Introduction

Mitochondrial function is of pivotal importance in cellular function. Subtle changes in mitochondrial function over time are thought to play a role in the development of several chronic diseases like Alzheimer’s disease and type 2 diabetes1. Mitochondrial dysfunction is also implicated to play an important role during critical illness, where oxygen demand, supply and consumption are often out of balance. For example, in sepsis, failure of the microcirculation and a diminished mitochondrial function is related to development of multi-organ failure and death, irrespective of age2,3. Measuring the balance between supply and demand of cellular oxygen might aid clinical evaluation of sepsis or guide patient-based care protocols4,5. The detrimental effects of sepsis on mitochondrial function were already shown in thrombocytes obtained from adult and pediatric patients3,6, showing an association between early changes in mitochondrial function and development of multi-organ failure. Direct, non-invasive measurement of the concentration of cellular oxygen and mitochondrial function in intact tissue reflects this balance between supply and demand of cellular oxygen levels7. Recently, the Cellular Oxygen METabolism (COMET) measuring system has been developed, which enables bedside measurement of mitochondrial oxygenation and respiration8.

The COMET device measures cutaneous mitochondrial oxygen tension (mitoPO2) by means of delayed fluorescence of mitochondrial protoporphyrin IX (PpIX)9. Microcirculatory flow can be stopped by applying pressure with the measuring probe on the skin, enabling the determination of the mitochondrial oxygen consumption (mitoVO2)10. The mitoVO2 measurement is a non-invasive technique to assess mitochondrial respiration in vivo. Using the COMET device, the technical feasibility to measure mitoPO2 and mitoVO2 in humans has been demonstrated in healthy volunteers and in clinical studies11,12.

In this study, we investigated the feasibility of the COMET system to detect changes in mitochondrial oxygenation and respiration during experimental human endotoxemia, a standardized well-controlled and reproducible model of systemic inflammation elicited by administration of E. coli lipopolysaccharide (LPS)13. We chose to use this model because previous work in rats has shown that LPS administration exerts detrimental effects on the mitochondrial oxygen consumption in skin, which was unrelated to alterations in mitochondrial oxygen tension14.

Materials and methods

Subjects and ethics

Data were collected from 52 healthy, nonsmoking, Dutch male volunteers, 48 of whom participated in an experimental endotoxemia study registered at ClinicalTrials.gov as NCT03240497 (12/04/2016). The endotoxemia study was performed at the Radboud university medical center in Nijmegen, the Netherlands. The time-control study, performed in four healthy volunteers was performed at the Erasmus university medical center in Rotterdam, the Netherlands after completion of the endotoxemia study. Both study protocols received institutional review board approval (CMO 2016–2312/toetsingonline NL56686.091.16 (11/04/2016) and MEC 2018-090/toetsingonline NL65767.078.18 (01/05/2018) for the endotoxemia and time-control studies, respectively). Our study protocol experiments were in accordance with the Declaration of Helsinki, including current revisions, and Good Clinical Practice guidelines and all subjects provided written informed consent. Subjects participating in the endotoxemia study were screened before the start of the experiment and had a normal physical examination, electrocardiography, and routine laboratory values. For all subjects, exclusion criteria were: lack of significant mitoPO2 signal at first measurement, febrile illness in the two weeks before the experiment, taking any prescription medication, history of spontaneous vagal collapse, participation in a drug trial or donation of blood three months prior to the experiment, porphyria, or participation in a previous trial where lipopolysaccharide (LPS) was administered.

The primary aim of the endotoxemia study was to investigate the effects of a training program consisting of a breathing exercise and/or exposure to cold on the inflammatory response. A two by two design was employed in which 48 participants were randomized to four different groups (n = 12 per group). Details of the training procedures and main findings are described elsewhere15. Data of all four groups were combined into a single LPS group for the purpose of the current study (see statistics section for justification).

