On-line version ISSN 1678-2674
Acta Cir. Bras. vol.18 suppl.5 São Paulo 2003
Behavior of cholinesterase and liver mitochondrial function in dogs submitted to normothermic ischemia and reperfusion.1
Colinesterase e função mitocondrial hepática em cães submetidos a isquemia normotérmica e reperfusão do fígado
Luis Pinto FernandesI; Ajith Kumar SankarankuttyII; Eduardo Garcia PachecoII; Sérgio CenturionII; Maria Cecília JordaniII; Orlando de Castro e Silva JrIII
IProfessor Doutor da Universidade do Distrito Federal, Brasília
IIPós Graduandos do Programa de Pós Graduação em Clínica Cirúrgica do Departamento de Cirurgia e Anatomia da FMRP-USP
IIIProfessor Associado do Departamento de Cirurgia e Anatomia
PURPOSE: The plasmatic activity of the cholinesterase (CHE) and the liver mitochondrial function, expressed by the ratio of respiratory control (RCR), were studied during normothermic ischemia.
METHODS: Sixteen adult mongrels, eight females and eight males were submitted to ischemia by clamping of the hepatic artery, portal vein and infrahepatic inferior vena cava, infra-hepatic, for two h, follwed by reperfusion for 1 h. The CHE and the mitochondrial function were evaluated at 60 and 120 min. of ischemia and at 15 and 60 minutes of reperfusion.
RESULTS: The CHE decreased, significantly, during ischemia and in reperfusion. The RCR was decreased at 120 min. of ischemia, returning to the initial values on reperfusion.
CONCLUSION: In this study, the CHE was a sensitive indicator of ischemic injury , suggesting irreversibility of ischemia injury. The RCR, by other side, showed a greater sensibility than the CHE in detection sense, during the studied period, the reversibility of the hepatic ischemic injury.
Key Words: Cholinesterase. Mitochondral function. Passive venous bypass. Liver transplantation. Ischemia. Reperfusion.
OBJETIVO: A atividade plasmática da colinesterase (CHE) e a função mitocondrial do fígado expressa pela RCR- razão de controle respiratório mitocondrial foram estudadas durante a isquemia/reperfusão hepáticas.
MÉTODOS: Dezesseis cães adultos sem raça definida (oito machos e oito fêmeas) foram submetidos a isquemia normotérmica por pinçamento do pedículo hepático e da veia cava inferior infra-hepática por 2 horas, seguida de 15 e 60 minutos de reperfusão.A CHE e a RCR foram avaliadas após 60 e 120 minutos de isquemia e após 15 e 60 minutos de reperfusão.
RESULTADOS: Os níveis de CHE diminuíram significativamente na isquemia e reperfusão.A RCR diminuiu após 120 minutos de isquemia retornando a níveis semelhantes ao controle após a reperfusão.
CONCLUSÃO: A CHE foi sensível para indicar a lesão isquêmica, sugerindo irreversibilidade da lesão. Já a RCR foi mais sensível no sentido de detectar a reversibilidade da lesão isquêmica após a reperfusão.
Descritores: Colinesterase. Função mitocondrial.Transplante de fígado.Isquemia. Reperfusão.
Several studies have been published regarding the hepatocellular biochemical and histological alterations during hepatic ischemia and reperfusion. Many of these alterations are studied in the hepatic tissue and, sometimes, alterations can be detected in the plasma 1,2,3,4,5,6,7,8.
To analyse, experimentaly, the ischemia and reperfusion injuries of the hepatocytes some parameters can be used: the mitochondrial function, capacity to produce ATP, and the intracellular enzimatic contents released into the plasma.
The reversibility of ischemic injuries is related to cellular alterations after reperfusion, to celular capacity of ATP production and to the intracelular calcium contents regulation9. The CHE, a enzyme produced in the liver by the endoplasmic reticulum, is usually in high concentrations in the human plasma10,11,12.
