Behavior of cholinesterase and liver mitochondrial function in dogs submitted to normothermic ischemia and reperfusion . 1

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.


INTRODUCTION
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.

ABSTRACT -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, infrahepatic, 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.

METHODS
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), infrahepatic 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 MgCl 2 , 2 mM KH2PO4, and 10 mM Hepes-KOH 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 respiration 18 .

Statistical analysis
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).

Results
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.

Discussion
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.

Fernandes
LP, Sankarankutty AK , Pacheco EG, Centurion S, Jordani MC, Castro e Silva Jr O. Behavior of cholinesterase and liver mitochondrial function in dogs submitted to normothermic ischemia and reperfusion.Acta Cir Bras [serial online] 2003 vol 18 suppl 5. Available in www.scielo.br/acb.