Hyperglycaemia-induced superoxide production decreases eNOS expression via AP-1 activation in aortic endothelial cells.

AIMS/HYPOTHESIS: Hyperglycaemia is a primary cause of vascular complications in diabetes. A hallmark of these vascular complications is endothelial cell dysfunction, which is partly due to the reduced production of nitric oxide. The aim of this study was to investigate the regulation of endothelial nitric oxide synthase (eNOS) activity by acute and chronic elevated glucose. METHODS: Human aortic endothelial cells were cultured in 5.5 mmol/l (NG) or 25 mmol/l glucose (HG) for 4 h, 1 day, 3 days or 7 days. Mouse aortic endothelial cells were freshly isolated from C57BL/6J control and diabetic db/db mice. The expression and activity of eNOS were measured using quantitative PCR and nitrite measurements respectively. The binding of activator protein-1 (AP-1) to DNA in nuclear extracts was determined using electrophoretic mobility-shift assays. RESULTS: Acute exposure (4 h) of human aortic endothelial cells to 25 mmol/l glucose moderately increased eNOS activity and eNOS mRNA and protein expression. In contrast, chronic exposure to elevated glucose (25 mmol/l for 7 days) reduced total nitrite levels (46% reduction), levels of eNOS mRNA (46% reduction) and eNOS protein (65% reduction). In addition, AP-1 DNA binding activity was increased in chronic HG-cultured human aortic endothelial cells, and this effect was reduced by the specific inhibition of reactive oxygen species production through the mitochondrial electron transport chain. Mutation of AP-1 sites in the human eNOS promoter reversed the effects of HG. Compared with C57BL/6J control mice, eNOS mRNA levels in diabetic db/db mouse aortic endothelial cells were reduced by 60%. This decrease was reversed by the overexpression of manganese superoxide dismutase using an adenoviral construct. CONCLUSIONS/INTERPRETATION: In diabetes, the expression and activity of eNOS is regulated through glucose-mediated mitochondrial production of reactive oxygen species and activation of the oxidative stress transcription factor AP-1.

Adenoviral-mediated transfer of tissue plasminogen activator gene into brain capillary endothelial cells in vitro.

Tissue plasminogen activator (tPA) has a critical role in fibrinolysis, converting plasminogen into active protease plasmin. Because intravenous tPA has only limited effectiveness as acute stroke therapy, enhancement of endogenous tPA represents a potential alternative to stroke treatment. Adenoviral-mediated gene transfer was used to enhance production of tPA in bovine brain capillary endothelial cells (BEC). Antigen and activity levels of tPA and plasminogen activator inhibitor-1 (PAI-1) in media from BEC infected with AdCMVtPA were analyzed. Conditioned media were analyzed for thrombomodulin, the integral membrane antithrombotic molecule that co-activates protein C. BEC infected with AdCMVtPA demonstrated enhanced expression of tPA antigen (40.2 +/- 0.4 ng/mL vs 1.1 +/- 1.5 ng/mL [p<0.001] and 0.3 +/- 0.5 ng/mL [p<0.0001], respectively) and increased tPA enzymatic activity (27.4 +/- 5.7 IU/mL vs 8.3 +/- 1.7 IU/mL [p<0.05] and 13.3 +/- 3.2 IU/mL [p<0.05], respectively) compared to BEC infected with the control adenovirus (Adl327) or uninfected BEC. There was a moderate increase in PAI-1 protein 4 days after transfection with AdCMVtPA, and the integral membrane protein thrombomodulin was released into media by transfected BEC. These results demonstrate that adenoviral-mediated delivery in vitro of the human tPA gene resulted in high levels of expression of tPA in BEC. Transient overexpression of tPA by gene transfer might be a useful strategy to protect against thrombotic occlusion during the period of risk of acute stroke.

In vivo interactions of apoA-II, apoA-I, and hepatic lipase contributing to HDL structure and antiatherogenic functions.

