Effect of polyenephosphatidylcholine on cholesterol uptake by human high density lipoprotein

527

Atherosclerosis, 39 (1981) 527-542 Elsevier/North-Holland Scientific Publishers,

Ltd.

EFFECT OF POLYENEPHOSPHATIDYLCHOLINE ON CHOLESTEROL UPTAKE BY HUMAN HIGH DENSITY LIPOPROTEIN

0. ZIERENBERG

I, G. ASSMANN 2, G. SCHMITZ 2 and M. ROSSENEU 3

1 A. Nattermann & Cie., Chemische Forschung, Abteilung Radiochemie, D-5000 Cologne (F.R.G.); 2 Zentrallaboratorium der Universitiitskliniken, D-4400 Miinster (F.R.G.) and 3 Algemeen Ziekenhuis St. Jan, B-8000 Brugge (Belgium) (Received 17 July, 1980) (Revised, received 29 October, 1980) (Accepted 3 February, 1981)

Summary The lipid and protein composition of human HDL was changed by incorporation of polyenephosphatidykholine (PPC) into HDL in vitro. HDL with incorporated PPC (HDL-PPC) had a higher molar PC/apoprotein ratio than native HDL. PPC accounted for up to 50% of the PC fraction of HDL. The fluidity of HDL-PPC was higher than that of native HDL but lower than that of PPC liposomes. Zonal ultracentrifugation separated HDL-PPC into a major and a minor component, The AI/AI1 ratio of the major fraction was reduced compared with native HDL . The storage capacity of HDL-PPC and native HDL for cholesterol was studied by incubation of these fractions with [14C]cholesterol-LDL. Significantly more cholesterol (56%) was taken up by HDL-PPC from LDL than by native HDL. The transfer of cholesterol from LDL to HDL in human serum was studied by .an in vitro [ 14C]cholesterol distribution test. In this test the lipoproteins of serum were labelled with [‘4C]cholesterol. An analytical procedure was developed to quantify the transfer of cholesterol from LDL to HDL after addition of PC. The transfer depended on the fluidity and the dose of the PC fraction

Correspondence: Dr. 0. Zierenberg. A. Nattermann & Cie., Abteflun~ Radiochemie, Postfach 360120. D-5000 Cologne 30. Abbreviations: PPC = polyenephosphatidylcholine, PC = phorphatidylchoke, HDL - high density lipoprotein. LDL = low denattylipoprotein. VLDL - very low density lipoprotein, CE = cholesterol ester. DTNB = B,Sdithio-bis-2-nitrobenzoic acid, LCAT = lecithin-cholesterolacyltransferase,DPH = Mphenylhexatriene.

0 021-9150/81/0000-0000/$02.50

@ Elsevier/North-Holland

Scientific Publishers,

Ltd.

528

used as well as on the initial LDL + VLDL/HDL ratio and was independent LCAT activity. Key words:

of

Cholesterol storage capacity of HDL - HDL-PPC - Microviscosity - Polyenephosphatidylcholine (PPC) - Zonal ultracentrifigation

Introduction Epidemiological studies show that plasma levels of HDL are negatively correlated with the incidence of atherosclerotic cardiovascular disease, In addition, there is probably an inverse correlation between levels of HDL cholesterol and tissue cholesterol pools [ 11. It was shown in cell culture studies that HDL can remove cholesterol from cells [2]. Cholesterol in HDL is esterified by LCAT, an enzyme which converts cholesterol from the surface of the HDL particle into cholesterol ester which is deposited in the core of HDL [3]. This enzymatic mechanism creates a gradient for the transfer of membrane cholesterol to HDL. It was postulated recently that the fluidity of HDL particles and their cholesterol uptake are linked together [4]. Consequently, the capacity of HDL for cholesterol incorporation may be limited by its protein and lipid compositions. In the present in vitro study we investigated the changes in the lipid and protein compositions of HDL after incorporation of polyenephosphatidylcholine (PPC), and the effect on the fluidity of HDL. PPC is a highly unsaturated PC species from soya beans (mainly dilinoleoyl-PC) which, while it cannot be synthesized in mammalian cells, is incorporated predominantly into HDL after oral or intravenous administration as Zierenberg et al. reported recently [ 5,6]. In an attempt to investigate the hypothesis that the fluidity of HDL regulates its role as cholesterol acceptor, we also studied the cholesterol transfer between LDL and HDL, either isolated or present in human serum, together with the cholesterol transfer from LDL to HDL with incorporated PPC (HDLPPC) .

