Jack Y. Vanderhoek

Normoxic Ventilation After Cardiac Arrest Reduces Oxidation of Brain Lipids and Improves Neurological Outcome

BACKGROUND AND PURPOSE Increasing evidence that oxidative stress contributes to delayed neuronal death after global cerebral ischemia has led to reconsideration of the prolonged use of 100% ventilatory O2 following resuscitation from cardiac arrest. This study determined the temporal course of oxidation of brain fatty acyl groups in a clinically relevant canine model of cardiac arrest and resuscitation and tested the hypothesis that postischemic ventilation with 21% inspired O2, rather than 100% O2, results in reduced levels of oxidized brain lipids and decreased neurological impairment. METHODS Neurological deficit scoring and high performance liquid chromatography measurement of fatty acyl lipid oxidation were used in an established canine model using 10 minutes of cardiac arrest followed by resuscitation with different ventilatory oxygenation protocols and restoration of spontaneous circulation for 30 minutes to 24 hours. RESULTS Significant increases in frontal cortex lipid oxidation occurred after 10 minutes of cardiac arrest alone with no reperfusion and after reperfusion for 30 minutes, 2 hours, and 24 hours (relative total 235-nm absorbing peak areas=7.1+/-0.7 SE, 17.3+/-2.7, 14.2+/-3.2, 16.1+/-1.0, and 14.0+/-0.8, respectively; n=4, P<0.05). The predominant oxidized lipids were identified by gas chromatography/mass spectrometry as 13- and 9-hydroxyoctadecadienoic acids (13- and 9-HODE). Animals ventilated on 21% to 30% O2 versus 100% O2 for the first hour after resuscitation exhibited significantly lower levels of total and specific oxidized lipids in the frontal cortex (1.7+/-0.1 versus 3.12+/-0.78 microg 13-HODE/g wet wt cortex., n=4 to 6, P<0.05) and lower neurological deficit scores (45.1+/-3.6 versus 58.3+/-3.8, n=9, P<0.05). CONCLUSIONS With a clinically relevant canine model of 10 minutes of cardiac arrest, resuscitation with 21% versus 100% inspired O2 resulted in lower levels of oxidized brain lipids and improved neurological outcome measured after 24 hours of reperfusion. This study casts further doubt on the appropriateness of present guidelines that recommend the indiscriminate use of 100% ventilatory O2 for undefined periods during and after resuscitation from cardiac arrest.

Inhibition of fatty acid oxygenases by onion and garlic oils: Evidence for the mechanism by which these oils inhibit platelet aggregation

Abstract Inhibition of in vitro platelet aggregation by onion and garlic oils was accompanied by decreased formation of thromboxane B 2 (TXB 2 ) and 12-hydroxyheptadecatrienoic acid (HHT) from [1- 14 C]arachidonic acid (AA). At intermediate concentrations (10–30 μg ml ), these oils also induced a redistribution of the products of the lipoxygenase pathway; at higher concentrations (30–60 μg ml ), they completely suppressed the formation of all oxygenase products. Measurements of oxygen consumption in antimycin-treated platelets indicated a direct correlation between inhibition of platelet fatty acid oxygenases and the anti-aggregating activity of these oils. Onion and garlic oils also inhibited fatty acid oxygenases from sheep vesicular gland preparations as shown both by decreased oxygen consumption and decreased formation of prostaglandin E 2 (PGE 2 ) and PGD 2 from [1- 14 C]AA. Onion oil was approximately ten times more effective in inhibiting the platelet oxygenases than the oxygenases from the vesicular gland. In addition, these oils suppressed the soybean lipoxygenase-catalyzed oxygenation of AA. In all three enzyme systems, onion oil exhibited greater inhibitory activity than garlic oil. Many nonsteroidal anti-inflammatory and anti-thrombotic drugs act by inhibiting the cyclooxygenase component of the prostaglandin and thromboxane synthetases. Our findings thus suggest that onion and garlic contain compounds that may have serendipitous pharmacological effects.

