In Vivo Mitochondrial Oxygen Tension Measured by a Delayed Fluorescence Lifetime Technique

Abstract

Mitochondrial oxygen tension (mitoPO(2)) is a key parameter for cellular function, which is considered to be affected under various pathophysiological circumstances. Although many techniques for assessing in vivo oxygenation are available, no technique for measuring mitoPO(2) in vivo exists. Here we report in vivo measurement of mitoPO(2) and the recovery of mitoPO(2) histograms in rat liver by a novel optical technique under normal and pathological circumstances. The technique is based on oxygen-dependent quenching of the delayed fluorescence lifetime of protoporphyrin IX. Application of 5-aminolevulinic acid enhanced mitochondrial protoporphyrin IX levels and induced oxygen-dependent delayed fluorescence in various tissues, without affecting mitochondrial respiration. Using fluorescence microscopy, we demonstrate in isolated hepatocytes that the signal is of mitochondrial origin. The delayed fluorescence lifetime was calibrated in isolated hepatocytes and isolated perfused livers. Ultimately, the technique was applied to measure mitoPO(2) in rat liver in vivo. The results demonstrate mitoPO(2) values of approximately 30-40 mmHg. mitoPO(2) was highly sensitive to small changes in inspired oxygen concentration around atmospheric oxygen level. Ischemia-reperfusion interventions showed altered mitoPO(2) distribution, which flattened overall compared to baseline conditions. The reported technology is scalable from microscopic to macroscopic applications, and its reliance on an endogenous compound greatly enhances its potential field of applications.

Oxygen‐dependent delayed fluorescence measured in skin after topical application of 5‐aminolevulinic acid

“…5-Aminolevulinic acid (ALA) enhanced PpIX is synthesized inside the mitochondria [11] and can act as mitochondrially located oxygen-sensitive dye. We successfully measured mitochondrial PO 2 (mitoPO 2 ) in cultured cells [10], rat liver [12] and rat heart [13].…”

“…The background of the PpIX delayed fluorescence technology is described in detail elsewhere [10,12]. In short, PpIX is the final precursor of heme in the heme biosynthetic pathway (Figure 1 upper panel).…”

“…Although the calibration constants have been determined in rat liver [12] and rat heart [13], t 0 and k q are not validated for our current application in skin. Especially the temperature dependency of t 0 and k q remain to be determined.…”

Oxygen delivery using engineered microparticles

“…The most obvious may be directly bubbling oxygen gas into the bloodstream, although this approach is impractical due to the low solubility of oxygen in plasma and the occurrence of intravascular bubble coalescence. Historically, direct infusion of free oxygen gas into the blood stream has resulted in pulmonary emboli at extremely low infusion rates (i.e., 0.25 mL·kg −1 ·min −1 , corresponding to only 8% of measured oxygen consumption of a healthy human adult) (4,6). Other groups have proposed the use of supersaturated oxygen suspensions to increase oxygen content of injected fluids, although even when the pO 2 reaches ∼1,000 mmHg, solutions only deliver ∼1 mL O 2 ·kg −1 ·h −1 in vivo (7).…”

Triplet Imaging of Oxygen Consumption During the Contraction of a Single Smooth Muscle Cell (A7r5)

“…Alternative probes for dissolved oxygen measurements include: 1), colorimetric probes (13); 2), methods based on fluorescence lifetime measurements of the very long-living fluorophore RTDP (14,15); and 3), endogenous protoporphyrin IX probes that emit a characteristic delayed fluorescence signal (16,17). The third type of alternative probe has been employed recently in vivo (18) to measure the mitochondrial oxygen tension of the heart, bringing this noninvasive technique closer to clinical application.…”