Measurement of adenine nucleotides in plasma

Abstract

The goal of this study was to identify the most important variables affecting bioluminescent ATP, ADP and AMP measurements in plasma and to develop an assay that takes these variables into account. Blood samples were drawn from conscious dogs. A 'stop solution' containing EDTA was prepared, which greatly retarded plasma ATP degradation by chelating Mg(+2) and Ca(+2) that are co-factors for many ATPases. Stop solution and blood were mixed using a two-syringe withdrawal system. Samples were centrifuged twice in order to remove red blood cells, and ATP was measured in the supernatant using the firefly luciferase assay. Sample pH was adjusted to the optimal range (7.75-7.95) and Mg(2+) (necessary for the luciferase reaction) was added back to the sample within the luminometer 2 s prior to luciferase addition. Four assay tubes were prepared for each plasma sample, containing standard additions of 0-15 pmol added ATP, in order to quantify native plasma ATP content. In separate plasma/stop solution samples ADP + ATP was measured after converting ADP to ATP via the pyruvate kinase reaction, and AMP + ADP + ATP was measured after addition of both myokinase and pyruvate kinase. Addition of forskolin and isobutylmethylxanthine (IBMX) to the stop solution to inhibit platelets resulted in lower ATP concentrations. Measurement of ATP and haemoglobin from lysed erythrocytes revealed that haemolysis exerts a strong influence on plasma ATP concentration that must be taken into account.

Human Plasma ATP Concentration

“…A plasma ATP assay (14 ) developed for the aforementioned dog study measured mean dog plasma ATP concentrations of 25-50 nmol/L (13 ). These concentrations are far lower than reported human venous plasma ATP concentrations, which are generally in the 1 mol/L range ( Table 1).…”

“…Hemolysis is another variable that may increase measured plasma ATP concentrations (14,21 ). Erythrocytes have millimolar cytosolic ATP concentrations, so that even a small amount of hemolysis can significantly increase plasma ATP concentration.…”

“…Plasma hemoglobin concentration is used to estimate the degree of sample hemolysis (28 ). The ATP and hemoglobin concentrations from freshly hemolyzed erythrocyte solutions are used to calculate the sample ATP concentration attributable to hemolysis (14 ).…”

“…However, EDTA does not prevent potential ATP release from erythrocytes or platelets during sample preparation. Gorman et al (14 ) collected blood samples in a stabilizing solution designed to minimize ATP release by platelets and erythrocytes and prevent plasma ATP catabolism. The stabilizing solution contains EDTA to inhibit ATPases and nitrobenzylthioinosine (NBTI) 3 to inhibit ATP release from erythrocytes (8 ).…”

Mechanisms for the control of local tissue blood flow during thermal interventions: influence of temperature‐dependent ATP release from human blood and endothelial cells

“…2003). Briefly, the stop solution contained 5 nmol l −1 S ‐(4‐nitrobenzyl)‐6‐thioinosine, 100 μmol l −1 3‐isobutyl‐1‐methylxanthine, 10 μmol l −1 forskolin, 4.15 mmol l −1 EDTA, 118 mmol l −1 NaCl, 5 mmol l −1 KCl and 40 mmol l −1 tricine buffer.…”