Free Radical Biology & Medicine 41 (2006) 1124–1132www.elsevier.com/locate/freeradbiomed

Original Contribution

Systematic study on ROS production induced by oleic, linoleic,and γ-linolenic acids in human and rat neutrophils

Elaine Hatanaka a,⁎, Adriana Cristina Levada-Pires a, Tania Cristina Pithon-Curi a,b, Rui Curi a

a Institute of Biomedical Sciences, Department of Physiology and Biophysics, University of São Paulo, Avenida Prof. Lineu Prestes,1524, 05508-900 Butantã, São Paulo, SP, Brazilb Cruzeiro do Sul University, São Paulo, Brazil

Received 12 February 2006; revised 23 June 2006; accepted 25 June 2006Available online 4 July 2006

Abstract

The effects of oleic, linoleic, and γ-linolenic acids on the production of ROS by unstimulated and PMA-stimulated neutrophils wereinvestigated by using five techniques: luminol- and lucigenin-amplified chemiluminescence, cytochrome c, hydroethidine, and phenol redreduction. Using lucigenin-amplified chemiluminescence, an increase in extracellular superoxide levels was observed by the treatment ofneutrophils with the fatty acids. There was also an increase in intracellular ROS levels under similar conditions as measured by thehydroethidine technique. An increment in the intra- and extracellular levels of H2O2 was also observed in neutrophils treated with oleic acid asmeasured by phenol red reduction assay. In the luminol technique, peroxidase activity is required in the reaction of luminol with ROS for lightgeneration. Oleic, linoleic, and γ-linolenic acids inhibited the myeloperoxidase activity in stimulated neutrophils. So, these fatty acidsjeopardize the results of ROS content measured by this technique. Oleic, linoleic, and γ-linolenic acids per se led to cytochrome c reductionand so this method also cannot be used to measure ROS production induced by fatty acids. Oleic, linoleic, and γ-linolenic acids do stimulateROS production by neutrophils; however, measurements using the luminol-amplified chemiluminescence and cytochrome c reductiontechniques require further analysis.© 2006 Elsevier Inc. All rights reserved.

Keywords: Fatty acids; ROS; Luminol; Lucigenin; Cytochrome c; Hydroethidine; Neutrophils; Free radicals

Neutrophils are the first cells that migrate to tissues inresponse to invading pathogens. The antimicrobial function ofthese phagocytes depends on the release of lytic enzymesstored in cytoplasmatic granules and on generation ofsuperoxide (O2

S−). In phagocytes, superoxide is mainlygenerated by the reaction of oxygen and NADPH throughthe NADPH oxidase complex [1]. This enzyme complex isformed by subunits found in intracellular granules and plasmamembrane. After activation, NADPH oxidase componentscontaining granules fuse to phagocytic vacuoles and generatesuperoxide. Also, granules may migrate to the cell surfaceand release superoxide into the extracellular space [2].Superoxide anion and hydrogen peroxide (H2O2) generated

⁎ Corresponding author. Fax: +55 11 3091 7285.E-mail address: ehata@usp.br (E. Hatanaka).

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by the NADPH oxidase give rise to other reactive oxygenspecies (ROS) that are strong cytolytic agents, such ashypochlorous acid (formed from H2O2 and chloride ions bythe action of myeloperoxidase (MPO) released from neutro-phil granules) and hydroxyl radical [1–3].

Superoxide can also be generated through the mitochon-drial electron transport chain, xanthine–xanthine oxidase,and cytochrome P450. Mitochondria generate superoxidemostly by the univalent reduction of oxygen in complexes Iand III of the electron transport chain [4]. Changes inmitochondrial generation of superoxide can be assessed bythe addition of mitochondria uncouplers that are depolariz-ing agents and inhibitors of the respiratory chain [5].

ROS generated by neutrophils play an important role in theinflammatory response by regulating other immune reactivecells [6,7]. Fatty acids have been used for the treatment ofvarious diseases that involve oxidative stress, such as coronary

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heart disease [8] and rheumatic arthritis [9]. However, theeffects of long-chain fatty acids on ROS production byneutrophils remain to be fully established. Some authorsshowed an increase [10–12], whereas others found adecrease, in ROS production by neutrophils in response tofatty acid treatment [13–16]. This discrepancy may be due tothe methods used to measure intra- and extracellular ROSlevels.

