Chemistry and Physics of Lipids 24 (1979) 45-55
© Elsevier/North-Holland Scientific Publishers Ltd.
CHOLESTERYL-PHOSPHORYL-CHOLINE IN LIPID BILAYERS
M. LYTE and M. SHINITZKY Department of Membrane Research, The Weizmann Institute of Science, Rehovot (Israel)
Received July 3rd, 1978
accepted December 5th, 1978
Cholesteryl-phosphoryl-choline (CPC), a hybrid between cholesterol and lecithin, is incorporated into sonicated liposomes and erythrocyte membranes similarly to cholesterol. The effect of CPC on lipid microviscosity and degree of order is smaller, but not significantly, than that of cholesterol. It is proposed that CPC may be employed as an efficient modulator of lipid dynamics.
I. Introduction Cholesterol, an integral constituent of most mammalian membranes, is distributed to varying degrees in the lipid bilayer. Its predominant function lies in increasing the rigidity and simultaneously increasing the degree of order in fluid lipid domains. Consequently, the dynamic properties of the lipid bilayer are directly related to the cholesterol/phospholipid mol ratio (C/PL). The effect of cholesterol is mediated primarily by its planar backbone, though a series of specific structural moieties optimize its effect. The most prominent are the/3-orientation of the OH at C-3 and the branched aliphatic chain at C-17 [1-71. One of the critical sites which determine the specific effect of cholesterol is the C-3 /3-OH group, Oxidation to ketone [ 1 - 3 ] , and even replacement with an a - O H group (epicholesterol), reduces markedly the specific effect o f cholesterol as determined by transport, osmotic fragility and lipid microviscosity [2,3]. These observations demonstrate that the OH group and its specific/3-orientation are critical for a proper interaction with the surrounding phospholipids [ 4 - 6 ] . This property is further demonstrated by the fact that only limited amounts of the common cholesterol esters can be incorporated into lipid bilayers [8]. However, charged esters, such as cholesterol hemisuccinate, may be incorporated into the lipid bilayer like a surfactant and not as specifically as cholesterol (Wilbrandt et al., unpublished results). Abbreviations: ANS, 1,8-analinonaphthalene sulphonate; cmc, critical micelle concentration;
CPC, cholesteryl-phosphoryl-choline; C/PL, cholesterol/phospholipid mol ratio; DPH, 1,6diphenyl hexatriene; HBS, HEPES-buffered saline; PC, phosphatidyl choline; PS, phosphatidyl serine. 45
46
M. Lyre and M. Shinitzky, Cholesteryl-phosphoryl-choline in lipid bilayers
The loss of the optimizing effect of the B-OH group upon esterification can in principle be regained by acylation with a hydrophilic group, which in itself is an integral part of the membrane. Such a group is phosphoryl-choline, which is the head group of lecithin and sphingomyelin, which together account for over 60% of the membrane phospholipids. It may be expected that the resulting special ester, cholesteryl-phosphoryl-choline (CPC), could exert similar effects on lipid dynamics when compared to cholesterol. In this study we have examined the incorporation of CPC into the lipid bilayer, and its effect on lipid fluidiy parameters. It is shown that CPC increases both the rigidity and degree of order in the lipid bilayer, however to a lesser extent than cholesterol.
c.3" t-£¢12
-o o-!-o-c..-c.,-..,-c.3 0
CH3
Scheme 1
11. Materials and methods CPC was synthesized and purified according to Hirt and Berchtold [9]. Thin layer chromatography on alumina-coated plates (Riedel - De Haen, AG), using chloroform/ methanol/0.01 M aqueous NaOH (65 : 25 : 4) as an effluent, gave a major band (> 98%) at R F = 0.45 and a minor trailing band (< 2%). A sample of CPC, which was kindly donated by Dr. Berchtold, showed similar chemical and chromatographical purity. 14C-Labelled CPC ([f4C] CPC), at the choline head group, was prepared by reacting [14C]methly iodide (Radiochemical Centre, Amersham, 500/~Ci) with N,N-dimethyl choles teryl-phosphoryl-ethanolamine [9] in ethanol, in the presence of excess sodium carbonate. The [14C]CPC was diluted with unlabelled CPC and kept as a stock solution of a specific activity of 320 000 cpm//ag CPC. Cholesterol of high purity was obtained from Sigma. Phosphatidylcholine (PC) and phosphatidylserine (PS), both from egg yolk and of grade 1, were purchased from Lipid Products, United Kingdom. All systems studied employed HEPES-buffered saline (HBS) as an isotonic medium (20 mM HEPES + 140 mM NaC1, pH 7.4). For the preparation of sonicated liposomes the proper volumes of organic solutions of the components were first mixed together and then evaporated under argon to complete dryness. HBS was added and the liposomes were prepared by ultrasonic irradiation at 100 W for 3 0 - 6 0 min, with a Branson sonifier (Model No. 136), under argon atmosphere at 4°C. The sonicated mixtures were centrifuged at 40 000 g for 15 min, and the upper and lower layers were discarded. Liposomes, prepared under such conditions, were shown to consist of over 91~o single-walled vesicles [10]. The vesicles were not purified any further. No detectable degradation of CPC was observed in the sonicated mixtures.
