Increases in serum carbonylated protein levels of dogs with hypercortisolism.

Protein carbonylation is an irreversible and degenerative modification that can be used to evaluate oxidative stress caused by glucocorticoids. In this study, we focused on protein carbonylation in dogs with hypercortisolism (HC). Sera samples were collected from 14 dogs diagnosed with HC and treated with trilostane, 12 dogs with inflammatory diseases (disease control group), and eight clinically healthy dogs. When the carbonylated protein levels were detected by the immunoblot analysis, one band of approximately 40 kDa was predominantly increased in the dogs with HC. The band was identified as haptoglobin using the liquid chromatography tandem mass spectrometry method. Furthermore, haptoglobin immune reactivity was higher in the dogs with HC. Although the average protein carbonylation level of the HC group was not significantly different from that of the other groups, the carbonylation level was significantly higher for the poorly controlled HC cases than for the well-controlled HC group. Additionally, the primary culture of canine hepatocytes was used to clarify the direct effect of glucocorticoids on protein carbonylation in dog livers. Both the carbonylated protein and haptoglobin clearly increased after 72 h. These findings suggest that haptoglobin and its carbonylated form are increased with canine HC, and that the protein carbonylation ratio and/or haptoglobin level could be related to disease management. These factors could be useful as biomarkers for an oxidative stress reaction, at least in the liver, and for treatment monitoring of HC.