In this study intra- and extracellular levels of ROS weremeasured in neutrophils treated with oleic, linoleic, and γ-linolenic acids. The luminol technique was used to measureintra- and extracellular ROS levels. Hydroethidine reductionwas employed to measure intracellular ROS production.Cytochrome c reduction and lucigenin-amplified chemilumi-nescence were used to determine extracellular superoxideanion levels. Phenol red reduction was employed to measureintra- and extracellular H2O2 levels.

Material and methods

RPMI 1640 medium, Hepes, penicillin, and streptomycinwere purchased from Invitrogen (Carlsbad, CA, USA). Fattyacids, glutamine, luminol, lucigenin, cytochrome c, hydro-ethidine, peroxidase type II, hydrogen peroxide, phenol red,Histopaque, oyster glycogen, and trypan blue were supplied bySigma Chemical Co (St. Louis, MO, USA). Fatty acids weredissolved in ethanol. The final concentration of ethanol in theassay medium did not exceed 0.05%. A preliminary experimentshowed that ethanol at this concentration is not toxic toneutrophils and does not interfere with the results obtained.Reagents, water, and plastic ware used in the experiments wereall endotoxin-free.

Rat neutrophil preparation

Male Wistar rats weighing 180 ± 20 g were obtained fromthe Department of Physiology and Biophysics, Institute ofBiomedical Sciences, University of São Paulo, Brazil. Therats were maintained at 23°C under a light:dark cycle of12:12 h. Food and water were given ad libitum. The AnimalCare Committee of the Institute of Biomedical Sciencesapproved the experimental procedure of this study. Rats werekilled by decapitation without anesthesia. Neutrophils wereobtained by intraperitoneal (ip) lavage using 40 ml sterilePBS, 4 h after the ip injection of 10 ml sterile oysterglycogen solution (Sigma; Type II) at 1% in PBS [17]. Thenumber of viable cells (>98%) was counted in a Neubauerchamber using a light microscope (Nikkon, Japan) and trypanblue solution at 1%.

Human neutrophil preparation

Human neutrophils were isolated from blood of healthyvolunteers, as previously described [18], using a commercialgradient of Ficoll–Hypaque (Histopaque). The Ethical Com-mittee of the Institute of Biomedical Sciences approved theexperimental procedure of this study.

Human and rat neutrophil treatment

Lucigenin (1 mM), luminol (1 mM), phenol red (0.28 mM),hydroethidine (1 μM), or cytochrome c (0.1 mM) was added toneutrophil (2.5 × 106 cells/ml) incubation medium whenrequired. Immediately afterward, cells were treated with variousconcentrations (0, 10, 25, 50, 100, and 200 μM) of oleic,linoleic, or γ-linolenic acid and phorbol myristate acetate(PMA) (54 ng/ml). ROS release was monitored for 20 min. Theassays were run in PBS buffer supplemented with CaCl2(1 mM), MgCl2 (1.5 mM), and glucose (10 mM), at 37°C, in afinal volume of 0.3 ml.

Lucigenin-enhanced chemiluminescence assay

Lucigenin is extensively used to measure the production ofreactive oxygen species by chemiluminescence. After beingexcited by superoxide anion, lucigenin releases energy in theform of light. Lucigenin-amplified chemiluminescence is aspecific method for studying the kinetics of superoxideproduction by neutrophils. In this method, the response toxanthine–xanthine oxidase presented a positive correlation withlight measurement and did not show augmentation ofchemiluminescence when MPO was added to the assay medium[19]. Also, neutrophil chemiluminescence induced by fMet-Leu-Phe (fMLP) is dose-dependently inhibited by scavengers ofsuperoxide anions but not by azide, catalase, mannitol, ortaurine; so this is a specific method to measure superoxide anionproduction [20]. The chemiluminescence response was mon-itored for 20 min, at 37°C, in a microplate luminometer (EG&GBerthold LB96V).

Luminol-enhanced chemiluminescence assay

Luminol (5-amino-2,3-dihydro-1,4-phthalazindione) is achemical light amplifier. One important point to be consideredin this technique is that MPO-derived metabolites areresponsible for the excitation of luminol [21]. Therefore,neutrophil degranulation can influence the results by releasingMPO found in the azurophil granules during the respiratoryburst. Neutrophils were treated as described above in thepresence of luminol (1 mM). Chemiluminescence was mea-sured as described above.

