Adenosine Measurement in humans in vivo

Adenosine, a degradation product of adenosinetriphosphate (ATP) accumulates in
many tissues following hypoxia, ischemia and inflammation. Known as the
*retaliatory* metabolite adenosine is able to modulate several physological
processes.

Adenosine is formed both extracellular as intracellular by dephosphorylation of
adenosinemonophosphate (AMP) by 5*-nucleotidase. The degradation of adenosine
is mainly intracellular, through adenosine deaminase and adenosine kinase. The
equilibrative nucleoside transporter (ENT) controles facillitated diffusion
between extra- and intracellular adensoine.

Over the past 60 years adenosine measurement has proven to be an extremely
difficult task. With a half life of approximately 1 second adenosine is rapidly
taken up and metabolised by erythrocytes.

In this study we describe an optimized method for the detection of adenosine in
blood. First, a syringe system enables us to withdraw blood and deliver blocker
solution to the sample at the same time. Secondly, a blocker solution
consisting of an adenosine re-uptake inhibitor, deaminase inhibitor, kinase
inhibitor and a 5`-nucleotidase inhibitor, paralyses the adenosine metabolism.

In order to create a reproducable and valid method for adenosine measurement we
tested blood several times within the same subject. Furthermore we used the
cold pressor test (CPT) as a local vasoconstrictor-inducing stimulus to
increase plasma levels of adenosine. Treatment with the well-known adenosine
re-uptake inhibitor dipyridamole was used to create higher levels of plasma
adenosine.

Creating a reproducible and valid method for adenosine measurement

methodological

Cold Pressor test:
The volunteers left hand will be held in ice water for 2 minutes

Forearm Bloodflow will be measured by venous occlusion plethysmography.

Time: screening 20 minutes
experiments day 1 and 7: 210 minutes

Bloodsampling: venous infusion on day 1 and 7 in both arms

Bloedcollection: total bloodsampling 50 ml.

Dipyridamole: Subjects will use dipyridamole for 7 days (twice daily Persantin
retard 200 mg). The *safety-analysis* ESPS-2 study showed that this
concentration is safe, besides a headache no other adverse effects are to be
expected. There are no health riks involved in the use of dipyridamole.