Methods (1) Materials and analytical

methods

Polyenephosphatidylcholine (PPC, Nattermann) is extracted from soya beans. It is esterified in the l- and 2-positions predominantly with linoleic acid. More than 50% of PPC consists of the di-linoleoyl-sn-glycerophosphocholine species. Saturated PC was obtained by catalytic (PtOz) hydrogenation of PPC in methanol. The fatty acid composition was as follows: 16 : 0 = 15.2%, 18 : 0 = 70.5%, 18 : lAgtrans = 6.4%, 18 : lAgcis = 4.7%, 18 : lA1lcis = 3.7%. Polyenephosphatidyl-N [C3H3]choline ([ 3H]PPC, specific activity 80 mCi/ mmol) and bis-[ l-14C]linoleyl-phosphocholine ([ 14C]PPC, specific activity 5 mCi/mmol) were synthesized according to established procedures [7] in our laboratory. [4-14C]cholesterol (specific activity > 50 mCi/mmol) was supplied by Amersham Buchler.

529

Human HDL was purified by sequential ultracentrifugation of human plasma (Medizinische Universititsklinik K61n) at densities 1.063 and 1.21, human LDL between densities 1.02 and 1.05 [S]. Both lipoprotein fractions gave a homogeneous band on agarose-gel electrophoresis after staining proteins as well as lipids. Agarose-gel electrophoresis was performed on ready-made plates using the Corning AC1 cassette electrophoresis cell (Palo Alto) with the constant power supply of 90 V. Electrophoresis was performed for 35 min at 25”C, with a 0.05 M barbital buffer, pH 8.6. Protein was determined according to Bradford [9] using the Bio Rad assay No. 5000001. Bovine serum albumin was used as a standard. The PC concentration was determined according to Rouser [lo]. Standards of PPC with known concentrations were included. The yield of the standard after scraping off the TLC plate and extraction was 60-80%. Radioactivity was measured with a Unisolve I scintillation cocktail (Zinsser) in a Packard liquid scintillation spectrometer 2425. Cholesterol was determined with Boehringer kit No. 124087 using the catalase method. Free cholesterol was measured with Boehringer kit No. 236691 using the CHOD/PAP method omitting cholesterol esterase. Electron-microscopic studies were performed on a Philips C 201 electron microscope after negative staining of the samples with 2% phosphotungstic acid. (2) In vitro incubation of human HDL with PPC [ 3H]PPC (2 /.&i/ml) was sonicated under nitrogen at a concentration of 25 mg PPC/ml 0.9% NaCl for 30 min at 4”C, 70 W. Ten mg human HDL were incubated with [‘H]PPC (l-20 mg) for 16 h at 37’C. The volume was adjusted to 2 ml with 0.9% NaCl. The incubation mixture was separated into HDL and non-reacted PC vesicles by KBr gradient ultracentrifugation according to Redgrave et al. [11] for 24 h, 286,000 Xg, 16°C. The gradients were fractionated into 1 ml fractions; protein concentration, radioactivity and density distribution were determined. The protein peak with density 1.15-1.08 was pooled, adjusted to d = 1.21 and subjected to a second centrifugation step using the same KBr gradient and centrifugal conditions as outlined above, in order to eliminate loosely attached vesicles from HDL. Protein concentration and incorporated radioactivity of the incubated HDL were determined. Subsequently an aliquot of the HDL was lyophilized and lipids were extracted with chloroform/methanol (2 : 1, v/v). The degree of extraction was controlled by determination of extracted radioactivity. The lipids were separated on thin-layer silica plates (Merck No. 11846) with the solvent system chloroform/methanol/water (65 : 25 : 4, v/v/v). The PC fraction and sometimes the lyso-PC fraction were scraped off. PC was eluted from the silica gel with chloroform/methanol/water (50 : 50 : 5, v/v/v). The specific activity of the PC fractions as well as the ratio pmol PC/mg HDL protein were calculated. The specific activities of the r3H]PPC liposomes used for incubation and of the non-reacted liposomes were determined after lyophilization and lipid extraction as outlined above, but without separation on TLC.

530

(3) Fluorescence polarization measurements The lipid phases of HDL, HDL-PPC and PPC liposomes were labelled with

1,6diphenyl-1,3,5-hexatriene (DPH) according to Inbar [ 121 and Rosseneu et al. [13]. The fluorescence polarization ratio (p) of DPH as a function of temperature was measured using an Elscint MV-1A microviscosimeter. This parameter is a measure of the fluidity of the lipid phase [13]. The molar ratio of DPH to lipids was in the range of l/500-1500 in all experiments, (4) Zonal ultracentrifugation HDL and PPC were incubated at a ratio 1 : 2 (w/w) as described above. The mixture was subsequently subjected to zonal ultracentrifugation in a Beckman L 8-80 ultracentrifuge with a Z 60 rotor at 260,000 X g for 15 h, 4°C. A nonlinear NaBr gradient d = 1.00-1.400 g/ml was used. The fractions were analyzed for density, absorbance and subjected to SDS-lo% acrylamide-gel electrophoresis. The electropherograms were scanned with a Beckman CDS-200 densitometer. (5) Transfer of [14C]cholesterol from LDL to HDL

