The initial phase of oxidative stress in a steroid-induced osteonecrosis rabbit model

Using a rabbit model, we examined N epsilon-(hexanoyl) lysine (HEL) levels in bone and urine to detect when peroxidative reaction first occurs after steroid administration. Japanese white rabbits weighing about 3.5 kg each were injected with a single intramuscular dose of methylprednisolone 40 mg/kg and divided into groups consisting of 10 rabbits each, which were killed after 1, 3, 5 and 14 days (groups A, B, C and D respectively). As a control, 10 untreated rabbits (group N) were also studied. The proximal femurs were examined histopathologically and immunohistochemically using monoclonal antibody HEL, which is a highly specific antibody against N epsilon-(hexanoyl) lysine, an early peroxidation marker. In addition, urinary levels of HEL were measured by enzyme-linked immunosorbent assay in group N, A, B and C. Osteonecrosis was detected only in group D (90%). Increase of positive reaction of HEL in the bone was observed in group A and D. HEL expression in group D was judged to be a secondary reaction resulting from the development of osteonecrosis. Urinary level of HEL showed a significant increase in only group A (P < 0.001). The present findings suggest that peroxidation in bone occurred within 24 hours after steroid administration in a rabbit model and that it is possible to noninvasively grasp the timing of this peroxidative reaction by measuring the urinary level of HEL.