Assays for Measuring Arachidonic Acid Release from Phospholipids

This chapter focuses on the various methods used to label cells with arachidonic acid and to quantify arachidonate release from phospholipids in the human neutrophil. Various assumptions and problems are encountered when using these methods are emphasized. These include the effects of arachidonic acid concentration and of incubation time on the ability to obtain equilibrium labeling conditions, the loss of labeled arachidonate from phospholipids, and the formation of radiolabeled eicosanoids. The major problem is the nonuniform uptake of exogenous arachidonic acid into phospholipid molecular species. In the neutrophil, the uptake of labeled arachidonic acid and its distribution into complex lipids are greatly influenced by (1) the amount of arachidonic acid provided to the cell, and (2) the time of exposure to arachidonic acid. The experiments presented in the chapter demonstrates that even after 120 min of incubation, there can be differences as great as 10-fold between the radiospecific activity of arachidonate in major phospholipid molecular species. Another assumption made when performing labeling or mass studies is that loss of arachidonate from a phospholipid can be equated to that phospholipid being a source of arachidonate for eicosanoids. In the experiments presented in the chapter, leukotrienes produced during cell activation represent only a small fraction of the total arachidonate lost from all phospholipids.