Luminescent study of proapo- and proferroptotic free radical reactions in the presence of phospholipids detected in atherosclerosis
One of the most modern issues in the study of the pathophysiological quantum-molecular basis of the development of a pathological process is the relationship between free radicals and regulatory lipidomics. Thus, over the course of the 21st century, a number of mechanisms have been discovered that determine various cellular process from the point of view of specific phospholipids that perform their (not only membrane-barrier or matrix) functions. For example, the famous phagocytic “eat me” signal, from a molecular point of view, indicates the presence of oxidized phosphatidylserine on the outer leaflet of the cell membrane, which is normally present only on the inner membrane. During apoptosis, oxidized cardiolipin appears in mitochondria, and the oxidation of arachidonic acid in phosphatidylethanolamine is a specific sign of ferroptosis.
In our work, we discovered a number of new properties which bind with the oxidation of cardiolipin in proapoptotic and proferroptotic reactions.
Thus, using surface plasmon resonance methods and using fluorescence quenching in the presence of cytochrome c, it was shown that the steady state association constant for cytochrome c with cardiolipin is about 10-7, but what is interesting is that the stoichiometry for this reaction goes far beyond the expected limits and is about 35 molecules of cardiolipin containing oleic fatty acids for every molecule of cytochrome c (instead of expected 4).
Using chemiluminescence, such complexes of cytochrome c with tetraoleoyl cardiolipin were found to have the ability to induce peroxidation of model biological membranes, and also only in a very narrow concentration range, which is close to the area with anomalous stoichiometry.
Chemiluminescent reactions showed that complexes of cytochrome c with tetraoleoyl caridolipin demonstrate high peroxidase activity, the intensification of which at increasing concentrations of peroxides leads to the release of iron from the heme, which means a switch from the pro-apoptotic to the pro-ferroptotic pathway. This source of iron, as we believe, can become the main one in the development of ferroptotic processes.
And today, we have discovered that such cytochrome c-dependent reactions sharply intensify with the development of atherosclerosis. Using mass spectrometry, we studied changes in the composition of membranes in the area of atherosclerotic plaques of patients after coronary artery bypass surgery and showed that in all cases the amount of oleic fatty acid significantly increases. And when cytochrome c is added to such “atherosclerotic” phospholipids, there is a sharp intensification of both the binding of cytochrome to oleyl-saturated membranes and a sharp increase in their peroxidation. Thus, we will explain the key role of oleic acid associated with the intensification of atherosclerotic processes.
Research in this area will not only help to identify the fundamental basis for the development of pathophysiological processes in atherosclerosis, but also, from an applied point of view, to identify a marker for the diagnosis of this process, through assessing the amount of oleic acid in the biological membranes of the endothelium of patients with coronary artery disease, which also manifests itself in the blood plasma.
German O. Stepanov
Department of General and Medical biophysics, Medical biological faculty, Pirogov Russian National Research Medical University