Reduced release of superoxide from isolated human neutrophils in response to high extracellular glucose

Background

Superoxide (O₂^-) - a key anti-microbial agent in phagocytes - is produced by the activity of NADPH oxidase. High concentrations of glucose may reduce O₂^- production through inhibition of glucose-6-phosphate dehydrogenase (G6PD) [1], which catalyzes the formation of NADPH.

Aims

To measure the acute effects of high glucose or the G6PD inhibitor, dehydroepiandrosterone (DHEA), on release of O₂^- from isolated human neutrophils.

Methods

Neutrophils were isolated from peripheral blood of healthy subjects by gradient centrifugation and incubated for 1 hour in Krebs-Ringer buffer containing 5, 10 or 25 mM glucose, 5 mM glucose with 0, 5 or 20 mM mannitol or 5 mM glucose with 1, 10 or 100 µM DHEA at 37°C. N-Formyl-methionyl-leucyl-phenylalanine (fMLP)-induced O₂^- release was measured by superoxide dismutase-inhibitable reduction of cytochrome c or luminol-enhanced luminescence. Scavenging of O₂^- by glucose or DHEA was assessed by the pyrogallol assay [2].

Results

Incubation with glucose or DHEA, but not glucose/mannitol, dose-dependently reduced fMLP-induced release of O₂^- as detected by either method. In a cell free system, neither glucose nor DHEA scavenged O₂^-.

Conclusions

Inhibition of G6PD may be the cause of acutely reduced O₂^- release from activated neutrophils in response to high extracellular glucose concentrations. If this occurs in vivo, microbial killing by neutrophils may be impaired in patients with acute hyperglycemia.

Figure 1

Superoxide release from fMLP-activated neutrophils - effect of high glucose or DHEA. Note: Mean (± SEM; n = 6) O₂^- realese. *P < 0.01 compared with 5 mM glucose or absence of DHEA (paired t-test).