P4.021: Mitigating Oxidative Stress in Barth Syndrome: A Novel Therapeutic Approach Using Bis-Allylically-Deuterated Arachidonic Acid

Background: Barth syndrome (BTS) is a rare X-linked-disorder caused by mutations in Tafazzin (Taz), which regulates remodeling of cardiolipin, a mitochondria-specific phospholipid. BTS affects the heart, skeletal muscles, and neurocognitive system; yet no mechanism-based therapies exist due to incomplete understanding of pathogenesis. We have put forward a new hypothesis of BTS-pathogenesis: accumulation of lyso-cardiolipin facilitates formation of a complex with cytochrome-c with aberrant peroxidase activity. This leads to widespread peroxidation of phospholipids. We proposed a mechanism-based-therapy using the kinetic isotopic effect, employing arachidonic-acid with fully deuterated bis-allylic positions (AA-D6) to suppress pathogenic phospholipid peroxidation by the complex. To test the success and specificity of our approach we tested the effect of non-bis-allylically-deuterated arachidonic-acid (AA-D11). To assess the resulting redox-lipidomics we used LC-MS/MS-protocols developed by us.

Methods: Using HT-22 (mouse hippocampal-neuronal-cells) we first evaluated the concentration dependence of AA-D6 for anti-ferroptotic activity after treatment with RSL3 (a GPX4 inhibitor) and Ferrostatin-1 (ferroptosis inhibitor as a control). Cell death was assessed using LDH-release. Next, cells were incubated with AA-D6 or AA-D11 to examine the effectiveness of integration of deuterated-AA into major phospholipids. Each group was treated with +RSL3± Ferrostatin-1 for an additional 17 hours. LC-MS/MS redox lipidomics was performed in negative ion mode using a Dionex Ultimate 3000 HPLC system with normal-phase silica column coupled to a Thermo Orbitrap Fusion Lumos mass spectrometer. Analysis of LC-MS/MS data was performed using the Compound Discoverer™ 2.0 software package. The species assignments were done by retention-time, exact mass, and fragmentation analysis.

Results: We demonstrated successful incorporation of AA-D6 and AA-D11 into various phospholipid classes, including cardiolipin (CL), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylcholine (PC). LC-MS/MS established effective AA-D6/AA-D11 integration into the phospholipids. Most importantly, cells loaded with AA-D6 (but not with AA-D11) exhibited full protection against phospholipid peroxidation and prevented ferroptotic death.

Conclusion: Detailed characterization of D6-AA vs D11-AA contents in different classes of phospholipids will provide important information on the mitochondria-related protective potential of this novel approach as a new therapeutic approach in Barth Syndrome.