Release of redox-active iron by muscle crush trauma: no liberation into the circulation.

After major skeletal muscle trauma, the iron-containing protein myoglobin and diverse other intracellular metabolites are liberated into the circulation from injured myocytes. Because chelatable iron should also be present in skeletal muscle cells, this redox-active, not tightly bound iron should be released from injured muscle tissue in addition to myoglobin and potentially account for oxidative tissue damage. The current study demonstrates in vitro the existence of 5 muM chelatable iron within the supernatant of a 1:10 homogenate of rat gastrocnemius muscle. This iron was almost exclusively associated with macromolecules greater than 30 kDa, most likely proteins. Presumably because of this association, only part of the chelatable iron could be scavenged by added apotransferrin. The chelatable iron was redox-active and thus responsible for the formation of thiobarbituric acid-reactive substances (TBARS) within the muscle homogenate. Correspondingly, using an in vivo model of closed trauma to the rat gastrocnemius muscle, a local TBARS formation in the damaged muscle tissue could be detected. Muscle trauma significantly increased plasma creatine kinase and myoglobin levels; however, no increase in serum non-transferrin-bound iron could be observed. Likewise, the serum parameters of iron-induced oxidative damage, TBARS, and protein carbonyls did not significantly increase after trauma. In conclusion, chelatable, redox-active iron is locally released by muscle destruction and responsible for lipid peroxidation within the damaged tissue. However, the liberation of chelatable iron into the circulation and its contribution to oxidative alterations of serum lipids and proteins could not be confirmed.

No evidence for protective erythropoietin alpha signalling in rat hepatocytes.

BACKGROUND: Recombinant human erythropoietin alpha (rHu-EPO) has been reported to protect the liver of rats and mice from ischemia-reperfusion injury. However, direct protective effects of rHu-EPO on hepatocytes and the responsible signalling pathways have not yet been described. The aim of the present work was to study the protective effect of rHu-EPO on warm hypoxia-reoxygenation and cold-induced injury to hepatocytes and the rHu-EPO-dependent signalling involved. METHODS: Loss of viability of isolated rat hepatocytes subjected to hypoxia/reoxygenation or incubated at 4 degrees C followed by rewarming was determined from released lactate dehydrogenase activity in the absence and presence of rHu-EPO (0.2-100 U/ml). Apoptotic nuclear morphology was assessed by fluorescence microscopy using the nuclear fluorophores H33342 and propidium iodide. Erythropoietin receptor (EPOR), EPO and Bcl-2 mRNAs were quantified by real time PCR. Activation of JAK-2, STAT-3 and STAT-5 in hepatocytes and rat livers perfused in situ was assessed by Western blotting. RESULTS: In contrast to previous in vivo studies on ischemia-reperfusion injury to the liver, rHu-EPO was without any protective effect on hypoxic injury, hypoxia-reoxygenation injury and cold-induced apoptosis to isolated cultured rat hepatocytes. EPOR mRNA was identified in these cells but specific detection of the EPO receptor protein was not possible due to the lack of antibody specificity. Both, in the cultured rat hepatocytes (10 U/ml for 15 minutes) and in the rat liver perfused in situ with rHu-EPO (8.9 U/ml for 15 minutes) no evidence for EPO-dependent signalling was found as indicated by missing effects of rHu-EPO on phosphorylation of JAK-2, STAT-3 and STAT-5 and on the induction of Bcl-2 mRNA. CONCLUSION: Together, these results indicate the absence of any protective EPO signalling in rat hepatocytes. This implies that the protection provided by rHu-EPO in vivo against ischemia-reperfusion and other causes of liver injury is most likely indirect and does not result from a direct effect on hepatocytes.