.024). However, 6 h after reperfusion, there was no significant difference in CFFA between the two groups. Post reperfusional CFFA was rapidly reduced during the first 12 h and gradually decreased thereafter (Fig. 2).
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Fig. 2. Changes in calculated serial CFFA values. Postreperfusional CFFA levels in the control group (n = 5, open bar) and LCT-treated group (n =5, closed bar) were demonstrated. * P =0.024.
3.3. AST and hyarulonic acid le6els In the LCT-treated group, elevation of serum AST levels 6 h after reperfusion was significantly improved compared with that in the control group (4539 59 vs. 671 9147 IU/l, P =0.006). However there were no significant differences in hyaruronic acid levels between the two groups (Table 1).
3.4. ATP content and HEC The mean ATP content markedly decreased during 4 h of cold preservation in all groups. Levels decreased to :30% that of the original ATP levels during 4 h of cold preservation. Significant improvement in ATP content and HEC in LCTtreated grafts was observed 1 h after reperfusion, (0.62 9 0.18 vs. 0.349 0.08, P=0.0064, Table 2).
Table 1 Serial change in AST and hyaluronic acid levels after reperfusion Group
Time after reperfusion (h) 1
Serum AST (IU/l) Control LCT Hyaluronic acid (ng/ml) Control LCT
2
341978 3029 84
384 993 296 970
16559773 13749873
1163 9667 862 9562
6
12
671 9147 453 9 59
1085 9 260 945 9277
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Table 2 Serial change in ATP content (mmol/g liver) and hepatic energy charge (HEC) in liver grafts Group
Before retrieval
Cold preservation (4 h)
1 h after reperfusion
ATP Control LCT-treated
806.89 136.4 923.49 321
218 964.6 231.2 9 73.9
336 979.1 623.2 9180.6a
HEC Control LCT-treated
0.3919 0.062 0.4559 0.053
0.260 9 0.060 0.263 90.061
0.336 90.051 0.477 90.032b
a b
P= 0.0064 versus control. P= 0.005 versus control.
3.5. Plasma endotoxin le6els Since plasma endotoxin values in the anhepatic phase in both groups were scattered over an extremely wide range, serial comparison of changes in plasma endotoxin levels was abandoned. After reperfusion, plasma endotoxin values began to decrease and converge in an assessable range. Both plasma endotoxin levels 1 and 2 h after reperfusion were significantly suppressed in the LCT-treated group (202.6 9149 vs. 29.3927.3, P =0.011 and 45.79 10.2 vs. 13.69 5.6, P= 0.003, Fig. 3).
3.6. Sur6i6al of recipients In the LCT-treated group, mean survival time was significantly prolonged in comparison with that of the control group (28.5 9 13.3 vs. 10.3 9 8.7 days, P = 0.013). Survival rates of 2 weeks in the LCT-treated and control groups were 83.3
Fig. 3. Plasma endotoxin levels 1 and 2 h after reperfusion. In comparison with the control group (n = 7, closed bar), postreperfusional endotoxemia was significantly improved in the LCT-treated group (n = 6, open bar). * P= 0.011; ** P= 0.003 vs. control.
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78 Table 3 Survival time Group
Survival (days)
MST (days)
2-week survival rate (%)
Control LCT
4, 7, 16, 17, 24a 8, 23, 29, 30, 32, 49
13.0 97.2* 28.5 913.3*
60 83.3
a
Two animals that died within 3 days were excluded in the control group. * P= 0.0452.
and 42.9%, respectively. Four recipients achieved 4-week survival in the LCTtreated group, while there were no 4-week survivors in the control group (Table 3). In the control group, three pigs died of septic events (two sepsis, one pneumonia) and the other two recipients died of acute rejection. But in the LCT-treated group, only one pig died of septic events and the most common cause of death was postoperative emerciation (Table 4).
