Liver tissue respiration, ATP content, caspase activity and urea synthesis in continuously oxygenated KH buffer. Liver specimens from a Taylor outbred mouse were incubated at 37°C in 50 mL KH buffer continuously gassed with 95% O2: 5% CO2 for up to ~6 h. At indicated time periods, samples were removed from the incubation medium and processed for measurements of O2 consumption, ATP content, caspase activity and urea synthesis as described in Methods. Panel A: Representative runs of cellular mitochondrial O2 consumption are shown; t = 0 corresponds to animal sacrifice. The rate of respiration (k, μM O2 min-1) was the negative of the slope of [O2] vs. t. The additions of 10 mM NaCN and 50 μg/ml glucose oxidase are shown. Panel B: The values of k
and ATP were plotted as a function of incubation time. Panel C: HPLC runs of caspase activity at 6 h. The retention time (Rt) for Ac-DEVD-AMC was ~2.5 min and AMC (representing caspase activity) ~4.8 min. Panel C insert: AMC peak areas are shown as a function of incubation time. Panel D: Liver specimens (~300 mg) were incubated at 37°C in 50 ml KH buffer supplemented with 10 mM NH4Cl and 2.5 mM ornithine and continuously gassed with 95% O2: 5% CO2. At t = 0, 3 and 6 h, an aliquot of the solution was taken for urea determination. Insert (error bars are standard deviation of 2 independent experiments), liver specimens were incubated at 37°C in 50 ml KH buffer continuously gassed as above. At indicated time points, specimens (~80 mg) were placed in 1.0 mL KH buffer supplemented with 10 mM NH4Cl and 2.5 mM ornithine. The reactions were allowed to continue at 37°C for 50 min with continuous gassing as above. At the end of the incubation period, the specimens were discarded and the solution was analyzed for urea.