Flow-cytometric measurement of reactive oxygen metabolitesusing hydroethidine

Hydroethidine has been widely used for the flow-cytometricmeasurement of intracellular ROS production. Hydroethidine, areduced derivative of ethidium bromide, easily penetrates intothe cells and shows weak fluorescence when excited by light at480-nm wavelength. Hydroethidine is intracellularly oxidizedby oxygen radicals, being converted into ethidium bromide thattightly binds to DNA and shows a strong red fluorescence[22,23]. One advantage of this method is the possibility ofevaluating the response of individual cells. It providesstatistically reliable distribution of cells according to the

Fig. 1. Cytochrome c reduction by oleic, linoleic, and γ-linolenic acids(100 μM) in assay medium without cells. Results are presented as means ± SEMof two experiments carried out in triplicate. *p < 0.05 for comparison betweencontrol condition and treatment with fatty acids.

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following activating states: dormant, primed, active, or resting[24]. Neutrophils were treated as described above in thepresence of hydroethidine (1 μM). The fluorescence wasmeasured using the FL3 channel in a FACSCalibur flowcytometer (Becton–Dickinson, San Jose, CA, USA). Tenthousand events were analyzed per experiment.

Cytochrome c reduction assay

The reduction of cytochrome c by reactive oxygen speciescan be measured by spectrophotometry. A control reactionwas carried out with 50 and 100 μM oleic, linoleic, and γ-linolenic acids and cytochrome c (0.1 and 1.0 mM) withoutcells. The reaction was run for 30 min in PBS in a finalvolume of 0.3 ml. After that, changes in absorbance weremonitored at 550 nm.

Hydrogen peroxide determination

For the measurement of hydrogen peroxide levels, a single,rapid, and inexpensive method described by Pick et al. [25] wasused. The assay is based on horseradish peroxidase (HRP)-mediated oxidation of phenol red by H2O2. The reaction leads toformation of a colored compound that shows absorbance at610 nm [25]. The phenol red assay allows the detection ofreactive oxygen species both inside and outside the cells.Neutrophils were treated as described above in the presence ofphenol red (0.28 mM) and 1 U/ml HRP type II. The reactionwas stopped by the addition of 10 μl of 1 N sodium hydroxideaqueous solution.

Measurement of MPO release from neutrophils

Neutrophils (2 × 106 cells/ml) were exposed for 30 min, at37°C, to oleic, linoleic, and γ-linolenic acids (0, 10, 25, 50, 100,and 200 μM) in the presence or absence of PMA. Afterincubation, the medium was immersed into ice and centrifugedat 500 g for 10 min, at 4°C, to separate the supernatant from thecells. The supernatant was used to measure MPO activity. Thereaction was run in PBS, H2O2 (0.1 mM) and luminol (1 mM),at 37°C, in a final volume of 0.3 ml. Chemiluminescence wasdetermined as described above [26].

Statistical analysis

Comparisons were performed using one-way ANOVAand the Dunnett test. The significance was set at p < 0.05.Results were obtained from three to five separate experi-ments and are expressed as means ± SEM.

Results

Appropriate controls were carried out using 10, 25, 50, 100,and 200 μM oleic, linoleic, and γ-linolenic acids in the assays(luminol, lucigenin, and phenol red) without cells. The threefatty acids did not directly affect the luminol, lucigenin, andphenol red assays. To test for possible interference by the fatty

acids in the reaction of ROS with the reagents, peroxide wasadded to the phenol red assay, and xanthine and xanthineoxidase were added to the lucigenin and luminol assays, withoutcells. There was no effect of the fatty acids on the lucigenin,luminol, and phenol red ROS-detecting systems.

Neutrophils treated with oleic, linoleic, and γ-linolenic acidsshowed an increase in superoxide levels as shown bycytochrome c reduction (data not shown). However, the controlreactions, containing 100 μMoleic, linoleic, or γ-linolenic acidswith cytochrome c (0.1 mM), without cells, also showed anincrease in absorbance (Fig. 1). The same result was obtainedwith 1.0 mM cytochrome c and 50 μM oleic, linoleic, or γ-linolenic acid (data not shown). Oleic, linoleic, and γ-linolenicacids per se led to cytochrome c reduction. Therefore, thismethod cannot be recommended as a reliable measurement ofROS production induced by fatty acids at the concentration usedin this study.

