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Evaluation of instrumental reproducibility is a primary component of quality control to quantify the precision and limit of detection of analytical procedures. A pre-analytical instrumental standard operating procedure (SOP) is implemented in high-resolution respirometry consisting of: (1) a daily SOP-POS for air calibration of the polarographic oxygen sensor (POS) in terms of oxygen concentration cO2 [µM]. This is part of the sensor test to evaluate POS performance; (2) a monthly SOP-BG (background) starting with the SOP-POS followed by the chamber test quantifying the instrumental O2 background flux. The chamber test focuses on the slope dcO2/dt [pmol∙s−1∙mL−1] to determine O2 consumption by the POS and O2 backdiffusion into the chamber as a function of cO2 in the absence of sample. Finally, zero O2 calibration completes the sensor test. We applied this SOP in a 3-year study using 48 Oroboros O2k chambers. Stability of air and zero O2 calibration signals was monitored throughout intervals of up to 8 months without sensor service. Maximum drift over 1 to 3 days was 0.06 pmol∙s−1∙mL−1, without persistence over time since drift was

Hypoxia is a condition of oxygen levels below normoxia and opposite to hyperoxia. We here define the normoxic reference state by three complementary precepts: (A) ambient normoxia at sea level in the contemporary atmosphere and corresponding dissolved O2 concentrations at air saturation of aqueous environments; (B) biological compartmental O2 levels at ambient normoxia under physiological activity of healthy organisms in the absence of environmental stress (e.g. stress in a diving human, a stranded whale, a thermally stressed fish); and (C) O2 levels above the respiratory oxygen control region. In the oxygen control region, the capacity for O2 consumption is compromised by hypoxic partial O2 pressure as evaluated by O2 kinetics of respiration or other critical functions. The ABC of hypoxia distinguishes deviations from these reference points caused by different mechanisms: (∆A) ambient alterations of oxygen levels; (∆B) biological O2 demand exceeding O2 supply under pathological or experimental limitations of convective O2 transport or O2 diffusion; and (∆C) critical oxygen pressure in oxygen kinetics shifted by pathological and toxicological effects or environmental stress. The ABC of hypoxia may be of help in the design and interpretation of in vitro and in vivo experimental studies.

For the advanced study of mitochondrial function, high-resolution respirometry is extended by fluorometric measurement of ATP production using the fluorophore Magnesium Green™ (MgG). A common problem with several fluorescent dyes is the inhibition of mitochondrial respiration. In the present study, a coupling control protocol was applied in combination with MgG to measure ATP production simultaneously with respiration for calculation of P»/O2 ratios. MgG at 1.1 µM did not affect respiration through the NADH-linked and succinate-linked pathways. Respiration was not inhibited in any of the coupling control states, hence coupling control efficiencies were not affected by MgG.

Redox states of the mitochondrial coenzyme Q pool, which reacts with the electron transfer system, reflect the balance between (1) reducing capacities of electron flow from fuel substrates converging at the Q-junction, (2) oxidative capacities downstream of Q to O2, and (3) the load on the OXPHOS system utilizing or dissipating the protonmotive force. A three-electrode sensor (Rich 1988; Moore et al 1988) was implemented into the NextGen-O2k to monitor continuously the redox state of CoQ2 added as a Q-mimetic simultaneously with O2 consumption. The Q-Module was optimized for high signal-to-noise ratio, minimum drift, and minimum oxygen diffusion. CoQ2 equilibrates in the same manner as Q at Complexes CI, CII and CIII. The CoQ2 redox state is monitored amperometrically with the working electrode, which is poised at CoQ2 redox peak potentials determined by cyclic voltammetry. The voltammogram also provides quality control of the Q-sensor and reveals chemical interferences. The CoQ2 redox state and O2 consumption were measured simultaneously in isolated mouse cardiac and brain mitochondria. CoQ2 ― and by implication mitochondrial CoQ ― was more oxidized when O2 flux was stimulated by coupling control: when load increased from LEAK to OXPHOS and electron transfer capacities in the succinate pathway. In contrast, CoQ2 was more reduced when O2 flux was stimulated by pathway-control of electron input capacities, increasing from the NADH (N)- to succinate (S)-linked pathway which converge at the Q-junction, with CI-Q-CIII and CII-Q-CIII segments, respectively. N- and S- respiratory pathway capacities were not completely additive, thus confirming partial pool behavior of Q intermediary between the solid-state and liquid-state models of supercomplex organization. The direct proportionality of CoQ2 reduction and electron input capacities through the CI-Q-CIII and CII-Q-CIII segments suggests that CoQ2 is accurately mimicking mitochondrial Q-redox changes.

Redox states of mitochondrial coenzyme Q (mtCoQ or Q) reflect the balance between (1) reducing capacities of electron flow from fuel substrates converging at the Q-junction, (2) oxidative capacities downstream of Q to oxygen, and (3) the load on the OXPHOS system utilizing or dissipating the protonmotive force. A three-electrode sensor (Rich 1988; Moore et al 1988) was implemented into the NextGen-O2k to monitor the Q redox state continuously and simultaneously with oxygen consumption. The Q-Module was optimized for high signal-to-noise ratio and minimum oxygen diffusion. CoQ2 is added as a redox probe equilibrating with Q at Complexes CI, CII and CIII and the detecting electrode. Q-sensors are poised with the CoQ2 redox peak potentials determined by cyclic voltammetry, which provides quality control of the Q-sensor and reveals chemical interferences. The Q redox state and oxygen consumption were measured simultaneously in isolated mitochondria. A coupling-control protocol was applied to analyze LEAK, OXPHOS, and electron transfer capacities (L, P, and E, respectively) in the succinate-pathway. In a second pathway-control protocol, NADH- and succinate-linked pathways (N and S) converge at the Q-junction. mtCoQ was more oxidized when O2 flux was stimulated in coupling-control states with load increasing from L to P and E. In contrast, mtCoQ was more reduced when O2 flux was stimulated with electron input capacities increasing from N-, S- to NS-pathway-control states. N- and S- pathway capacities were not completely additive, thus confirming partial pool behavior of Q as proposed in the plasticity model of supercomplex organization.