Measurement of oxygen consumption by murine tissues in vitro

Introduction: A novel in vitro system was developed to measure O-2 consumption by murine tissues over several hours. Methods: Tissue specimens (7-35 mg) excised from male Balb/c mice were immediately immersed in ice-cold Krebs-Henseleit buffer, saturated with 95% O-2:5% CO2. The specimens were incubated at 37 degrees C in the buffer, continuously gassed with O-2:CO2 (95:5). [O-2] was determined as a function of time from the phosphorescence decay rates (1/tau) of Pd(II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. The values of 1/tau were linear with [O-2]: 1/tau = 1/tau o + kq [O-2]; 1/tau o = the decay rate for zero O-2, kq = the rate constant in s(-1) mu M-1. Results: NaCN inhibited O-2 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 mu M O-2 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. Discussion: This approach allows accurate assessment of tissue bioenergetics in vitro. (C) 2010 Elsevier Inc. All rights reserved.