Metabolism of arachidonic acid by human neutrophils. Characterization of the enzymatic reactions that lead to the synthesis of leukotriene B4.

Human neutrophils stimulated with calcium ionophore A23187 synthesized 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4. Time-course studies showed that the concentrations of both products reached a maximum after 2 min after which the products were rapidly removed. In longer incubations, 5-HETE was esterified into membrane lipids, and leukotriene B4 was converted to 20- hydroxyleukotriene B4 and/or 20- carboxyleukotriene B4. The reaction is apparently self-limiting. After the maximum was reached, addition of fresh ionophore, Ca2+ or oxygen had little effect. Fresh arachidonic acid increases the yields of 5-HETE and delta 6-trans-leukotriene B4 but not additional leukotriene B4. Only the addition of fresh neutrophils gave additional leukotriene B4. This finding suggests that leukotriene B4 synthesis is limited by both substrate availability and enzyme inhibition by hydroperoxide intermediate. Exogenous arachidonic acid added with ionophore had different effects on the syntheses of leukotriene B4, delta 6-trans-leukotriene B4, and 5-HETE. As the arachidonic acid concentration increases, product formation increases in the following order: 5-HETE greater than delta 6-trans-leukotriene B4 greater than leukotriene B4. At a high concentration (more than 10 microM) of arachidonic acid, the synthesis of delta 6-trans-leukotriene B4 was greater than leukotriene B4 itself. Since delta 6-trans-leukotriene B4 represents the nonenzymatic decomposition of leukotriene A4, we suggest that one of the rate-limiting steps in the synthesis of leukotriene B4 is the leukotriene A4 hydrolase. Our data suggest the synthesis of leukotriene B4 is under the control of three factors: (1) substrate availability; (2) limited capacity of the leukotriene A4 hydrolase, and (3) enzyme inactivators generated during the reaction, such as hydroperoxide intermediate. The tightly controlled system assures only a finite amount of this powerful bioactive substance will be produced under most conditions.