M. Lyre and M. Shinitzky, Cholesteryl-phosphoryl-choline in lipid bilayers
47
Membranes of human red blood ceils were prepared from freshly-drawn blood by the method of Dodge et al. [11]. Phospholipid and cholesterol analyses were performed on the final preparations. Free cholesterol was analyzed according to the method of Chiamori and Henri [ 12]. Phospholipids were determined by the HC104 modification [ 13] of Bartlett's method [14]. Lipid microviscosity, ~, in both liposomes and membranes was determined by fluorescence depolarization of 1,6-diphenyl hexatriene (DPH). The degree of fluorescence polarization and the excited state lifetime (determined by pulse excitation and deconvolution analysis) were used for the calculation o f ~ , as described previously (refs. 15 and 16; for a review see ref. 17). The dependence on temperature o f ~ was presented as log ~ versus l i t according to equation I: f? = A e x p ( A E / R t )
(I)
where AE is the flow activation energy [16,17]. The critical micelle concentration (cmc) of CPC in water was measured by the degree of fluorescence polarization of 1,8-anilino naphthalene sulfonate (ANS, 2 X 10 - s M) excited at 365 nm [18]. For osmotic fragility experiments with CPC, an ethanolic solution of CPC (10 mg/ml) was first evaporated to a thin layer in a beaker, and heat-inactivated human serum in HBS was added for reconstitution. The mixture was stirred at 56°C for up to 20 min, at which point the CPC had completely dissolved. To 5 ml of mixtures containing increasing amounts of CPC and constant amounts of serum, 20 ~1 of washed human red blood ceils were added and incubated at 25°C for 10 min. After centrifugation, the light absorption of the released hemoglobin in the supernatant was read at 540 nm and the optical density reading was converted to % hemolysis.
III. Results Sonicated liposomes, made of 1 : 9 PS/PC (mol/mol), and increasing amounts of either cholesterol or CPC of up to 1.5 sterol to phospholipid mol ratio were used as model systems to compare the effects of CPC and cholesterol on lipid fluidity parameters. The fluorescence polarization and intensity of DPH-labelled liposomes of final phospholipid concentration of 0.2 mg/ml were then measured simultaneously, and the apparent lipid microviscosity, ~, was evaluated (see equation I and ref. 17). Figures 1 and 2 present the results obtained with cholesterol and CPC-containing liposomes, respectively. As shown, both cholesterol and CPC increase ~ to a similar extent. In Fig. 3 a direct comparison between CPC and cholesterol is illustrated. Both sterols progessively increase ~, though cholesterol is approx. 20% more effective than CPC. The flow activation energy, AE, a parameter which inversely correlates with the degree of order in the system [ 16,17], is also reduced by both sterols; however, cholesterol is more effective than CPC (see Fig. 4). By increasing the sterol to phospholipid mol ratio from 0 to 1.3, cholesterol reduces the AE from 12 to 7.5 kcal/mol whereas CPC reduces it to 8.8 kcal/mol.
48
M. Lyre and M. Shinitzky, Cholesteryl-phosphoryl-choline in lipid bilayers
20.0
--
IO.O
O Q.
5.0 o
o o
1.0
0.5
3.20
5:50 3.40 350 I/T( x 103,*K"1 ) Fig. I. Temperature prof'de of lipid microviscosity in single-walled liposomes, made of 1 : 9 PS/PC (mol/mol) of the total concentration of 0.2 mg/ml and different amounts of cholesterol. Cholesterol/phospholipid mol ratios are: 0 (o o); 0.22 (o 4 ) ; 0.43 (X ×);0.69(o o);0.92 (• zx); 1.12 (o o);and 1.50 (+ +).
