A luminometric method for the determination of ATP and phosphocreatine in single human skeletal muscle fibres

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Abstract
A sensitive method for the analysis of ATP and phosphocreatine (PCr) in single human skeletal muscle fibres is described. Muscle tissue was freeze‐dried and single fibres were dissected free with the aid of low‐power microscopy. The fibres were then extracted in trichloroacetic acid and neutralized with KHCO3. The assay is based on the continuous monitoring of light produced as a result of ATP degradation in the firefly luciferase reaction. PCr is measured as the amount of ATP formed in the creatine kinase reaction. The coefficient of variation was less than 4% for both ATP and PCr determination. The amount of tissue required for the assay is approximately 0.5 μg (dry weight). The assay showed good agreement with spectrophotometric and high‐performance liquid chromatographic (HPLC) measurements made upon extracts of whole muscle tissue.