Stepwise assessment of the relation between glycocalyx dimensions, oxidative stress and level of glycaemia

Endothelial dysfunction is a hallmark of diabetic vascular complications.
Hyperglycemia induced formation of reactive oxygen species (ROS) is thought to
induce endothelial dysfunction by enhancing four mechanisms of tissue damage.
Also, oxidative stress may have a direct effect on the glycocalyx, a network of
membrane-bound proteoglycans and glycoproteins exerting vasoprotective effects.
It has been shown that patients with diabetes mellitus have higher levels of
oxidative stress and a diminished glycocalyx volume compared to healthy
controls. However, little is known about the degree of glycemia at which these
changes occur and whether damage is influenced by glycemic swings.

To assess at which level of glycemia the process of endothelial damage,
represented by ROS formation and glycocalyx damage begins and to determine
whether there is a glycemic threshold value for oxidative stress formation and
glycocalyx damage. To assess whether there is a direct relationship between
increased ROS formation and loss of glycocalyx. To investigate the relation
between level of glycemia and activation of coagulation parameters.

After a screening visit the actual study consists of a stepwise hyperglycemic
clamp at glucose levels of fasting, 6, 8 and 10 mmol/l. Each plasma glucose
concentration will be held constant for 120 minutes. At each glucose level
glycocalyx thickness and will be measured every 30 minutes by OPS recordings
and shedding of glycocalyx components and ROS formation by determination of
plasma malondialdehyde and nitrotyrosine. After this, glucose infusion will be
increased to reach the next level of glycemia. In total, the clamp will take
420 minutes. The day after the clamp, the participants will come to the AMC for
visit 3 for evaluation of glycocalyx restoration and ROS formation.

Hyperglycaemic clamp. The plasma glucose concentration will be elevated in a
stepwise manner by administration of somatostatin, glucose and insulin
(fasting, 6 mmol/l, 8 mmol/l and 10 mmol/l).

Included subjects will visit the AMC hospital three times:
Visit 1 (Screening visit): informed consent, medical history, vital signs, vena
puncture (7.5 ml in total).
Visit 2: stepwise hyperglycemic clamp with a total duration of 420 minutes:
Placement of two intravenous catheters; venous blood sampling for glucose
measurement every 5 minutes and venous blood sampling for determination of ROS
formation and coagulation activation at 14 time points (294 ml in total);
somatostatin, insulin and glucose infusion; OPS recordings every 30 minutes (14
in total);
Visit 3: OPS recording and vena puncture (9 ml in total)
Burden for the subjects consists of coming to the hospital in a fasting state
on three different occasions, a period of lying down during the clamp and two
venapunctures as well as the placement of two intravenous catheters. The
infusion of somatostatin may cause nausea. The overall risk of this study is
minimal.