One hundred mg LDL-cholesterol in 5 ml 0.9% NaCl, 0.01% NaNa, 0.01% EDTA, pH 7.4, were labelled with [4J4C]cholesterol (Amersham Buchler) by injecting 10 @i [i4C]cholesterol, dissolved in 30 ~1 ethanol, into the LDL solution. The mixture was incubated for 6 h at 37°C and thereafter dialyzed against the above-mentioned buffer (0.01% NaN3, 0.01% EDTA) for 4 days at 4°C with several renewals of buffer. The preparation gave a single band on agarose-gel electrophoresis after staining or after precipitation of the lipoprotein and counting of the radioactivity. HDL was incubated with PPC as described in section (1) with an HDL-protein/PPC ratio of 1 : 2 (w/w). The cholesterol/protein ratios of HDL-PPC and native HDL were not identical. 1.25 mg HDL-cholesterol (or HDL-PPC cholesterol) were incubated with 3.75 mg [14C]LDL cholesterol, adjusted to 2.5 ml with 0.9% NaCl, for 16 h at 37°C in a shaking water bath. The incubation mixture was adjusted to d = 1.150. A discontinuous gradient was prepared with the following KBr solutions: 1.0 ml of d 1.100, 4.0 ml of d 1.063, 4.5 ml of d 1.006. The gradients were centrifuged in a Beckman L 5-75 ultracentrifuge with an SW 41 rotor, 286,000 X g, 24 h, at 16°C. The gradients were fractionated; density, radioactivity, cholesterol and protein concentration in each fraction were determined. Only the 2 peak fractions (HDL, LDL) were further analyzed by calculating the specific activity of cholesterol as dpm/mM cholesterol or dpm/mg protein. The lipoproteins of these fractions (HDL or LDL) were not contaminated by each other as checked by agarose electrophoresis. Two different HDL and LDL preparations were used (experiments I and II). At least 5 incubations were performed with each HDL and LDL preparation. (6) [‘4C]cholesterol distribution test One ml human serum was incubated with 1.5 I.tCi [ 14C]cholesterol for 2 h

531

according to Kupke [ 14 1. The serum was centrifuged for 3 min in a Beckman microfuge. The supernatant was subsequently divided. 0.5 ml served as a blank, and to the other 0.5 ml, 0.05 mg PC (PPC, egg-PC, saturated PC) was added in a liposomal form (1 mg/ml 0.9% NaCI). The mixture was incubated for 4-16 h at 37°C in a shaking water bath. The incubation mixtures and the blanks were diluted 1 : 1 with 0.9% agarose. Thirty ~1 of the diluted mixtures was separated by agarose-gel electrophoresis according to Kupke et al. [ 151. A lipoprotein separation film, type P-7R (Folex GmbH, Dreieich) was used. Electrophoresis was performed for 90 min, 4”C, at constant 150 V. The lipoproteins were precipitated by a solution of dextran sulfate (6 g/l) and CaCIZ (0.33 mol/l). Gel fractions (5 mm) were cut off, digested with 1 ml HCl at 60°C overnight, cooled down and counted with 10 ml of an Unisolve I scintillation cocktail. In some experiments heat-inactivated serum was used for incubation. For this purpose the serum was preincubated at 56°C for 30 min. The precipitates were spun down at 3,000 X g for 10 min at 4°C. In other experiments 1 ml serum was supplemented with 200 ~1 Ellman’s reagent (5,5dithio-bis-2nitrobenzoic acid, 1.4 mM, dissolved in 0.2 ml phosphate buffer, pH 7.1) according to Stokke et al. [ 161. (7) Statistical analysis Statistical analysis was performed using Student’s+test. Results (1) Incorporation of PPC into human HDL

PPC was taken up by human HDL after incubation at 37°C for 16 h. After separation of the incubation mixture by KBr gradient ultracentrifugation (Fig. 1) most of the PPC from the incubation mixture (l-20 mg PPC) was incorpo-

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A

1:

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2.0

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1.0

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1

I

fraction’’1 nr.

I

5

*ts 10

I*

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s

% __N$#.& 1

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Fig. 1. Separation of HDL and PPC mixtures after incubation for 16 h at 37’C, by KBr+radient ultracenMfwation. A: 10 mg HDL + 1 mg C3HlPPC, first gradient: B: 10 mg HDL + 20 mg 13HlPPC, tirst gradient; C: Recentrifugation of HDL peak (fractions 4-6 of Fig. 1B). a, cpm/lOO M; ?? , d20; a, mg protein.