Regulation of T-lymphocyte mitogenesis by the leukocyte product 15-hydroxy-eicosatetraenoic acid (15-HETE)

Abstract Synthesis of lipoxygenase metabolites of [14C]arachidonic acid by mouse spleen lymphocyte cultures was inhibited by the leukocyte product 15-hydroxy-eicosatetraenoic acid (15-HETE) in a dose-dependent manner. In parallel experiments, the influence of 15-HETE on mitogenesis in spleen lymphocyte cultures was examined. 15-HETE at concentrations similar to those which inhibited cellular lipoxygenases progressively inhibited mitogenesis induced by the T-cell mitogen PHA but had no significant effect on the mitogenic response to the B-cell mitogen LPS. The inhibitory response was maximal when 15-HETE was added within 8 hr of exposure to PHA. Several analogs of 15-HETE having progressively fewer double bonds were tested in the same systems. 15-OH,20:3 had approximately the same potency as 15-HETE in inhibiting both mitogenesis and formation of metabolites from [14C]arachidonic acid. 15-OH, 20:2 and 15-OH,20:0 were much less active in either assay. Mitogenesis, induced in spleen cell cultures by the tumor promoter phorbol myristate acetate, was also blocked by 15-HETE. These experiments indicate that lipoxygenase metabolites of arachidonic acid may play an important role in T-lymphocyte blastogenesis and suggest that 15-HETE, via its ability to selectively inhibit cellular lipoxygenases, may function as an endogenous regulator of T-lymphocyte responses.

Regulation of leukocyte and platelet lipoxygenases by hydroxyeicosanoids

During allergic and inflammatory reactions, arachidonic acid is oxidized by lipoxygenases to a variety of biologically active products, including leukotrienes. The mechanisms for regulation of the different lipoxygenase activities are not well defined. We report here that [14C]arachidonic acid metabolism by the 5- and 15-lipoxygenase activities in rabbit leukocytes and the 12-lipoxygenase in rabbit platelets is inhibited by various hydroxyeicosatetraenoic acids (HETEs). 15-HETE was the most effective inhibitor of the 5- and 12-lipoxygenases, whereas similar inhibitory potencies were observed for 5-HETE and 12-HETE acting on the 15-lipoxygenase. These three enzyme pathways were all least sensitive to their own products HETEs. To determine which structural characteristics of 15-HETE are essential for inhibition of the 5-lipoxygenase, various derivatives were prepared and purified by high pressure liquid chromatography, and their structures were confirmed by gas chromatography-mass spectrometry. The inhibitory potencies of 15-HETE analogs with different degrees of unsaturation were in the order of three double bonds greater than 4 greater than 2 greater than 0. 15-Hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) was four times more potent than 15-HETE. The 15-acetoxy, 15-keto and methyl ester derivatives were of comparable activity to 15-HETE, and the 15-acetoxy methyl ester derivative was less potent. Based upon the observed patterns of inhibition, we postulate that complex interregulatory relationships exist between the various lipoxygenases, and that cells containing these lipoxygenases may interact with each other via their lipoxygenase metabolites.

Antiplatelet effects of conjugated linoleic acid isomers.

Conjugated diene isomers of linoleic acid (CLA) are normal constituents of certain foods and exhibit anticarcinogenic and antiatherogenic properties. In the present study, the effects of several CLA isomers on human platelet aggregation and arachidonic acid metabolism were examined. It was found that 9c,11t-CLA, 10t, 12c-CLA and 13-hydroxy-9c,11t-octadecadienoic acid (13-HODE) inhibited arachidonic acid- and collagen-induced platelet aggregation with I50s in the 5-7 microM range. The nonconjugated 9c, 12c-LA was about 300% and 50%, respectively, less potent an inhibitor with these aggregating agents. Using either thrombin or the calcium ionophore A23187 as aggregating agents, a CLA isomer mix was also found to be more inhibitory than 9c,12c-LA. The 9c,11t- and 10t,12c-CLA isomers as well as the CLA isomer mix inhibited formation of the proaggregatory cyclooxygenase-catalyzed product TXA2, as measured by decreased production of its inactive metabolite [14C]TXB2 from exogenously added [14C]arachidonic acid (I50s=9-16 microM). None of the CLA isomers tested inhibited production of the platelet lipoxygenase metabolite [14C]12-HETE. The additional presence of a hydroxyl group gave opposite results: 13-HODE (I50=3 microM) was about 4-fold more potent a cyclooxygenase inhibitor than the 9c,11t-CLA isomer but 9-HODE was 2- to 3-fold less effective an inhibitor (I50=34 microM) of [14C]TXB2 formation than the corresponding 10t,12c-CLA. In both the aggregation and arachidonic acid metabolism experiments, the inhibitory effects of CLA on platelets were reversible and dependent on the time of addition of either the aggregating agent or the [14C]arachidonic acid substrate. These studies suggest that CLA isomers may also possess antithrombotic properties.

Mechanism of action of gonadotropin releasing hormone: Role of lipoxygenase products of arachidonic acid in luteinizing hormone release

The mechanism of action of gonadotropin-releasing hormone (GnRH) upon pituitary luteinizing hormone (LH) secretion has not yet been elucidated, but recent evidence has suggested that arachidonic acid or its metabolites are involved in GnRH action. In cultured rat pituitary cells, arachidonic acid and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) elicited concentration-dependent release of LH with EC50 of about 12 microM. Other lipoxygenase derivatives including 11-, 12- and 15-HETE, had no consistent effect on LH release, and leukotrienes (B4 and C4) exerted only minor stimulatory actions on LH release. The lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosatetraynoic acid (ETYA), and 3-amino-1-(3-trifluoromethyl phenyl)-2-pyrazoline hydrochloride (BW 755C) caused dose-dependent inhibition of GnRH-induced LH release, with IC50 values of 5, 8.5, and 175 microM, respectively. In contrast, the cyclooxygenase inhibitor, indomethacin, had a biphasic action on GnRH-stimulated LH release, with potentiation of GnRH action at low doses (up to 25 microM) and no effect at higher concentrations. These findings are consistent with the potential role of a 5-lipoxygenase product of arachidonic acid in the mechanism of action of GnRH on pituitary gonadotropin release.

Conjugated linoleic acid exhibits stimulatory and inhibitory effects on prostanoid production in human endothelial cells and platelets

In addition to their reported antitumorigenic properties, various conjugated linoleic acid (CLA) isomers have also been shown to decrease prostanoid synthesis as a result of inhibiting the cyclooxygenase (COX) enzyme. We have previously reported that several CLA isomers inhibited both platelet aggregation and formation of thromboxane A(2) (TXA(2)), a proaggregatory and vasoconstrictive agent. Since the interaction between platelets and vascular endothelial cells is essential to maintaining vascular homeostasis, we decided to investigate the effects of various CLA isomers on the production of endothelial prostacyclin (PGI(2)), a potent vasodilator and inhibitor of platelet function. Using interleukin 1-beta (IL1-beta)-stimulated human umbilical vein endothelial cells (HUVECs), we initially established that HUVECs of passage #2 should be used since these cells were most responsive to thrombin-induced conversion of endogenous arachidonic acid to PGI(2), as monitored by the formation of its stable, inactive metabolite, 6-ketoPGF(1alpha). In the first part of the study, the effects of CLA isomers in the free fatty acid form were tested. The 10(E), 12(Z)- and 9(Z), 11(E)-CLA isomers inhibited thrombin-induced 6-ketoPGF(1alpha) formation with I(50)s of 2.6 and 5.5 microM, whereas the 9(Z), 11(Z)- and 9(E), 11(E)-CLA were ineffective at concentrations up to 60 microM. The inhibitory effect of the 10(E), 12(Z)-CLA was irreversible. Next, the effects of CLA incorporation into HUVECs on PGI(2) generation was determined. An average 8-fold stimulation of 6-ketoPGF(1alpha) formation was obtained with quiescent IL1-beta-exposed HUVECs pretreated for 18 h with 25 microM 9(Z), 11(Z)-CLA, whereas cells preincubated with the 10(E), 12(Z) isomer enhanced this eicosanoid 3-fold. Such IL1-beta-treated HUVECs prelabeled with 25 microM 9(Z), 11(Z)-CLA became refractory to thrombin stimulation, as measured by 6-ketoPGF(1alpha) production, whereas a small, statistically insignificant, inhibition was observed upon thrombin treatment of HUVECs prelabeled with the 10(E), 12(Z) isomer. Qualitative similar results were obtained with resting or thrombin-stimulated platelets containing these esterified CLA isomers indicating that these effects occur with cells that contain either the COX-1 or COX-2 isozymes. The results of this in vitro study indicate that the effects of CLA on cellular prostanoid formation in endothelial cells and platelets can be either inhibitory or stimulatory, and this seems to depend not only on the specific CLA isomer and whether or not the CLA is in the free fatty acid form or esterified into cellular lipids, but also whether cells are in the resting or stimulated state. These findings suggest that in vivo, CLA might have multiple, complex effects on vascular homeostasis.

