Advancing a sensitive method for measuring mitochondrial ATP production in small muscle biopsy samples

We present an optimised luminometric method for measuring muscle mitochondrial ATP production rate (MAPR), adapted to a 96-well microplate format. The enhanced assay enables quantification of ATP production from 12 or more substrate combinations within 15 min, using only 10 μL of isolated mitochondria. The method demonstrates high accuracy and precision, with a validated measurement range of 0.3-70 nmol/min/L. To support clinical interpretation, a reference dataset was established from 92 individuals aged seven months to 79 years. All these individuals were referred for muscle biopsy but were subsequently deemed unlikely to have a mitochondrial disorder following comprehensive clinical evaluation. An overview of the current version of our assays for oxidative phosphorylation (OXPHOS) enzymes is also provided. As proof of concept, we present three patients carrying pathogenic variants in mitochondrial DNA (ATP6 and MT-TL1) and the nuclear PDHA1 gene. All exhibited decreased MAPR with one or more substrates, along with additional clinical, biochemical, and morphological features consistent with mitochondrial disease. Furthermore, we illustrate the age-dependent development of MAPR in muscle across the human lifespan, demonstrating a 60-80 % higher maximal capacity for oxidative ATP production in adults compared with young children. In contrast, MAPR supported by fatty acid-derived substrates remains unchanged over the same period. In conclusion, the improved MAPR assay offers a robust and efficient tool for assessing mitochondrial function in both clinical diagnostics and research. Its high-throughput format and reliable performance make it particularly well-suited for the investigation of suspected mitochondrial disorders.