Studies with mice have revealed that increased expression of apolipoprotein A-II (apoA-II) results in elevations in high density lipoprotein (HDL), the formation of larger HDL, and the development of early atherosclerosis. We now show that the increased size of HDL results in part from an inhibition of the ability of hepatic lipase (HL) to hydrolyze phospholipids and triglycerides in the HDL and that the ratio of apoA-I to apoA-II determines HDL functional and antiatherogenic properties. HDL from apoA-II transgenic mice was relatively resistant to the action of HL in vitro. To test whether HL and apoA-II influence HDL size independently, combined apoA-II transgenic/HL knockout (HLko) mice were examined. These mice had HDL similar in size to apoA-II transgenic mice and HLko mice, suggesting that they do not increase HDL side by independent mechanisms. Overexpression of apoA-I from a transgene reversed many of the effects of apoA-II overexpression, including the ability of HDL to serve as a substrate for HL. Combined apoA-I/apoA-II transgenic mice exhibited significantly less atherosclerotic lesion formation than did apoA-II transgenic mice. These results were paralleled by the effects of the transgenes on the ability of HDL to protect against the proinflammatory effects of oxidized low density lipoprotein (LDL). Whereas nontransgenic HDL protected against oxidized LDL induction of adhesion molecules in endothelial cells, HDL from apoA-II transgenic mice was proinflammatory. HDL from combined apoA-I/apoA-II transgenic mice was equally as protective as HDL from nontransgenic mice. Our data suggest that as the ratio of apoA-II to apoA-I is increased, the HDL become larger because of inhibition of HL, and lose their antiatherogenic properties.

Adenoviral delivery of a leukocyte-type 12 lipoxygenase ribozyme inhibits effects of glucose and platelet-derived growth factor in vascular endothelial and smooth muscle cells.

The lipoxygenase (LO) pathway has been implicated as an important mediator of chronic glucose and platelet-derived growth factor (PDGF)-induced effects in the vascular system. Endothelial cells treated with 12LO products or cultured in high glucose showed enhanced monocyte adhesion, an important step in atherogenesis. We have previously reported that PDGF increased HETE levels in porcine aortic smooth muscle cells. Although several pharmacological inhibitors to the LO pathway are available, most lack specificity and may harbor undesirable side effects. Therefore, we developed a recombinant adenovirus expressing a hammerhead ribozyme (AdRZ) targeted against the porcine leukocyte-type 12LO mRNA to investigate the involvement of LO in glucose- and PDGF-mediated effects in vascular cells. Infection of porcine aortic endothelial cells with AdRZ reduced the level of glucose-enhanced 12LO mRNA expression as determined by quantitative, real-time reverse transcriptase-polymerase chain reaction. Reverse-phase HPLC and RIA analysis also revealed a corresponding decrease in glucose-stimulated 12HETE production in both the cellular and supernatant fractions. In the ribozyme-treated porcine aortic endothelial cells, there was marked inhibition of high glucose-stimulated monocyte adhesion. Infection with AdRZ also reduced PDGF-induced porcine aortic smooth muscle cell migration by approximately 50%. These studies demonstrate the efficacy of recombinant adenovirus expressing 12LO ribozyme in studying the effects of 12LO in vascular wall cells. They document an important role for the 12LO pathway in regulating inflammatory changes in endothelial cells and smooth muscle cells.

Role for peroxisome proliferator-activated receptor alpha in oxidized phospholipid-induced synthesis of monocyte chemotactic protein-1 and interleukin-8 by endothelial cells.

The attraction, binding, and entry of monocytes into the vessel wall play an important role in atherogenesis. We have previously shown that minimally oxidized/modified LDL (MM-LDL), a pathogenically relevant lipoprotein, can activate human aortic endothelial cells (HAECs) to produce monocyte chemotactic activators. In the present study, we demonstrate that MM-LDL and oxidation products of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) activate endothelial cells to synthesize monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8). Several lines of evidence suggest that this activation is mediated by the lipid-dependent transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha), the most abundant member of the PPAR family in HAECs. Treatment of transfected CV-1 cells demonstrated activation of the PPARalpha ligand-binding domain by MM-LDL, Ox-PAPC, or its component phospholipids, 1-palmitoyl-2-oxovalaroyl-sn-glycero-phosphocholine and 1-palmitoyl-2-glutaroyl-sn-glycero-phosphocholine; these lipids also activated a consensus peroxisome proliferator-activated receptor response element (PPRE) in transfected HAECs. Furthermore, activation of PPARalpha with synthetic ligand Wy14,643 stimulates the synthesis of IL-8 and MCP-1 by HAECs. By contrast, troglitazone, a PPARgamma agonist, decreased the levels of IL-8 and MCP-1. Finally, we demonstrate that unlike wild-type endothelial cells, endothelial cells derived from PPARalpha null mice do not produce MCP-1/JE in response to Ox-PAPC and MM-LDL. Together, these data demonstrate a proinflammatory role for PPARalpha in mediation of the activation of endothelial cells to produce monocyte chemotactic activity in response to oxidized phospholipids and lipoproteins.

