Spectrofluorimetric Quantification of Malondialdehyde for Evaluation of Cyclooxygenase-1/Thromboxane Synthase Inhibition

Abstract

The in vitro assay developed by Hartmann and Ledergerber (1995)[1] utilizing the Spectrofluorimetric quantification of malondialdehyde after reaction with thiobarbituric acid was modified and used for further investigations. The human whole blood was replaced by a platelet suspension of pig blood, and calcium ionophore A23187 was used instead of collagen for inducing the arachidonic acid cascade. The modified assay represents a simple, time and cost saving method for the evaluation of cyclooxygenase‐1/thromboxane synthase inhibition. The reproducibility and comparability of results is given. Additional experiments allow classification of selective phospholipase A2, cyclooxygenase‐1, and thromboxane synthase inhibitors. Further studies of malondialdehyde formation show that the cyclooxygenase and/or the thromboxane synthase are competitively inhibited by reaction products of the cyclooxygenase pathway by a negative feedback mechanism.

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“…For a rapid screening for the inhibition of the arachidonic acid cascade, the spectrofluorimetric quantification of malondialdehyde (MDA) formation in pig blood platelets after addition of calcimycin A23187 according to Dannhardt et al [12] was used. Pig blood was obtained from the local slaughterhouse and platelets were isolated as described [12]. The volume of the assay was modified to be run in microtitre plates: 140 μ L of a platelet suspension was mixed with 2 μ L of DMSO solution of test compounds and incubated for 10 min at 37°C.…”