Effects of onion (allium cepa) extract on platelet aggregation and thromboxane synthesis

Oral administration of onion and garlic reportedly decreases platelet aggregation in both human and animal subjects. An oily chloroform extract of onion (Allium Cepa) was prepared and separated by column chromatography on silicic acid into six fractions by elution with solvents of increasing polarity. The least polar fraction contained most of the inhibitory activity towards platelet aggregation induced by either ADP or arachidonic acid. Further purification was afforded by thin-layer chromatography. The specific activity of this major active fraction (I50 per ml of PRP) was approximately 7 units per milligram. Platelets incubated in the presence of onion inhibitor and (1-14C)-arachidonic acid showed striking changes in the pattern of arachidonic acid metabolites formed. Thromboxane B2 synthesis was almost completely suppressed without significant decreases in total hydroxy fatty acid formation. It was concluded that the observed antiplatelet activity of onion relates to the presence of a non-polar, heat stable inhibitor of thromboxane synthesis. This appears to be the first demonstration of this type of inhibitor present in significant quantities in a common foodstuff.

Nonsteroidal anti-inflammatory drugs stimulate 15-lipoxygenase/leukotriene pathway in human polymorphonuclear leukocytes

[14C]Arachidonic acid metabolism in human polymorphonuclear (PMN) leukocytes proceeds predominantly through the 5- and 15-lipoxygenase pathways. The major products are 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and 15-HETE. Three nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit 5-HETE production. Concentrations of drugs required for 50% inhibition of 5-lipoxygenase were 0.17mM for indomethacin, 0.60mM for ibuprofen, and 3.4mM for aspirin. A surprising result was that the human PMN leukocyte 15-lipoxygenase/leukotriene pathway was selectively activated by 1mM to 5mM ibuprofen. Metabolites were identified by gas chromatography/mass spectrometry or by retention times on high-performance liquid chromatography in comparison with authentic standards. The major product was 15-HETE; in all 19 human donors tested, 15-HETE formation was stimulated up to twentyfold by 5mM ibuprofen. Other identified products include 5,15-DiHETE, 8,15-DiHETE, and 12-HETE. This ibuprofen-induced activation of 15-HETE formation occurred even in the presence of 10% serum. When the effects of aspirin, indomethacin, and ibuprofen were compared in PMN leukocytes from six donors, ibuprofen caused an average ninefold stimulation of 15-lipoxygenase, whereas aspirin and indomethacin exhibited an average 150% and 200% enhancement, respectively. Results suggest that ibuprofen acts at the postphospholipase stage and may mimic an endogenous activator, initiate a physiologic activation process, or displace a naturally occurring inhibitor of the 15-lipoxygenase. The capacity of NSAIDs to activate the 15-lipoxygenation of arachidonic acid provides a novel mechanism for the stimulation of 15-HETE production, which may indirectly stimulate the generation of leukotrienes by tissue mast cells.

Arachidonic acid metabolism in gonadotroph-enriched pituitary cells

Control of pituitary hormone secretion by hypothalamic-releasing peptides appears to involve unidentified products of the cyclooxygenase and lipoxygenase pathways, as well as the adenylate cyclase system. To identify the patterns of arachidonic acid metabolism in specific pituitary cell types, the labeled products formed from [14C]-arachidonic acid were analyzed in rat pituitary cells separated by centrifugal elutriation into fractions enriched in gonadotrophs, somatotrophs and lactotrophs. Gonadotroph-enriched cell fractions metabolized arachidonic acid to 11-, 12- and 15-HETE, HHT, PGD2, PGE2 and TXB2. The products were characterized by high performance liquid and thin-layer chromatography, together with gas chromatographic-mass spectrometric identification of 12- and 15-HETE. In cells preincubated with indomethacin, the formation of 11-HETE, HHT, PGD2, PGE2 and TXB2 was markedly reduced. In gonadotroph-enriched cell fractions, the production of cyclooxygenase metabolites was 3 to 4 times greater than that of lipoxygenase products. The somatotroph- and lactotroph-enriched cell fractions produced only very small amounts of oxygenated arachidonic acid metabolites under the conditions studied, but all cell fractions incorporated [14C]-arachidonate into mono-, di- and tri-glycerides, as well as into phospholipids. These results demonstrate the differential capacities of the individual pituitary cell populations for metabolizing arachidonic acid, and emphasize the relative prominence of the oxidation pathways for arachidonate metabolism in the gonadotroph-enriched cell fraction of the rat pituitary gland.