Its reduced activity is considered a sign of hepatic perfusion reduction, shortage of proteic synthesis and associated with liver necrosis, with parenchyma reduction11, mainly in chronic liver diaseases, as cirrhosis and Kwashiorkor. The CHE would be related to the hepatic synthesis activity as well as the seric albumin10.
The hepatocyte mitochondrial function and its capacity to restore the energh production by the activation of glicolitic pathway during ischemia, has been studied as important indicators of irreversibility cellular injury2,13,14 .Its known that maintenance of cellular membrane potentialises an energy dependent process and that during ischemia, the content of hepatocyte ATP is reduced in minutes 15.
Rhodes et al.13 showed that the hepatic ischemia needed to cause cellular death involves irreversible alterations in mitochondrial energetics functions. There is controversy whether irreversible mitochondrial injury precedes or whetherit is a late manifestation of cellular death. Its clear that duration of ischemic is a crucial factor in the reversibility of the process. 1
The purpose of this work is to investigate the alterations on CHE plasmatic activity, as well as the hepatocyte mitochondrial function during 2h of ischemia and after 1h of reperfusion, relating them to cellular viability. These parameters can be used as prognostic indicators in surgical procedures where the blood supply should be temporality interrupted, was well as for evaluating hepatic metabolic restoration after reperfusion in liver transplantation, hepatectomies and livers injuries.
Sixteen adult mongrels, eighth females and eight males, weighing between 8 and 30 kilograms, were studied. All the surgical procedures were made in a normal temperature of 25°C, with non-sterilised, but clean instruments. The animals were kept fasting for 24 hours before the surgery, with water "ad libitum" until 2 hours before anesthesia. The anesthetics used was sodium ethyl barbiturate (Nembutal Abbot Lab.), 30 mg per kilogram of body weight, intravenously.
The hepatic ischemia was produced by temporary clamping of the hepatic artery (HA), infra-hepatic cava inferior vein, above the renal veins (CIV), porta vein (PV), with 2 hours duration. All dogs received heparine, 150 U per kilogram of weight, endovenous, 10 min. before the beginning of ischemia14.
The portal and inferior vena caval descompression was achieved by bypass of the femoral vein and the lateral and cranial branch of the splenic vein to the jugular vein, with the use of a siliconized and heparinized of polyvinyl tubing. At the end of the ischemic period, the splenic flux to the porta vein was kept by ligadure of its branch, keeping the splenic vein permeability. The arterial mean pressure and the central venous pressure were monitored by cateters introduced into femoral artery and into the external jugular, connected to the Physiograph MK IV, from Narco Bio Systems.
Liver biopsies were made before the ischemia, at 60 and 120 min. of ischemia, and at15 and 60 min. of reperfusion to study the mitochondrial function. Samples of venous blood were collected in heparinized glass tubes (0,05 mL Liquemine, Roche Lab, 5000 U/mL) at the same periods.
Isolation of dog liver mitochondria. Mitochondria were isolated by conventional differential centrifugation16. The liver was immediately removed, washed in cold saline and homogenized three times at 1 min intervals in a Potter-Elvehjem homogenizer in 10 mL of a medium containing 250 mM sucrose, 1 mM EGTA, 10 mM Hepes-KOH at pH 7.2. Homogenates were centrifuged at 770g for 5 min and the resulting supernatant further centrifuged at 9.800g for 10 min. Pellets were suspended in 10 mL of a medium containing 250 mM sucrose, 0.3 mM EGTA and 10 mM Hepes-KOH at pH 7.2, and centrifuged at 4.500g for 15 min. The final mitochondrial pellet was suspended in 0.5 mL of a medium containing 250 mM sucrose, and 10 mM Hepes-KOH at pH 7.2. All procedures were conducted at 4°C and all solutions were prepared using glass-distilled and deionized water.
Protein determination. Mitochondrial protein content was determined by biuret reaction17.
Mitochondrial respiration. Mitochondrial respiration was monitored polarographically with an oxygraph equipped with a Clarck-type oxygen electrode (Gilson Medical Electronics, Middlenton, WI, USA). Assays were performed at 30°C using mitochondria (1 mg protein/mL) energized by 5 mM a-ketoglutarate. Respiration media contained 125 mM sucrose, 65 mM KCl, 0.1 mM EGTA, 1mM MgCl2, 2 mM KH2PO4, and 10 mM HepesKOH at pH 7.4. The state 3 respiration was induced with 400 nmol ADP and state 4 respiration was determined after the phosphorylation of ADP. These respiratory parameters were expressed in n at. O/min/mg mitochondrial protein. The Respiratory Control Ratio (RCR),was determined by relation between the state 3 respiration and state 4 respiration18.
Determination of cholinesterasis activity. The plasma cholinesterasis activity study was made by colorimetric method18.
The Wilcoxon pared test was used with the significance level of 5%. The absolute values of each experiment were enunciated as mean (x) and standard error (se).
The figure 1 shows the plasma cholinesterasis activity (U/L), mean and standard error, as well as the ratio of velocity the states 3 and 4 of mitochondrial respiration expressed by RCR, in animals before ischemia (C) at 60 (I1) and 120 (I2) minutes of ischemia and after 15 (R1) and 60 (R2) minutes of reperfusion.
The CHE plasmatic activity mean in the studied times was: 1278, 1093, 938, 777 and 736 U/L. There was significant difference between the mean before ischemia (C) and the times I1, I2, R1, and R2 (p < 0,05). There wasn't difference, in statistical point of view, between R1 and R2 (p > 0,05). In relation to the RCR, the means were: 4,8; 4,4; 3,4; 3,8 and 3,9. Significant reduction of RCR between times C and I2 (p< 0,05) was observed. There wasn't also statistical differences between times C, I1, R1 and R2 (p> 0,05).
In this study, accentuated reduction in the plasmatic levels of the CHE was observed during ischemia and after reperfusion of the liver. This can be explained by the injuries caused during the two periods, with a consequent reduction of its synthesis. After reperfusion there was a significant reduction of the CHE, characterizing the injuries of ischemia and reperfusion.
The change in plasmatic enzymatic concentration expresses the cellular injury without relating to it is reversibility5. Consistent mitochondrial alterations occur after an interruption of the blood flow to different organs and the impossibility to reverse these disturbances are related to the incapacity of restructuring the cellular function 4,9,20.
The injuries from the reperfusion depends of the reestablishment of the blood flow and the amount of oxygen available to an organ previously ischemic or anoxic. There are tissue microcirculatory changes, just after the reperfusion, which result in some areas having no blood flow
Another aspect reperfusion injuries are the nocives chemical reactions involving free radicals derivated from oxygen that is introduced in to ischemic areas, causing cellular injury 8,10.
The RCR showed a decrease in I2 with return to the initial values in R2, characterizing the reversibility of the injuries.
After half an hour of ischemia few or no evidence of histological cellular death was observed after 24 hours of reperfusion3. Nevertheless, after 2 to 3 hours of ischemia there was extensive necrosis compromising the membrane function, calcium pump and the capacity to produce ATP. These same authors showed that ischemia induces disturbances in phospholipid metabolism which can be detected as membrane disfunction.
The present study showed that CHE is a sensitive indicator of ischemia and reperfusion injury, in the time studied. Probably, 60 minutes was insufficient to the liver could resyntesized the CHE, by other side, the mitochondrial function showed a greater sensitivity than the CHE, during the period studied, to detect the reversibility of the hepatic ischemic injury.
