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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
figure1

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).

References

  1. 1.

    Zhang , et al.: J Biol Chem (in press)

  2. 2.

    Marklund , et al.: Eur J Biochem 1974, 47: 469. 10.1111/j.1432-1033.1974.tb03714.x

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Perner, A., Nielsen, S. & Rask-Madsen, J. Reduced release of superoxide from isolated human neutrophils in response to high extracellular glucose. Crit Care 5, P063 (2001). https://doi.org/10.1186/cc1130

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Keywords

  • Superoxide
  • NADPH
  • Hyperglycemia
  • NADPH Oxidase
  • DHEA