532

Fig. 2, Electron microscopic picture after negative staining of A: PPC liposomes; B: native HDL; C: HDLPPC (10 mg HDL were incubated with 20 mg PPC; HDL-PPC was purified by 2 KBr-gradient ultracentrifugetions). The bar indicates 100 nm.

rated into HDL. Part of the HDL protein was solubilized by unbound PPC and floated with non-reacted vesicles (Fig. 1, fraction 8). The HDL-peak shifted to lower density with increasing PPC concentration in the incubation mixture. After incubation of 10 mg HDL with 1 mg PPC, HDL reached its peak at d = 1.16 g/ml and with 20 mg PPC at d = 1.08 g/ml. The HDL-PPC peak was recentifuged. The homogenicity of the HDL-PPC fraction after two gradient ultracentrifugations was demonstrated by electron microscopy (Fig, 2C). No liposomes (Fig. 2A) could be detected in the HDLPPC preparation (Fig. 2C). HDL-PPC particles had a diameter of 9.7 f 1.5 nm

dpm/mM

PC

X109 A

Fig, 3. Incubation HDL-PC.

of 10 mg HDL with increasing amounts of PPC. Specific activity (dpm/mM PC) of

533 TABLE 1 INCUBATION

OF HDL WITH PPC: ANALYSIS

OF HDL-PPC AND PPC LIPOSOMES

Mean of 2 experiments with 3 replicates each. 1

2

3

4

5

6

Incubation medium

Spec. act. HDL-PC [dpm 3H/mM1 (X109)

Spec. act. vesicles [dpm 3H/mMl

Mel total PC/m01 HDL protein

Mel [3HJPPC/ mol HDL protein

Mol PC/mol HDL protein (incubated) +

(mf3 PPC)

(X109)

mol[3HlPPC/mol HDL protein (column 5)

1 2 5 7 10 20 Incubated

1.1 2.2 4.5 4.3 6.3 7.5

4.2 6.0 8.2 8.1 9.0 10.4

53 57 68 96 59 76

13.5

13.5

49

4 9 23 36 28 37

53 58 72 85 77 86

whereas native HDL formed 8.1+ 1.3 nm particles. Some bigger aggregates could be seen in HDL-PPC as well as in native HDL preparations. A detailed analysis of the HDL-PC fractions is shown in Fig. 3 and Table 1. The specific activity of HDL-PC rose with increasing PPC in the incubation medium. Up to a concentration of 5 mg PPC in the incubation mixture the increase in specific activity of HDL-PC was proportional to the increase in PPC concentration in the incubation mixture (Fig, 3), at higher PPC concentrations the uptake into HDL was disproportional and exceeded a maximum, Up to 37 mol PPC were incorporated into HDL per mol protein (Table 1, column 5); in addition, the molar ratio of total PC to protein increased to a maximum of 76 in comparison with normal HDL (49 mol PC/mol protein - ; Table 1, column 4). Consequently, a maximum enrichment of the HDL-PC fraction by PPC was obtained with approximately 50% PPC. The total phospholipid concentration in HDL was calculated by arithmetic addition of the native HDL-PC (49 mol/mol prot&n) plus the incorporated PPC as well as by phosphorus determination (Table 1, column 4). The arithmetically calculated total phospholipid concentration (Table 1, column 6) exceeded the measured concentration (Table 1, column 4). This indicates that PPC increased the PC concentration in HDL and partially exchanged with the native PC. This conclusion was further supported by the specific activity of the nonreacted PC vesicles (Table 1, column 3). During incubation the specific activity of administered PPC was reduced by liberated HDL phospholipids, i.e. a phospholipid transfer from HDL to liposomes took place. This was more pronounced with low liposomal concentrations (l-7 mg) than with higher ones (10-20 mg). (2) Fluorescence polarization measurements The Buidity of a lipid phase is inversely proportional to the ffuorescence polarization ratio of DPH [13 3. The fluorescence polarization ratio is plotted

534 P

Fig. 4. Fluorescence depolarization measurements of human HDL, HDL-PPC and PPC liposomes after incorporation of DPH as a marker. p = fluorescence polarization ratio of DPH. 1

AE _

Tube Number Fig. 6. Zonal ultracentrifugation of 100 mg HDL aud 200 mg PPC liposomes after incubation for 16 h, 3’I’C. E = absorbance at 280 nm; gradient, d: 1.0-1.4 g/ml: fraction I. non-reacted liposomes; fractions II-IV, HDL-PPC: major fraction; fractious V-VIII, HDL-PPC: miuor fraction. Insert: SDS-10% acrylamide gel electzophoresis; protein pattern of the different fractions and of HDL and VLDL atendards. The ape-AI/&I ratios obtained by densitometric analydn of the gels are In fraction II: 0.91; III: 1.09; V: 2.8; VI: 6.9; VII: 5.9: VIII: 13.1; HDL: 2.6.