As indicated in the Fig. 2, oleic and linoleic acids stronglyinhibited basal and PMA-stimulated ROS production by ratneutrophils in a dose-dependent manner. The inhibitory effect ofoleic acid on ROS release by unstimulated neutrophils was 15%for 5 μM and varied from 68 to 86% for 10, 25, 50, 100, and200 μM. In PMA-stimulated cells, ROS release was decreasedby 56–93% for the same fatty acid concentrations. Theinhibitory effect of linoleic acid on neutrophil ROS releasewas 25% for 5 μM, 41% for 10 μM, and approximately 90% for25, 50, 100, and 200 μM. In PMA-stimulated neutrophils, theinhibition induced by linoleic acid was 39% for 5 μM, 43% for10 μM, and about 95% for 25, 50, 100, and 200 μM (Fig. 2). Theinhibitory effect of γ-linolenic acid on PMA-induced neutrophilROS release was less pronounced than that of oleic and linoleicacids. γ-Linolenic acid caused a significant effect at the 200 μMconcentration only (64.5% reduction compared to control).

Oleic, linoleic, and γ-linolenic acids inhibited MPOactivity in the incubation medium of PMA-stimulatedneutrophils (Fig. 3). Taking into consideration that the light-generating reaction is peroxidase-dependent [21], ROSproduction by neutrophils is underestimated by using theluminol-amplified chemiluminescence technique in neutro-phils treated with fatty acids.

The increase in superoxide production induced by fatty acidsin neutrophils using the lucigenin-amplified chemiluminescence

Fig. 3. Changes in MPO activity in the incubation medium of PMA-stimulatedrat neutrophils treated with oleic, linoleic, and γ-linolenic acids. Results arepresented as means ± SEM of at least three experiments carried out in triplicate.*p < 0.05 for comparison between control condition and treatment with PMAand #p < 0.05, ##p < 0.01, for comparison between PMA and fatty acids.

Fig. 2. Intra- and extracellular ROS levels in rat neutrophils (2.5 × 106 cells/ml)as measured by the luminol-amplified chemiluminescence method, in theabsence and in the presence of various concentrations of oleic, linoleic, and γ-linolenic acids (0, 10, 50, 100, and 200 μM) with or without PMA. Results arepresented as means ± SEM of at least three experiments carried out in triplicate.

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technique is a reproductive finding. There was no significantinterference or cross-reaction of fatty acids with lucigenin itself.Kinetic studies showed that induction of superoxide productionin human neutrophils is a fast event that occurs within minutesafter neutrophil treatment with oleic, linoleic, and γ-linolenicacids (50 μM) (Fig. 4).

By using the lucigenin-amplified chemiluminescence assay,an increase in extracellular O2

S− levels was observed bytreatment of neutrophils with the three fatty acids. Oleic,linoleic, and γ-linolenic acids (in human neutrophils) and oleicand linoleic acids (in rat neutrophils) raised the chemilumines-cent signal to a magnitude expected for classical stimuli, such asPMA or opsonized particles, 3 to 4 orders of magnitude higherthan in unstimulated cells (Fig. 5) [27].

An additive effect on fatty acid-induced superoxide produc-tion by neutrophils was observed when PMAwas added to theassay medium (Fig. 5). The additive effect of the fatty acids andPMA on superoxide production was more pronounced in humanthan in rat neutrophils and occurred mainly by the treatment ofthe cells with γ-linolenic acid. Human neutrophils treated withPMA plus oleic (50 and 100 μM), linoleic (50 and 100 μM), orγ-linolenic (10, 50, 100, and 200 μM) acid showed an additiveincrease in superoxide production. For oleic acid, this incrementwas of 3.2 and 4.3 times higher compared with control

Fig. 5. Superoxide anion levels in the incubation medium of (A–C) rat and (D–F) habsence and in the presence of various concentrations of oleic, linoleic, and γ-linolenias means ± SEM of at least three experiments carried out in triplicate. *p < 0.05, **p <and #p < 0.05, ###p < 0.001, for comparison between columns of the same concen

Fig. 4. Kinetics of light emission by unstimulated neutrophils (2.5 × 106 cells/ml) treated with oleic, linoleic, and γ-linolenic acids (50 μM) in the presence oflucigenin (1 mM).

