FIGURE 5.

Calcium-independent PLA₂ activity is increased in membrane fractions of neutrophils from diabetic subjects. A, Neutrophils (4 × 10⁸/ml) were sonicated in a homogenization buffer supplemented with protease inhibitor. Cells were then fractionated to isolate the membrane components. The reaction was initiated by the addition of sample to the substrate arachidonyl thiophosphatidyl choline. The reactions were stopped by the addition of 5',5'-dithio-bis(2-nitrobenzoic acid)/EGTA, and the plate was read at 415 nm. The buffers for this assay were calcium free and contained EDTA to chelate all calcium, thus activating calcium-independent PLA₂. Results are expressed as mean ± SEM for n = 3. B, The membrane fraction of neutrophils from diabetic subjects demonstrated a significant increase in calcium-independent PLA₂ activity. Data represent percent increase of iPLA₂ activity over healthy controls and are expressed as mean ± SEM for n = 4. p, 0.05.