A number of cell functions directly rely on the capacity of mitochondria to utilize oxygen through the mitochondrial respiratory chain (RC) [1
]. Accordingly, in a number of clinical conditions, measuring the ability of mitochondria to use oxygen can shed light on the disease mechanism and/or help in establishing a diagnosis [2
]. An impaired capacity for oxygen uptake is in particular observed in most primary mitochondriopathies of genetic origin. These are relatively rare disorders, but encompass numerous medical specialties [3
]. In addition, both primary and secondary impairments of mitochondrial function are now regarded as instrumental in the course of a set of common diseases, including different cancers [4
] and age-related neurodegenerative diseases [5
]. This comes as no surprise given the role of mitochondria as a crucial turntable for the overall cell metabolism, acting as determining actor for cell differentiation, proliferation, and death. Finally, mitochondria represent a cellular sink for numerous toxins [6
] to which organisms are exposed, potentially affecting their own function [7
Significant defects of the RC generally result in most tissues in an elevation of the redox status of the matrix pyridine nucleotides and a reduced capacity of mitochondria to oxidize pyruvate [8
]. This unused pyruvate is instead reduced to lactate by cytosolic lactate dehydrogenase and is excreted from the cells. Accordingly, the suspicion of an RC defect can be reinforced by the demonstration of abnormal acetoacetate/hydroxybutyrate (tracing the redox status of the mitochondrial pyridine nucleotide pool) and lactate/pyruvate ratios in the body fluids [2
An impairment of the RC activity might also reduce mitochondrial ATP production. Under these circumstances, as to match a cellular unsatisfied demand for ATP, an activation of glycolysis, an alternative way to produce ATP (yet less efficient than the RC), will take place producing both ATP and pyruvate, thus again favoring lactate production and excretion [9
Starting with the pioneer work of Otto Heinrich Warburg during the last century [10
], a number of devices have been developed to quantify the capacity of biological samples to consume oxygen. Successively using gas pressure in a closed chamber (Warburg apparatus [10
]), oxygen-dependent current flow at the surface of an electrode (Clark oxygen electrode [11
]), or oxygen-sensing fluorophore (oxygen extracellular fluxes; Seahorse technology [12
]), methods have substantially increased in sensitivity, reducing volumes to be used from several milliliters to a few tens of microliters; however, price varied inversely, from a few to now more than €150,000. As a sensitive, versatile, and cheap alternative, we describe here the use of the Redflash technology (FireSting O2; PyroScience; Aachen, Germany) to measure oxygen uptake by various biological systems in an aqueous medium. The method measures the luminescence of an oxygen-sensitive sensor molecule covalently attached to a polymer membrane, which covers the tip of an optic fiber connected to a PC-controlled meter (Figure 1
). The luminescence measurement uses red light excitation and lifetime detection in the near infrared. This represents a quite sensitive, very low-cost alternative in terms of quantifying oxygen uptake by intact cells or isolated mitochondria.
In addition, thanks to the convenient flexibility offered by the optic fiber, this device was fitted to the cuvette of a spectrophoto- or spectroflurometer, allowing for concurrent measurement of oxygen uptake plus an additional optical signal. Using such a configuration, it was possible to concomitantly and continuously measure mitochondrial substrate oxidation and membrane potential, or cell respiration and glycolysis (specifically through lactate excreted).
The comprehensive diagnostic of suspected oxidative phosphorylation (OXPHOS) defect requires the complementary assays of RC complex activity and of mitochondrial oxygen consumption [2
]. Similarly, the significance of numerous base changes in the several hundred genes encoding OXPHOS components revealed by systematic sequencing can only be established by an extensive characterization of OXPHOS activities [3
]. In addition to the determination of the activity of OXPHOS complexes, when possible, this includes the study of the cell respiration, the mitochondrial oxidation of various respiratory substrates, the determination of the ADP/O and respiratory control values. A complete investigation of oxidative properties supposes the use of an adaptable device allowing for the addition of multiple substrates and inhibitors in the assay medium and to register the oxygen consumption in real time. To this end, the Clark oxygen electrode that replaced the previous Warburg apparatus represented major progress, allowing for the use of much less precious material.
Here we have shown that it is possible to use RedFlash technology to reduce (by at least two-thirds) the amount of biological sample to be studied, as compared with previous devices. This represents similar progress in terms of the biological material required and the ease of use. The device is stable for months/years, as long as the probe is kept dry and not exposed to strong light. Various optodes have been used for several years in other fields of biology [25
] and a careful comparison between these devices and the Clark electrode already been reported [28
In the context of screening for OXPHOS defects, an immediate benefit of using this technology is smaller muscle biopsies or blood samples needed to be taken from patients and a reduction of the amount of cultured cells to be used, i.e., fewer traumas for patients and a lower cost in terms of cell cultures. The flexibility of the optic fiber allows one to adapt the device to various specific environments, such as spectrophoto- or spectrofluorometer cuvettes. As such, it is suitable for the simultaneous determination of cell respiration and lactate cell excretion. More specific than suspending medium acidification [29
], an increased rate of lactate excretion can be taken as an indication of a reduced rate of mitochondrial pyruvate oxidation or increased pyruvate production by glycolysis [22