My initial understanding of how permeabilization worked was that when the permeabilizing agent of recombinant cytolysin protein was injected to create large pores in the leukocytes outer membrane, we would see a normal OXPHOS blueprint from each of the working complexes and a flatline for the impaired complex.
However on reflection, once the leukocytes outer membrane was permeabilized and the cells contents leaked out into the cytosol, there would be no further substrates of any type left in the cell to fuel any of the enzyme complexes CI-CIV.
What I now expect to see is that once the leukocytes membrane is permeabilized, and a specific substrate is injected, the only enzyme complex that will be activated to process the substrate and produce ATP, will be the complex that can oxidise the substrate injected into the cell.
As an example, by selectively injecting succinate we can test complex II’s ability to produce ATP. Any increase or change in OCR will indicate production of ATP indicating that Complex II is working normally. However if there is no OCR consumption, then Complex II has been identified as impaired suggesting that CII, may be the cause of the PD mitochondrial bioenergetic impairment. This then gives us a target for more specific clinical and pharmaceutical investigation.
