Unlocking the Power of Mitochondrial Function: Real-time Assessment of
Mitochondrial ATP contents and Response Against Inhibiting and
Stimulating Substrates (MitoRAISE)
Abstract
Purpose The objective is to develop a real-time Mitochondrial ATP
contents and Response Against Inhibiting and Stimulating Substrates
(MitoRAISE) assay and assess its potential to evaluate the mitochondrial
oxidative phosphorylation function. Methods MitoRAISE measures the total
ATP contents, ATP synthesis capacity, and the response to inhibitory
substrates. The measurements were all quantified with an ATP standard
curve and validated with in vitro testing. MitoRAISE was then applied
using peripheral blood mononuclear cells (PBMCs) from 35 healthy
volunteers and 20 breast cancer patients. Result Results from isolated
mitochondria, cells with different permeabilization percentages, and
cells with damaged mitochondria demonstrated the ability of capturing
ATP signals specifically from the mitochondria. Breast cancer PBMCs
exhibited a significant increase in glutamic acid- and malic
acid-induced mitochondrial ATP synthesis capacity yet a significant
decrease in mitochondrial DNA copy number (mtDNA CN) compared to healthy
PBMCs. Furthermore, breast cancer PBMCs mostly showed negative
correlation between mtDNA CN and parameters from MitoRAISE but healthy
individuals showed positive correlation. Conclusion We developed a quick
and easy method to detect real-time mitochondrial activity in live
cells. Monitoring mitochondrial ATP synthesis capacity and sensitivity
to inhibitory substrates could aid in assessing the functional status of
mitochondrial oxidative phosphorylation.