Measurement of arachidonic acid release from human polymorphonuclear neutrophils and platelets: Comparison between gas chromatographic and radiometric assays
| Autor: | Chakkodabylu S. Ramesha, Leslie A. Taylor |
|---|---|
| Rok vydání: | 1991 |
| Předmět: |
Blood Platelets
Chromatography Gas Neutrophils Biophysics Ionophore Phospholipid chemistry.chemical_element Arachidonic Acids Calcium Tritium Biochemistry Phospholipases A chemistry.chemical_compound Phospholipase A2 Humans Bovine serum albumin Derivatization Molecular Biology Calcimycin Chromatography biology Acetophenones Serum Albumin Bovine Cell Biology Enzyme assay Kinetics Phospholipases A2 chemistry Fatty Acids Unsaturated biology.protein Arachidonic acid |
| Zdroj: | Analytical Biochemistry. 192:173-180 |
| ISSN: | 0003-2697 |
| DOI: | 10.1016/0003-2697(91)90203-6 |
| Popis: | A simple gas chromatographic method for the assay of phospholipase A2 (PLA2) has been described in which arachidonic acid released from endogenous phospholipid pools is measured following its extraction and derivatization to pentafluorobenzyl esters. Using this assay, PLA2 activities in control and calcium ionophore-stimulated human neutrophils, as well as in control, thrombin, and calcium ionophore stimulated human platelets, have been measured. These values are compared with those obtained by monitoring the release of radioactivity from [3H]- or [14C]arachidonic acid prelabeled cells. While the radiometric assay measures only the release of exogenously incorporated radioactive arachidonic acid, the gas chromatographic assay measures arachidonic acid released from all the endogenous pools. Thus, the apparent increase in PLA2 activity in stimulated cells measured by the gas chromatographic assay is four- to fivefold higher than that by the radiometric assay. Inclusion of fatty acid free bovine serum albumin in the reaction buffer significantly increases the amount of arachidonic acid that is measured by gas chromatography. The gas chromatographic method has also been successfully utilized for measuring PLA2 activity in cell-free preparations derived from physically disrupted human neutrophils. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|