The role of glutamine in skeletal muscle ischemia/reperfusion injury in the rat hind limb model.
OBJECTIVE
Ischemia/reperfusion injury is a commonly occurring event with severe pathologic consequences. Reperfusion initiates both the local and systematic damage in part through rapid oxygen generation. The glutathione system is a major mechanism of reducing this oxidative stress. If this system can be maintained or augmented during this stress then less damage may occur. Glutamine provides the source of glutamate to this system and has been shown to preserve total glutathione levels after injury/ischemia to both hepatic and gut models. To test this effect, we looked at glutamine and its role in ischemia/reperfusion injury in a rat hind limb model.


METHODS
Fifty male HSD/Holtzman rats weighing 350-400 g were randomized to receive glutamine (3% sol) or normal saline via intraperitoneal injections. The groups were then subjected to 2 hours of ischemia to their hind limbs using the Tourni-Cot method. Animals were then randomized to reperfusion groups of 30 minutes, 2 hours, and 4 hours. Muscle tissue assays were performed for lipid peroxidation (LPO), total glutathione (GSH), and myeloperoxidase (MPO). Peripheral blood was analyzed for creatinephosphokinase levels (CPK).


RESULTS
Animals that received glutamine showed a general trend of less lipid peroxidation products than the normal saline groups. In animals that received glutamine and underwent 2 hours of ischemia and reperfusion times of 0 minutes, 30 minutes, and 2 hours, there were significantly less percent changes in lipid peroxidation products from controls (4.6% vs 48.2%, P <0.05), (18.9% vs 123%, P <0.05), (12.6% vs 115%, P <0.05). A general trend upward was noted in CPK levels in both groups. In animals receiving 2 hours of ischemia and 30 minutes of reperfusion, there was a significantly greater level of creatinephosphokinase (CPK) calculated as percent change from control in the normal saline group as compared with the glutamine group (209.2% vs 92.7%). Myeloperoxidase assay of muscle tissue revealed a progressive increase as the reperfusion times grew. In animals receiving 2 hours of ischemia and 30 minutes of reperfusion, the normal saline group had a significantly larger percent increase from controls than the group that received glutamine (1126.4% vs 108%, P <0.05). Also, in those animals receiving 4 hours of reperfusion, the normal saline group had a significantly higher percent increase in MPO content than the glutamine group (6245% vs 108%, P <0.05). Total glutathione levels decreased rapidly as reperfusion occurred in both the normal saline and glutamine groups. No significant difference between the groups was noted.


CONCLUSIONS
Total glutathione levels during reperfusion were not significantly different in the groups receiving glutamine versus normal saline. Glutamine may provide an initial protective effect on reperfusion injury after moderate reperfusion times in the hind limb model as defined by CPK and LPO levels. Glutamine may blunt neutrophil recruitment after longer reperfusion times (4 hours) in the ischemic hind limb. Total glutathione levels decreased significantly after moderate levels of ischemia (2 hours) and reperfusion (30 minutes, 2 hours).
Prem
J T
JT
Department of Surgery, Wright State University School of Medicine, Dayton, Ohio 45409-2793, USA.
Eppinger
M
M
Lemmon
G
G
Miller
S
S
Nolan
D
D
Peoples
J
J
D000818:Animals; D003402:Creatine Kinase / Q000097:blood; D004195:Disease Models, Animal; D018698:Glutamic Acid / Q000378:metabolism; D005973:Glutamine / Q000008:administration & dosage / Q000627:therapeutic use*; D005978:Glutathione / Q000096:biosynthesis; D006614:Hindlimb / Q000098:blood supply; D007274:Injections, Intraperitoneal; D007511:Ischemia / Q000378:metabolism / Q000503:physiopathology; D015227:Lipid Peroxidation / Q000187:drug effects; D008297:Male; D018482:Muscle, Skeletal / Q000098:blood supply / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D009504:Neutrophils / Q000187:drug effects / Q000473:pathology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D009195:Peroxidase / Q000378:metabolism; D020011:Protective Agents / Q000008:administration & dosage / Q000627:therapeutic use*; D011897:Random Allocation; D051381:Rats; D011919:Rats, Inbred Strains; D015427:Reperfusion Injury / Q000378:metabolism / Q000503:physiopathology / Q000517:prevention & control*; D016037:Single-Blind Method; D013997:Time Factors
D020011:Protective Agents; D005973:Glutamine; D018698:Glutamic Acid; D009195:Peroxidase; D003402:Creatine Kinase; D005978:Glutathione