Endoscopic Measurements of Mitochondrial Oxygen Tension: a dose-finding study

Adequate oxygen supply to the tissues is a required condition for human life,
moreover for life of all mammalians. Oxygen supply starts with inhaling of
oxygen and subsequently the inhaled oxygen will be transported via the
circulating blood to the tissues. Many pathophysiological mechanisms lead to
insufficient oxygen supply: on one hand a decreased oxygen delivery due to for
example a decreased cardiac output, obstruction of the blood flow, anemia or
poor oxygenation, low flow state for example due to systemic vasodilatation in
sepsis, on the other hand an increased metabolic demand in, for example,
critically ill patients. Therefore, adequate and reliable measurements of
tissue oxygenation are important for diagnosis and treatment decisions of a
broad spectrum of diseases. Many techniques have been developed for oxygen
measurements in vivo but the ultimate goal is to measure oxygen at the level
where it is used by the mitochondria. Mik et al. introduced the protoporphyrin
IX-triplet state lifetime technique (PpIX-TSLT) for measuring PO2 in
mitochondria. The in vivo experiments with this technique of measuring
mitochondrial oxygen (mitoPO2) were performed in animals and humans.

The technique resulted in the development of the COMET monitor, a clinical
monitor for assessment of Cellular Oxygen METabolism, allows cutaneous mitoPO2
measurements to be made in humans. COMET measures mitoPO2 by means of the
Protoporphyrin IX-Triplet State Lifetime Technique (PpIX-TSLT). Cutaneous
application of 5-aminolevulinic acid (ALA) is necessary to induce enough
mitochondrial PpIX for detection of the weak delayed fluorescence signal. Since
the first publication of this technique in 2006, extensive research has been
done to develop an instrument for clinical use. The technique has been tested
en calibrated for use in isolated organs and in vivo in experimental animals.
The positive results of a feasibility test in healthy volunteers have been
published.

This technique is recently tested in the stomach and small intestine during
upper endoscopy in our center. We performed mitoPO2 measurements of the
gastrointestinal tract. Possibly, mitoPO2 measurements can be used in the
work-up of chronic mesenteric ischemia (CMI) in the future. CMI is the result
of insufficient blood supply to the gastro intestinal tract. Three aortic
branches provide the arterial blood supply of the gastro intestinal tract: the
celiac artery (CA), the superior mesenteric artery (SMA) and the inferior
mesenteric artery (IMA). Between these vessels is an extensive collateral
circulation. The main cause of CMI is occlusive disease of one or more
supplying arteries, most common due to atherosclerosis. In some cases, the
mesenteric blood flow is diminished due to a non-occlusive cause like decreased
cardiac output, hypovolemia and hypotension: non-occlusive mesenteric ischemia
(NOMI).

The diagnosis of CMI remains a clinical challenge because this diagnosis is
difficult to distinguish by the frequent incidence of chronic abdominal pain
and asymptomatic stenosis of the mesenteric arteries, due the presence of
abundant collateral circulation.
The standard diagnostic work up includes assessment of clinical symptoms,
radiological imaging and a functional test6,7 as visible light spectroscopy
(VLS) or tonometry. The diagnosis of CMI is based on three main components. The
first main component concerns assessment of medical history, clinical symptoms
and physical examination. Radiological imaging of the mesenteric arteries is
the second component. Finally, the third component consists of detection of
mucosal ischemia by a functional test. The sensitivity and specificity of VLS
is respectively 90% and 60% with positive and negative predictive values of 88%
and 67%, respectively. The sensitivity and specificity of 24-hours tonometry is
respectively 76% and 94% with positive and negative predictive values of 76%
and 94%, respectively. These three main components will be discussed in a
multidisciplinary team consisting of a gastroenterologist, a vascular surgeon
and an interventional radiologist, all specialized in CMI. This results in an
expert-based consensus diagnosis.
If the patient fulfills two of the following criteria, the diagnosis CMI is
made:
1. Distinctive clinical presentation including presence of postprandial pain
and otherwise unexplained weight loss of >5% of the normal body weight.
2. Significant stenosis of >70% of at least one of the mesenteric arteries
demonstrated by radiological evaluation.
3. Mucosal ischemia detected by tonometry or visible light spectroscopy (VLS).

When relief of symptoms is persistent after treatment, a definitive diagnosis
of CMI is made. In case of one-vessel disease, three of the above criteria has
to be fulfilled for the diagnosis of CMI.