Study procedures

In both the endotoxemia and time-control study protocols, subjects refrained from consuming caffeine or alcohol 24 h before the start of the experiment, and from food 10 h before the start of the experiment. An ALAcare 8 mg plaster (Photonamic GmbH & Co KG, Wedel, Germany) was applied on the sternum 8–10 h before the first mitochondrial respiration measurement with the COMET (off-label use approved by the institutional review board). At the end of the experiment, the measurement site was covered by a plaster to prevent exposure to light for a day. The following morning, debriefing of the volunteers followed with attention to possible side effect of LPS and the plaster. A schematic overview of the procedures is provided in Fig. 1.

Fig. 1
figure 1

Schematic overview of the experimental procedures. After fasting for 10 h, subjects were prehydrated during 1 h before intravenous administration of LPS (LPS groups) or no administration (control group). Mitochondrial respiration and blood gas parameters were measured at 4 time points during the experiment. The x-axis represents hours relative to LPS/no LPS administration.

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In the endotoxemia protocol, purified LPS (Clinical Center Reference Endotoxin derived from Escherichia coli O:113, obtained from Pharmaceutical Development Section of the National Institutes of Health (Bethesda, MD, USA)) supplied as a lyophilized powder, was reconstituted in 5 mL saline 0.9% for injection and vortex mixed for at least 20 min after reconstitution. The LPS solution was administered as an i.v. bolus injection at a dose of 2 ng/kg body weight in 1 min at T0.

In both protocols, a cannula was placed in an antecubital vein to permit infusion of 2.5% glucose / 0.45% solution; all subjects received 1.5 L 2.5% glucose / 0.45% saline during one hour, starting one hour before T0 (prehydration) as part of the standard endotoxemia protocol12, followed by 150 mL/h for six hours and 75 mL/h until completion of the experiment. All the volunteers remained in bed during the entire experiment. Baseline COMET measurements were performed before the start of prehydration. Heart rate (three-lead electrocardiogram), blood pressure, and oxygen saturation (pulse oximetry) data were recorded, starting one hour before administration of LPS until discharge eight hours post-LPS, and at corresponding timepoints in the control group. Body temperature was measured using an infrared tympanic thermometer (FirstTemp Genius 2; Sherwood Medical, Mansfield, UK). In the endotoxemia study, the radial artery was cannulated using a 20-gauge arterial catheter (Angiocath; Becton Dickinson, Sandy UT, USA) and connected to an arterial pressure monitoring set (Edwards Lifesciences, Irivine, CA, USA) to allow continuous monitoring of blood pressure (see Fig. 1). In the control group, blood pressure was measured noninvasively using a cuff.

Measurement of mitoPO2 and mitoVO2 using the COMET monitor

Oxygen-dependent quenching of the delayed fluorescence lifetime of mitochondrial PpIX is the first known method to measure mitoPO2 in living cells and tissues, in a non-invasive and feasible manner in humans. PpIX is the final precursor of heme in the heme biosynthetic pathway. PpIX is synthesized in the mitochondria and administration of 5-aminolevulinc acid (ALA) substantially enhances the PpIX concentration. We used ALAcare plasters for PpIX enhancement. Photoexcitation of PpIX populates the first excited triplet state and causes the emission of red delayed fluorescence. The delayed fluorescence lifetime is inversely related to the mitoPO2 according to the Stern–Volmer equation. The background of the delayed fluorescence lifetime technique is extensively described elsewhere9,16,17,18.

The light source and the detection system are the two core components of the COMET monitor (Photonic Healthcare, Utrecht, the Netherlands). A 515 nm pulsed laser, pulse duration 60ns, with a 10 Hz repetition rate illuminates the intra cellular accumulated PpIX. The fluorescent signal is projected on a gated red-sensitive photomultiplier tube. The light emitted by the sensor is divergent and safe for eyesight at any distance. A detailed description of the COMET measuring system can be found elsewhere8.

