Release of Free F2-isoprostanes from Esterified Phospholipids Is Catalyzed by Intracellular and Plasma Platelet-activating Factor Acetylhydrolases

Abstract

F 2 -isoprostanes are produced in vivo by nonenzymatic peroxidation of arachidonic acid esterified in phospholipids. Increased urinary and plasma F 2 -isoprostane levels are associated with a number of human diseases. These metabolites are regarded as excellent markers of oxidant stress in vivo. Isoprostanes are initially generated in situ, i.e. when the arachidonate precursor is esterified in phospholipids, and they are subsequently released in free form. Although the mechanism(s) responsible for the release of free isoprostanes after in situ generation in membrane phospholipids is, for the most part, unknown, this process is likely mediated by phospholipase A 2 activity(ies). Here we reported that human plasma contains an enzymatic activity that catalyzes this reaction. The activity associates with high density and low density lipoprotein and comigrates with platelet-activating factor (PAF) acetylhydrolase on KBr density gradients. Plasma samples from subjects deficient in PAF acetylhydrolase do not release F 2 -isoprostanes from esterified precursors. The intracellular PAF acetylhydrolase II, which shares homology to the plasma enzyme, also catalyzes this reaction. We found that both the intracellular and plasma PAF acetylhydrolases have high affinity for esterified F 2 -isoprostanes. However, the rate of esterified F 2 -isoprostane hydrolysis is much slower compared with the rate of hydrolysis of other substrates utilized by these enzymes. Studies using PAF acetylhydrolase transgenic mice indicated that these animals have a higher capacity to release F 2 -isoprostanes compared with nontransgenic littermates. Our results suggested that PAF acetylhydrolases play key roles in the hydrolysis of F 2 -isoprostanes esterified on phospholipids in vivo.2 are a family of prostaglandin (PG)-like compounds produced in vivo primarily by a nonenzymatic, free radicalinduced oxidation of arachidonic acid (1). These compounds are generated in situ esterified to phospholipids through the formation of PGH 2 -like bicyclic endoperoxide intermediates. These intermediates can either undergo rearrangement to E 2 /D 2 -IPs, isothromboxanes, and highly reactive isoketals or become reduced to F 2 -IPs that have been characterized in detail. Following in situ oxidation, IPs are released in free form by the action of phospholipase(s) activity.The measurement of F 2 -IPs in biological fluids has become one of the most accepted quantitative assessments of lipid peroxidation and oxidant stress reactions that occur in vivo (2). This approach provides the advantage of being noninvasive, as F 2 -IPs circulate freely in plasma and are excreted in urine. Increased IP production has been observed in a number of human diseases associated with oxidant stress, including atherosclerosis, pulmonary and liver diseases, habitual smoking, neurological disorders, and diabetes (3-5). In addition, exposure of experimental animals to increased oxidant stress also results in remarkable increases in the levels of free IPs in the urine and in plasma (2, 6). P...

Formation of Highly Reactive Cyclopentenone Isoprostane Compounds (A3/J3-Isoprostanes) in Vivo from Eicosapentaenoic Acid

“…Like other classes of IsoPs, the A 3 /J 3 -IsoPs are formed in situ esterified in phospholipids and are then presumably released in the free form by a phospholipase(s). Recently, it has been shown that platelet-activating factor acetyl hydrolases are capable of hydrolyzing F 2 -IsoPs from phospholipids, and, thus, it is conceivable that these enzymes also hydrolyze phospholipids containing A 3 /J 3 -IsoPs (12). Previously, we have demonstrated that another class of IsoP-like molecules, F 3 -IsoPs, is also formed from the oxidation of EPA in vivo (16).…”

Protection against Oxidative Stress-induced Hepatic Injury by Intracellular Type II Platelet-activating Factor Acetylhydrolase by Metabolism of Oxidized Phospholipids in Vivo

“…It has been generally considered that F 2 -isoprostanes are initially formed in situ from arachidonoyl chains attached to phospholipids and then released in the free form into the circulation (65,66). Stafforini et al (24) recently showed that PAF-AH (II) hydrolyzes esterified F 2 -isoprostanes in vitro. Because F 2 -isoprostanes are initially formed in situ from esterified arachidonic acids in phospholipids (65), we can estimate the content of esterified 8-iso-PGF 2␣ in phospholipids by subtracting the amount of free 8-iso-PGF 2␣ from the total amount 8-iso-PGF 2␣ .…”

Oxidized glycerophosphocholines as biologically active mediators for ultraviolet radiation-mediated effects

“…This is important, as serum PAF-AH exhibits high substrate specificity for phospholipids with short sn-2 acyl chains; enzymatic activity is rapidly lost with saturated acyl chain lengths greater than 2 carbons, with 10-carbon acyl groups demonstrating essentially no activity 19,91,92 . However, as seen in figure 3 for AzPC, serum PAF-AH exhibits potent activity for oxidized phospholipids with sn-2 residues of up to 9 carbons in length that contain ω-oxy functions 19,[91][92][93] . Thus, while PAF-AH has been proposed to limit the actions of un-regulated ox-GPC production on PAF-R activation 19,92 , it is also quite likely that it serves to limit the ability of ox-GPCs to activate PPARγ.…”