Resumo

Introduction: Membranes play a key role in all systems, especially in the central nervous system, due to plasticity, receptors, cellular signaling precursors, among other functions. Phospholipases A2 are a group of hydrolases which cleaves phospholipids, resulting in lysophospholipids and fatty acids, such as arachidonyl, and is involved in apoptosis, cell remodeling and signaling. PLA2 activity alterations have been reported in brain and blood cell is many neuropsychiatry disorders, such as Schizophrenia and Alzheimer disease. Aims: To develop an assay to determine the activity of the PLA2 subgroups. We optimized a radioenzymatic method for the three main groups of PLA2 (intracellular calcium independent PLA2, iPLA2; cytosolic calcium dependent PLA2, cPLA2 and extracellular calcium dependent PLA2, sPLA2) activities determination on leukocytes. Two independent assays (performed on triplicate) of each critical variable for enzymatic action were performed: protein (1 - 3 mg/mL); radioactive substrate (0.06 - 0.11 μCi); incubation time (15 - 45 min); pH (7 - 8.5); calcium concentration (iPLA2: 10 - 1000 μM; cPLA2: 5 - 500 μM; sPLA2: 0.5 - 50 mM); BEL inhibitor concentration (1 - 0.02 mM). Analysis of variance followed by Tukey HSD test was performed to verify each activity peak. The significance level adopted was 0.05. Results: The best conditions for PLA2 activity determination were: 1 mg/mL of protein (p < 0.001, n =6); 0.075 μCi of radioactive substrate (p < 0.001, n =6); 15 min for iPLA2 (p = 0.037, n=6) and 45 min for cPLA2 and sPLA2 (p= 0.001 and p=0.009, respectively; n=6); 8.5 as pH for all subtypes (p < 0.001, n=6); calcium: 100 μM for iPLA2 (p < 0.008, n=6), 50 μM for cPLA2 (p < 0.001, n=6) and 5 mM for sPLA2 (p <0.005, n=6); BEL 0.02 mM for iPLA2 (p <0.001,n=6). Conclusion: A suitable method for PLA2 subtype activities in leukocytes was optimized. Leukocytes could be an useful as peripheral biomarker for the study of PLA2 alterations in psychiatryic disorders.