535

as a function of temperature in Fig. 4. The fluidity of HDL-PPC particles was higher than that of native HDL but lower than the fluidity of PPC liposomes. The incorporation of PPC into HDL therefore reduced the microviscosity of the HDL lipid phase. (3) Zonal ultracentrifugation HDL and PPC mixtures were subjected to zonal ultracentrifugation. The gradient profile is shown in Fig. 5. A small portion of PPC liposomes from the incubation mixtures (identified by a separate run with PPC liposomes) was not incorporated into the HDL particle (fraction I). HDL-PPC split into a major (fractions II-IV) and a minor peak (fractions V-VIII). Densitometric analysis of the gels showed that fractions II-IV had an apo-AI/AI1 ratio of approximately 1 whereas fractions V-VIII had an apo-AI/AI1 ratio of 2.8-13.9 and were enriched with C-apoproteins and apo-AIII. Native HDL had an apo-AI/AI1 ratio of 2.5. (4) Transport of cholesterol from LDL to HDL LDL was labelled with [ 14C]cholesterol. [ 14C]LDL, HDL-PPC and native HDL were incubated with a ratio of cholesterol (LDL) to cholesterol (HDL) of 3 : 1, resembling physiological conditions of human plasma (approx. 120 mg/dl LDL cholesterol, 40 mg/dl HDL cholesterol). After incubation of (14C]LDL with HDL or HDL-PPC and separation of both lipoproteins by KBr-gradient ultracentrifugation, part of the LDL cholesterol had shifted to HDL. When [14C]LDL was centrifuged without HDL no radioactivity was measured in the density range 1.07-1.20 g/ml, Analysis of the peak fractions (HDL or LDL) of the gradients is presented in Tables 2 and 3. HDL shifted to lower density (Table 2) after incorporation of PPC, which confirmed the results of section (1). The specific activity of HDL cholesterol (dpm/mM total cholesterol) was significantly higher in HDL-PPC than in native’HDL. Using the specific activity of [ 14C]LDL cholesterol, we calculated that in experiments I and II 283 and 397 nmol cholesterol, respectively., TABLE 2 HDL ANALYSIS:

[14C]LDL/HDL

incubation; [I4 C]LDL/HDGPPC

INCUBATION

Means of incubatione with SEM are given.

Number of incubations d2’ (s/ml) Spec. act. (dpm/mM

chol.)

nM chol. traneferred/mM HDL-choL

nM

chol. trenefemedlmg HDL protein

Experiment I

Experiment II

5

7

HDL HDL-PPC

1.109 * 0.002 1.083 * 0.004

1.108 1.087

i 0.09 f 0.010

HDL HDGPPC

2.74 4.59

1.04 1.16

f 0.420 f 0.361

HDL HDL-PPC A%

P

169 283 67
267 397 49
HDL HDL-PPC A%

132 194 47

230 246 7

f 0.718 * 0.922

X 10’ X 10’

X lo6 X 10’

536 TABLE 3 LDL ANALYSIS:

[14Cl LDL/HDL INCUBATION:

[14Cl LDL/HDL-PPC INCUBATION

Mean of incubations with SEM is given. [14C1LDL incubated with

Experiment I

Experiment II

5

1

dpmlmg protein

HDL HDL-PPC

2.28 * 0.145 x 105 1.86 i 0.183 X 10s

4.76 f 0.929 X lo4 3.72 f 0.462 X lo4

MMchol./mg protein

HDL HDL-PPC

1.41 * 0.09 1.15 f 0.116

1.63 1.21

18
21 n.s.

1.60 X 103 1.76 X 10’3 1.62 X 106

n.d. n.d. 2.92 x 107

Number of incubations

A%

P Spec. act. (dpm/mM chol.)

HDL HDL-PPC LDL incubated

were taken up per nmol HDL-PPC cholesterol. In experiments I and II HDLPPC consequently accepted more cholesterol from LDL (67% and 49%, respectively) than native HDL. Similar data was obtained by expressing the cholesterol transfer as nmol cholesterol/mg HDL protein. An uptake of cholesterol by HDL-PPC or HDL is likely to result in a cholesterol depletion of LDL. Due to the different cholesterol affinities there will obviously be differences in cholesterol depletion of LDL after HDL-PPC and HDL incubation, This is shown in Table 3. After incubation of [14C]LDL with HDLPPC, LDL had lost significantly more cholesterol/mg protein than after incubation with native HDL. On the other hand, the specific activity of LDL cholesterol did not differ significantly between the pre- and post-incubation periods, i.e., there was no redistribution between the unlabelled HDL cholesterol pool and the labelled LDL cholesterol pool. (5)

[‘4C]Chole8terol

After incubation NaCl

distribution

test

of [14C]PPC with serum (1 mg PPC/ml serum) about 70% SerlJ,Tl

Fig. 6. Distribution of radioactivity between lipoproteins after separation of human serum, incubated with [14C]PPC, by agaroseael electrophoreais and precipitation of lipoproteins (right). Distribution of radioactivity after separation of PPC Iiposomes by agarose electrophoresis (left).

