Abstract
Accurate determination of in vivo circulating concentrations of extracellular adenosine in blood samples is challenging due to the rapid formation and rapid clearance of adenosine in blood. A blood collection protocol was developed based on direct sampling of venous blood into, and instant mixing with, a STOP solution developed to conserve in vivo adenosine concentrations by completely preventing both its formation and clearance in collected blood. Stable isotope labeled AMP and adenosine spiked into blood ex vivo were used in combination with mass spectrometry to evaluate conservation of adenosine and prevention of its formation. A number of approved drugs, including the P2Y12 antagonist ticagrelor, have been described to increase extracellular adenosine. This may contribute to its clinical profile, highlighting the importance of accurate measurement of in vivo adenosine concentrations.A high sensitive ultra performance liquid chromatography–tandem- mass spectrometry (UPLC-tandem-MS) analytical method for plasma adenosine was developed and validated with a lower limit of quantification of 2 nmol/L. The method demonstrated plasma adenosine stability during sample processing and analytical method performance relevant to human blood samples. The final STOP solution proved able to conserve exogenous adenosine and to prevent adenosine formation from exogenous AMP added in vitro to human blood over 15 minutes. The mean endogenous adenosine concentration in plasma prepared from venous blood collected from 10 healthy volunteers was 13 ± 7 nmol/L. Finally, the method was used to demonstrate the previously described concentration-dependent ability of ticagrelor to conserve extracellular adenosine at clinically relevant exposures. In conclusion, we report an optimized sampling protocol and a validated analytical method for accurate measurement of in vivo circulating adenosine concentrations in human blood, suitable for use in clinical trials.
Highlights
Adenosine is an endogenous molecule that has been attributed a number of important physiological effects mediated by its four G-protein coupled receptors (A1, A2a, A2b, A3) including vasodilation, anti-platelet, anti-inflammatory and cardioprotective effects [1]
Effects of perchloric acid extraction versus filtration on plasma adenosine concentrations Sample preparation for removal of plasma proteins by the filtration method validated here was compared to classical perchloric acid (PCA) extraction followed by neutralization after 5 minutes or after 12 hours at 4 ̊C to test the stability of adenosine and AMP during strong acidic conditions occurring during PCA extraction
Exogenous adenosine was not detectable in samples collected into NO STOP whereas about 100% of added adenosine could be detected in samples collected into STOP solution
Summary
Adenosine is an endogenous molecule that has been attributed a number of important physiological effects mediated by its four G-protein coupled receptors (A1, A2a, A2b, A3) including vasodilation, anti-platelet, anti-inflammatory and cardioprotective effects [1]. Cell uptake of adenosine is primarily mediated by the equilibrative nucleoside transporter 1 (ENT1) which acts to keep the adenosine extracellular and intracellular concentrations equal. ENT1 expression on red blood cells in the circulation effectively acts as a sink of extracellular adenosine due to the rapid metabolism of adenosine once entering the cells (Fig 1). The action of adenosine is locally restricted to sites of tissue injury, hypoxia or inflammation where high local concentrations of ATP and ADP will be released whereas the systemic circulating concentrations are generally too low to induce a response. There is high variability in the reported concentrations as measuring adenosine concentrations is technically very challenging mainly due to its short half-life in blood [5]. A plasma adenosine UPLC-tandem-MS analytical assay was validated and in vitro experiments with ticagrelor confirmed prior data supporting an adenosine mode of action
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