1. Bernelli-Zazzera A, Gaja G. Some aspects of glycogen metabolism following reversible of irreversible liver ischemia. Exp Mol Pathol 1964; 3: 351-68. [ Links ]
2. Farkouh EF, Daniel AM, Beaudoin JG. Predictive value of liver biochemistry in auto hepatic ischemia. Surg Gynecol Obstet 1971; 132: 832-38. [ Links ]
3. Chien KR, Farber JL. Microsomal membrane dysfunction in ischemic rat liver cells. Arch Biochem Biophys 1977; 180: 191-8. [ Links ]
4. Farber LJ, Martin JT, Chien KR. Irreversible ischemic cell injury: prevention by chlorpromazine of the aggregation of the intramembranous particles of rat liver plasma membranes. Am J Pathol 1978; 92: 713-32. [ Links ]
5. Fredericks WM, Myagkaya GL, Bosch KS. The value of enzyme leakage for the prediction of necrosis in the liver ischemia. Histochemistry 1983; 78: 459-72. [ Links ]
6. Reichling JJ, Kaplan MM. Clinical use of serum enzymes in liver disease. Dig Dis Sci 1988; 33: 1601-14. [ Links ]
7. Radziuk J. Metabolic function of liver: carbohydrates. In: Liver Function 1990; (Cramp DG, Carson ER, eds.), London, Chapman and Hall, 106-51. [ Links ]
8. Kobayashi, H, Nonami, T, Kurokawa T. Mechanism and prevention of ischemia-reperfusion- induced liver injury in rats. J Surg Res 1991; 51: 2400-4. [ Links ]
9. Mittnacht Jr S, Farber JL. Reversal of mitochondrial of cholinesterase. J Biol Chem 1981; 256: 3199-206. [ Links ]
10. Kutty KM. Review: biological function of cholinesterase. Clin Biochem 1980; 193: 265-75. [ Links ]
11. Schmidt E. Estratégias e avaliação das determinações enzimáticas no soro em doenças do fígado e do sistema biliar. In: Avaliação da doença hepática 1980; (Demers LM, Shaw LM, eds.), Rio de Janeiro, Interamericana, 71-91. [ Links ]
12. Berninsone, P, Katz E, Napp M. Acetylcholinesterase and nonspecific cholinesterase activities in rat liver: subcellular localization, molecular forms, and some extraction properties. Biochem Cell Biol 1989; 67: 817-22. [ Links ]
13. Rhodes RS, DePalma RG, Druet RL. Reversibility of ischemically induced mitochondrial dysfunction with reperfusion. Surg Gynecol Obstet 1977; 145: 719-24. [ Links ]
14. Kono I, Ozawa K, Tanaka J. Significance of mitochondrial enhancement in restoring hepatic energy charge after revascularization of isolated ischemic liver. Transplantation 1982; 33: 150-5. [ Links ]
15. Jennische E. Effects of ischemia on the hepatic all membrane potential in the rat: differences between fed and fasted animals. Acta Phisiol Scand 1983; 118: 69-73. [ Links ]
16. Pedersen PL, Greenawalt JW, Reynafarje B, Hullihen J, Dercker GL, Soper JW, Bustamente E. Preparation and characterization of mitochondria and submitochondrial particles of rat liver and liver derived tissues. Methods Cell Biol, 1978 20: 411-81. [ Links ]
17. Cain K, Skilleter, DN. Preparation and use of mitochondria in toxicological research.In: Snell K, Mullock B. Biochem Toxicol 1987.Oxford IRL Press, 217-54. [ Links ]
18. Chance B, Williams GR The respiratory chain and oxidative phosphorilation. Adv Enzimol 1956; 17: 65- 134. [ Links ]
19. Ellma GL, Courtney KD, Featherstone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961; 7: 88-95. [ Links ]
20. Fath JJ, Cyr JAS, Konstantinides FN. Alteration of amino acid clearence during ischemia predict hepatocellular ATP changes. Surgery 1985; 98: 396-404. [ Links ]
Orlando Castro-e-Silva Jr
Rua Campos Salles 890, 9 andar
Centro, Ribeirão Preto, São Paulo, Brazil
55 16 602 2871/ 55 16 6100626
Conflito de interesse: nenhum.
Fonte de financiamento:FAPESP
1. Estudo realizado no Laboratório de Bioquímica e Transplante Hepático do Departamento de Cirurgia e Anatomia da Faculdade de Medicina de Ribeirão Preto-Universidade de São Paulo (FMRP-USP)