Phospholipid bilayers enhance the stability of leukotriene A4 and epoxytetraenes: Stabilization of eicosanoids by liposomes

Vol. 159, No. 2, 1989 March 15, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 477-481

PHOSPHOLIPID BILAYERS ENHANCE THE STABILITY OF LEUKOTRIENE A, AND EPOXYTETRAENES: STABILIZATION OF EICOSANOIDS BY LIPOSOMES Stefano Fiore and Charles N. Serhan”

Brigham

Received

December

Hematology Division, Department of Medicine and Women’s Hospital and Harvard Medical Boston, MA 02115

21,

School

1988

The hydrolysis of ieukotriene A, and two epoxytetraenes was examined in the presence and absence of liposomes. When added to iiposomes in suspension, the stability of LTA, was increased in a time- and dose-dependent fashion. At 10 min, the half-life of LTA was increased 67.1 + 6.8% in the presence of liposomes which was comparable to that observed with albumin (10 me/ml): 68.3 -+ 6.9%. Phosphatidylcholine, in a non-bilayer configuration, was also effective in enhancing the half-life of LTA,, albeit to a lesser extent than liposomes. At equal molar concentrations, the enhanced stability of eicosanoid epoxides with iiposomes gave a rank order with leukotriene A, > 5(6)epoxytetraene > 14(15)epoxytetraene. Results indicate that phospbolipid bilayers can protect leukotriene A and 5(6)epoxytetraene from non-enzymatic hydrolysis. Moreover, they suggest that the biological h alf-life of intermediates involved in the formation of both leukotrienes and lipoxins can be increased by their association with membranes. 0 1989 Academic Press, Inc.

Lipoxygenaseproductsof arachidonicacid are involved in activation of a wide rangeof cell types and various aspectsof inflammatory reactions(1,2). Leukotrienesand lipoxins, two seriesof lipoxygenaseproducts,areeachgeneratedfrom epoxide-containingintermediates(3-5). Aside from its role as an intermediate in the formation of leukotriene B, or leukotriene C, within its cell of origin, it is now recognizedthat the epoxide LTA, can alsobe releasedby or escapefrom human neutrophilsto be transformedvia transcellularroutes. For example,LTA, is stabilizedby albumin (6) and can be transformedto LTB, by either a plasmaprotein (7) or red cells (8). It can alsobe transformedto LTC, by either endothelialcells (9), platelets(10) or mastcells(11). Recently, we have found that, in addition to transcellularmetabolism,both LTA, and the 5(6)epoxytetraenecanstimulateCa*+mobilizationin neutrophils,which canantecedetheir metabolic transformation(12). The 5-lipoxygenasedisplaysa dual action and it can generateboth 5-HPETE

* Addressall correspondenceto CharlesN. Serhan,HematologyDivision, Brigham and Women’s Hospital, 75 Francis Street, Boston,MA 02115, Abbreviaw RP-HPLC, reverse phasehigh pressureliquid chromatography; leukotriene A, (LTA,), SS-trans-5(6)-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid; PC, phosphatidylchoiine; dicetyiphosphate; 5(6)-epoxytetraene, 15S-hydroxy-5,6-oxido-7,9,13-trans-I l-cisDcp, eicosatetraenoic acid, 14(15)epoxytetraene. SS-hydroxy-14,15-oxido-6,10,12-trans-S-ciseicosatetraenoicacid, LTB,, SS,12R-dihydroxy-6,14-cis-8,lO-trans-eicosatetraenoicacid. 0006-291x/89 477