4. Discussion Hydrolysis of fat emulsion is essential for the degradation of triglycerides before further metabolism. The activity of lipoprotein lipase (LPL) on capillary endothelial cells plays a key role in the catalysis of exogenous triglycerides. There are two types of lipase which are involved in the degradation of triglycerides. One is well known as a hormone (insulin)-sensitive lipase in adipocytes and contributes to the degradation of pooled triglycerides and FFA induction into the blood stream. Insulin inhibition of the enzyme terminates the release of FFAs from adipose tissue and subsequently decreases the levels of circulating FFAs [5]. The other is known as a heparin-releasable lipase and is responsible for the hydrolysis of triglyceride-rich particles in the bloodstream and heparin-induced hyper free fatty acidemia [6]. Serum FFA levels in the control group did not rise, despite a 100 U/kg heparin challenge and massive surgical stress induced by transplantation surgery and neither LPL was shown to have been activated during the operation due to maintenance of blood glucose levels by continuous infusion of a 10% glucose solution. Significant elevation of FFA levels in the LCT-treated group, which were sustained for the duration of LCT administration, was derived from FFAs degraded from exogenous LCTs. FFA, one of the major energy substrates for various organs, has a very short half-life (1.7 – 3.1 min) and turnover ratio (23–41% per minute). A total of 20–40% of FFAs were trapped and used by the liver as basic components for de novo triglycerides or phospholipid synthesis and as a source of ketone bodies, which are utilized as an energy source essentially in the muscle. We previously reported that reperfused livers demand a large amount of energy substrates, including intracellular glycogen deposits and FFAs, after cold preservation [1,2]. Although the arterial ketone body ratio (AKBR) is an index of direct energetic substrates from FFAs [7],
No.
Survival (days)
Control 1 4 2 7 3 16 4 17 5 24 LCT-treated 6 8 7 23 8 29 9 30 10 32 11 49
Plasma endotoxin levels at 1 h after reperfusion
Cause of death in autopsy
Proven micro-organisms in blood or tissue
195 168 498 146 121
Sepsis Sepsis Acute rejection Acute rejection Pneumonia
Enterococcus a6ium Acinetobacter spp.
Pneumonia Acute rejection Emerciation Unknown Emerciation Emerciation
Pseudomonous spp.
21 22.8 7.8 15.7 16.8 32.4
Infectious death ratio (%)
60 Xanthomonoas maltphilia
16.70
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Table 4 Cause of death in autopsy
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it failed to assess energy metabolism in the reperfused graft because of its instability in the present study (data not shown). Increased serum FFA levels, hepatic CFFA and improvement of parenchymal ATP content/HEC in the LCT-treated group suggested that the function of intraoperatively administered LCTs might be glucose- and insulin-independent and an ‘energy booster’ for the reperfused liver graft in OLT. Hepatic impairment in total parenteral nutrition (TPN) is commonly reported [8] and it has been suggested that exogenous LCTs is one of the key substances for the injury [9]. However, some investigators reported that the infusion of triacylglycerol emulsions at a rate of :0.1 g/kg of body weight per hour seems optimal to prevent reticulo-endothelial dysfunction and the inhibition of the essential functions of HCs [10]. In the present study, LCTs were administered at a rate of 0.5 ml/kg per hour (0.1 g/kg per hour) in order to promote their endotoxin-absorbing effect. However, the fact that there were no differences between the two groups in hyaluronic acid levels after reperfusion reconfirmed that 90 min of LCT administration at these rates did not cause endothelial dysfunction. Forced administration of glucose-rich solution using high-dose insulin suppresses FFA release from peripheral adipose tissues. Therefore, muscles and liver that ordinarily metabolize FFAs as energy sources are starved in such conditions. Since the ‘energy booster’ effect of FFA was not affected by blood sugar level, hormonal environment or the lipo-degrading drug, LCTs may also be helpful energy substrates for types of hepatic injury other than reperfusion injury, e.g. cirrhosis. The adversability of using lipid emulsion in TPN for cirrhotic patients is still controversial, but a considerable amount of fat as emulsion can be hydrolyzed by patients with hepatic cirrhosis [11]. However, care should be taken that cirrhotic patients demonstrate a significantly lower level of plasma apolipoproteins. In an endotoxin challenge rat model, Read et al. reported that synthetic triglyceride-rich lipid emulsions significantly improve the survival of rats when given after a