Short-term feeding of atherogenic diet to mice results in reduction of HDL and paraoxonase that may be mediated by an immune mechanism.

Short-term feeding (up to 7 days) of an atherogenic diet to C57BL/6 low density lipoprotein receptor-deficient mice did not result in decreased hepatic paraoxonase (PON) mRNA but caused a dramatic decrease in plasma PON activity and mass. The decreased activity and mass were temporally related to an increase in plasma and high density lipoprotein (HDL) lipid hydroperoxides and to a decrease in HDL cholesterol and native apolipoprotein A-I (apoA-I) and apolipoprotein A-II (apoA-II). As the native apoA-I protein disappeared from the circulation, higher molecular weight forms of apoA-I appeared, some of which contained epitopes recognized by an antibody (EO6) that recognizes oxidized phospholipids. After mice consumed an atherogenic diet for 1 or 3 days, switching the mice to a low fat chow diet for 3 days resulted in a return to baseline levels of lipid hydroperoxides but only a small return toward baseline for HDL cholesterol, with no significant increase in apoA-I mass or PON activity and mass. After mice consumed an atherogenic diet for 3 days, switching to the chow diet for 3 days did not significantly alter the high molecular weight forms of apoA-I or the signal generated by EO6. In marked contrast, after mice consumed an atherogenic diet for 7 days, switching to the chow diet for 3 days resulted in a dramatic increase in native apoA-I to baseline levels, with virtual disappearance of the high molecular weight forms of apoA-I, including the form recognized by EO6. After mice consumed an atherogenic diet for 7 days, switching to the chow diet for 3 days also resulted in significant increases in HDL cholesterol and PON mass and activity, although baseline levels were not reached. IgG and IgM antibodies were found to be associated with apoA-I in control animals, were minimally decreased after the 3-day atherogenic diet, were dramatically decreased after the 7-day atherogenic diet, and returned to near or above baseline levels after a return to the chow diet for 3 days. We conclude that the atherogenic diet rapidly induces lipid hydroperoxide formation and apoA-I oxidation with the formation of high molecular weight forms of apoA-I. Concomitant with these changes in apoA-I levels, HDL cholesterol and PON activity and mass declined without changes in mRNA levels for apoA-I or PON, suggesting increased clearance of these altered HDL particles. We further conclude that between the third and seventh day of the atherogenic diet, an as-yet-unidentified mechanism for clearing the high molecular weight forms of apoA-I is induced and that this mechanism may be related to the clearance of immune complexes.

Glycation impairs high-density lipoprotein function.

AIMS/HYPOTHESIS: To examine the effects of incubation of high-density lipoprotein (HDL) under hyperglycaemic conditions on several functions of HDL in vitro. METHODS: Human HDL (5 mg protein) was incubated for 1 week at 37 degrees C in the presence or absence of 25 mmol/l glucose. Additional samples of human HDL were incubated in butylated hydroxytoluene to control for oxidation. RESULTS: High-density lipoprotein incubated for 1 week in 25 mmol/l glucose had significant increases in the glycation product, fructoselysine and in the advanced glycation end product, N epsilon-(carboxymethyl)-lysine. High-density lipoprotein apolipoprotein AI and AII concentrations were not altered but glycated HDL had a 65% reduction in paraoxonase enzymatic activity. Glycated HDL did not inhibit monocyte adhesion to human aortic endothelial cells in response to oxidised low-density lipoprotein in vitro (43 +/- 4 monocytes bound vs 21 +/- 2 monocytes for control HDL, p < 0.0001). Hepatic lipase-mediated non-esterified fatty acid release from HDL lipids was enhanced in glycated HDL compared with control HDL (25 +/- 1 vs 16 +/- 1 nmol non-esterified fatty acid hydrolysed/min, respectively, p < 0.0001). Direct glycation of purified paraoxonase protein by incubation in 25 mmol/l glucose caused a 40% reduction in enzymatic activity. This glycated paraoxonase did not inhibit monocyte adhesion to human aortic endothelial cells in vitro (68 +/- 3 monocytes vs 49 +/- 2 monocytes bound for control paraoxonase, respectively, p < 0.001). We also measured a 40% reduction in paraoxonase activity in patients with Type II (non-insulin-dependent) diabetes mellitus and documented coronary artery disease compared with non-diabetic subjects, p < 0.0001. CONCLUSIONS/INTERPRETATION: Alterations in function of HDL caused by exposure to hyperglycaemic conditions could contribute to the accelerated atherosclerosis observed in Type II diabetes.

