Cutaneous mitochondrial respirometry: non-invasive monitoring of mitochondrial function

The recently developed technique for measuring cutaneous mitochondrial oxygen tension (mitoPO(2)) by means of the Protoporphyrin IX-Triplet State Lifetime Technique (PpIX-TSLT) provides new opportunities for assessing mitochondrial function in vivo. The aims of this work were to study whether cutaneous mitochondrial measurements reflect mitochondrial status in other parts of the body and to demonstrate the feasibility of the technique for potential clinical use. The first part of this paper demonstrates a correlation between alterations in mitochondrial parameters in skin and other tissues during endotoxemia. Experiments were performed in rats in which mitochondrial dysfunction was induced by a lipopolysaccharide-induced sepsis (n = 5) and a time control group (n = 5). MitoPO(2) and mitochondrial oxygen consumption (mitoVO(2)) were measured using PpIX-TSLT in skin, liver and buccal mucosa of the mouth. Both skin and buccal mucosa show a significant mitoPO(2)-independent decrease (P < 0.05) in mitoVO(2) after LPS infusion (a decrease of 37 and 39 % respectively). In liver both mitoPO(2) and mitoVO(2) decreased significantly (33 and 27 % respectively). The second part of this paper describes the clinical concept of monitoring cutaneous mitochondrial respiration in man. A first prototype of a clinical PpIX-TSLT monitor is described and its usability is demonstrated on human skin. We expect that clinical implementation of this device will greatly contribute to our understanding of mitochondrial oxygenation and oxygen metabolism in perioperative medicine and in critical illness. Our ultimate goal is to develop a clinical monitor for mitochondrial function and the current results are an important step forward

Non-invasive monitoring of mitochondrial oxygenation and respiration in critical illness using a novel technique

Introduction: Although mitochondrial dysfunction is proposed to be involved in the pathophysiology of sepsis, conflicting results are reported. Variation in methods used to assess mitochondrial function might contribute to this controversy. A non-invasive method for monitoring mitochondrial function might help overcome this limitation. Therefore, this study explores the possibility of in vivo monitoring of mitochondrial oxygen tension (mitoPO(2)) and local mitochondrial oxygen consumptionin in an endotoxin-induced septic animal model. Methods: Animals (rats n = 28) were assigned to a control group (no treatment), or to receive lipopolysaccharide without fluid resuscitation (LPS-NR) or lipopolysaccharide plus fluid resuscitation (LPS-FR). Sepsis was induced by intravenous LPS injection (1.6 mg/kg during 10 min), fluid resuscitation was performed by continuous infusion of a colloid solution, 7 ml kg(-1) h(-1) and a 2-ml bolus of the same colloid solution. MitoPO(2) and ODR were measured by means of the protoporphyrin IX-triplet state lifetime technique (PpIX-TSLT). Kinetic aspects of the drop in mitoPO(2) were recorded during 60s of skin compression. ODR was derived from the slope of the mitoPO(2) oxygen disappearance curve. Measurements were made before and 3 h after induction of sepsis. Results: At baseline (t0) all rats were hemodynamically stable. After LPS induction (t1), significant (p < 0.05) hemodynamic changes were observed in both LPS groups. At t0, mitoPO(2) and ODR were 59 +/- 1 mmHg, 64 +/- 3 mmHg, 68 +/- 4 mmHg and 5.0 +/- 0.3 mmHg s(-1), 5.3 +/- 0.5 mmHg s(-1), 5.7 +/- 0.5 mmHg s(-1) in the control, LPS-FR and LPS-NR groups, respectively; at t1 these values were 58 +/- 5 mmHg, 50 +/- 2.3 mmHg, 30 +/- 3.3 mmHg and 4.5 +/- 0.5 mmHg s(-1), 3.3 +/- 0.3 mmHg s-1, 1.8 +/- 0.3 mmHg s(-1), respectively. At t1, only mitoPO(2) showed a significant difference between the controls and LPS-NR. In contrast, at t1 both LPS groups showed a significantly lower ODR compared to controls. Conclusion: These data show the feasibility to monitor alterations in mitochondrial oxygen consumption in vivo by PpIX-TSLT in a septic rat model. These results may contribute to the development of a clinical device to monitor mitochondrial function in the critically ill

Validation of the protoporphyrin IX-triplet state lifetime technique for mitochondrial oxygen measurements in the skin

Mitochondrial oxygen tension can be measured in vivo by means of oxygen-dependent quenching of delayed fluorescence of protoporphyrin IX (PpIX). Here we demonstrate that mitochondrial PO2 (mitoPO(2)) can be measured in the skin of a rat after topical application of the PpIX precursor 5-aminolevulinic acid (ALA). Calibration of mitoPO(2) measurements was done by comparison with simultaneous measurements of the cutaneous microvascular PO2 This was done under three different conditions: in normal skin tissue, in nonrespiration skin tissue due to the application of cyanide, and in anoxic skin tissue after the ventilation with 100% nitrogen. The results of this study show that it is feasible to measure the mitoPO(2) after the topical application of ALA cream by means of the PpIX-triplet state lifetime technique. (C) 2012 Optical Society of Americ

Validation of the protoporphyrin IX-triplet state lifetime technique for mitochondrial oxygen measurements in the skin