Preliminary observations have indicated that CPC, at concentrations above 50/lg/ml ir~ the presence o f 10% serum in HBS, hemolyses human red blood cells. This may be expected from the amphipatic structure of CPC which can act as a mild detergent similar to bile acids. The micelles formed by CPC in aqueous solution could be expected to have an intermediate nature between micelles o f lysolecithin and bile acids. The pattern of micelle formation by CPC in water, as reflected by the increase in fluorescence polarization of ANS, is shown in Fig. 5. The gradual increase in Ill/I.L , rather than a sigmoid curve (see legend to Fig. 5), can be accounted for by incorporation o f ANS into increasingly larger sized aggregations. The transition point observed at ~80/~g/ml, which may be taken as the cmc, is probably between metastable oligomers and thermodynamically stable micelles. Figure 6 presents osmotic fragility profdes o f human red blood cells (4/ll/ml) in the presence o f increasing concentrations of CPC and constant amounts o f serum. In Fig. 7 the osmotic fragility prof'de o f CPC, in the absence o f serum, is shown. As expected, the presence of serum reduces the hemolytic activity of CPC presumably by increasing partitioning into the serum lipoproteins. Thus, an increase in serum
M. L yte and M. Shinitzky, CholesteryLphosphoryl-choline in lipid bilayers
49
20.0 I0.0
5.0
0 U
U
1.0
05
320
3.30 3.40 I/T(xlO3,OK -')
3.50
Fig. 2. Temperature profile of lipid microviscosity in single-wailed liposomes, made of I : 9 PS/PC (mol/mol) of the total concentration of 0.2 mg/ml and different amounts of CPC. CPC/phospholipid mol ratios are: 0 ( 0); 0.14 ( e e); 0.25 (X ×); 0.41 (o -u);0.47 (A ~);0.60(o 0);0.72(= m);0.87 (+ +);and 1.28 O' *)I
I
60
I
/ ~
5.0 eA
.-~ 4.0 0 >" 30 I~
2.0
I.O. I
l
I
0.5 1.0 1.5 Sterol / phospholipid (mole/mole) Fig. 3. Increase in lipid microviscosity at 35°C with cholesterol/phospholipid (e CPC/phospholipid (× ×), as obtained from Figs. 1 and 2.
e) and
M. Lyre and M. Shinitzky, Cholesteryl-phosphoryl-choline in lipid bilayers
50 15.0
I
[
i
-~ IO.C
v t,i
© Elsevier/North-Holland Scientific Publishers Ltd.
CHOLESTERYL-PHOSPHORYL-CHOLINE IN LIPID BILAYERS
M. LYTE and M. SHINITZKY Department of Membrane Research, The Weizmann Institute of Science, Rehovot (Israel)
Received July 3rd, 1978
accepted December 5th, 1978
Cholesteryl-phosphoryl-choline (CPC), a hybrid between cholesterol and lecithin, is incorporated into sonicated liposomes and erythrocyte membranes similarly to cholesterol. The effect of CPC on lipid microviscosity and degree of order is smaller, but not significantly, than that of cholesterol. It is proposed that CPC may be employed as an efficient modulator of lipid dynamics.
I. Introduction Cholesterol, an integral constituent of most mammalian membranes, is distributed to varying degrees in the lipid bilayer. Its predominant function lies in increasing the rigidity and simultaneously increasing the degree of order in fluid lipid domains. Consequently, the dynamic properties of the lipid bilayer are directly related to the cholesterol/phospholipid mol ratio (C/PL). The effect of cholesterol is mediated primarily by its planar backbone, though a series of specific structural moieties optimize its effect. The most prominent are the/3-orientation of the OH at C-3 and the branched aliphatic chain at C-17 [1-71. One of the critical sites which determine the specific effect of cholesterol is the C-3 /3-OH group, Oxidation to ketone [ 1 - 3 ] , and even replacement with an a - O H group (epicholesterol), reduces markedly the specific effect o f cholesterol as determined by transport, osmotic fragility and lipid microviscosity [2,3]. These observations demonstrate that the OH group and its specific/3-orientation are critical for a proper interaction with the surrounding phospholipids [ 4 - 6 ] . This property is further demonstrated by the fact that only limited amounts of the common cholesterol esters can be incorporated into lipid bilayers [8]. However, charged esters, such as cholesterol hemisuccinate, may be incorporated into the lipid bilayer like a surfactant and not as specifically as cholesterol (Wilbrandt et al., unpublished results). Abbreviations: ANS, 1,8-analinonaphthalene sulphonate; cmc, critical micelle concentration;
CPC, cholesteryl-phosphoryl-choline; C/PL, cholesterol/phospholipid mol ratio; DPH, 1,6diphenyl hexatriene; HBS, HEPES-buffered saline; PC, phosphatidyl choline; PS, phosphatidyl serine. 45
46
M. Lyre and M. Shinitzky, Cholesteryl-phosphoryl-choline in lipid bilayers
The loss of the optimizing effect of the B-OH group upon esterification can in principle be regained by acylation with a hydrophilic group, which in itself is an integral part of the membrane. Such a group is phosphoryl-choline, which is the head group of lecithin and sphingomyelin, which together account for over 60% of the membrane phospholipids. It may be expected that the resulting special ester, cholesteryl-phosphoryl-choline (CPC), could exert similar effects on lipid dynamics when compared to cholesterol. In this study we have examined the incorporation of CPC into the lipid bilayer, and its effect on lipid fluidiy parameters. It is shown that CPC increases both the rigidity and degree of order in the lipid bilayer, however to a lesser extent than cholesterol.
