Measurement of arachidonic acid release from human polymorphonuclear neutrophils and platelets: comparison between gas chromatographic and radiometric assays

Journal Article · Mon Dec 31 23:00:00 EST 1990 · Analytical Biochemistry; (United States)

DOI:https://doi.org/10.1016/0003-2697(91)90203-6· OSTI ID:5509800

Ramesha, C S; Taylor, L A ^[1]

---

1. Department of Inflammation Biology, Syntex Research, Palo Alto, California (USA)

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 {sup 3}H- or {sup 14}Carachidonic 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.

• OSTI ID: 5509800
• Journal Information: Analytical Biochemistry; (United States), Journal Name: Analytical Biochemistry; (United States) Vol. 192:1; ISSN ANBCA; ISSN 0003-2697
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALBUMINS
ANIMALS
ARACHIDONIC ACID
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BLOOD PLATELETS
BODY FLUIDS
CARBON 14 COMPOUNDS
CARBON COMPOUNDS
CARBOXYLESTERASES
CARBOXYLIC ACIDS
CHROMATOGRAPHY
COMPARATIVE EVALUATIONS
ENZYME ACTIVITY
ENZYMES
ESTERASES
GAS CHROMATOGRAPHY
HYDROGEN COMPOUNDS
HYDROLASES
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LEUKOCYTES
LIPASES
MAMMALS
MAN
MATERIALS
MONOCARBOXYLIC ACIDS
NEUTROPHILS
ORGANIC ACIDS
ORGANIC COMPOUNDS
PRIMATES
PROTEINS
RADIOASSAY
SECRETION
SEPARATION PROCESSES
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES