Detection of cytochrome c in serum of chemotherapy-treated cancer patients.

Sera (3 mL) obtained from different cancer patients (Table 1) before and during chemotherapy were precleared from residual immunoglobulins with protein G. Cytochrome c was immunoprecipitated and detected by Western blotting. A sample from a healthy control person was incorporated into each Western blot and served as an internal standard to normalize the data obtained from the different patients. The relative levels of cytochrome c in the sera were quantified by densitometric analysis and calculated as the ratio relative to the value of the control, which was set to 1. Day 0 indicates a sample taken before the start of the therapy. The first day of chemotherapy was considered as day 1; at day 1 the serum was withdrawn 8 to 12 hours after the start of infusion with chemotherapeutic drugs. The patients were loosely classified according to their serum cytochrome c levels. Panel A depicts patients with at least 2-fold elevated cytochrome c levels, and panel B patients with cytochrome c levels less than 2-fold increased in comparison to controls. Healthy control persons showed only low detectable levels of serum cytochrome c. The gray horizontal bar indicates the range of measured serum cytochrome c in the controls, which did not show marked interindividual differences (< 9%). The top panel of each graph shows a Western blot of a representative patient. The symbol in the left upper corner of each Western blot indicates the corresponding densitometric analysis.