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and LTA,, aswell asepoxytetraeneswith appropriatesubstrates(13). The activity of this enzyme is increasedwhenassociated with membranephospholipids(14). Therefore, the factors that influence the biological half-life of its products (e.g. epoxides)are of interest. In this report, we present evidenceindicatingthat the stability of epoxide-containingeicosanoids (i.e. LTA,, 5(6)epoxytetraene) can be dramatically increasedwith phospholipidbilayers. Materials and Methods HEPES,L-a phosphatidylcholine,dicetyl phosphate,cholesteroland humanalbumin(fatty acid free) were from SigmaChemicalCo., St. Louis, MO, SephadexG-50 was purchasedfrom Pharmacia,Inc., Piscataway,NJ andHPLC solventswerefrom J.T. Baker, Phillisbury, NJ. Synthetic LTA waspurchasedfrom Biomol ResearchLabs, Philadelphia,PA and the 5(6)epoxytetraeneand 14(153epoxytetraene were provided by Prof. K.C. Nicolaou,University of Pennsylvania. Liposomes (multilamellarvesicles;MLV’s) were preparedasdescribed(15) in KC1 (145mM), and HEPES(10 mM) buffer at pH 7.45. Liposomescontaining PC 7:DCP 2:Chol 1 were isolated following chromatographyon SephadexG-50 (15). IncubationsandHPLC analvsis.The free acidsof LTA,, 5(6)epoxytetraene,and 14(15)epoxytetraene were preparedby LiOH saponificationof their correspondingmethyl estersin tetrahydrofurane (4). The epoxideswere addedto either buffer (145mM KCI, IO mM HEPES)alone,albumin(10 mg/ml) or liposomesand incubatedfor various time intervals (pH 7.45, at RT). Incubationswere stopped by addition of 2 vol of acidic MeOH (HCI) in order to generatemethoxy trapping products from epoxideswhich remain intact (seeResults). Sampleswere rapidly neutralized followed by addition of PGB, asinternal standard. Following extraction and chromatographyon SepPack C cartridges (WatersAssociates,Millipore Co., Milford, MA) (12), samples were analyzedby RP-H&C (Waters Associates). The column (Altex, Ultrasphere-ODS,dp 5 PM, 4.6 mm x 25 cm) was eluted with methanokwatecaceticacid (75:25:0.01,vol/vol) at 1mlfmin, approx. 1900psi. The UV detector was set at either 301 nm to monitor conjugatedtetraenesor 270 nm for conjugatedtriene-containing products. The recovery of PGB, in theseexperimentswas83.3 f 4.6% (n=5 separateexperiments with 91 determinations). Productswere identified by coelution with synthetic standards.

Resultsand Discussion LTA, is highly reactive for nucleophilic substitution (half-life < IO set in aqueous environmentspH 7.4 at 25°C) (16) and can be stabilized in the presenceof albumin (6). Nonenzymatic hydrolysis of LTA, leadsto the formation of two major products, 6-trans-LTB4 and 12-epi-6-trans-LTB, (3). Upon addition of acidic methanol,two major 12-O-methyl derivatives areformed from the intact epoxide(3). Therefore, in the presentstudy, we haveevaluatedthe levels of intact epoxidespresentin varioussuspensions by monitoring the amountsof 6-trans-LTB4, 12epi-6-trans-LTB4 and the 12-O-methyl-derivatives (trapping products)formed following addition of acidic methanol. Addition of LTA, to suspensions containing either albuminor liposomesled to the formation of 12-O-methyl derivatives of LTA, whenthe incubationswere stopped(IO min) by addition of acidic methanoland analyzedby RP-HPLC (Figure 1). In the absenceof either liposomes or albumin, LTA, underwenthydrolysis to give 6-trans-LTB4 and 12-epi-6-trans-LTB4 Previous resultshave shownthat LTA, ($15 PM) is effectively stabilized by albumin (IO mg/ml) (6). When liposomeswere comparedto the stabilizing action of albumin(10 mg/ml), time coursestudiesrevealedthat liposomeswere comparableto the action of albumin(Figure 2, cf. ref. 6). Here, the appearanceof trapping productsandaqueoushydrolysisproductsof LTA, (5 seconds60 min) following analysisby RP-HPLC gave secondorder kinetics. A linear regressionfor the reciprocal of concentrationvs. time gave an r value of 0.95 f 0.07 (n=5). Therefore, the half-life valueswere calculatedfrom T,,, = l/aK[A,,] where[Alo wasthe initial concentrationand aK value (stoichiometriccoefficient and rate constant,respectively) wasgiven by the slopeobtained. 478

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A. LIPOSOMES

AUF 0.02

0 c; d

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A Time

B. ALBUMIN

0

40

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Figure 1. RP-HPLC analysis of products obtained following incubation of LTAg with liposomes (panel A) and huyan albumin (panel B). LTA, (8 PM) was added to either albumm (10 mg/ml) or liposome (1 x IO- M: total liposomal lipid) containing suspensions and incubated for 10 min at RT. Incubations were stopped by addition of acidic methanol and products were extracted and analyzed as described under “Materials and Methods.” Retention times were: I, 14.5 min; II, 16.7 min; III, 33.8 min; and IV, 36.6 min. respectively. The identity of each of the materials beneath the peaks was determined by coelution with synthetic standards and is indicated by arrows.

