Franny Jongbloed

120 CHAPTER 5 RESULTS Single EAA deprivation protects against hepatic IRI To disentangle the role of single EAA in the protection against hepatic IRI, mice were administered a Met-free, Leu-free or Trp-free diet for three days. The results were compared with a control diet and diets that are known to either protect or not protect against IRI 17 . We have demonstrated that three days of a protein-free diet induces protection against renal IRI, while others also showed these results in other IRI models, including hepatic IRI 1 . On the contrary, three days of 30% DR does not induce resistance against renal IRI 17 , which makes it a control group for reduced calorie intake and concomitant body weight loss observed during feeding of EAA free diets. Mice fed a protein- or EAA-free diet during three days restricted themselves on calorie intake (Figure 1A). Mice fed the Met-free diet restricted food intake by 44%, while the Leu-free diet resulted in a restriction of 45%. A Trp-diet resulted in the smallest decrease in calorie intake, namely an average of 9% fewer calories than mice fed a control diet. Mice fed three days of a protein-free diet lost on average 9.8% of their body weight, while mice fed the non-protective 3-days of 30% DR showed a similar pattern in body weight loss (Figure 1B). Mice fed a single EAA-free diet also lost body weight: a Met-free diet resulted in 9.5% body weight loss, a Leu-free diet 6.2% and a Trp-free diet 8.5% (Figure 1C). After induction of hepatic IRI, mice on a protein-free diet resumed intake of the control food within 24 hours and showed no further weight loss, while ad libitum fed mice or mice restricted for 30% during three days lost weight after surgery. Mice fed the EAA-free diet resumed food intake postoperatively and stabilized their body weight (Figure 1C). Serum levels of liver enzymes ALT (Figure 2A) and LDH (Figure 2B) were significantly lower in the Leu-free and Trp-free groups than in the control group at six and 24 hours postoperatively. At 24 hours after hepatic IRI, all EAA-free diets showed significantly lower liver enzymes (Figure 2A/B). Histological analysis of the livers supports these findings. At six hours after reperfusion, the percentage of hemorrhagic necrosis was significantly lower in the Leu-free and Trp-free diets than in the control group (Figure 2C). At 24 hours postoperatively, the percentage of necrosis was increased compared to six hours, but still a significant lower percentage of necrosis was seen in the Leu-free and Met-free diet, while the reduction did not reach significance in het Trp-free diet. The number of neutrophils, as a marker for the acute inflammatory response in the liver, was significantly higher in het Leu-free and Trp-free diets than in the control group at six hours (Figure 2D), despite the lower percentage of hemorrhagic necrosis.