Project Abstract Long-chain fatty acid oxidation disorders (LC-FAODs) are a heterogenous group of disorders characterized by the inability to break down long-chain fatty acids in the mitochondria for energy. The primary tissues affected are liver, heart, and muscle. These disorders are identified at birth through newborn screening programs. Treatment consists of fasting avoidance and replacing long-chain fats in the diet with medium-chain fatty acids (MCFA). Despite decades of orally dosing patients with MCFA-containing oils, it is not understood how MCFA are metabolized by liver, muscle, and heart. Further, the rigor of the experimental evidence regarding the therapeutic efficacy of oral MCFA is low. In mouse models of LC-FAOD, oral MCFA do not improve cardiomyopathy or the capacity for exercise. Human patients likewise still suffer from muscle symptoms and rhabdomyolysis. In the current proposal it is postulated that there are two major problems with current MCFA-based therapies. First, muscle and heart are not equipped to metabolize exogenous MCFA. Second, orally-administered MCFA are nearly completely absorbed by the liver and do distribute to heart and muscle. It is hypothesized that MCFA therapy can be optimized to treat cardiomyopathy and rhabdomyolysis through the exploration of alternative medium-chain lipid species and alternative routes of delivery. The hypothesis is supported by preliminary data showing that heart and muscle prefer carnitine conjugates of MCFA (MC-carnitines) over free MCFAs, and a demonstrated improvement in muscle function of LC-FAOD mice upon subcutaneous injection of an MC- carnitine. This hypothesis will be fully explored in three Specific Aims: 1) Determine the optimal medium-chain lipid species for liver, heart and muscle; 2) Determine the bioavailability and biodistribution of orally versus subcutaneously-administered medium-chain lipids; and 3) Determine the therapeutic efficacy of medium-chain lipids in LC-FAOD mice. Aim 1 is expected to show that liver prefers free MCFA as substrates, while muscle and heart prefer MC-carnitines. The differential preference is proposed to be due to the presence of mitochondrial medium-chain acyl-CoA synthases in liver but not heart or muscle. Aim 2 is expected to demonstrate that subcutaneous delivery of medium-chain lipids greatly increases bioavailability and subsequent biodistribution to the periphery. Finally, in Aim 3, pre-clinical testing of LC-FAOD mouse models is expected to document the therapeutic advantage of the optimized substrates from Aim 1 and the subcutaneous delivery from Aim 2. The results of this project will lay the groundwork for more personalized, symptom-specific application of MCFA- based therapies in LC-FAOD patients.