c.3" t-£¢12
-o o-!-o-c..-c.,-..,-c.3 0
CH3
Scheme 1
11. Materials and methods CPC was synthesized and purified according to Hirt and Berchtold [9]. Thin layer chromatography on alumina-coated plates (Riedel - De Haen, AG), using chloroform/ methanol/0.01 M aqueous NaOH (65 : 25 : 4) as an effluent, gave a major band (> 98%) at R F = 0.45 and a minor trailing band (< 2%). A sample of CPC, which was kindly donated by Dr. Berchtold, showed similar chemical and chromatographical purity. 14C-Labelled CPC ([f4C] CPC), at the choline head group, was prepared by reacting [14C]methly iodide (Radiochemical Centre, Amersham, 500/~Ci) with N,N-dimethyl choles teryl-phosphoryl-ethanolamine [9] in ethanol, in the presence of excess sodium carbonate. The [14C]CPC was diluted with unlabelled CPC and kept as a stock solution of a specific activity of 320 000 cpm//ag CPC. Cholesterol of high purity was obtained from Sigma. Phosphatidylcholine (PC) and phosphatidylserine (PS), both from egg yolk and of grade 1, were purchased from Lipid Products, United Kingdom. All systems studied employed HEPES-buffered saline (HBS) as an isotonic medium (20 mM HEPES + 140 mM NaC1, pH 7.4). For the preparation of sonicated liposomes the proper volumes of organic solutions of the components were first mixed together and then evaporated under argon to complete dryness. HBS was added and the liposomes were prepared by ultrasonic irradiation at 100 W for 3 0 - 6 0 min, with a Branson sonifier (Model No. 136), under argon atmosphere at 4°C. The sonicated mixtures were centrifuged at 40 000 g for 15 min, and the upper and lower layers were discarded. Liposomes, prepared under such conditions, were shown to consist of over 91~o single-walled vesicles [10]. The vesicles were not purified any further. No detectable degradation of CPC was observed in the sonicated mixtures.
M. Lyre and M. Shinitzky, Cholesteryl-phosphoryl-choline in lipid bilayers
47
Membranes of human red blood ceils were prepared from freshly-drawn blood by the method of Dodge et al. [11]. Phospholipid and cholesterol analyses were performed on the final preparations. Free cholesterol was analyzed according to the method of Chiamori and Henri [ 12]. Phospholipids were determined by the HC104 modification [ 13] of Bartlett's method [14]. Lipid microviscosity, ~, in both liposomes and membranes was determined by fluorescence depolarization of 1,6-diphenyl hexatriene (DPH). The degree of fluorescence polarization and the excited state lifetime (determined by pulse excitation and deconvolution analysis) were used for the calculation o f ~ , as described previously (refs. 15 and 16; for a review see ref. 17). The dependence on temperature o f ~ was presented as log ~ versus l i t according to equation I: f? = A e x p ( A E / R t )
(I)
where AE is the flow activation energy [16,17]. The critical micelle concentration (cmc) of CPC in water was measured by the degree of fluorescence polarization of 1,8-anilino naphthalene sulfonate (ANS, 2 X 10 - s M) excited at 365 nm [18]. For osmotic fragility experiments with CPC, an ethanolic solution of CPC (10 mg/ml) was first evaporated to a thin layer in a beaker, and heat-inactivated human serum in HBS was added for reconstitution. The mixture was stirred at 56°C for up to 20 min, at which point the CPC had completely dissolved. To 5 ml of mixtures containing increasing amounts of CPC and constant amounts of serum, 20 ~1 of washed human red blood ceils were added and incubated at 25°C for 10 min. After centrifugation, the light absorption of the released hemoglobin in the supernatant was read at 540 nm and the optical density reading was converted to % hemolysis.