lethal dose of endotoxin. LCTs, like chylomicron, protect animals from endotoxic death via two complementary mechanisms: (1) by binding endotoxin and inhibiting its activity directly; and (2) by accelerating endotoxin clearance by HCs. Chylomicron- or LCT-endotoxin complexes are rapidly removed from plasma by HCs, whereas unbound endotoxin is cleared more slowly by Kupffer cells [3]. Endotoxins immediately bind to lipopolysaccharides binding protein (LPSBP) and are usually trapped by Kupffer cells, which express CD14, a ligand of LPS-LPSBP complex. This CD14-Kupffer cell mediated pathway is a major disposal system that manages 90% of LPS in the blood stream. However, apolipoprotein-binded LCTs not only inactivate LPS-LPSBP complex as absorbents, but also increase the disposal activity of HCs. After administration of a favorable amount of LCTs, HCs are able to trap the complexes and excrete them via the biliary tract [12,13]. It is interesting that the LPS-disposal rate, reported by Read et al., was similar to our endotoxin rate between the control and LCT-treated groups at 1 h after reperfusion (1:9). In our study, plasma endotoxin levels in the anhepatic phase were unstable (156 to \20 000 pg/ml) and did not correlate with recipient outcome as reported in humans. The discrepancy in outcomes between our study and the Pittsburgh series
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may have been due to our shorter preservation time (4 h) and higher graft viability. The anhepatic phase in liver transplantation exposes recipients to an unavoidable, iatrogenic spillover endotoxemia. Therefore, administration of LCTs during liver transplantation surgery may be a potent and convenient protective procedure from septic complications among recipients. Surprisingly, recipients in the LCT-treated group had a definitely longer mean survival time (\4 weeks) compared with the control group. These facts are not explained by the aforementioned beneficial effects of LCTs in hepatic energy status and endotoxemia because these occurred in the acute phase reaction. Since infection-related death ratio in the LCT-treated group was lower than that in the control group, it was recognised that LCT-treatment was contributing to the avoidance of lethal septic complications in early after OLT. Moreover, incidence of optimal rejection was low even in long survivors in the LCT-treated group. Any immunosuppressants, except for intraoperative steroids, were not administered to the recipients, therefore, a kind of immunosuppressive activity of LCTs by which early phase-survivors were protected from rejection-related hepatic failure in late after OLT was suspected. Recent reports revealed an immunosuppressive effect of LCTs caused a fall of lymphokine-activated killer activity in LCT-treated patients by inhibition of the binding of IL-2 to its receptor [14,15]. Although individual allogenic reactivity between the paired animals was not assessed, the initial immunosuppressive effect of LCTs on the reperfused liver graft might contribute to prolonged survival of recipients in the LCT-treated group. In conclusion, supplementation of lipid emulsion in the peri-anhepatic phase induced a better energy status in liver grafts without SEC damage, improvement of serum endotoxin levels and better recipient outcome.
References [1] Ishikawa T, Yagi T, Ishine N, Sadamori H, Sasaki H, et al. Energy metabolism of the grafted liver and influence of preretrieval feeding process on swine orthotopic liver transplantation. Transplant Proc 1997;29(Suppl. 1–2):397 – 9. [2] Ishikawa T, Yagi T, Sadamori H, Ishine N, Sasaki H, et al. Kupffer cell activation in the survival discrepancy between liver grafts from enterally and parenterally fed donors. Transplant Int 1998;(Suppl. 11):410–416. [3] Read TE, Grunfeld C, Kumwenda Z, Calhoun MC, Kane JP, et al. Triglyceride-rich lipoproteins improve survival when given after endotoxin in rats. Surgery 1995;117(Suppl. 1):62 – 7. [4] Yokoyama I, Todo S, Miyata T, Selby R, Tzakis AG, et al. Endotoxemia and human liver transplantation. Transplant Proc 1989;21(Suppl. 5):3833 – 41. [5] Lewis GF, Uffelman KD, Szeto LW, Weller B, Steiner G. Interaction between free fatty acids and insulin in the acute control of very low density lipoprotein production in humans. J Clin Invest 1995;95(Suppl. 1):158–66. [6] Chen X, Ruiz J, Boden G. Release, oxidation and reesterification of fatty acids from infused triglycerides: effect of heparin. Metabolism 1995;44(Suppl. 12):1590 – 5. [7] Yamamoto Y, Ozawa K, Okamoto R, Kiuchi T, Maki A, et al. Prognostic implications of postoperative suppression of arterial ketone body ratio: time factor involved in the suppression of hepatic mitochondrial oxidation-reduction state. Surgery 1990;107(Suppl. 3):289 – 94.
