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. 2018 Oct 25;13(10):e0205707.
doi: 10.1371/journal.pone.0205707. eCollection 2018.

Accurate Measurement of Endogenous Adenosine in Human Blood

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Free PMC article

Accurate Measurement of Endogenous Adenosine in Human Blood

Lars Löfgren et al. PLoS One. .
Free PMC article

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.

Conflict of interest statement

I have read the journal's policy and the authors of this manuscript have the following competing interests: L. L., S. P. and S. N. are employees of AstraZeneca. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
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.
Fig 2
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
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
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
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
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
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.

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Grant support

The study was funded by AstraZeneca. The funder provided support in the form of salaries for authors L.L., S.P. and S.N., all employees of AstraZeneca, and payment for the method validation work performed by Q&Q Labs AB, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of the authors are articulated in the ‘author contributions’ section.
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