III. Results Sonicated liposomes, made of 1 : 9 PS/PC (mol/mol), and increasing amounts of either cholesterol or CPC of up to 1.5 sterol to phospholipid mol ratio were used as model systems to compare the effects of CPC and cholesterol on lipid fluidity parameters. The fluorescence polarization and intensity of DPH-labelled liposomes of final phospholipid concentration of 0.2 mg/ml were then measured simultaneously, and the apparent lipid microviscosity, ~, was evaluated (see equation I and ref. 17). Figures 1 and 2 present the results obtained with cholesterol and CPC-containing liposomes, respectively. As shown, both cholesterol and CPC increase ~ to a similar extent. In Fig. 3 a direct comparison between CPC and cholesterol is illustrated. Both sterols progessively increase ~, though cholesterol is approx. 20% more effective than CPC. The flow activation energy, AE, a parameter which inversely correlates with the degree of order in the system [ 16,17], is also reduced by both sterols; however, cholesterol is more effective than CPC (see Fig. 4). By increasing the sterol to phospholipid mol ratio from 0 to 1.3, cholesterol reduces the AE from 12 to 7.5 kcal/mol whereas CPC reduces it to 8.8 kcal/mol.
48
M. Lyre and M. Shinitzky, Cholesteryl-phosphoryl-choline in lipid bilayers
20.0
--
IO.O
O Q.
5.0 o
o o
1.0
0.5
3.20
5:50 3.40 350 I/T( x 103,*K"1 ) Fig. I. Temperature prof'de of lipid microviscosity in single-walled liposomes, made of 1 : 9 PS/PC (mol/mol) of the total concentration of 0.2 mg/ml and different amounts of cholesterol. Cholesterol/phospholipid mol ratios are: 0 (o o); 0.22 (o 4 ) ; 0.43 (X ×);0.69(o o);0.92 (• zx); 1.12 (o o);and 1.50 (+ +).
Preliminary observations have indicated that CPC, at concentrations above 50/lg/ml ir~ the presence o f 10% serum in HBS, hemolyses human red blood cells. This may be expected from the amphipatic structure of CPC which can act as a mild detergent similar to bile acids. The micelles formed by CPC in aqueous solution could be expected to have an intermediate nature between micelles o f lysolecithin and bile acids. The pattern of micelle formation by CPC in water, as reflected by the increase in fluorescence polarization of ANS, is shown in Fig. 5. The gradual increase in Ill/I.L , rather than a sigmoid curve (see legend to Fig. 5), can be accounted for by incorporation o f ANS into increasingly larger sized aggregations. The transition point observed at ~80/~g/ml, which may be taken as the cmc, is probably between metastable oligomers and thermodynamically stable micelles. Figure 6 presents osmotic fragility profdes o f human red blood cells (4/ll/ml) in the presence o f increasing concentrations of CPC and constant amounts o f serum. In Fig. 7 the osmotic fragility prof'de o f CPC, in the absence o f serum, is shown. As expected, the presence of serum reduces the hemolytic activity of CPC presumably by increasing partitioning into the serum lipoproteins. Thus, an increase in serum
M. L yte and M. Shinitzky, CholesteryLphosphoryl-choline in lipid bilayers
49
20.0 I0.0
5.0
0 U
U
1.0
05
320
3.30 3.40 I/T(xlO3,OK -')
3.50
Fig. 2. Temperature profile of lipid microviscosity in single-wailed liposomes, made of I : 9 PS/PC (mol/mol) of the total concentration of 0.2 mg/ml and different amounts of CPC. CPC/phospholipid mol ratios are: 0 ( 0); 0.14 ( e e); 0.25 (X ×); 0.41 (o -u);0.47 (A ~);0.60(o 0);0.72(= m);0.87 (+ +);and 1.28 O' *)I
I
60
I
/ ~
5.0 eA
.-~ 4.0 0 >" 30 I~
2.0
I.O. I
l
I
0.5 1.0 1.5 Sterol / phospholipid (mole/mole) Fig. 3. Increase in lipid microviscosity at 35°C with cholesterol/phospholipid (e CPC/phospholipid (× ×), as obtained from Figs. 1 and 2.
e) and
M. Lyre and M. Shinitzky, Cholesteryl-phosphoryl-choline in lipid bilayers
50 15.0
I
[
i
-~ IO.C
v t,i
