Quantitating reactive oxygen species (by Flow Cytometry) in healthy volunteers day-by-day.

Hyperoxia stands for a high concentration of oxygen in blood. Hyperoxia leads
to adverse effects, for example to important circulatory effects. The
hypothesis is that hyperoxia causes vasoconstriction in the microcirculation,
which causes an increase in systemic vascular resistance. The increase in
vascular resistance causes a reduction of the cardiac output to keep the blood
pressure constant.

Optimal PaO2 levels are unknown. To determine an optimal PaO2 range, it is
important to quantify oxidative stress because it appears related to
hyperoxia. Oxidative stress is defined as an imbalance between the production
of ROS and anti-oxidants defence system. ROS has the potential to cause damage
to lipids, proteins and DNA.

To investigate pathologies involving oxidative stress, it is important to
reliably measure ROS production. Indirect methods are generally used, which
measure end products of oxidative stress (F2-isoprostanes). Direct methods for
ROS production are preferred but are difficult to perform, because ROS are
highly reactive and results are hard to reproduce (analytical and
pre-analytical factors are of great influence).

Currently, we have an assay that is capable of directly measuring ROS under
strict conditions. We are in progress of optimizing said assay, so that it can
be applied in clinical trials investigating oxidative stress.

To validate a flowcytometry assay which can measure ROS day-by-day with healthy
volunteers and CABG patients.

To evaluate the assay we will perform measurements in 5 healthy volunteers and
in 5 CABG patients. Of the healthy volunteers, 3 blood samples will be
collected over 3 consecutive days. Since these healthy volunteers are expected
to have a low and steady production of ROS, we anticipate to see this reflected
in our assay. Additionally, these measurements will give us insight into the
inter-assay variation. In the CABG patients, a total of 3 blood samples will be
taken perioperatively (before, during and 12 hours after surgery) and the
results will be compared with our current assay. To determine inter-assay
variation, all measurements will be performed in triplicate.

Study subjects will undergo three venipunctures. Stress and risks are
negligible.