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[8] Hamawy KJ, Moldawer LL, Georgieff M, Valicenti AJ, Babayan VK, et al. The Henry M. Vars Award. The effect of lipid emulsions on reticuloendothelial system function in the injured animal. Jpn J Parenter Enteral Nutr 1985;9(Suppl. 5):559 – 65. [9] Nugent KM. Intralipid effects on reticuloendothelial function. J Leukoc Biol 1984;36(Suppl. 2):123–32. [10] Iriyama K, Carpentier YA. Clinical significance of transfer of apolipoproteins between triacylglycerol-rich particles in lipid emulsions and plasma lipoproteins. Nutrition 1994;10(Suppl. 3):252 – 4. [11] Muscaritoli M, Cangiono C, Cascino A, Caputo V, Serra P, et al. Exogenous lipid clearance in compensated liver cirrhosis. JEPN 1986;10(Suppl. 6):599 – 603. [12] Harris HW, Grunfeld C, Feingold KR, Read TE, Kane JP, et al. Chylomicrons alter the fate of endotoxin, decreasing tumor necrosis factor release and preventing death. J Clin Invest 1993;91(Suppl. 3):1028–34. [13] Read TE, Harris HW, Grunfeld C, Feingold KR, Calhoun MC, et al. Chylomicrons enhance endotoxin excretion in bile. Infect Immunol 1993;61(Suppl. 8):3496 – 502. [14] Sedman PC, Ramsden CW, Brennan TG, Guillou PJ. Pharmacological concentrations of lipid emulsions inhibit interleukin- 2-dependent lymphocyte responses in vitro. Jpn J Parenter Enteral Nutr 1990;14(Suppl. 1):12–7. [15] Sirota L, Straussberg R, Notti I, Bessler H. Effect of lipid emulsion on IL – 2 production by mononuclear cells of newborn infants and adults. Acta Paediatr 1997;86(Suppl. 4):410 – 3.
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Short communication
Hepatic energy booster effect and lipo-modulatory effect on postreperfusional endotoxemia by intraoperative lipid infusion in porcine liver transplantation Takahito Yagi *, Takashi Ishikawa, Masahiro Oishi, Hiroaki Matsuda, Akira Endo, Hiroyoshi Matsukawa, Atsunori Nakao, Yutaka Okada, Hiroshi Sadamori, Masaru Inagaki, Norihisa Takakura, Noriaki Tanaka First Department of Surgery, Okayama Uni6ersity Medical School, 2 -5 -1 Shikata-Cyo, Okayama City, Okayama 700 -8558, Japan Received 21 April 1999; received in revised form 8 July 1999; accepted 16 July 1999
Abstract We investigated whether intraoperative infusion of fat emulsion could suppress postoperative endotoxemia and improve energy status in the transplanted liver graft using a swine orthotopic liver transplantation (OLT) model. Intraoperative free fatty acid (FFA) concentration, hepatic FFA clearance, serum hyaluronic acid levels, ATP content and hepatic energy charge (HEC) in liver grafts, postreperfusional endotoxin levels and recipient outcome were compared between a fat emulsion-treated group (LCT-treated group, n= 6) and a saline infused-group (control group, n= 7). Independent of massive surgical stress and administration of heparin, FFA concentration was significantly elevated in the LCT-treated group (P= 0.003). Since FFA clearance, ATP contents and HEC were also increased in the LCT-treated group (P =0.024, 0.006, 0.005, respectively), intraoperative LCT-treatment was shown to increase FFA concentration and improve energy status of liver grafts. Because
* Corresponding author. Tel.: +81-86-235-7257; fax: +81-86-221-8775. E-mail address: [email protected] (T. Yagi) 1386-6346/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 3 8 6 - 6 3 4 6 ( 9 9 ) 0 0 0 5 6 - X
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there were no significant differences in postoperative hyaluronic acid levels, infused fat emulsion (0.1 g/kg per hour) prevented endothelial cell injury. Significant improvement of postoperative endotoxemia was obtained at 1 and 2 h after reperfusion (P =0.01 and 0.003, respectively). Energy booster effect and antiseptic effect led to prolonged survival of recipients in the LCT-treated group (28.5 913.3 days, P= 0.013 vs. control). We concluded that the hepatic energy booster effect and the lipo-modulatory effect on postreperfusional endotoxemia caused by intraoperative infusion of fat emulsion may be of great use in human liver transplantation. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Liver transplantation; Lipid emulsion; ATP; Energy charge; Endotoxin; Free fatty acid; Clearance
1. Introduction We previously reported that reperfused liver grafts require excessive FFA as an energy source when the graft is exposed to mild–moderate reperfusion injury [1,2]. Synthetic lipid emulsion, which is an extrinsic source of FFA, was recently reported by Read et al. to have a potent antiendotoxic property [3]. The accepted concept that lipid emulsion as a foreign body impairs non-perenchymal cell functions and leads to irreversible hepatic injury, is once again debatable. Liver transplantation recipients are inevitably exposed to iatrogenic ‘spill over endotoxemia’ because they have complete porta-caval shunts during the anhepatic-phase. Since a plasma endotoxin value of \100 pg/ml in the anhepatic phase was reported to be a strong risk factor for postoperative graft death in human liver transplantation in the Pittsburgh series [4], suppression of intraoperative endotoxemia was supposed to provide some favorable effects on the outcome of liver transplantation recipients. Therefore, we investigated whether intraoperative administration of synthetic lipid emulsion could prevent ‘spill over endotoxemia’ during transplantation surgery and improve the energy status in the graft using a pig OLT model.