Interleukin-10 blocks atherosclerotic events in vitro and in vivo.

Atherosclerosis can be viewed in part as an inflammatory disease process and may therefore be susceptible to manipulation of the immune state. Interleukin 10 (IL-10) is an inhibitory cytokine produced by activated lymphocytes and monocytes. These studies present evidence that IL-10 can inhibit minimally oxidized LDL (MM-LDL)-induced monocyte-endothelium interaction as well as inhibit atherosclerotic lesion formation in mice fed an atherosclerotic diet. Pretreatment of human aortic endothelial cells (HAECs) for 18, but not 4, hours with recombinant IL-10 caused a significant decrease in MM-LDL-induced monocyte binding. IL-10 was found to be maximally effective at 10 ng/mL. Transfection of HAECs with adenovirus expressing viral bcrf-1 IL-10 (Ad-vIL-10) in a sense but not antisense orientation completely inhibited the ability of MM-LDL to induce monocyte binding. Similar results were obtained with IL-10 or Ad-vIL-10 in HAECs stimulated with oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC). We have previously shown increases in cAMP associated with MM-LDL activation of endothelial cells. The MM-LDL-induced increase in cAMP levels was not inhibited by preincubation with IL-10. In vivo studies demonstrated that mice with a murine IL-10 transgene under the control of the human IL-2 promoter have decreased lesions versus controls on an atherogenic diet (5433+/-4008 mm(2) versus 13 574+/-4212 mm(2); P<0.05), whereas IL-10 null mice have increased lesions (33 250+/-9117 mm(2); P<0.0001) compared with either controls or IL-10 transgenic mice. These studies suggest an important role for IL-10 in the atherosclerotic disease process.

Lipoxygenase products increase monocyte adhesion to human aortic endothelial cells.

The development of atherosclerosis is accelerated in individuals with type 2 diabetes. Adhesion of monocytes to the vascular endothelium is a key initial step in atherogenesis. We have previously shown that monocyte adhesion to human aortic endothelial cells (HAECs) cultured long-term in high-glucose medium (25 mmol/L, 2 passages) is increased compared with cells grown in normal glucose (5 mmol/L). One potential mechanism for increased monocyte adhesion to HAECs under hyperglycemic conditions is via the 12-lipoxygenase (12-LO) pathway. In this study, we demonstrated in HAECs that the major LO metabolite of arachidonic acid was the 12-LO product, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], which was increased severalfold in HAECs cultured under high-glucose conditions. Furthermore, treatment of HAECs with 12(S)-HETE induced monocyte, but not neutrophil, adhesion an average of 3-fold (range of 1.5- to 5-fold) compared with untreated cells (75+/-5 versus 26+/-1 monocytes per field, respectively, P<0.001). Expression of the adhesion molecules vascular cell adhesion molecule-1, E-selectin, and intercellular adhesion molecule-1 was not significantly increased. However, both glucose and 12(S)-HETE induced a 60% increase in HAEC surface expression of connecting segment-1 (ie, CS-1) fibronectin, a ligand for very late-acting antigen-4 (VLA-4). The antibodies used to block monocyte integrin VLA-4 and leukocyte function-related antigen-1, a monocytic counterreceptor for intercellular adhesion molecule-1, inhibited the ability of both 12-LO products and high glucose to induce monocyte adhesion. These results definitively demonstrate for the first time in HAECs that the 12-LO pathway can induce monocyte-endothelial cell interaction and that the effects of glucose may be mediated, at least in part, through this pathway. Thus, these results suggest that the 12-LO pathway may play a role in the increased susceptibility of diabetics to atherosclerosis.

12-Lipoxygenase products increase monocyte:endothelial interactions.