Concluding, current diagnostic work-up of CMI is quite a process with various
investigations in the absence of just one specific test to diagnose CMI. There
is need of a reliable non-invasive functional test for chronic mesenteric
ischemia to assess the oxygenation of the gastrointestinal tract with high
accuracy. The protoporphyrin IX-triplet state lifetime technique for measuring
mitoPO2 in the skin, liver4 and heart is fast, non-invasive and reliable.
Recently, we have proven he feasibility of endoscopic MitoPO2 measurements in
healthy volunteers during a previous pilot-study in our center. However, these
measurements were performed with a dose of 20 mg/kg 5-aminolevulinic acid
(Gliolan). We recorded a very high signal of the 5-aminolevulinic acid induced
PpIX, which means that a lower dose of Gliolan will be effective as well for
adequate MitoPO2 measurements during upper endoscopy. Further, a lower dose of
Gliolan will provide less dose-dependent side effects as photo toxicity.

* To determine the minimum effective dose of 5-aminolevulinic acid (Gliolan)
for adequate MitoPO2 measurements during upper endoscopy.

Inclusion
Healthy volunteers with no gastrointestinal complaints and unremarkable medical
history will be asked to participate in our study through information folders
in the Erasmus MC, Rotterdam. This folder will provide information about the
study and the study procedure, and also how to contact the research
investigator. If people are interested, they can contact the coordinating
investigator for a consult to obtain further information. They will receive the
patient information folder. If a healthy volunteer decides to participate in
the study, he or she will sign the Informed Consent Form and blood sampling to
exclude renal and liver impairment and the upper endoscopy will be scheduled.

Intervention
The healthy volunteers will drink 5-aminolevulinic acid (ALA, Gliolan 30 mg/ml)
4 hours before the upper endoscopy. The dose of Gliolan will be 0 mg/kg weight
for the first volunteer and 1 mg/kg for the 2nd and third volunteer. If the
signal of the 5-aminolevulinic acid induced PpIX is too low with 1 mg/kg, a 4th
and 5th volunteer will be included an they will receive Gliolan with a dose of
5 mg/kg. If the signal of the 5-aminolevulinic acid induced PpIX is too low
with 5 mg/kg, a 6th and 7th volunteer will be included and they will receive
Gliolan with a dose of 10 mg/kg. Four hours after the administration of Gliolan
the upper endoscopy will be performed.
Healthy volunteers can choose if they want sedation or not during the
endoscopy. Sedation will be 2.5-5 mg midazolam combined with 0.05 mg fentanyl
intravenously prior to the endoscopy. The MitoPO2 measurements will be
performed with the COMET measurement system during upper endoscopy using a
sterile single use fiberoptic-catheter (MUCS000001, LightGuideOptics, Germany)
that can be passed through the accessory channel of the endoscope. Measurements
of the MitoPO2 will be performed at three sites in the stomach and duodenum:
antrum of the stomach, descending duodenum and duodenal bulb. Three repeated
readings will be taken at different areas of each location. The average of the
three readings per location will be regarded as the actual measurement of that
specific location. Furthermore, on each location the probe will gently pressed
on the tissue to demonstrate the oxygen dependence of the measurements. To
prevent luminal spasms butylscopalamin 20mg is admitted intravenously before
the start of the measurements.

Afterwards, healthy volunteers with sedation will be brought to the endoscopy
recovery room to sleep off. If no sedation is used, healthy volunteers are
required to go immediately after the endoscopy. It is important to notice that
they should avoid exposure to strong light sources (eg. direct sunlight or
brightly focused indoor light) of eyes and skin for 24 hours after
administration of Gliolan. The total duration of the upper endoscopy will be 15
minutes maximally.

Follow-up
There is no follow-up of the healthy volunteers in this study. Obviously, if
during upper endoscopy findings are detected, the healthy volunteer will be
referred to our outpatient clinic for further analysis and treatment.

An upper endoscopy with MitoPO2 measurements will be performed in healthy
volunteers. The risks of upper endoscopy without invasive intervention are very
low, especially when no sedation is used. Oral administration of Gliolan (for
PpIX induction) is safe. The risk of phototoxicity after PpIX induction is
considered low, because the COMET uses short-pulsed excitation and very low
total light dosage. To limit the potential effects of phototoxicity, healthy
volunteers will be instructed to avoid exposure to strong light sources (eg.
direct sunlight or brightly focused indoor light) of eyes and skin for 24 hours
after administration of Gliolan.