Discussion
Since the discovery of ATP in 1929 and its function as an energy carrier
in the late 1930s,[18] the importance of
synthesizing ATP through OXPHOS system has never been questioned
.[19] Among the co-working protein complexes in
the mitochondria, mitochondrial complex I is the largest, and its
dysfunction is directly linked to mitochondrial malfunction in dementia
and cancer.[20, 21] Mitochondrial complex II, on
the other hand, is the smallest protein complex involved in both the
tricarboxylic acid cycle and OXPHOS. Complex II complications can lead
to cancer,[22] cell death, and
necrosis.[23]
Currently, assays are available to only estimate the rate of ATP
production. Agilent Seahorse XF Analyzer is the most widely used
platform for estimating the total ATP produced through calculating
oxygen consumption and extracellular acidification
rate.[24, 25] Oroboros Oxygraph-O2k also measures
mitochondrial respiration to estimate ATP
production.[26] Oxygen consumption and ATP
synthesis are not directly associated and may be masked by other
factors, such as mitochondrial concentration or ATP
concentration.[27] Since measuring oxygen
consumption rate may not be an accurate measure of ATP production, there
is a need to investigate the actual energy
metabolism.[28]
Our assay focuses on measuring the actual change of ATP in the
mitochondria in response to activating or inhibiting substrates. Using
an agent nontoxic to mitochondria, we were able to permeabilize the
cells and directly deliver mitochondrial complex-activating substrates.
The protocol can be applied to both adhesion and suspension cells and
showed high reproducibility in both cell types. During the development
process, the A549 cell line was tested on different dates, yet the
GM-induced ATP synthesis capacity was consistently 160 ± 20 nm/min,
whereas the S-induced ATP synthesis capacity was always 294 ± 70 nM/min,
which is an error range of only 10%–20% (data not shown). Notably, we
were also able to obtain reproducible data from PBMCs. The samples
tested immediately after blood collection and after 6 hours of
incubation at room temperature only had a 10–20% error range (data not
shown).
In addition to the novel measurement of mitochondrial ATP synthesis
capacity, MitoRAISE can measure the ATP contents and sensitivity to
inhibiting substrates. Finding the sensitivity to inhibiting substrates
can be helpful in quickly finding the sensitivity of a patient to a
specific drug. For example, when treating a patient with a drug with
known inhibitory effects on the mitochondria, testing the patient PBMCs
with MitoRAISE would be a non-invasive method to screen the drug. One of
the greatest advantages of MitoRAISE is that it is not limited to just
one specific type of activating substrates or inhibiting substrates. We
utilized GM and S to activate mitochondrial complexes I and II, but
other activating substrates can be utilized. The ATP synthesis capacity
can also signify the mitochondrial sensitivity or reactivity to specific
substrates. As such, we can utilize MitoRAISE to measure the
mitochondrial reactivity to inhibitory substrates.
PBMCs are readily available sources of patient samples and are currently
receiving attention for their potential use as predictive risk
biomarkers for multiple diseases.[29] Myriad of
studies have shown the use of PBMCs as potential tools to determine the
inflammatory and metabolic status in various disease states, including
chronic fatigue syndrome,[30] and type 2
diabetes.[31] Animal model studies have also shown
that the metabolic responses of PBMCs can correlate with tissues such as
the liver and brain. [32,33] Data from our
preliminary clinical study showed disease-linked disparity in PBMC
mitochondrial activity parameters, in which breast cancer patients
showed higher ATP synthesis capacity yet lower mtDNA copy number and
lower sensitivity to inhibiting substrates. Also, healthy females showed
positive correlation between mitochondrial DNA copy number and ATP
synthesis capacity and inhibitory substrate sensitivity meaning more
mitochondria meant more ATP synthesis capacity and more reaction to the
inhibitory substrates, but breast cancer patients showed slightly
negative correlation. When testing the newly developed assay for ATP
synthesis capacity, we found differences in the mitochondrial function
between gender and between diseased and healthy. It has been shown that
upregulated mitochondrial complex I is required for high metastatic
features of colorectal cancer cells.[34] Likewise,
the upregulated ATP producing capacity in the PBMCs of breast cancer
patients could signify the high need of energy in cancer cells. Thus,
this newly developed MitoRAISE assay can be a prospective tool in
determining mitochondrial function of patients.