In summary, we suggest that hyperglycemia causes upregulation of 12-lipoxygenase activity. The increased production of 12-LO products, 12(S) and 15(S)-HETE, activates monocyte integrins which result in enhanced adhesion of monocytes to endothelium. The binding of monocytes to endothelium is a key early event in development of atherosclerosis. Upregulation of this process by vascular cells exposed to chronic elevations in glucose may be one explanation for the accelerated atherosclerosis observed in patients with Type 2 diabetes.

High density associated enzymes: their role in vascular biology.

Enzymes associated with circulating HDL include lecithin: cholesterol acyl transferase, phospholipid transfer protein, cholesterol ester transfer protein, paraoxonase 1 and platelet activating factor acetylhydrolase. Together with lipoprotein lipase and hepatic lipase these enzymes produce important lipoprotein remodeling and modulate their structure and function and therefore their role in artery wall metabolism.

Overexpression of apolipoprotein AII in transgenic mice converts high density lipoproteins to proinflammatory particles.

Previous studies showed that transgenic mice overexpressing either apolipoprotein AI (apoAI) or apolipoprotein AII (apoAII), the major proteins of HDL, exhibited elevated levels of HDL cholesterol, but, whereas the apoAI-transgenic mice were protected against atherosclerosis, the apoAII-transgenic mice had increased lesion development. We now examine the basis for this striking functional heterogeneity. HDL from apoAI transgenics exhibited an enhanced ability to promote cholesterol efflux from macrophages, but HDL from apoAII transgenics and nontransgenics were not discernibly different in efflux studies. In contrast with HDL from nontransgenics and apoAI transgenics, HDL from the apoAII transgenics were unable to protect against LDL oxidation in a coculture model of the artery wall. Furthermore, HDL taken from apoAII-transgenic mice, but not HDL taken from either the apoAI transgenics or nontransgenic littermate controls, by itself stimulated lipid hydroperoxide formation in artery wall cells and induced monocyte transmigration, indicating that the apoAII-transgenic HDL were in fact proinflammatory. This loss in the ability of the apoAII-transgenic HDL to function as an antioxidant/antiinflammatory agent was associated with a decreased content of paraoxonase, an enzyme that protects against LDL oxidation. Reconstitution of the apoAII transgenic HDL with purified paraoxonase restored both paraoxonase activity and the ability to protect against LDL oxidation. We conclude that overexpression of apoAII converts HDL from an anti- to a proinflammatory particle and that paraoxonase plays a role in this transformation.

Endogenous natural killer enhancing factor-B increases cellular resistance to oxidative stresses.

Natural killer-enhancing factor (NKEF) was identified and cloned on the basis of its ability to increase NK cytotoxicity. Two genes, NKEF-A and -B, encode NKEF proteins and sequence analysis presented suggests that each belongs to a highly conserved family of antioxidants. To examine the antioxidant potential of NKEF, we transfected the coding region of NKEF-B cDNA into the human endothelial cell line ECV304. The stable transfectant, B/1, was found to overexpress NKEF-B gene transcript and protein. We subjected B/1 to oxidative stress by either culturing them with glucose oxidase (GO), which continuously generates hydrogen peroxide, or by direct addition of hydrogen peroxide. We found that B/1 cells were more resistant than control cell lines. Resistance to hydrogen peroxide was originally thought to be mediated mainly by catalase and the glutathione cycle. Therefore, we used inhibitors to block the two pathways and found that B/1 cells were more resistant to oxidative stress than control cells when we used inhibitors to preblock either pathway. We also examined the cellular inflammatory responses to oxidized low-density lipoprotein (LDL) and bacterial lipopolysaccharide (LPS) by measuring monocyte adhesion to endothelial cells in vitro and found that B/1 cells were resistant to such responses. Lastly, we found that B/1 cells were more resistant to a novel chemotherapeutic agent CT-2584, which appears to kill tumor cells by stimulating production of reactive oxygen intermediates in mitochondria. These results demonstrate that the NKEF-B is an antioxidant that protects cells from oxidative stress, chemotherapy agents, and inflammation-induced monocyte adhesion. Furthermore, its expression may mediate cellular responses to proinflammatory molecules.

Apolipoprotein A-II: a protein in search of a function.