537

1%- dpm

of total“k-dpl

n-IllI1 30

***

20

10

PPC

control

PPC

Fig. 7. [14CICholesterol distribution test. Conditions: 2nd incubation: 16 h. 37’C: n = 7. *** P < 0.001.

c

PF

-

1 mg PPC/ml serum; 1st incubation:

2 h. 37’C;

of PPC was incorporated into HDL and approximately 30% into VLDL and LDL (Fig. 6, right). This observation confirms our results [5,6] obtained after incubation of human serum with PPC and separation of the incubation mixture by ultracentrifugation. On the other hand, [ 14C]PPC liposomes do not migrate in the electric field (Fig. 6, left), enabling the separation of lipoproteins and unbound PPC liposomes by agarose electrophoresis. An example of the analysis is presented in Fig. 7. After incubation of serum with PPC (1 mg/ml serum) the [ 14C]cholesterol concentration in HDL was

Fig. 8. [14C]Cholesterol distribution test. Correlation of LDL + VLDL/HDL incubation and cholesterol uptake by HDL. (Same conditions as in Fig. 7.)

ratio from serum before

538 ~ALDL+vL~L)

%K~HDL

[‘“cl

total14C-dpm

HDL

Of

I

I

10

10

5

5

con1rol

control

PPC

Fig. 9. [ 14ClCholesterol

PPC

distribution test with Tangier serum. (Same conditions as in Fig. 7.)

increased in comparison with the control whereas [14C]cholesterol in LDL and VLDL. was decreased, resulting in a significant decrease in the ratio of [14C]LDL + VLDL/[ 14C]HDL. The increase in HDL cholesterol after incubation of serum with PPC was positively correlated with the initial LDL + VLDL/HDL ratio (Fig. 8). The linear regression line is best described by the equation y = 0.15 + 3.96x with a correlation coefficient of 0.95. Serum without HDL had to be used to check the possibility that other acceptors for cholesterol which migrate with HDL on agarose electrophoresis, such as albumin, were activated by PPC or its metabolites to bind more cholesterol. Therefore serum obtained from a patient (J.S.) with Tangier disease [17], an autosomal recessive disorder characterized by a total deficiency of native

p”&LDL c’“C]

3

+VLDL)

cl”4 total

HDL

HDL

of

“C-dpm

f

2

1

contml

hydr-

egg-

PC

PC

PPC

control

hydr-

egg

PC

PC

PPC

Fig. 10. [14C]Cholesterol distribution test. Cholesterol uptake by HDL af%er incubation of serum with PPC, egg-PC, hydrogenated PC. (Same conditions as in Fig. 7.)

539

I

015

I!0

115

i.0mg

Fe/mlserum

Fig. 11. [14C]Choleeterol distribution test. Effect of PPC concentration in the incubation medium on LDL + VLDL/HDL ratio. let incubation: 2 h. 370% 2nd incubation: 16 h. 37’C.

HDL, was incubated with PPC. After agarose-gel electrophoresis no redistzibution of cholesterol from LDL and VLDL to other protein fractions could be observed (Fig. 9). Consequently, albumin or other serum constituents do not account for the cholesterol shift measured in the cholesterol distribution test. The cholesterol uptake by HDL was investigated with respect to (a) the fluidity of different PC species, (b) the second incubation time, (c) the PPC concentration used, (d) the LCAT mechanism. (a) The results plotted on Fig. 10 suggest that the cholesterol uptake capacity of HDL was limited by the composition of the HDL lipid phase, After incubation of serum with PPC more cholesterol was shifted from LDL and VLDL to HDL than after incubation with more saturated PC species. The uptake of cholesterol by HDL decreased in the order PPC > egg-PC > saturated PC (Fig. lob). As a result of the cholesterol uptake by HDL the ratio of LDL + VLDL/HDL decreased with increasing unsaturation of the PC species (Fig. 1Oa). (b) The effect which the duration of incubation with PPC had on serum TABLE 4 [14ClCHOLESTEROL

-DISTRIBUTION

TEST

Effect of time of second incubation.

Assay

j”Cl

4h

(LDL + VLDL) [‘4C1HDL 6h

Control

1.73

1.96

+ PPC

1.27

1.34

A%

26

31

16 h 1.38 1.30 31

540 TABLE 5 [14ClCHOLESTEROL

-

DISTRIBUTION

TEST

Effect of enzymes.

