Citric Acid → 5-Hydroxyeicosatetraenoic Acid

Participants

Reaction Steps

A suspension of human neutrophils in buffer is incubated for 3 min at 30° C. with (14C)-arachidonic acid (AA) and calcium ionophore A23187. Citric Acid (2M) /NDGA* (10 mM) is used to quench the reaction. Following the addition of a trace amount of (3H)-5-HETE together with an excess of unlabeled 5-HETE to each tube, the mixture is extracted with chloroform/methanol. The organic layer is washed with dilute hydrochloric acid and the total volume is transferred to glass tubes and dried in vacuo. The residue is dissolved in a small volume of chloroform and is spotted on silica gel TLC sheets which are developed with an ethyl acetate/isooctane/water/acetic acid solvent system

Provenance & Evidence