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(neutrophils plus PMA), respectively, for 50 and 100 μM. Forlinoleic acid at 50 and 100 μM, the increment was of 3.6 timeshigher compared to control, whereas for γ-linolenic acid theincrease was 1.5-, 2.6-, 3.8-, and 2.4-fold for 10, 50, 100, and200 μM, respectively. Rat neutrophils treated with γ-linolenicacid did not show any change in superoxide production;however, when PMA was added to the assay medium anadditive effect on superoxide anion production was observed,2.2 times higher for 10 and 50 μM compared to control.

The treatment of rat neutrophils with oleic, linoleic, and γ-linolenic acids increased the intracellular levels of ROS asindicated by the reduction of hydroethidine (Fig. 6). For oleicacid (50 and 100 μM), the increment in ROS production was3.6-fold higher compared to control. For linoleic acid, theincrement was over three times higher compared to control for

uman neutrophils (2.5 × 106 cells/ml) as measured by the lucigenin assay in thec acids (0, 10, 50, 100, and 200 μM) with or without PMA. Results are presented0.01, and ***p < 0.001, due the effects of the fatty acids compared with control

tration of fatty acids, in the presence or absence of PMA.

Fig. 6. Intracellular ROS production by rat neutrophils (2.5 × 106cells/ml), asmeasured by the hydroethidine technique in the absence and in the presence ofvarious concentrations of oleic, linoleic, andγ-linolenic acids (0, 10, 50, 100, and200 μM) with or without PMA. Results are presented as means ± SEM of at leastthree experiments carried out in triplicate. *p < 0.05, **p < 0.01, for comparisonbetween the treatment with oleic, linoleic, and γ-linolenic acids and control.

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both 50 and 100 μM. For γ-linolenic acid the increment was2.0-fold for the 200 μM concentration.

Oleic, linoleic, and γ-linolenic acids did not significantlyincrease the intra- and extracellular basal levels of H2O2 inhuman and rat neutrophils as showed by the phenol red reductionassay. However, there was a positive dose–response correlationbetween fatty acid concentrations and H2O2 production. ThePearson correlation found was r = 0.97 and p = 0.004 for oleicacid and r = 0.92 and p = 0.02 for linoleic and γ-linolenic acids(Fig. 7). An additive effect on H2O2 production was observedwhen PMA was added to the assay medium in both rat andhuman neutrophils treated with oleic acid (Fig. 7).

Discussion

ROS production by neutrophils is primarily associated withphagocyte defense against foreign organisms and occurs

mainly through the NADPH oxidase complex. NADPHoxidase is assembled and activated either in the plasmamembrane or in the membrane of internalized phagosomes.The reactive oxygen species generated will then either bereleased from the cells (activation in the plasma membrane)or be retained inside the phagocyte (activation in thephagosomal membrane) [1,2,28,29]. Mitochondria are alsoconsidered to be an important intracellular site for superoxidegeneration, which occurs mostly by the univalent reduction ofoxygen in complexes I and III of the electron transport chain[4,5].

Evidence is presented herein that oleic, linoleic, and γ-linolenic acids cause a marked increase in intra- andextracellular ROS levels in incubated rat and human neutro-phils. One important point observed is that these fatty acidsinterfered with cytochrome c reduction and luminol-amplifiedchemiluminescence assays. Thus, the contradictory findings ofthe fatty acid effects on ROS production found in the literaturecan be in part due to the methods used.

The fatty acids tested caused a direct reduction ofcytochrome c. Therefore, the effects of fatty acids on superoxideproduction cannot be measured by cytochrome c reductionassay under the conditions herein used. A similar effect wasobserved by Hardy et al. [30]. Another important point of thistechnique is that H2O2 may also interfere with the assay.Accumulation of H2O2 in the measuring system can result in areoxidation of Fe2+ cytochrome c back to the Fe3+ form, thusgiving underestimated results [29]. In addition to the directeffect on cytochrome c reduction, fatty acids can also jeopardizethe light emission techniques (luminol/isoluminol and luci-genin) when the concentration used affects the turbidity of themedium. Measurements of intracellular ROS levels can also beunderestimated because neutrophils rapidly release oxygenspecies to the extracellular medium.

