A bioluminescent assay for detection of fatty acid oxidation

Research scientists measure fatty acid oxidation (FAO) to assess how efficiently cells and tissues convert fats into energy and how this process shifts under different physiological or pathological conditions. Because FAO is central to mitochondrial energy metabolism, its measurement reveals insights into metabolic flexibility, mitochondrial health, and substrate preference. Altered FAO is implicated in many diseases including diabetes, obesity, heart failure, and cancer, making it an important marker for both mechanistic studies and drug screening. Additionally, FAO measurements help evaluate the effects of exercise, diet, or pharmacological interventions on energy balance and cellular metabolism. Radiolabeled fatty acids and oxygen consumption assays are the most common methods for measuring fatty acid oxidation (FAO), but these approaches can be labor-intensive and technically demanding. To simplify FAO measurement, we developed a bioluminescent assay based on a fatty acid-linked pro-luciferin substrate. The substrate readily enters cells, where FAO enzymes remove the fatty acid moiety, releasing a modified luciferin precursor. Addition of a detection reagent converts this intermediate to luciferin, generating luminescence proportional to FAO activity. The signal scales with cell number and incubation time and is compatible with both cancer cell lines and primary cells. With just 20,000 cells and a 1-hour incubation, the assay generates strong signal-to-background ratios - about 200 in HEK293 cells and 100 in primary human hepatocytes. The assay is sensitive to inhibition by etomoxir, confirming dependence on carnitine palmitoyltransferase (CPT1) activity, a key regulatory step in mitochondrial FAO. After media removal, all steps are performed in an add-and-read format suitable for 96- or 384-well plates, enabling convenient, high-throughput quantification of FAO. AI was used to help construct this abstract.