Measurement of oxygen consumption by murine tissues in vitro

IntroductionA novel in vitro system was developed to measure O2 consumption by murine tissues over several hours.MethodsTissue specimens (7–35 mg) excised from male Balb/c mice were immediately immersed in ice-cold Krebs–Henseleit buffer, saturated with 95% O2:5% CO2. The specimens were incubated at 37 °C in the buffer, continuously gassed with O2:CO2 (95:5). [O2] was determined as a function of time from the phosphorescence decay rates (1 / τ) of Pd(II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. The values of 1 / τ were linear with [O2]: 1 / τ = 1/τo + kq [O2]; 1 / τo = the decay rate for zero O2, kq = the rate constant in s−1 μM−1.ResultsNaCN inhibited O2 consumption, confirming oxidation occurred in the mitochondrial respiratory chain. The rate of respiration in lung specimens incubated in vitro for 3.9 ≤ t ≤ 12.4 h was 0.24 ± 0.03 μM O2 min−1 mg−1 (mean ± SD, n = 28). The corresponding rate for the liver was 0.27 ± 0.13 (n = 11, t ≤ 4.7 h), spleen 0.28 ±  0.07 (n = 10, t ≤ 5 h), kidney 0.34 ± 0.12 (n = 7, t ≤ 5 h) and pancreas 0.35 ± 0.09 (n = 10, t ≤ 4 h). Normal tissue histology at hour 5 was confirmed by light and electron microscopy. There was negligible number of apoptotic cells by caspase 3 staining.DiscussionThis approach allows accurate assessment of tissue bioenergetics in vitro.