Assay

[14CI(LDL + VLDL) [14ClHDL Control

+ PPC

A%

Serum + DTNB

1.82 1.73

1.38 0.92

24 41

Serum heat-inactivated

3.50 3.11

1.97 1.73

44 44

labelled with [14C]cholesterol was investigated (Table 4). No difference was found between 16 and 6 h and only a slight difference between incubation times of 4 and 6 h. (c) There was a dosedependent shift of cholesterol from LDL and VLDL to HDL. The maximum was obtained with approximately 1 mg PPC/ml serum. Thus the ratio of VLDL + LDL/HDL reached its minimum level at this PPC concentration (Fig. 11). (d) Finally it was investigated whether serum enzymes might be limiting factors for cholesterol uptake in HDL. Heat-inactivated serum was employed to eliminate enzymatic activities. No significant difference in the lipoprotein cholesterol ratio was found between normal and heat-inactivated serum. In particular, no effect of LCAT on the cholesterol uptake into HDL was observed by selective inhibition of its enzymatic activity with DTNB (Table 5). Discussion The interaction of PPC liposomes with human HDL at 37°C led to a net transfer of PPC molecules to HDL. The resultant HDL-PPC particles had an increased phospholipid/protein ratio, a decreased apo-AI/AI1 ratio and an increased diameter compared with native HDL. By means of these experimental data we can speculate on the possible mechanisms of PPC transfer to HDL. Interaction of PPC liposomes with HDL resulted in a loss of apo-AI from HDL because we measured a decreased apo-AI/AI1 ratio in HDL-PPC. The displaced apo-AI had probably been incorporated into the unbound PPC liposomes (see Fig. 1A). A similar apo-AI transfer from HDL to liposomes was observed by other authors [ 181 who investigated the transfer of egg-PC from liposomes to HDL. The apo-AldePleted HDL may be unstable. These particles therefore fuse to larger ones with an increased surface coat to core ratio, as was shown by Tall et al. [l] for a dimiristoyl-PC/HDL system. In consequence the phospholipid/protein ratio must increase after fusion. Indeed we measured an increased phospholipid/protein ratio and diameter of HDL-PPC. Our data are also consistent with a displacement of apo-AI/phospholipid complexes from HDL with a substitution for PPC and/or fusing of the destabilized particles. This interpretation is based on the analysis of the specific activity of the unbound liposomal fraction (see Table 1): the specific activity of the

541

unbound PC liposomes was reduced by native HDL phospholipids. Another possibility is that either the native HDL or the fused HDL particles exchanged part of their phospholipids for liposomal PPC. As a result of either one of these mechanisms the HDL-PC fraction was enriched up to 50% with PPC. The change in the PC and protein compositions might account for the measured increase in the fluidity of HDL-PPC in comparison with native HDL. The in-vitro incubation of HDL with PPC may resemble the physiological mechanisms of PC transfer to HDL in vivo. HDL was the main acceptor for i.v. injected PC liposomes which was shown for some animal species [5,19,20]. Also, during chylomicron clearance, phospholipid/apo-AI complexes are liberated, which are integrated into HDL to build up particles with an increased surface to volume ratio. This was shown, for example, by Redgrave et al. [21] who found phospholipid-rich HDLz particles after injection of chylomicrons into rats. The hypothesis was in agreement with the findings after oral administration of PPC to rats [ 221 and man [ 231: PPC was absorbed into serum chylomicrons and was measured in the HDL fraction after clearance of the chylomicrons [ 51. These examples demonstrate that the lipid and protein compositions of HDL are partly dependent on dietary regimens or can be influenced by iv. infusion of PC. The cholesterol concentration of HDL in plasma is regulated by its fractional catabolic rate as well as by the synthetic rate of HDL. A third parameter probably involved in changing the cholesterol acceptor function of HDL is the composition of the circulating HDL, that is to say, its lipid/protein ratio and the fluidity of its phospholipid species. This possibility was ruled out in the second part of our investigations. As a model we studied the transfer of cholesterol from LDL to HDL as well as to HDL-PPC in vitro. We found that, calculated per mole total HDL cholesterol, HDL-PPC took up approximately 50% more cholesterol from LDL than native HDL. The results accord with those of Adams et al. [24] who reported a faster cholesterol crystal dissolution after incubation with a mixture of HDL and PPC compared with native HDL. We interpret these results as an enhanced incorporation of cholesterol into HDL-PPC particles. In order to study the cholesterol transfer from LDL to HDL under treatment with PPC or more saturated PC species in human serum we developed the [ 14C]cholesterol distribution test. In this test the cholesterol transfer from LDL to HDL depended, apart from other factors, on the fluidity of the PC species used: transfer was optimal for PPC whereas cholesterol transfer decreased with increasing fatty acid saturation of the PC-species. The transfer was not dependent on the LCAT activity. Therefore the surface properties of HDL were the limiting factor for cholesterol uptake and not the ability of LCAT to clear the surface from cholesterol. In conclusion, our experiments support the hypothesis that the HDL cholesterol pool in plasma is not regulated solely by synthesis and catabolism of HDL. The phospholipid/protein ratio of circulating HDL and the degree of unsaturation of its phospholipids are probably also major factors in this process.

