Studies of ischemia and reperfusion in muscle and liver on glutathione and amino acid metabolism in man

Abstract

Oxidative stress increases the formation of reactive oxygen species (ROS), which can induce damage and breakdown of enzymes and lipoproteins. This leads to impaired cellular function and ultimately to death of cells and eventually organ failure. ROS are thought to be responsible for the damage during reperfusion following ischemia. ROS are counteracted by scavengers (anti-oxidants) among which glutathione is one of the most important.

Glutathione and oxidative stress during ischemia and reperfusion have been extensively investigated in animal models earlier. However, the relevance for human physiology especially during disease is not clear. The aim of the present studies was to characterise glutathione and amino acid metabolism in human skeletal muscle and liver during ischemia and reperfusion using surgical interventions as potential human models.

In the first study abdominal aneurysm surgery with an ischemia time of about 65 minutes was used. Biopsies from the thigh muscle were obtained before ischemia, at maximal ischemia and after 10 min, 4, 24 and 48 hours of reperfusion. In a second study aorto bi-femoral surgery patients were studied because of the longer ischemia times up to 120 minutes with this procedure. Biopsies from both legs were obtained before ischemia, at maximal ischemia and at 10 min. and 24 h of reperfusion. In the third study knee replacement surgery was used as a model since the obtained ischemia is more complete than in the first 2 studies and because no severe metabolic stress due to abdominal surgery is present in this model. Muscle biopsies were taken before ischemia, at maximal ischemia and 24 h into reperfusion. In the last study the effect of ischemia and reperfusion on human liver was studied during liver resection surgery. In this study biopsies were obtained before ischemia, at maximal ischemia and at 5, 10, 15, 20, 25 and 30 minutes of reperfusion.

Results from the first two studies show that both reduced and total glutathione are decreased at 24 and 48 hours of reperfusion. No changes were observed at maximal ischemia or in oxidised glutathione at any point. The changes were however very similar to those previously obtained in muscle following abdominal surgery without ischemia to the muscle. And therefore the observed changes are mainly due to the surgical trauma. In the knee surgery model reduced and total glutathione were also only decreased after 24h of reperfusion. However in this model a complete ischemia is obtained without the metabolic stress of abdominal surgery. The decrease here is therefore more likely to be due to ischemia and reperfusion in combination with a minor surgical trauma. Ischemia of the human liver had no effect on glutathione levels at all. Changes in amino acid levels during reperfusion were very similar as those seen in skeletal muscle following surgery without ischemia reperfusion. However the changes in amino acids seen at maximal ischemia are specific for the ischemia. Alanine is increased and glutamate decreased most likely to remove pyruvate to maintain the flux in the glycolysis. The branched chain amino acids behave differently in liver and muscle during maximal ischemia with and increase in liver and a slight decrease in muscle. Glutamate is decreased at ischemia in both muscle and liver, and decreases in reperfusion in muscle, while an increase is seen in liver at reperfusion. Glutamine is not affected by ischemia but decreases in muscle and increases in liver at reperfusion.

In conclusion, ischemia times of up to 120 minutes for skeletal muscle and about 30 minutes for liver do not affect glutathione levels in humans. During early reperfusion phase in the liver no changes are seeneither. In skeletal muscle at 1 to 2 days after reperfusion decreases in glutathione can be observed which seem to a large extend to be caused by the surgical stress rather then the ischemic insult. However in a human model with minimal surgical stress also a decrease is observed suggesting a contribution of the IR. Amino acids are used during the ischemia to maintain the energy status of both skeletal muscle and liver in humans.