Local oxygen consumption is measured as mitochondrial oxygen consumption (mitoVO2), by pressure-induced occlusion of the microcirculation thereby stopping local oxygen supply. mitoVO2 and mitoPO2 were measured with the COMET at baseline, 1.45 h, 4 h and 7 h post-LPS administration, and at corresponding timepoints in the control group (Fig. 1). These time points were chosen to match.

the start of the experiment (baseline), the peak of cytokine release (1.45 h after LPS) the peak in temperature rise (4 h after LPS) and the end of the experiment (7 h after LPS). All COMET measurements were performed in the supine position measuring on the sternum and were performed by the first author (Fig. 2A). At baseline the volunteers were rested supine for at least 30 min after venous and arterial puncture to minimize the effect of stress on the baseline measurement. The measurement probe was held directly above the ALA-treated skin by hand. Occlusion of the microcirculation in the skin was achieved by manual firm pressure with the measurement probe (Fig. 2B). This simple procedure repeatedly created a measurable mitoVO2, due to cessation of the microvascular oxygen supply and ongoing cellular oxygen consumption. The mitochondrial oxygen concentration was measured before and during application of pressure at an interval of 1 Hz, using two laser pulses per measurement. The mitoVO2 is analyzed using Michaelis-Menten kinetics (Fig. 3). We previously described these principles in detail and provided a working implementation of the technique for mitoVO2 measurements18.

Fig. 2
figure 2

COMET measurements. (A) Dynamic measurement with COMET setup in the experiment. Microcirculation of subject’s skin was stopped by direct pressure with the measurement probe by the researcher. (B) COMET monitor display showing an ongoing dynamic measurement of mitoVO2. Raw data of COMET monitor was captured from the COM-port using a laptop. The subject gave permission to use his photograph.

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Fig. 3
figure 3

Michaelis Menten kinetics of mitochondrial oxygen concentration (mitoPO2). Dynamic measurement of mitochondrial respiration, sampling rate of 1 Hz. Baseline measurement for 10 s where P0 is determined. After a baseline measurement, local pressure by probe is applied which cuts oxygen supply. The cellular respiration causes the observed decrease in oxygen levels. At 38 s pressure is released and MitoPO2 recovers.

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Statistics

Baseline characteristics are represented as median with range. Experimental data are represented as mean ± standard error of mean based on their normal distribution (assessed using the Shapiro-Wilk test). Between-group differences over time were tested using repeated measures two-way analysis of variance (ANOVA, time*group interaction term) followed by Šídák’s multiple comparisons tests on individual timepoints. Two-sided p values of < 0.05 were considered statistically significant. Statistical calculations were performed using Graphpad Prism version 10.4.0 (GraphPad Software, La Jolla, CA, USA).

Results

Subject characteristics and vital parameters

A sufficient PpIX signal was obtained in all four participants of the control group and in 42 out of the 48 subjects participating in the endotoxemia study. Four subjects did not follow instructions and applied the ALA-plaster later than instructed, while in the other two subjects an insufficient signal was observed despite the fact that the plaster was applied in a timely fashion. Furthermore, the training interventions in the endotoxemia study (i.e. breathing exercise and/or exposure to cold) did not influence the main study outcomes of the current work (group*time interaction effects: p = 0.78 for mitoPO2 and p = 0.15 for mitoVO2). Therefore, data of the four groups were combined into a single LPS group. Baseline characteristics of the study subjects are listed in Table 1 and revealed no relevant differences between the LPS and control groups. Expectedly, LPS administration led to significant changes in all vital parameters compared to the control group, with MAP decreasing (p = 0.007, Fig. 4A) and temperature and HR increasing (both p < 0.0001, Fig. 4B,C) over time in the LPS group compared with the control group.