2. Materials and methods
2.1. Transplantation surgery and protocol Thirteen pairs of female large-white pigs, aged 4–6 months and weighing 20–30 kg, were used as donors and recipients. After 9 h of fasting for both donors and recipients, OLT was performed. After irrigation with cold (4°C) Euro-Collins’ solution (EC), retrieved grafts were cold-preserved in EC for 4 h. The recipients were divided into two groups according to intraoperative lipid emulsion treatment. In group 1 (control group, n = 7), saline was administered at 0.5 ml/kg per hour from 30 min prior to reperfusion up to 60 min after reperfusion. In group 2 (LCT-treated group, n =6), an emulsion of long-chain triglycerides (20% LCTs; Intralipid®; Otsuka Pharmaceutical, Tokyo, Japan) was administered instead of saline (0.5 ml/kg per hour). In both groups, intraoperative blood glucose level was
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monitored every 45 min and maintained at 150–200 mg/dl by continuous infusion of 10% glucose solution. In order to keep FFA induction from the peripheral adipose tissues, insulin was not used during collection of blood samples in this study. None of the recipients manifested life-threatening hyperglycemia during transplant surgery. Before induction of veno-venous bypass using the centrifugal pump in the anhepatic phase, recipients were given 100 U/kg of heparin sodium as an anticoagulant for use in non-heparin-coated bypass circuit. Extracorporeal circulation was maintained at a flow rate of 25 ml/min per kilogram. No immunosuppressive reagents, except for bolus steroids and heparin antagonists, were used in the present study. Cephazolin sodium (20 mg/kg) was administered up to postoperative day 2 for prophylaxis of postoperative infection.
2.2. E6aluation Serial serum FFA levels were measured and compared in the two groups. Free fatty acid levels in the portal vein (PFFA, mEq/l) and in the hepatic vein (VFFA, mEq/l) were also measured in order to calculate hepatic FFA clearance (CFFA), as shown in Fig. 1, which reflected hepatic FFA consumption as an energy source. CFFA was defined as CFFA = (100− Hct)/100 × PBF× (PFFA − VFFA), where PBF is the portal blood flow (ml/min). PBF was measured using ultrasound transit time flowprobes and a flowmeter (Transonic Systems, Ithaca, NY). Samples from the hepatic and portal veins were obtained via a liver-wedged infantile Swan-Gantz catheter and a cannula into the ileo-cecal vein. The serum asparate aminotransferase (AST) and hyarulonic acid (HA) levels were determined as functional indices of hepatocyte (HC) and sinusoidal endothelial cell (SEC) damage, respectively.