Mitochondrial oxygen tension can be measured in vivo by means of oxygen-dependent quenching of delayed fluorescence of protoporphyrin IX (PpIX). Here we demonstrate that mitochondrial PO2 (mitoPO(2)) can be measured in the skin of a rat after topical application of the PpIX precursor 5-aminolevulinic acid (ALA). Calibration of mitoPO(2) measurements was done by comparison with simultaneous measurements of the cutaneous microvascular PO2 This was done under three different conditions: in normal skin tissue, in nonrespiration skin tissue due to the application of cyanide, and in anoxic skin tissue after the ventilation with 100% nitrogen. The results of this study show that it is feasible to measure the mitoPO(2) after the topical application of ALA cream by means of the PpIX-triplet state lifetime technique. (C) 2012 Optical Society of Americ

Validation of the protoporphyrin IX-triplet state lifetime technique for mitochondrial oxygen measurements in the skin

Mitochondrial oxygen tension can be measured in vivo by means of oxygen-dependent quenching of delayed fluorescence of protoporphyrin IX (PpIX). Here we demonstrate that mitochondrial PO2 (mitoPO(2)) can be measured in the skin of a rat after topical application of the PpIX precursor 5-aminolevulinic acid (ALA). Calibration of mitoPO(2) measurements was done by comparison with simultaneous measurements of the cutaneous microvascular PO2 This was done under three different conditions: in normal skin tissue, in nonrespiration skin tissue due to the application of cyanide, and in anoxic skin tissue after the ventilation with 100% nitrogen. The results of this study show that it is feasible to measure the mitoPO(2) after the topical application of ALA cream by means of the PpIX-triplet state lifetime technique. (C) 2012 Optical Society of Americ

Microvascular and mitochondrial PO2 simultaneously measured by oxygen-dependent delayed luminescence

Measurement of tissue oxygenation is a complex task and various techniques have led to a wide range of tissue PO2 values and contradictory results. Tissue is compartmentalized in microcirculation, interstitium and intracellular space and current techniques are biased towards a certain compartment. Simultaneous oxygen measurements in various compartments might be of great benefit for our understanding of determinants of tissue oxygenation. Here we report simultaneous measurement of microvascular PO2 (mu PO2) and mitochondrial PO2 (mitoPO2) in rats. The mu PO2 measurements are based on oxygen-dependent quenching of phosphorescence of the near-infrared phosphor Oxyphor G2. The mitoPO2 measurements are based on oxygen-dependent quenching of delayed fluorescence of protoporphyrin IX (PpIX). Favorable spectral properties of these porphyrins allow simultaneous measurement of the delayed luminescence lifetimes. A dedicated fiber-based time-domain setup consisting of a tunable pulsed laser, 2 red-sensitive gated photomultiplier tubes and a simultaneous sampling data-acquisition system is described in detail. The absence of cross talk between the channels is shown and the feasibility of simultaneous mu PO2 and mitoPO2 measurements is demonstrated in rat liver in vivo. It is anticipated that this novel approach will greatly contribute to our understanding of tissue oxygenation in physiological and pathological circumstances. (C) 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Cutaneous Mitochondrial P o 2, but Not Tissue Oxygen Saturation, Is an Early Indicator of the Physiologic Limit of Hemodilution in the Pig

Background: Hemodilution is a consequence of fluid replacement during blood loss and is limited by the individual ability to compensate for decreasing hemoglobin level. We tested the ability of a novel noninvasive method for measuring cutaneous mitochondrial Po-2 (mitoPo(2)) to detect this threshold early. Methods: Anesthetized and ventilated pigs were hemodynamically monitored and randomized into a hemodilution (n = 12) or a time control (TC) group (n = 14). MitoPo(2) measurements were done by oxygen-dependent delayed fluorescence of protoporphyrin IX after preparation of the skin with 20% 5-aminolevulinic acid cream. Tissue oxygen saturation (StO(2)) was measured with near infrared spectroscopy on the thoracic wall. After baseline measurements, progressive normovolemic hemodilution was performed in the hemodilution group in equal steps (500 ml blood replaced by 500 ml Voluven (R); Fresenius Kabi AG, Germany). Consecutive measurements were performed after 20-min stabilization periods and repeated 8 times or until the animal died. Results: The TC animals remained stable with regard to hemodynamics and mitoPo(2). In the hemodilution group, mitoPo(2) became hemoglobin-dependent after reaching a threshold of 2.6 +/- 0.2 g/dl. During hemodilution, hemoglobin and mitoPo(2) decreased (7.9 +/- 0.2 to 2.1 +/- 0.2 g/dl; 23.6 +/- 2 to 9.9 +/- 0.8 mmHg), but StO(2) did not. Notably, mitoPo(2) dropped quite abruptly (about 39%) at the individual threshold. We observed that this decrease in mitoPo(2) occurred at least one hemodilution step before changes in other conventional parameters. Conclusions: Cutaneous mitoPo(2) decreased typically one hemodilution step before occurrence of significant alterations in systemic oxygen consumption and lactate levels. This makes mitoPo(2) a potential early indicator of the physiologic limit of hemodilution and possibly a physiologic trigger for blood transfusion