Fig 1.
Regulation of blood plasma concentrations of adenosine.
The extracellular concentrations of adenosine are depending on the balance between its generation and metabolism. Adenosine is generated by the ecto-5′-nucleotidase CD73 mediated degradation of AMP which is proceeded by the ectonucleoside triphosphate diphosphohydrolase-1 CD39 mediated hydrolysis of ATP and ADP to AMP. The processes regulating adenosine metabolism is more complex and is largely restricted to the intracellular space as adenosine deaminase (ADA) and adenosine kinase (AK) enzyme activity predominantly exist intracellularly [3]. Hence, adenosine transport into cells is a key regulatory step in adenosine metabolism. Cell uptake of adenosine is primarily mediated by the equilibrative nucleoside transporter 1 (ENT1) which acts to keep the adenosine extracellular and intracellular concentration 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.
Table 1.
Composition of inhibitor solutions (μmol/L).
Table 2.
Mass spectrometry settings on Agilent 6540 UPLC-tandem-MS system.
Fig 2.
Effects of inhibitors of adenosine formation and clearance on plasma concentrations of exogenous 13C5-adenosine (A), 15N5-adenosine from exogenous 15N5-AMP (B), 15N5-hypoxanthine from exogenous 15N5-AMP (C), and endogenous adenosine (D). Venous blood from healthy donors was collected into blood collection tubes pre-filled with inhibitor solutions containing final blood concentrations of 1 μmol/L 13C5-adenosine and 10 μmol/L 15N5-AMP. Plasma was prepared within 5 min of blood collection. Data expressed as means ± SD, n = 5.
Fig 3.
Stability over time of exogenous 13C5-adenosine in blood collected into STOP solution.
Venous blood from healthy donors was collected into blood collection tubes pre-filled with STOP solution containing a final blood concentration of 1μmol/L 13C5-adenosine. Plasma was prepared at 4, 15 and 30 min after blood collection. Data expressed as means ±SD, n = 5.
Fig 4.
15N5-adenosine formation over time by degradation of exogenous 15N5-AMP in blood collected into STOP solution.
Venous blood from healthy donors was collected into blood collection tubes pre-filled with the STOP solution containing a final blood concentration of 10 μmol/L 15N5-AMP. Plasma was prepared at 4, 15 and 30 min after blood collection. Data expressed as means ±SD, n = 5.
Fig 5.
Comparing STOP and STOPx2 performance in conservation of exogenous 13C10-15N5-adenosine (A) and measured endogenous adenosine concentrations (B) Venous blood was collected into blood collection tubes pre-filled with STOP and STOPx2 containing 100 nmol/L 13C10-15N5-adenosine final blood concentrations. Plasma was prepared at 4, 15 and 30 min after blood collection. Data expressed as means ± SD, n = 5.
Fig 6.
Endogenous adenosine in human blood plasma.
Venous blood was collected into blood collection tubes pre-filled with STOP solution. Plasma was prepared at 4, 15 and 30 min after blood collection. Data expressed as means ±SD, n = 10.
Fig 7.
Ticagrelor effects on exogenous 13C5-adenosine clearance in blood.
Venous blood was pre-incubated in vitro with ticagrelor for one h. Pre-incubated blood was spiked with a final blood concentration of 1 μmol/L 13C5-adenosine and mixed by gently inverting the tubes eight times. After 1 min, blood was transferred into the STOP solution and plasma was directly prepared. Data expressed as means ± SD, n = 4. p value by one-tailed paired t-test.