Table 1 Demographic characteristics.
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Fig. 4
figure 4

Vital parameters over time. (A) Mean arterial pressure (MAP, mmHg). (B) Heart rate (HR, BPM). (C) Temperature (Temp, C). Data are presented as mean and SEM. P-values represent the interaction term (time*group) of a 2-way repeated measures ANOVA. * indicates P < 0.05 and *** indicates P < 0.0001 calculated using Sidaks post-hoc tests comparing group differences on individual time points. BL: baseline, one hour before LPS administration or the corresponding timepoint in the control group.

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Mitochondrial function parameters

At the debriefing, no side effects of the ALA plaster were observed or reported. Figure 3 shows a representative example of the mitoVO2 tracing for a single time point, analyzed using an adapted Michaelis-Menten fit procedure16. Analysis of the tracing with Michaelis-Menten kinetics yield an mitoVO2 of -8 mmHg*s− 1, value for oxygen concentration change per second, and a baseline mitoPO2 of 73 mmHg.

Compared to the control group, mitoPO2 significantly decreased over time in the LPS group (p = 0.002), with the nadir observed at 1.45 h post-LPS administration (45.8 ± 1.8 vs. 75.2 ± 2.6 mmHg, Fig. 5A). LPS administration did not result in significant changes in mitoVO2 over time compared to the control group (p = 0.58, Fig. 5B).

Fig. 5
figure 5

Mitochondrial oxygen parameters over time. (A) Mitochondrial oxygen concentration (mitoPO2). (B) Dynamic mitochondrial oxygen consumption (mitoVO2). Data are presented as mean and SEM. P-values represent the interaction term (time*group) of a 2-way repeated measures ANOVA. *Indicates P < 0.05 calculated using Sidaks post-hoc tests comparing group differences on individual time points. BL: baseline, one hour before LPS administration or the corresponding timepoint in the control group.

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Discussion

In the present study, we were able to obtain mitoPO2 and mitoVO2 measurements in 46 out of the 52 enrolled subjects. The LPS-induced systemic inflammatory response resulted in decreased mitoPO2 values, whereas mitoVO2 was not affected. Thereby, we demonstrate the feasibility of using the COMET measurement system to perform sequential mitochondrial respiration measurements at the bedside in a dynamic setting during experimental human endotoxemia.

Due to the set-up of the experiments (volunteers arrived at the hospital early in the morning), we choose to apply the ALA-plaster longer before the first measurement than in earlier studies. In six subjects, a reliable PpIX signal could not be obtained. Four subjects did not comply with the instruction to apply the plaster in time (i.e. a minimum of 8 h before the first measurement), so a relevant rise in PpIX was not expected. Henceforth, the measurement did not succeed in two out of the 48 volunteers who correctly followed the application instructions of the ALA plaster, yielding a success rate of 96%.

To the best of our knowledge, we are the first to show changes in in vivo mitochondrial oxygenation during experimental human endotoxemia. LPS administration resulted in an early significant decrease in mitoPO2 in the LPS group. In earlier experimental work in rats, a sharp decline in mitoPO2 following LPS administration was also observed, which was reversed by aggressive fluid resuscitation14,19. However, LPS administration also decreased mitoVO2 by 30% in rats, an effect which was not observed in the present study10,19. This discrepancy with the results of the current study may be explained by the use of a much higher dosage of LPS in rats, resulting in a more pronounced systemic inflammatory response14,19.

MitoPO2 represents a measure of the balance between oxygen demand and supply in tissue. A decrease in mitoPO2 may indicate a decrease in oxygen supply to the measurement site or an increase of oxygen usage while supply is maintained. Combined with the relatively minor change in mitoVO2, the decreased mitoPO2 observed after LPS administration in the present study is more likely indicative of decreased oxygen supply and thus blood supply to the skin, marking the first step of centralization of blood supply during endotoxemia.

