Critical ATP parameters associated with blood and mammalian cells: Relevant measurement techniques

We have developed an integrated package that includes software programs and algorithms for measuring in vivo total blood ATP, extracellular blood ATP, blood ATP release rates, and plasma ATP breakdown rates. This clinically tested methodology improves upon existing luciferin/luciferase and high performance liquid chromatography assay techniques in that it involves the multidetector luminometric system for determination of blood ATP parameters with simultaneous monitoring of ATP standards and standard curve generations with r(2) > 0.99 and increased speed of preparation and assay time (time necessarily for 96-well plate preparation, assay itself, and analysis). The system and associated software program permits analysis blood ATP parameters in mol, mol/L, or mol/red blood cell (RBC) and has been extensively tested with samples measured in clinics and with samples obtained and measured at the patient bedside. The mobility of the system and ability to perform measurements on location is an important consideration and feature because of the fact that ATP blood levels are rapidly hydrolyzed and hence extremely sensitive to any perturbations associated with any vigorous manipulations during the processing and analysis. EDTA inhibition of extracellular ATP hydrolysis permits determination of extracellular ATP (plasma) and ATP release rates levels performed by iterative measurements performed on sequentially diluted blood aliquots. Back-extrapolation permits close approximation of the in vivo blood ATP parameters. The assay system is capable of detecting RBC ATP release rates as low as 10,000 ATP molecules (.) RBC-1 min(-1). (C) 2003 Wiley-Liss, Inc.