The percent increase in the Tl,g of LTA, wasdose-dependentin the presence of liposomes (Figure 3). Phosphatidylcholineprepared in a non-bilayer configuration was lesseffective than liposomessuggestingthat the bilayer configuration providesa suitableenvironment to protect the epoxide from aqueoushydrolysis. Approximately 5 molecules of PC were required (in the outer leaflet (15)) to protect LTA, from aqueoushydrolysis. Next, to determine the generality of these findings demonstratedfor LTA,, two other epoxide-containingeicosanoids wereexamined.Both the 5(6)epoxytetraeneand 14(15)epoxytetraene

14c

60-

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Phosphatidyl 0

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2: Time course of LTA, stabilization: Comparison between albumin and liposomes. The Increase m half-life of LTA was calculated as described under “Materials and Methods.” The data are expressed as the mean f s .E.M. for three separate experiments. Figure

Dose response for LTA, stabilization: Comparison between liposomes and Figure 3. phosphatidylcholine. LTA (8 PM) was incubated with various concentrations of liposomes (expressed as total liposomaliipid) or PC for 10 min at 25°C. Incubations were stopped and products were analyzed as described under Materials and Methods. The data are expressed as the mean f S.E.M. of three separate experiments. 479

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ECOOH

J!rLsEY

5(6)-

epoxytetraene

14(15)epoxytetraene

Figure4. Comparison betweenLTA4.5(6)-epoxytetraene andI4(I 5)-epox-3tetraene for stabilization by liposomes. Theepoxides (5 PM)wereeachaddedto lipesomes (I x 10 M: total lipesomal lipid) andincubatedfor 10 minat RT. Incubationswerestoppedandproductswereanalyzedby RPHPLC as described under “Materials and Methods.” The data represent meanf S.E.M.for three determinations.

can be enzymatically generatedby appropriate lipoxygenases(1,13,17), and evidence has been presentedfor the enzymatic transformationof S(6)epoxytetraeneto lipoxin A, (5). When both of theseepoxideswere addedto liposomesand their hydrolysis comparedto that of LTA, utilizing identical conditions, a rank order for stabilization wasestablished: LTA, > 5(6)epoxytetraene> 14(15)epoxytetraene(Figure 4). Thesefindingssuggestthat the stabilizationof LTA, by bilayerscan, in part, be attributed to its ability to intercalate within the hydrophobic region of the bilayer. Although the 5(6)epoxytetraeneis a planar structure (4) the presenceof the alcoholgroup at carbon 15may hinder its ability to enter the bilayer. Likewise, by substitutingan alcoholgroup at carbon 5 rather than an epoxide asin the caseof the 14(15)epoxytetraene,a lesssuitablestabilization was observedwhen the epoxideswere comparedat equimolarconcentrations(Figure 4). Nevertheless, it is clear from theseresultsthat theseallylic epoxidescanbe shieldedfrom non-enzymatic hydrolysis by phospholipidbilayers. When liposomeswere added to A23187-stimulatedhuman neutrophils, enhancementwas observedin LTA, derived products(47.4 f 18.2%[meanf S.E.M.]) indicating that liposomescan increasethe stability of LTA, generatedby activated cells. Recent results indicate that the 5-lipoxygenaseandits LTA, synthaseactivity require Ca*+anda membranestimulatoryfactor which canbe replacedby PC-containingvesicles(18). The presentfindings demonstratethat phospholipid bilayers can also enhancethe stability of labile allylic epoxides(Figs. l-4). Upon activation, leukocytes may translocatetheir 5-lipoxygenase to the plasmamembrane,which leads to full expressionof its activity (19,20)namely, at a site where its products(i.e. LTA,, 5(6)epoxytetraene) canalsobestabilizedby membranecomponents.LTA, isknown to undergotranscellularmetabolism (7-11) (i.e. shuttling from its cell of origin to enzymatic transformation in a secondcell type). Whethermembranephospholipidscan directly contribute to this event or the transport of epoxidecontainingeicosanoidsby increasingtheir biological half-life in various cell systemsremainsto be determined. 480

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In summary,the presentresultsprovide an additional meansfor stabilizing eicosanoids(i.e. with liposomes),which may be usefultools for the cellular delivery and study of labile productsof arachidonicacid and their bioactions.

Acknowledaments Wethank Mary Halm Smallfor skillful assistance in preparationof this manuscript. This work wassupportedby National Institutes of Health grant AI26714-01. CNS is a 1988Pew Scholarin the BiomedicalSciences.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

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