The Interrelationship between Leukotriene B4 and Leukotriene-A4-Hydrolase in Collagen/Adjuvant-Induced Arthritis in Rats
During a 2-min incubation of leukotriene A4 (LTA4) with human liver microsomes, 1.7 mol% was converted into leukotriene B4 (LTB4). The reaction was dependent on protein concentration, time, and substrate concentration, was not supported by heat-inactivated microsomes, and did not require NADPH. Kinetic analysis of the reaction revealed apparent Michaelis-Menten type behavior (app Km approximately 20 microM). Production rates varied widely among three patients examined. Piperonyl butoxide, propanethiol, and cyclohexene oxide (1 mM) inhibited LTB4 formation by microsomal LTA4-hydrolase by 52, 40, and 60%, respectively. The latter two compounds were shown not to inhibit cytosolic LTA4-hydrolase activity. The activity of microsomal and cytosolic LTA4-hydrolase was decreased in the presence of 100% O2 by 45 and 64%, respectively. Direct chemical ionization mass spectrometry was used to obtain a mass spectrum of 50 ng of underivatized synthetic LTB4 free acid and show that this spectrum is identical with that of 10 ng of the product isolated from LTA4 hydrolysis by human liver microsomes. The authenticity of the biologically generated LTB4 was confirmed by functional characterization in a receptor displacement assay. Displacement of [3H]LTB4 from the high affinity receptors of LTB4 on human neutrophils revealed KD50 values of 8.2 and 5.1 nM for human liver microsome derived and synthetic LTB4, respectively. The nearly two-fold higher KD50 of the microsomally generated LTB4 is suggested to result from an epimeric mixture of the active 5(S),12(R)- and the less active 5(S),12(S)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid.