Chemistry and Physws of Ltptds 14 (1975) 309-312 © North-Holland Pubhshlng Company SHORT COMMUNICATION
THE SIMILARITY BETWEEN MESOMORPHIC DROPLETS IN A T H E R O S C L E R O T I C L E S I O N S AND CHOLESTERYL ESTER SUSPENSIONS Recewed February 4, 1975,
acceptedMarch 20, 1975
A great accumulation of hplds IS the most striking feature of the atherosclerotlc lesions In the early lesion llplds accumulate mainly in the form of lntracellular droplets [ 1] These occur in mLxtures of two kinds, amsotroplc (mesomorphlc) and lSOtropic (true liquid) [2] The anlsotroplc droplets (spherulltes) contain a polarization cross with arms parallel to the planes of polarLzatlon and a posltwe sign of blrefrlngence indicating a smectlc mesophase [3] The spherulates have proved to be very rich In cholesteryl esters [4,5] The cholesteryl esters are nonpolar hplds which are insoluble m water and do not spread at air-water interfaces Therefore their behavlour in water environment has attracted very little attention However, in the light of the existence of droplets composed mainly of cholesteryl esters m the basically aqueous milieu of the arteries, model systems with cholesteryl esters in water would be of Interest In the present work the formation of stable water suspensions of cholesteryl esters in mesomorphlc state is reported and the similarity of these to spheruhtes in early atherosclerotlc lesions in pointed out The cholesteryl esters used for the preparation of suspensions (stearate, oleate, and hnoleate) were proved to be chromatographically pure by TLC analyses The water used was quartz-distilled Suspensions of the cholesteryl esters were prepared by vigorous shaking of cholesteryl ester-water mixtures (0 1 - 0 5%) at a temperature above the melting point of the cholesteryl ester The suspensions were then held at room temperature and studied at different times by polarizing microscopy Material for the hlstological study was obtained from atheromatous vessels of human autopsy and surgical cases The tissue was embedded in polyethylene glycol or frozen and then sectioned During the polarizing microscopic examination the sections were kept at 37°C by a hot stage Spheruhtes were isolated in the following way the atherosclerotlc tissue was &ssected and minced in water and the droplets then collected by centrifugation as a layer on the top of the homogenate Freeze-etching electron microscopy was carried out with an apparatus manufactured by Balzers Drops of cholesteryl ester and spherullte suspensions were placed on copper discs and rapidly frozen In hquid freon and nitrogen
310
Short communlcatton
fqg 1 Spheruhtes m section of aorta with early atherosclerotlc lesion Polarized hght Ftrst-order red compensator >(400
Ftg 2 Globules in a cholesteryl ester suspension Polarized hght Ftrst-order red compensator ×400
Short commumcatton
311
Fig 3 Freeze-etchingelectron microscopicpicture of a globule m a cholesteryl ester suspension The larnellar fracture ISclearly seen × 50,000
Examined m polartzlng microscope sections of arteries with early atherosclerotlc lesions showed spheruhtes with a diameter of about 1/a By use of a first-order red compensator the sign of blrefrmgence was found to be posltwe (fig 1) Studied m the same way suspensions of cholesteryl ester droplets showed the same polarizing microscopic pattern (fig 2) Tins pattern re&cares a smectlc mesophase m whach the molecules are arranged m layers In agreement w~th th~s conception both spheruhtes and cholesteryl ester globules showed a lamellar structure at freeze-etching electron microscopic examinations (fig 3) In bulk systems long chain cholesteryl esters on coohng from lsotroplc melt exhibit both cholestenc and smectlc mesophases In the suspended state the cholesteryl esters stud~ed showed only the smectlc mesophase One outstandmg feature of the smectlc cholesteryl ester suspensions is their high stability also at temperatures far below that of the smect~c to crystalhne trans~tlon This property to be supercooled is also vahd m bulk systems especially for esters w~th unsaturated fatty acids but It Is much enhanced in suspensions However, from the thermodynamic pomt of view the crystalhne state is the most stable one The smectlc globules thus have to be regarded as being in a metastable state In accordance to this crystaUtzatlon gradually takes place mainly at the air-water interface Within the suspension the larger globules crystallize at first whde the smaller globules are very stable (fig 4) Dke m bulk systems the mesophase of the unsaturated esters (oleate and hnoleate) was more stable than that of stearate
312
Short communwatton
Fxg 4 Photomicrograph showing large globules m the stage of crystalhzatlon and smaller intact globules Polarized hght ×400
There is a growing interest m using model systems to elucidate the physlcochemlcal bas~s of the deposition of llplds in atherosclerotlc vessels [6,7] The present results showing globules in cholesteryl ester suspensions with the same morphology as the spherulltes m early atherosclerotlc lesions are obviously slgmficant when discusstag atherogenesls starting from model systems
Research Institute, .~bo A kademt Foundatton
References [1] 12] [3] [4] [5] [6] [71
J W Bahs, M D Haust and R H More, Exptl Mol Pathol 3 (1964) 511 RD Weller, J Path Bact 94(1967) 171 GT Stewart, J Path Bact 81 (1961) 385 P D Langand W Insull, J Chn Invest 49 (1970)1479 B Lundberg, Acta Acad Aboensls, Ser B34 (4) (1974) D M Smalland G G Shlpley, Science 185 (1974) 222 B Lundberg, Academic Dissertation, Abo Akademl 1974
B LUNDBERG
THE SIMILARITY BETWEEN MESOMORPHIC DROPLETS IN A T H E R O S C L E R O T I C L E S I O N S AND CHOLESTERYL ESTER SUSPENSIONS Recewed February 4, 1975,
acceptedMarch 20, 1975
A great accumulation of hplds IS the most striking feature of the atherosclerotlc lesions In the early lesion llplds accumulate mainly in the form of lntracellular droplets [ 1] These occur in mLxtures of two kinds, amsotroplc (mesomorphlc) and lSOtropic (true liquid) [2] The anlsotroplc droplets (spherulltes) contain a polarization cross with arms parallel to the planes of polarLzatlon and a posltwe sign of blrefrlngence indicating a smectlc mesophase [3] The spherulates have proved to be very rich In cholesteryl esters [4,5] The cholesteryl esters are nonpolar hplds which are insoluble m water and do not spread at air-water interfaces Therefore their behavlour in water environment has attracted very little attention However, in the light of the existence of droplets composed mainly of cholesteryl esters m the basically aqueous milieu of the arteries, model systems with cholesteryl esters in water would be of Interest In the present work the formation of stable water suspensions of cholesteryl esters in mesomorphlc state is reported and the similarity of these to spheruhtes in early atherosclerotlc lesions in pointed out The cholesteryl esters used for the preparation of suspensions (stearate, oleate, and hnoleate) were proved to be chromatographically pure by TLC analyses The water used was quartz-distilled Suspensions of the cholesteryl esters were prepared by vigorous shaking of cholesteryl ester-water mixtures (0 1 - 0 5%) at a temperature above the melting point of the cholesteryl ester The suspensions were then held at room temperature and studied at different times by polarizing microscopy Material for the hlstological study was obtained from atheromatous vessels of human autopsy and surgical cases The tissue was embedded in polyethylene glycol or frozen and then sectioned During the polarizing microscopic examination the sections were kept at 37°C by a hot stage Spheruhtes were isolated in the following way the atherosclerotlc tissue was &ssected and minced in water and the droplets then collected by centrifugation as a layer on the top of the homogenate Freeze-etching electron microscopy was carried out with an apparatus manufactured by Balzers Drops of cholesteryl ester and spherullte suspensions were placed on copper discs and rapidly frozen In hquid freon and nitrogen
310
Short communlcatton
fqg 1 Spheruhtes m section of aorta with early atherosclerotlc lesion Polarized hght Ftrst-order red compensator >(400
Ftg 2 Globules in a cholesteryl ester suspension Polarized hght Ftrst-order red compensator ×400
Short commumcatton
311
Fig 3 Freeze-etchingelectron microscopicpicture of a globule m a cholesteryl ester suspension The larnellar fracture ISclearly seen × 50,000
Examined m polartzlng microscope sections of arteries with early atherosclerotlc lesions showed spheruhtes with a diameter of about 1/a By use of a first-order red compensator the sign of blrefrmgence was found to be posltwe (fig 1) Studied m the same way suspensions of cholesteryl ester droplets showed the same polarizing microscopic pattern (fig 2) Tins pattern re&cares a smectlc mesophase m whach the molecules are arranged m layers In agreement w~th th~s conception both spheruhtes and cholesteryl ester globules showed a lamellar structure at freeze-etching electron microscopic examinations (fig 3) In bulk systems long chain cholesteryl esters on coohng from lsotroplc melt exhibit both cholestenc and smectlc mesophases In the suspended state the cholesteryl esters stud~ed showed only the smectlc mesophase One outstandmg feature of the smectlc cholesteryl ester suspensions is their high stability also at temperatures far below that of the smect~c to crystalhne trans~tlon This property to be supercooled is also vahd m bulk systems especially for esters w~th unsaturated fatty acids but It Is much enhanced in suspensions However, from the thermodynamic pomt of view the crystalhne state is the most stable one The smectlc globules thus have to be regarded as being in a metastable state In accordance to this crystaUtzatlon gradually takes place mainly at the air-water interface Within the suspension the larger globules crystallize at first whde the smaller globules are very stable (fig 4) Dke m bulk systems the mesophase of the unsaturated esters (oleate and hnoleate) was more stable than that of stearate
312
Short communwatton
Fxg 4 Photomicrograph showing large globules m the stage of crystalhzatlon and smaller intact globules Polarized hght ×400
There is a growing interest m using model systems to elucidate the physlcochemlcal bas~s of the deposition of llplds in atherosclerotlc vessels [6,7] The present results showing globules in cholesteryl ester suspensions with the same morphology as the spherulltes m early atherosclerotlc lesions are obviously slgmficant when discusstag atherogenesls starting from model systems
Research Institute, .~bo A kademt Foundatton
References [1] 12] [3] [4] [5] [6] [71
J W Bahs, M D Haust and R H More, Exptl Mol Pathol 3 (1964) 511 RD Weller, J Path Bact 94(1967) 171 GT Stewart, J Path Bact 81 (1961) 385 P D Langand W Insull, J Chn Invest 49 (1970)1479 B Lundberg, Acta Acad Aboensls, Ser B34 (4) (1974) D M Smalland G G Shlpley, Science 185 (1974) 222 B Lundberg, Academic Dissertation, Abo Akademl 1974
B LUNDBERG
