Structural studies of cholesteryl acetate - phosphatidylcholine complexes

Chemistry and Phy~es of Lipids 20 t i 977) 253--262 ©Fa~vier/Norttl-Hotland Setentffic Pubhshers. Ltd.

STRUCTURAL STUDIES OF CHOLESTERYL ACETATE PHATIDYLCLUOLINE COMPLEXES*

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S.C. GOHEEN, LJ. LIS** and J.W. KAUFFMAN Bhemedieal Engineering Center"Northwestern University, The Technological Institute. Evanston, ill. 60201. USA R¢oeived May ! 5th, 1977

accepted May 22nd, 1977

The sl~'uctural order of cholestetyi acetate-dimyrjstoylphosphatidylcholine (DMPC)--v~ater mixtures was examined at room temperature usang Raman spectra in the C - H and C -C stretch l~gions, intensit~ ratios i~s~o[12tso, [2s~/1:,:0 and [to~4/I,0~9 were u~d as structural parameters to measure the order o! DMPC aL3,1 chains. Fhese three rattos pass ~hrough a well defined minimum at ca I4 mo!*~ chogesteryl acetate, Th¢.~ data al.~ ~ o w that cholesteryl acetate disrupts the packing order o f the DMPC acyl chains front 0 to ca 35 mol% cholestery! acetate. As the concentration of cholesteryl aoctate is int:teased from ca. t4 to ca. 35 molS~ th~ DMPC acy| chains become more ordered and chot~steryl acetate appears te form a crystalline phase that remains m su~ension with the lecithin. As more cholcstery! acetate is added, these crystalhne regtons become "larger and begin to form a precipitate which ts structuralt~ Identical to those pal ides v, hich remain m suspension.

I. Introduction

Chotesteryl ester-lipid complexes are major components of atherosclerotic les~ons. The effect of a cholesteryl ester, cholesteryl linolenate, oft the liquid crystalhmty of !ecithin-water systems has been recently described with both fixed [ I l and varied water content [2]. Or~ the basis of these as well as other studies on the components in choiestetot-cholestcryl ester--lipid-water systems [3,4], Small and Shipley [5] d¢scnbed the possible role of cholesteryl esters in athetosderosis. Lundberg [6] has also observed some structural similarities between cholesterol ester suspensions and athotmdetoti¢ lesions when viewed under polarized light, Bulkin and Kdshaan [71 were the first to examine the ¢ffect of liquid crystallinity on various ehtdesterol liquid crystalline materials using Raman spectroscopy. These authors [71 classified the liquid crysta/tine state of valious cholesterol derivatives on * Presell1 at the Biophysical gociety 21st Annual Meeting, February 15 - t8, 1977, New Orleans,

-** Present address: DeCarar~at of Btolo#cat S~nces, Brock University,St. Catltannes, L2S 3AI, Ontario, Caaada, 253

254

$.c Goheen et ai., Structural ~tudies o f dmlesteryl acetate

the basis of the features in the Raman spectral range 2~s00-3000 em"1 and found the least amount of interference from hydrocarbon side chain C-H stretch, which also occurs in this szectral region, in the eholestewI acetate system. Phospholipids ha,~e been extensively examined using l~nmn spectroscopy to determine the effect of environment on the liquid crystallinlty ofthe sample [8- .t3]. In particular, the Sl~ctra[ region 2800-3000 cm-x has also been found to be informative i~zexeminingphos. phatidylcholine liquid crystallinity [8, 9, 11~13] Receatly, l~,e effect of s~r,all amounts of proteins on the phosphatidylcholine packing structure has been examined using Raman spectroscopy [13-15] by studying changes in the peak heisht intensity ratios I2sgo/l~so0 a monitor for hydrocarbon chain order [8,9] and l~o/I~rjo, an independent monitor for polarity of the hydrocarbon chain environment as well as hydrocarbon chain order [13]. in particular as the lipid goes through a thermal (or similar) phase transition the intensity ratios 12~se/12~o and 1~/12s~o undergo dramatic decreases primarily due to the !oss of intensity by the 2890 cm"t band. These changes are due to increased fluidity which is a combination of decreased acyl cl'~n order and increased polarity of the hydrocarbon chain environment. In this study we have used Raman spectroscopy to examine the effect ofchoiestery~ acetate on the liquid crysta~inity of the dimyds~oylphosphatidytcholine-water system. We report and discuss environmental changes which occur around the hydrocarbon region of the dimyristoytphospt~tidylcholin~ (DMPC) molecule as cholesteryl acetate is progre~oiv~lyadded to the DMI~-water mixture.

Ii. Procedure

1,2 L-a dimyristoylphosphatidylcholine (DMPC) was obtained from Calbtochem and ¢holesteryl acetate (ChromatigraphicaUy pure) from General Bioc~mica!s. Water was doubly distilled and reagents were spectre quality. All lipid samples were stored dry, under nitrogen at --15QC until mixing; l~fixingw u performed 24 h before observing the Raman spectra. Ten milligrams of DMt~ was wetghted~to each ~ dram g l ~ vial. A known weight of cholesteryl acetate in chloroform-methanol (2 : ~, v/v) was added ~toeach vial to provide the desired tool ratio ofDMPC-~holeste~l ~lcetate,Thechlorofoml-.n~ethanol was allowed to evaporate at room temperature with Oxeremaining solvent removed by placing the dry samples in a moderate vacumn (i0 -4 Ton)fo~ 30rain. Enough water was added to each samp)e go obtain a final weight ratio Of totaI~lipkl to water of I : 4. ,~llsamples were then incubated at 600Cforoae hem to tnmre:comple~tewater mixing. Samples were allowed to equilibrate overnight before -~bservtngtheir g~eetra, The Raman spectrometer has been described elsewhere [ 16], Typictlly th~ 488.~ and 514.5 nm lasel lines from an Argon ion laler were u~e~d. LaW~pewer WU kept at 500 mW with monochrometer slits sefat 2 0 0 / J M . o ~ n g w a $ d0t~ei~Kim~x ~ capillary tubes and all samples were eg~rtiaedat 2 3 C The ~ e k height in tei1~ty rati~ are at least good to -+0.1 when compared between indepen~nt eX~dmeatt,

~£C Goheen et aI., Structural studies o/'chotesten,l a,~ztate

255

HI. Results and Discussion The Raman spectra of the region around 2800 cm -~ fi~r a range of DMPC, c holestery. I acetate and water mixtures, are shown in fig. 1. As the concentration of cholesleryl acetate is increased from 0 to 100%, the Raman active vibratmns ofchotesteryl acetate increase in intensity relative to those of DMPC. It can be seetl from tile s | ~ e c t t - a of pure ¢holestt~W! aoctate crystals(fig, It) and pure DMPC in water (fig. I a) that some of the DMPC and ehotesteryl acetate peak*, overlap. Since peak height Raman intenslttes are measured from the base of the C - H stretch region, the peaks at ca. 2940 C t n -1 , 2900 cm -t and 2850 cm -1 in chotestery! acetate wilt interfere with the lntenmtv measurements of the 2930 cm -x . 2890 cm -1 and 2850 cm -t peaks of DMPC respectively when ehotesteryl acetate is present in significant quantity ~ca. 60 mode). At tile bottom of fig. 1, all three spectra corresponding to DMPC-cho}esteryl acetate ratios of l : 5, I • 10, and dry cho!esteryl acetate crystals appear nearly identical. This indicates that neither water nor DMPC disrupt the cholester~ ! acetate liquid crystalline hydrocarbon structure at high concentrations of the cholesteryl ester. Conversely, Rainan spectral features independently change in the DMPC spectrum as cholestew1 acetate is progressively added, it appears from fig. I that the 2890 c m - t peak first decreases as more cholesteryl acetate is added, and then increa~s until ca. 40 reel% cholesteryl acetate. At this point, the 2890 cm -l arid 2850 cm -~ peaks t~,adually diminish until the cholesteryl acetate spectrum begins to dominate at ca. 80 reel% cholesteryl acetMe. This phenomena is illustrated mote accurately in fig. 2 where the peak height in-tensity ratios Ixsgo/12ss~, and 12s~o/l~ versus molC/~ of cholesteryl acetate are present. It has been shown by others that both of these ratios are indicative of the order of DMPC hydrocarbon chains [8.9.13]. At low concelmalions of eholeste~,l acetate, these ratios both decrease indicating that the DMPC hydrocarbon chains are becoming more loosely packed. However, near the reel ratio of 5 " 1, DMPC-eholesteryt acetate, the DMPC Raman spectrum dramatically changes with an increase in the 12s~ll:~ase intensity ratio indicating that the phosphatidylchohne hydro¢afbon chains have become more ordered. At a ratio of ca. 1 " 2, DMPC-cholesteryt acetate the Dldt~ spectrum disappears with the now predominant chotesteryl acetate spectrum. One further striking feature of fig. 2 is the leveling of the lugo/lzsso ratio and the linear decrease of the l~so/12~o ratio with respect to composition. We suggest that those linear effects are due to the emergence of Raman peaks from cholesteryl acetate near those a~sociated with DMPC. We no|e that each of the limiting intensity ratios in fig. 2 at¢ intercepts of lines which can be extended through most of the data points/This linear eff~:t indicates that both intensity ratios l:sg0/l:sso and 12sso/129.~o are tnf!ueneed by some component which is directly proportional to the cholesteryl acetate concentration. This component cannot be a function of any structural perturbatiom became the data taken from both pure chotesteryl acetate and pure DMPC in water are merely extensiom of the linear data. Certainly, DMPC cannot be affected by ~ ¢ ~ t acetate at 100~ DMPC and vice versa, Therefore all points which lie on the line between these limits must represent the original ordered state of DMPC, To

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provide further evidence for our interpretation, we have observed iden.cal changes in the DMPC hydrocarbon chain C-C ~tretch Raman peak heigh~ intensity ratios !io~/ il0m (see fig. 3) with a decrease in th~s intensity ratio also indicating a decrease in the acyl chain ordcl [10,1 I]. The 1089 ~an"1 and 1064 cm -~ peaks are le~ intense and therefore noisier than either the 29:30 cm "j , 2890 cm -I , or 2850 cm -1 peaks in the C-H stretch ~egion. However, the peak height intensity .ratio I m~/1 ms9 yields information on the relative amount of all trans (IO64 cm "~ peak) to random (4089 cm -1 peak) acyt chain confomlations~ These independent observations support our belief that the obsened SlpeCtralchanges between 0 and 35 tool% cholesteryt acetate are due to sfflg:tural changes in the ~ acyl chains and not due to spectral hiterference from the dmlestery| acetate Raman spectrum. Further, the~ changes cannot be due to the effect o f a sttuetund r¢~ranl~ment in cholesteryl acetate since a change in its ttquidctystaUhte stat~ should not affect the peak height intensity ratio 12s~/12sso [7]. The llha~ of the c u n ~ in fig. 2 can therefore be attributed to two causes. First 'at low ¢ ~ e t l t | ~C¢late ¢on~tRtatlom. the 12SSe/I~L~, I2SSO/I~jo and lton4[llo~ l,attoS a~¢ ktreeleddue !o decicased acyl chain order and increased polarity around the aCyl chUM of DMPC. liars apl~ent that smadl amounts of choleste~! acetate (less t t ~ 14 m ~ ) ~ l i ~ the I)MI~ acy| chain matrix. After ca. 14 tool% chnlesteryl

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acetate the peak height intensity ratios I~o/la~e, I~ae/I~3o and llec~]Itew increase. Thus the order of the DMPC acyl chains begins to return to tha~t af ~Iie ordered or gel phase, One explanation for this behavior h that beyond ca. 14 tool% cholesteryi acetate, a crystalline phase of the ester emerges from the original DMPC + water phase. At 35 tool% cholesteryl acetate, the peek height intensity ratios I~ge/Izeso, 12sse/lzsr~ and Im64/11~ indicate that DMPC is the get state which implies that cholesteryl acetate is excluded from the DMPC acyl chain region, Therefore between 35 and 60 tool% cholesteryl acetate, two phases ale present, the DMPC gel phase, gnd another phase containing cholesteryl acetate. As the concentration of ¢holest¢,-yl acetate increases greater than ca. 60 tool%, the intensity ratios of Raman peaks associated with DbLPC in 88. 2 are affected by the spectral interference from the cholesteryt acetate Raman spectrum. At very large concentrations of chotesteryl acetate, it becomes clear that chotesteryl acetate exists in the c r y s ~ phase by the similarity in sl?ectra representhag DMPC-cholesteryl acetate ratios 1 : 5, 1 : 10 and crystalline cholesteryl a~tate in fig. It. Durin~ these experiments we made two additional observations. Ftrst we could not dissolve enough crystalline cholesteryt acetate in distilled water to obtain any spectral featares at room temperature. Second in the presence of DMPC, a spectrum of cholestcryl acetate was easily obtained from the fluid phase. It appears from these observa, tions tl'et cholesteryl acetete must be protected from the water environment in order

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to tmnain in solution. Th/s is coherent with a model in which cholesteryl acetate is by s monol~er of DMPC, while the bulk of DMPC remains in its vrdered AtIow D M I ~ ~ Y l acetate tool ratios (50 mol~ DMPC and below), a precipitnte f ~ in ;Klditioa tolhe supcmatant. The spectra from both the fluid and precipitate were nearly identical, Some iadication of the similarities is reflected in the inten-

'260 cipttate and supernatant of the same ~mp, Ostt~o~t.~Bothof th~spect~a |a fig.4 at~" Since these speCuaof supematantand p~ecipi~ m so . ~ , we ~ ~ ~ is little o~ no structural difference between them. We su88e~ f ~ / t h a t t h e ~ cipitate contains particles similar to fh~'~ deputed in fig. 5 ~ r ~ !ergeg~ancrtherefore too dense to remain in su~pe~on. ,'-

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Our testtlts indicate that at low concentrations, cholesteryl acetate disrupts the hy~bon ~haim o~DMPC by perietratingand remaining between the acyl chains of the ~ t ~ h o l i t t e bil~yet~(fig, Sb). As the concentration of cholesteryl acetate is ine~a~ed to the molar ratio DMPC : dtolesteryl acetate of ca, 6 : 1 (fig. 5c), the DMP~ bilayer begins to tetttm to the ordered gel state probably due to a separate mote stable ~ pha~ of ester forming between the DMPC bflayers (fig. 5d). The enw,lopi~ ~ of DMl~ may eventually become void of trapped cholesteryl aceta~ (fl$: 5,) ff the biltdi~ enexgy of the DMPC-cholesteryt aceta~ complex is much 10wet,than the binding energy between the ester molecules. This has been shown to be tree in related systems [ 1 ~]. .When the lipid bilayer is totally saturated with cholesteryl acetate (fig. 5¢) the system is at the boundary between two phase regions. At higher concentrations of chote~ter}4 acetate,the free energy of the cholestery! acetate, crysta_!~nephase becomes lower than-that of the cho!estery! ester*DMl?C complex. This is probably due to a combination ofeffec~ts iachuling the difference in binding energy between cholesteryl acetate molecules and the stable DMPC-acetate complex, the local enviromnental fffects of disrupted DMPC-hydrocarbon chains, and the difference in the entropy of both the ¢empkm and crystalline phases. As the more stable crystalline phase (fig. 5e) becomes larger, some o~ the aggregates probably become more dense and therefore form p~cA#tates. The precipitate .~nd supc~natent howeve~ arc ~tructurally s~m'lar as the tpect~a in fig. 4 indicate. Tt~ in~rpretation of our data is similar to that for the previously mentioned tel~ted systom {I ,2[. We have demonstrated here that not only is this stzuctural interpretation consistent with our results, but-our Ramim data directly defines the natu~ of the irrmtediate envirommnt amtmd the IIMPC molecules, Slxcif'tcaUy the ~yl chaim. .~-kNwJed~ments We thauk the No/~zw~ern Univ~n~ty Ma~rlah Resn~ch Cewter for the use of the l~,z~aa~ I ~ work w~s supportedin partby NIH g~am No. GM00874.

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