Gnaiger IOC62-Introduction

Gnaiger E (2011) The elements of high-resolution respirometry: An introduction to the Oroboros Oxygraph-2k. IOC62.

Link: IOC61

Gnaiger E (2011)

Event: IOC62

High-resolution respirometry (HRR) provides a quantitative approach to bioenergetics and mitochondrial physiology with the Oroboros O2k (Oroboros Instruments) offering several sole-source features.

• O2k-Network Lab: AT Innsbruck Oroboros

Full text

Gnaiger E (2011) The elements of high-resolution respirometry: An introduction to the Oroboros Oxygraph-2k.

High-resolution respirometry (HRR) provides a quantitative approach to bioenergetics and mitochondrial physiology with the Oroboros O2k (Oroboros Instruments) offering several sole-source features.

Hardware and software developments are based on long-term expertise with polarographic oxygen sensors (POS) [1] and continuous evaluation relative to alternative sensors. The POS is superior in the range from zero oxygen to pure oxygen at about 1 mM dissolved O₂, yielding a 500,000-fold dynamic range with a digital resolution of 2 nM in the O2k [2]. Oxygen flux is measured in the closed system as the negative time derivative of oxygen concentration, calculated and displayed on-line with correction for instrumental background oxygen flux, yielding a resolution of 1 pmol O₂.s^-1.ml^-1. Minimization, experimental evaluation, and automatic correction (DatLab) of instrumental background oxygen flux are integral to HRR [2], as emphasized in practical tests and data analysis during the basic O2k-Workshop. Alternatively, oxygen flux can be measured in an open system mode of operation, using the Titration-Injection microPump (TIP2k) for feedback control of oxygen levels by matching oxygen supply to demand, particularly at graded levels of hypoxia in studies of oxygen kinetics [3].

Most applications of HRR take advantage of the high stability and sensitivity of the O2k in coupling-control protocols with intact cells, or substrate-uncoupler-inhibitor titration (SUIT) protocols with isolated mitochondria, permeabilized cells or tissues, in particular permeabilized muscle fibres [4]. Physiological temperatures (electronic Peltier control, within 0.001 °C), optimized incubation media (MiR06), and a rationale for the design of tested SUIT protocols [5] are the hallmark of quantitative and comparative mitochondrial respiratory physiology. Coupling-control ratio and substrate control of mitochondrial respiration are expressed as flux control ratios, resolving some confusion related to the respiratory control ratio (RCR) [6,7]. Applicaton of CI+II substrate combinations in SUIT protocols extends conventional bioenergetic studies to the level of mitochondrial physiology, some principles of which are applied and discussed at the O2k-Workshop, whereas an in-depth introduction is provided at the MiPsummer School.

Compared to the long tradition of applications of polarographic oxygen sensors, replacing the classical manometric (Warburg) apparatus, HRR is a recent development, which now provides a widely applied tool for routine and specific analyses of mitochondrial function/dysfunction where (1) reliability and quality control are important (clinical studies, functional diagnosis), (2) the amount of biological material is limited (<0.5 mill cultured cells, 1-2 mg of fresh tissue from biopsies; <0.05 mg of mitochondrial protein), (3) pathological effects result in reduced respiration, and (4) effects need to be tested at physiological, low intracellular oxygen levels [8].

Finally, the basic O2k-Workshop provides an overview on O2k-MultiSensor System applications, for the simultaneous measurement of respiration and mitochondrial membrane potential (TPP⁺ electrode), acidification (pH electrode), nitric oxide (amperometric), spectrophotometry (cytochrome spectra), and spectrofluorimetry (Amplex red, safranin, etc.). In parallel sessions, a hands-on introduction is provided to the application of the TPP⁺ electrode for advanced users [9]. Taken together, HRR integrates metabolic and physicochemial concepts on fluxes and forces in open and closed systems [10].