Fig. 1. Serum FFA levels in both groups. The open triangle connected to a broken line and the chain line connecting closed squares demonstrate the LCT-treated group and the control group, respectively. The dark hatched area indicates the duration of LCT-administration (30 min before to 60 min after reperfusion). The black arrow indicates intravenous, bolus injection of 100 U/kg heparin sodium in both groups. Heparin-independent, significant elevation of serum FFA levels was observed in the LCT-treated group (P= 0.003, by repeated ANOVA). aRPF, after reperfusion
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Serial biopsy specimens \ 1 cm3 were obtained from the graft before organ harvesting, during cold preservation and 1 h after reperfusion. The concentrations of adenin nucleotides in the liver graft were determined in perchloric acidextracted tissues. They were separated from other nucleotides by high-performance liquid chromatography and the concentrations were determined by comparison of absorbance with a standard. HEC was calculated as (ATP+ ADP/2)/AMP +ADP + ATP. Plasma endotoxin assay was performed by the Endospecy method using Endotoxin test® kit (Wako Pure Chemical Industries, Osaka, Japan). Autopsy and postmortem blood sampling from the right atrium were immediately commenced after death of the recipients. All atrial blood samples and surgically diagnosed abscess formation and/or inflammatory tissues were cultured for diagnosis of septic complication. All liver grafts were removed and histologically investigated in order to detect the presence of acute rejection. The individual which had an optimal body weight loss \ 20% without rejection and proof of septic events was diagnosed as having emerciation.
2.3. Statistical analysis Values are presented as means9 S.D. The significance of differences was tested using repeated ANOVA and Student’s t-test. Findings of PB 0.05 indicated significant difference.
3. Results
3.1. FFA le6els in peripheral blood The mean preanhepatic FFA level was 0.4579 0.28 mEq/l. In the anhepatic phase, serum FFA levels in the control group did not increase despite a 100 U/kg heparin challenge, whereas in the LCT-treated group, FFA levels significantly increased (0.2790.13 vs. 1.45 9 0.38 mEq/l, Fig. 1). Significant elevation of FFA levels in the LCT treated group lasted for up to 60 min after reperfusion (0.4190.13 vs. 1.019 0.16 mEq/l, P=0.003).
3.2. CFFA (li6er FFA clearance) FFA clearance of the liver graft was significantly increased in the LCT-treated group soon after reperfusion (up to 2 h, P= 0.024). However, 6 h after reperfusion, there was no significant difference in CFFA between the two groups. Post reperfusional CFFA was rapidly reduced during the first 12 h and gradually decreased thereafter (Fig. 2).
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Fig. 2. Changes in calculated serial CFFA values. Postreperfusional CFFA levels in the control group (n = 5, open bar) and LCT-treated group (n =5, closed bar) were demonstrated. * P =0.024.
3.3. AST and hyarulonic acid le6els In the LCT-treated group, elevation of serum AST levels 6 h after reperfusion was significantly improved compared with that in the control group (4539 59 vs. 671 9147 IU/l, P =0.006). However there were no significant differences in hyaruronic acid levels between the two groups (Table 1).
3.4. ATP content and HEC The mean ATP content markedly decreased during 4 h of cold preservation in all groups. Levels decreased to :30% that of the original ATP levels during 4 h of cold preservation. Significant improvement in ATP content and HEC in LCTtreated grafts was observed 1 h after reperfusion, (0.62 9 0.18 vs. 0.349 0.08, P=0.0064, Table 2).
Table 1 Serial change in AST and hyaluronic acid levels after reperfusion Group
Time after reperfusion (h) 1
Serum AST (IU/l) Control LCT Hyaluronic acid (ng/ml) Control LCT
2
341978 3029 84
384 993 296 970
16559773 13749873
1163 9667 862 9562
6
12
671 9147 453 9 59
1085 9 260 945 9277
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Table 2 Serial change in ATP content (mmol/g liver) and hepatic energy charge (HEC) in liver grafts Group
Before retrieval
Cold preservation (4 h)
1 h after reperfusion
ATP Control LCT-treated
806.89 136.4 923.49 321
218 964.6 231.2 9 73.9
336 979.1 623.2 9180.6a
HEC Control LCT-treated
0.3919 0.062 0.4559 0.053
0.260 9 0.060 0.263 90.061
0.336 90.051 0.477 90.032b
a b
P= 0.0064 versus control. P= 0.005 versus control.
3.5. Plasma endotoxin le6els Since plasma endotoxin values in the anhepatic phase in both groups were scattered over an extremely wide range, serial comparison of changes in plasma endotoxin levels was abandoned. After reperfusion, plasma endotoxin values began to decrease and converge in an assessable range. Both plasma endotoxin levels 1 and 2 h after reperfusion were significantly suppressed in the LCT-treated group (202.6 9149 vs. 29.3927.3, P =0.011 and 45.79 10.2 vs. 13.69 5.6, P= 0.003, Fig. 3).
