Background:Glucose is the main substrate for the generation
of NADPH, the cofactor of the oxidative burst enzyme NADPH-oxidase of blood
neutrophils. Changes in blood glucose are thus expected to modify the
generation of reactive oxygen species (ROS). The new blood ROS generation assay
(BRGA) quantifies ROS changes induced by blood glucose concentrations as they
are found in diabetes mellitus. Material and Methods:Citrated or EDTA blood of 6 healthy donors
were analyzed in the BRGA: 10 μl sample in black polystyrene F-microwells
(Brand781608) were incubated in
triplicate with 125 μl Hanks’ balanced
salt solution, 40 μl 0 - 200 mM glucose in 0.9% NaCl (final added conc.: 0 - 41
mM; final basal glucose conc.: about4 mM),
10 μl5 mMluminol,
and 10 μl zymosan A (final conc.: 1.9 μg/ml) in 0.9% NaCl. The plates were
measured within 0 - 250 min (37℃) in a photons-multiplyer microtiter plate
luminometer (LUmo) with an integration time of 1 s. Results: Up to about 30 min reaction time the
mean ROS generation was 50% inhibited by about1
mMadded glucose (= approx. IC50). At ≥80 min reaction time (possibly
necessary for full phosphorylation of glucose to glucose-6-phosphate (G6P), the
substrate metabolized by G6P-dehydrogenase to generate NADPH, the cofactor of
the NADPH-oxidase) the mean ROS generation approximately doubled at about1 mMadded glucose (= approx. SC200) in citrated blood. Discussion:Elevated glucose concentrations not only increase systemic
thrombin generation, they can also diminish cellular fibrinolysis and increase
systemic inflammation, resulting in a chronic pro-thrombotic state. The
fascinating importance of NADPH-oxidases not only in phagocytes but also in the
beta cells of pancreas points towards a new pathogenesis explication of
diabetes mellitus type 1: whatever stimulus (e.g. a pancreas-tropic virus)
could activate the beta cell’s autodestructive NADPH-oxidase.
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