For the purpose of comparing our mitoVO2 data to known cell consumption found in cell cultures we provide the following model calculation. The diameter of the illuminated spot on the skin is approximately 5 mm. While the penetration depth of the green excitation light is approximately 0.5 mm, the use of ALA plasters limits the actual measurement depth to the epidermis. The epidermal thickness on the arm is typically 40 μm20. The effective measuring volume is therefore approximately 0.8 µL. Since the average volume of skin cells is around 800 cubical microns20 the number of skin cells in the measuring volume is in the range of 1 million. The temperature of the skin during the measurements is around 30 degrees Celsius (continuously measured with the COMET) and the solubility coefficient of oxygen in physiological solutions at this temperature is approximately 0.032 ml O2/L/mmHg21, equivalent to 1.4 µmol/L/mmHg. Therefore, a unit of 1 mmHg mitoPO2 in the COMET measurement is about equivalent to 1.0 pmol O2 in the measuring volume. A mitoVO2 of 8 mmHg/s therefore reflects an oxygen consumption rate of roughly 0.5 nmol O2/min per 1 million cells. This value is lower than previously reported values from standard cell cultures. For example, Gasparrini et al. reported an oxygen consumption rate of 0.9 nmol O2/min/106 cells in human dermal fibroblasts22. However, our measurements compare favorably to the reported value of 0.7 nmol O2/min/106 cells in 3D cultures of fibroblasts23, especially considering the somewhat lower temperature during our measurements in in vivo skin.

Although our previous studies in rats showed stable values of mitoPO2 and mitoVO2 in the control groups over time14,19, the control group in the current study showed a relatively high mitoPO2 at baseline, followed by a decline. In a recent clinical study in patients under anesthesia, a small decline of mitoPO2 was also observed during a similar timeframe24.

The mean arterial blood pressure in the LPS group was noticeably higher than in the control group. It might be speculated that the breathing exercises and/or cold exposure that the subjects of the LPS group practiced influenced baseline cathecholamine levels. As adrenalin is known to stimulate the Na-K-ATP pump, glycolysis and ATP consumption, this may have influenced the mitochondrial oxygen consumption rate. However, as described in publication of the primary endpoints of the endotoxemia study15, circulating adrenaline concentrations were low at baseline. Therefore, this factor is not likely to have been of influence on the mitochondrial oxygen consumption rate.

Mitochondrial dysfunction is suggested to play a major role in the development of organ failure in sepsis25. Although the presence of pathophysiological changes in mitochondrial function in sepsis is well described26, the role of mitochondrial dysfunction in sepsis remains controversial4. Nevertheless, changes in mitochondrial function in blood cells, platelet and peripheral mononuclear blood cells are associated with mortality and morbidity in sepsis in adults and children3,27. Therefore, an in vivo bedside monitor that enables frequent longitudinal monitoring can be of importance in determining the pathophysiological role of mitochondrial dysfunction in sepsis. However, the translation of these findings to sepsis patients has to be treated with caution for several reasons. We studied healthy volunteers between 20 and 35 years of age, while most sepsis patients are much older, with a recent sepsis study reporting nearly a quarter of the study population being older than 75 years28. Moreover, the inflammatory stimulus in the model we used is relatively mild and only short-lived, as opposed to an ongoing and often severe infection in patients. Nevertheless, the COMET was monitor was still able to detect changes in mitochondrial oxygen tension. Furthermore, experimental human endotoxemia is a highly controlled and reproducible model used to investigate the inflammatory response and therapeutic modalities for sepsis29,30,31.

Conclusion

We show feasibility of measuring kinetics of mitochondrial oxygen tension and cellular oxygen usage in a model of systemic inflammation in humans in vivo, using a method that was previously established in rats10,14,19. We demonstrate a significant decrease in mitoPO2, whereas mitoVO2 did not change following LPS administration. Thereby, these findings show technical feasibility of frequent measurements of mitochondrial function parameters with the COMET monitor, paving the way for clinical implementation of in vivo bedside monitoring of mitochondrial respiration.