3.6. Sur6i6al of recipients In the LCT-treated group, mean survival time was significantly prolonged in comparison with that of the control group (28.5 9 13.3 vs. 10.3 9 8.7 days, P = 0.013). Survival rates of 2 weeks in the LCT-treated and control groups were 83.3
Fig. 3. Plasma endotoxin levels 1 and 2 h after reperfusion. In comparison with the control group (n = 7, closed bar), postreperfusional endotoxemia was significantly improved in the LCT-treated group (n = 6, open bar). * P= 0.011; ** P= 0.003 vs. control.
T. Yagi et al. / Hepatology Research 17 (2000) 72–82
78 Table 3 Survival time Group
Survival (days)
MST (days)
2-week survival rate (%)
Control LCT
4, 7, 16, 17, 24a 8, 23, 29, 30, 32, 49
13.0 97.2* 28.5 913.3*
60 83.3
a
Two animals that died within 3 days were excluded in the control group. * P= 0.0452.
and 42.9%, respectively. Four recipients achieved 4-week survival in the LCTtreated group, while there were no 4-week survivors in the control group (Table 3). In the control group, three pigs died of septic events (two sepsis, one pneumonia) and the other two recipients died of acute rejection. But in the LCT-treated group, only one pig died of septic events and the most common cause of death was postoperative emerciation (Table 4).
4. Discussion Hydrolysis of fat emulsion is essential for the degradation of triglycerides before further metabolism. The activity of lipoprotein lipase (LPL) on capillary endothelial cells plays a key role in the catalysis of exogenous triglycerides. There are two types of lipase which are involved in the degradation of triglycerides. One is well known as a hormone (insulin)-sensitive lipase in adipocytes and contributes to the degradation of pooled triglycerides and FFA induction into the blood stream. Insulin inhibition of the enzyme terminates the release of FFAs from adipose tissue and subsequently decreases the levels of circulating FFAs [5]. The other is known as a heparin-releasable lipase and is responsible for the hydrolysis of triglyceride-rich particles in the bloodstream and heparin-induced hyper free fatty acidemia [6]. Serum FFA levels in the control group did not rise, despite a 100 U/kg heparin challenge and massive surgical stress induced by transplantation surgery and neither LPL was shown to have been activated during the operation due to maintenance of blood glucose levels by continuous infusion of a 10% glucose solution. Significant elevation of FFA levels in the LCT-treated group, which were sustained for the duration of LCT administration, was derived from FFAs degraded from exogenous LCTs. FFA, one of the major energy substrates for various organs, has a very short half-life (1.7 – 3.1 min) and turnover ratio (23–41% per minute). A total of 20–40% of FFAs were trapped and used by the liver as basic components for de novo triglycerides or phospholipid synthesis and as a source of ketone bodies, which are utilized as an energy source essentially in the muscle. We previously reported that reperfused livers demand a large amount of energy substrates, including intracellular glycogen deposits and FFAs, after cold preservation [1,2]. Although the arterial ketone body ratio (AKBR) is an index of direct energetic substrates from FFAs [7],
No.
Survival (days)
Control 1 4 2 7 3 16 4 17 5 24 LCT-treated 6 8 7 23 8 29 9 30 10 32 11 49
Plasma endotoxin levels at 1 h after reperfusion
Cause of death in autopsy
Proven micro-organisms in blood or tissue
195 168 498 146 121
Sepsis Sepsis Acute rejection Acute rejection Pneumonia
Enterococcus a6ium Acinetobacter spp.
Pneumonia Acute rejection Emerciation Unknown Emerciation Emerciation
Pseudomonous spp.