An increase in ROS production induced by fatty acids inneutrophils was found by using lucigenin-amplified chemilu-minescence, hydroethidine, and phenol red reduction techni-ques, whereas a decrease was observed with luminol-enhancedchemiluminescence assay. To address this discrepancy, theeffects of oleic, linoleic, and γ-linolenic acids on MPO activityin the neutrophil incubation medium were examined. Oleic,linoleic, and γ-linolenic acids reduced MPO activity in theincubation medium of PMA-stimulated neutrophils. Our resultsagree with those of Naccache et al. [31]. These authors reportedthat oleic, linoleic, and linolenic acids inhibit neutrophil granulesecretion in response to addition of fMLP [31]. Taking intoconsideration that the light-generating reaction is peroxidase-dependent [21], ROS production by neutrophils is under-estimated by using the luminol-amplified chemiluminescencetechnique in fatty acid-treated neutrophils. The influence ofneutrophil degranulation on ROS production through therespiratory burst by using the luminol technique was alsoestimated by Dahlgren and Stendahl [32]. In this study, NADPHoxidase activity was measured in cytoplasts and in normal cells.The cytoplasts lacking all granules were then deficient in MPOactivity. Under this condition, there was a decrease inchemiluminescence [32]. Therefore, luminol-enhanced chemi-

Fig. 7. Hydrogen peroxide levels in incubation medium of (A–C) rat and (D–F) human neutrophils (2.5 × 106 cells/ml) as measured by the phenol red method inthe absence and in the presence of various concentrations of oleic, linoleic, and γ-linolenic acids (0, 10, 50, 100, and 200 μM) with or without PMA. Results arepresented as means ± SEM of at least three experiments carried out in triplicate. *p < 0.05, **p < 0.01, for comparison between the treatment with the oleic,linoleic, and γ-linolenic acids and the control and #p < 0.05, ###p < 0.001, for comparison between columns of the same concentration of the fatty acids in thepresence and absence of PMA.

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luminescence is not an appropriate technique to measure ROSrelease by neutrophils treated with fatty acids under theconditions herein used.

As mentioned above, oleic, linoleic, and linolenic acidsinhibit neutrophil granule secretion by fMLP- and PMA-stimulated neutrophils. However, in the absence of stimuli,Bates et al. [33] showed that fatty acids increase neutrophildegranulation as follows: linolenic > linoleic > oleic. In general,as the number of double bonds in the 18-carbon fatty acidmolecule increases, so does its ability to stimulate degranulationand oxidative burst in unstimulated neutrophils [33,34]. Thismay explain the fact that the inhibitory effect of γ-linolenic acidon PMA-induced ROS production as measured by the luminoltechnique is less pronounced than that caused by linoleic andoleic acids.

PMA addition to the assay medium caused an additive effecton superoxide and hydrogen peroxide production induced byoleic, linoleic, and γ-linolenic acids. These results support theproposal of a possible priming effect of fatty acids on neutrophiloxidative burst. Neutrophils exist in various states of activationsuch as primed, activated, and spent. Priming and activation arebiochemically integrated events. Priming is a state of preactiva-tion of dormant neutrophils that enables a prompt response ofthe cells to a microbicidal activity [35]. Small biochemicalchanges may trigger priming and large changes may lead to fullactivation including degranulation. Priming agents such ascytokines (TNF-α, IL-8) [36,37], phospholipase A2 [38],platelet-activating factor [39], and serum amyloid A [27]cause an increment in oxygen consumption when a secondstimulus occurs [40].

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Primed oxygenation activity may result from the activationof one or more of the components of the neutrophiltransduction pathways induced by fatty acids. This mayinclude fluxes of free cations (Na+, K+, and Ca2+), changes inmembrane potential, activation of intracellular proteases,increases in arachidonic acid and phospholipid metabolism,phosphorylation of specific proteins (oxidase components),and increase in the intracellular concentrations of cyclicnucleotides [40,41]. Fatty acids support the structural basis ofthe modulation of the membrane lipid organization and thesubsequent regulation of G-protein-coupled receptor signaling[42]. Hardy et al. demonstrated that pretreating neutrophilswith arachidonic, eicosapentaenoic, and docosahexanoic acidsenhances their capacity to respond to either fMLP or PMA,thereby producing more superoxide than when challengedwith the stimulators only [34,43].

In the present study, oleic, linoleic, and γ-linolenic acidsshowed a modulatory effect on inflammation by stimulatingROS production. However, ROS measurement can be jeopar-dized by using luminol-amplified chemiluminescence andcytochrome c reduction techniques.

Acknowledgments

The authors are indebted to the Fundação de Amparo àPesquisa do Estado de São Paulo and to the Conselho Nacionalde Desenvolvimento Científico e Tecnológico for financialsupport.

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