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.