21 22.8 7.8 15.7 16.8 32.4
Infectious death ratio (%)
60 Xanthomonoas maltphilia
16.70
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Table 4 Cause of death in autopsy
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it failed to assess energy metabolism in the reperfused graft because of its instability in the present study (data not shown). Increased serum FFA levels, hepatic CFFA and improvement of parenchymal ATP content/HEC in the LCT-treated group suggested that the function of intraoperatively administered LCTs might be glucose- and insulin-independent and an ‘energy booster’ for the reperfused liver graft in OLT. Hepatic impairment in total parenteral nutrition (TPN) is commonly reported [8] and it has been suggested that exogenous LCTs is one of the key substances for the injury [9]. However, some investigators reported that the infusion of triacylglycerol emulsions at a rate of :0.1 g/kg of body weight per hour seems optimal to prevent reticulo-endothelial dysfunction and the inhibition of the essential functions of HCs [10]. In the present study, LCTs were administered at a rate of 0.5 ml/kg per hour (0.1 g/kg per hour) in order to promote their endotoxin-absorbing effect. However, the fact that there were no differences between the two groups in hyaluronic acid levels after reperfusion reconfirmed that 90 min of LCT administration at these rates did not cause endothelial dysfunction. Forced administration of glucose-rich solution using high-dose insulin suppresses FFA release from peripheral adipose tissues. Therefore, muscles and liver that ordinarily metabolize FFAs as energy sources are starved in such conditions. Since the ‘energy booster’ effect of FFA was not affected by blood sugar level, hormonal environment or the lipo-degrading drug, LCTs may also be helpful energy substrates for types of hepatic injury other than reperfusion injury, e.g. cirrhosis. The adversability of using lipid emulsion in TPN for cirrhotic patients is still controversial, but a considerable amount of fat as emulsion can be hydrolyzed by patients with hepatic cirrhosis [11]. However, care should be taken that cirrhotic patients demonstrate a significantly lower level of plasma apolipoproteins. In an endotoxin challenge rat model, Read et al. reported that synthetic triglyceride-rich lipid emulsions significantly improve the survival of rats when given after a lethal dose of endotoxin. LCTs, like chylomicron, protect animals from endotoxic death via two complementary mechanisms: (1) by binding endotoxin and inhibiting its activity directly; and (2) by accelerating endotoxin clearance by HCs. Chylomicron- or LCT-endotoxin complexes are rapidly removed from plasma by HCs, whereas unbound endotoxin is cleared more slowly by Kupffer cells [3]. Endotoxins immediately bind to lipopolysaccharides binding protein (LPSBP) and are usually trapped by Kupffer cells, which express CD14, a ligand of LPS-LPSBP complex. This CD14-Kupffer cell mediated pathway is a major disposal system that manages 90% of LPS in the blood stream. However, apolipoprotein-binded LCTs not only inactivate LPS-LPSBP complex as absorbents, but also increase the disposal activity of HCs. After administration of a favorable amount of LCTs, HCs are able to trap the complexes and excrete them via the biliary tract [12,13]. It is interesting that the LPS-disposal rate, reported by Read et al., was similar to our endotoxin rate between the control and LCT-treated groups at 1 h after reperfusion (1:9). In our study, plasma endotoxin levels in the anhepatic phase were unstable (156 to \20 000 pg/ml) and did not correlate with recipient outcome as reported in humans. The discrepancy in outcomes between our study and the Pittsburgh series
T. Yagi et al. / Hepatology Research 17 (2000) 72–82
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may have been due to our shorter preservation time (4 h) and higher graft viability. The anhepatic phase in liver transplantation exposes recipients to an unavoidable, iatrogenic spillover endotoxemia. Therefore, administration of LCTs during liver transplantation surgery may be a potent and convenient protective procedure from septic complications among recipients. Surprisingly, recipients in the LCT-treated group had a definitely longer mean survival time (\4 weeks) compared with the control group. These facts are not explained by the aforementioned beneficial effects of LCTs in hepatic energy status and endotoxemia because these occurred in the acute phase reaction. Since infection-related death ratio in the LCT-treated group was lower than that in the control group, it was recognised that LCT-treatment was contributing to the avoidance of lethal septic complications in early after OLT. Moreover, incidence of optimal rejection was low even in long survivors in the LCT-treated group. Any immunosuppressants, except for intraoperative steroids, were not administered to the recipients, therefore, a kind of immunosuppressive activity of LCTs by which early phase-survivors were protected from rejection-related hepatic failure in late after OLT was suspected. Recent reports revealed an immunosuppressive effect of LCTs caused a fall of lymphokine-activated killer activity in LCT-treated patients by inhibition of the binding of IL-2 to its receptor [14,15]. Although individual allogenic reactivity between the paired animals was not assessed, the initial immunosuppressive effect of LCTs on the reperfused liver graft might contribute to prolonged survival of recipients in the LCT-treated group. In conclusion, supplementation of lipid emulsion in the peri-anhepatic phase induced a better energy status in liver grafts without SEC damage, improvement of serum endotoxin levels and better recipient outcome.
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.