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

Abstract

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 highperformance 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 Á RBC À1 min À1 . Drug Dev. Res. 59:152-160, 2003.

Simultaneous determination of adenosine triphosphate and its metabolites in human whole blood by RP-HPLC and UV-detection

“…Erythrocyte ATP concentrations exceed plasma ATP concentrations, which are in the nanomolar range, by over 10,000-fold [32]. A frequently used method to measure ATP is the luciferinluciferase assay [33,34]. This method has the sensitivity needed for the detection of low plasma ATP levels.…”

“…However, its main disadvantage compared to our method is that only ATP can be determined, whereas we can quantify the complete adenylate pool in one run. Ion-exchange methods often require more extensive pretreatment of samples and have difficulties in separating nucleobases, nucleosides and nucleotides in one run, given the charged nature of the nucleotides in the operating pH range (pH 2-7) [33,[36][37][38][39].…”

Dynamics of shear-induced ATP release from red blood cells

“…2C versus the duration of the applied shear stress. Below the activation time of 3-6 ms there is little or no additional ATP released compared with the baseline, but there is a maximum at ᐉ c ϭ 800 m. These data can be interpreted in terms of the available intracellular ATP concentration, which for RBCs is Ϸ10 mM (5,27,28). Only 1% or less of the intracellular ATP molecules is believed to be released for signaling purposes (5).…”

Acute Effect of MCRC on Selected Blood Parameters - A Placebo-controlled Acute Clinical Study

“…Indirectly, RBC can affect vasodilation, platelet function, and functioning of endothelial and smooth muscle cells mainly by releasing ATP to extracellular milieu [25,33]. Also, it has been reported that extracellular ATP may exert pronounced effects on a variety of biological processes including neurotransmission, muscle contraction, cardiac function, immune cell function, platelet function and vasodilatation [25,34]. Interestingly, the flow of ATP released from RBC determines plasma levels of extracellular ATP [34] and this process may depend on the intracellular amount of ATP in RBC.…”