Determination of ATP and ADP in blood platelets: A modification of the firefly luciferase assay for plasma

Abstract

A method for ATP-ADP determination in plasma by ethanol extraction and firefly luminescence has been modified for platelets. EDTA had to be included in the ethanol for proper inactivation of platelet adenylate kinase. Presence of EDTA inhibited conversion of ADP to ATP (prior to luminescence measurement), presumably through a drop in pH caused by the interaction between H 2EDTA 2− and the Mg 2+ of the pyruvate-kinase system employed. Introduction of Tris maleate buffer gave 100% ADP-ATP conversion. Optimal amounts of platelet ATP (99%) and ADP (80–90%) were solubilized by 50% EDTA ethanol; less EDTA ethanol failed to inactivate ATP-ADP degrading platelet enzymes whereas increasing EDTA ethanol concentrations gave decreasing solubilization of the nucleotides. ATP and ADP could be extracted from the EDTA ethanol-insoluble material by 0.6 M HClO 4. ADP and ATP added to such EDTA-ethanol and HClO 4 extracts of both platelet-rich plasma and suspensions of washed platelets gave luminescence responses per mole nucleotide equaling that of ATP-ADP standards. Optimal conditions for nucleotide determination are described as well as the beneficial use of the DuPont luminescence biometer.