OBJECTIVE: To summarize current findings relevant to the function of apolipoprotein A-II (apoA-II), a major protein component of high density lipoproteins (HDL). STUDY SELECTION: Studies relating to the structure, regulation and genetics of apoA-II are reviewed. These include studies of apoA-II expression in humans and mouse animal models. All studies cited were published between 1972 and 1993. CONCLUSIONS: Since its discovery over 20 years ago, apoA-II has been a protein in search of a function. In fact, it has often been stated that apoA-II has no major function in plasma lipoprotein metabolism. While it is clear that apoA-II does not serve an essential function in HDL metabolism, the evidence overwhelmingly indicates that apoA-II importantly influences HDL functional states and suggests strongly that apoA-II is likely to contribute to atherogenesis.

Influence of mouse apolipoprotein A-II on plasma lipoproteins in transgenic mice.

The role of apolipoprotein A-II (apoA-II) in the metabolism of high density lipoproteins (HDL) is poorly understood. Previous studies of naturally occurring variations of apoA-II in mice have, however, suggested that apoA-II expression influences HDL size and concentration. We now provide definitive evidence of this using transgenic mice overexpressing mouse apoA-II. Thus, as compared with nontransgenic littermates, transgenic mice possessing 4-fold elevations of apoA-II mRNA and 2-3-fold elevations in plasma apoA-II levels exhibited more than a 2-fold increase in HDL levels as well as about a 25% increase in average HDL diameter. Overexpression of mouse apoA-II also resulted in a substantial decrease in the levels of apolipoprotein E associated with HDL, suggesting that apoA-II can displace apoE from HDL. The apoA-II transgenic mice also exhibited a 2-4-fold increase in plasma very low density and low density lipoprotein-cholesterol levels, suggesting novel interactions among the classes of plasma lipoproteins.

Atherosclerosis in transgenic mice overexpressing apolipoprotein A-II.

Concentrations of plasma high density lipoprotein (HDL) are inversely correlated with atherosclerotic coronary artery disease. The two most abundant protein constituents of HDL are apolipoproteins A-I and A-II (apoA-I and apoA-II). ApoA-I is required for assembly of HDL and, when overexpressed in transgenic mice, confers resistance to early atherosclerosis. The present studies reveal that transgenic mice that overexpress mouse apoA-II had elevated HDL-cholesterol concentrations but, nevertheless, exhibited increased atherosclerotic lesion development as compared to normal mice. The HDL in the transgenic mice was larger and had an increased ratio of apoA-II to apoA-I. Thus, both the composition and amount of HDL appear to be important determinants of atherosclerosis.

Superoxide generation by the human polymorphonuclear leukocyte in response to latex beads.

In this study, superoxide formation was not immediately detected when polymorphonuclear leukocytes were treated with linoleyl alcohol-coated, carboxy-modified latex beads. However, all other measures of neutrophil activation were present. Superoxide was not detected until 30 min after the initial exposure to beads. However, an O2(-)-producing, NADPH-dependent oxidase is active 15 min after exposure. When polymorphonuclear leukocytes are pretreated with cytochalasin B, superoxide production is detected immediately after exposure to the beads. Superoxide secretion after treatment with linoleyl alcohol-coated latex beads is compared with the response to other latex beads. The results imply that neutrophils form a phagolysosome around linoleyl alcohol-coated latex beads that is tightly sealed and does not allow superoxide to escape into the medium where it could be detected by the reduction of ferricytochrome c.

Role of free radical processes in stimulated human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes produce large quantities of superoxide when they attack and kill bacteria. However, superoxide is a weak oxidizing and reducing agent, and other more reactive oxygen species derived from reactions of superoxide are suggested to participate in the killing processes. To test the hypothesis that a reactive free radical or singlet oxygen is involved in bactericidal activity, human polymorphonuclear leukocytes were exposed to phagocytozable particles containing lipids that contain the easily autoxidized 1,4-diene moiety. After incubation the preparations were extracted and the extracts reduced with NaBH4 to convert hydroperoxides to stable alcohols. Using gas chromatography/mass spectrometry to analyze the extracts, we were unable to detect products unless iron salts were added to the medium. The products obtained by extraction are those that would be expected if both free radical chain autoxidation and 1O2 oxidation were taking place. In summary, we find that polymorphonuclear leukocytes do not cause peroxidation, implying that formation of strongly oxidizing free radicals is not an intrinsic property of the leukocyte. Added iron catalyzes peroxidation by activated leukocytes yielding an unusual distribution of hydroxylated products.