Spectrophotometric Quantitation of the Lipoxygenase Derived Conjugated 1,3-Diene (12-Hete) in Biological Systems

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A simple TLC/UV spectrophotometric method is described for the quantitation of the lipoxygenase derived product 12-HETE released by the rat testis, lung and platelets .Tissues were incubated in Krebs' solution containing reduced glutathione. The tissues were chopped and incubated at 37°C for 30 min. The medium was acidified and extracted with diethyl ether. The ethereal residue was dissolved in alcohol, applied to silica gel plates and the latter developed using a mixture of n-hexa-ne : diethyl ether : glacial acetfcl acid (60: 40 : 1 v/v/v/). Standard arachidonic acid and hydroxya-cids were applied on the same plate to locate zones of biological products. 12-HETE zones were eluted with ethanol, centrifuged and the extinction of the clear alcoholic supernatant was measured at 239 nm. 12-HETE content in each zone was calculated using a standard 12-HETE curve. The production of 12-HETE by the testis and lungs was 20.3 ±1.9 and 15.4 ± 0.9 ng/mg wet tissue whereas the production by the platelets was 1.3 ± 0.05 ng/106 platelets. Pretreatment of the tissues with mepacrine and phen id one significantly suppressed the production of 12-HETE whereas indo-methacin either did not affect or stimulated the release. The described mathod may facilitate the discovery of potential lipoxygenase inhibitors.

Quantitation; Lipoxygenase; Biological