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Acknowledgement The authors thank Professor H.E. SchPfer (Cologne) for performing the electron-microscopic studies. References 1 Tall, A.R. and Small, D.M.. Body cholesterol removal - Role of plasma HDL. Adv. Lip. Res., 17 (1980) 2. 2 Stein, 0.. Vanderhoek, J. and Stein, Y., Cholesterol content and sterol synthesis in human skin fibroblasts and rat aortic smooth muscle cells exposed to lipoprotein-depleted serum and HDL/phospholipid mixtures, Biochim. Biophys. Acta. 431(1976) 347. 3 Stoffel, W., Zierenberg, O., Tunggal. B. and Schreiber, E.. 13C-NMR-spectroscopic evidence for hydrophobic lipid-protein interactions in human HDL, Proc. Nat. Acad. Sci. (USA), 71 (1974) 36963100. 4 Rosseneu, M., Declerq, B.. Vandamme. D.. Vercaemst. R., Soetewey. F., Peeters, Ii. and Blaton. V., Influence of oral polyunsaturated and satura?d phospholipid treatment on the lipid composition and fatty add profile of chimpanzee lipoproteins, Atherosclerosis. 32 (1979) 141. 5 Zierenberg. 0.. Odenthal, J. and Betzing, H., Incorporation of PPC into serum lipoproteins after oral or i.v. administration, Atherosclerosis. 34 (1979) 269. 6 Zierenberg, 0. and Betzfng, H., Lipoprotein pattern in animals after application of labelled PPC. In: L.A. Carlson (Ed.), International Conference on Atherosclerosis, Raven Press, New York, 1978, D. 531. I Zierenberg. 0. and Betzing, H., Phannacokinetics and metabolism of i.m. injected PPC liposomes, Drug Eiesearch, 29(1979)494. 8 Scanu, A.M. and Wisdon. C., Serum lipoproteins - Structure and function, Ann. Rev. Biochem.. 41 (1972) 704. 9 Bradford, M.M.. Protein way by dye binding, Anal. Biochem.. 72 (1976) 248. 10 Rouser, G., Flebcher. S. and Yamamoto, A., Twodimensional thin-layer chromatographic separation of polar lipids and determtnation of phospholipids by phosphorus analysisof spots, Lipids, 5 (1970) 494. 11 Redgrave, T.G., Roberts, D.C.K. and West, C.E., Separation of plasma lipoproteins by den&y-gradient ultracentrifugation. Anal. Biochem., 66 (1975) 42. 12 Shinitzky, M. and Inbar. M., Microviscosity parameters and protein mobility in biological membranes, Biochim. Biophyd. Acta, 433 (1976) 133. 13 Rosseneu. M.. Soetewey. P., Vercaemst. R.. Lievens. M.-J. and Peeters, H. In: H. Peeters (Ed.), Protides of the Biological Fluids, Pergamon. New York, 1978. p. 47. 14 Kupke. J.R., New Principle for the separation of plasma lipoprotein lipids without ultracentifugation, J. Chromatogr., 162 (1979) 414. 16 Kupke, J.R., Zeugner, S. and Gottschalk. A., Comparison of two micromethods for determination of lipoprotein cholesterol in plasma, Clin. Chem.. 26 (1979) 1795. 16 Stokke, K.T. and Norum, K.R., Determination of LCAT in human plasma, Stand. J. Clin. Lab. Invest., 27 (1971) 21. 17 Assmann, G., Tangier-Krankheit. In: G. Schettler et al. (Eds.), Handbuch der inneren Medizin. Vol. VII, Part 4, Spriqger-Verlag.. Berlin, 19’76, D. 461. 18 Chobanian, J.V., TaU, A.R. and Brecher. P.J.. Interaction between unilamellar egg yolk ledthln vesicles and human HDL, Biochemistry, 18 (1979) 180. 19 Kmpp, L., Chobanian. A.V. and Brecher, P.J.. The in-viva degradation of phoapholipid vesicles to a particle resembling HDL in the rat. Biochem. Biophys. Res. Comm.. 72 (1976) 1261. 20 Scherphof, 0.. Roerdink, F., Waite, M. and Parks, J.. Disintegration of phosphatidylcholine liposomes in plasma as a result of interaction with HDL, Biochim. Biophvs. Acta, 642 (1978) 296. 21 Redgrave. T.G. and Small, D.M., Quantitation of the transfer of surface phospholtpid of chylomiu’ons to the HDL fraction during the catabolism of chylomicrons in the rat, J. Clin. Invest., 64 (1979) 162. 22 Lekim, D. and Betzing, H.. Intestinal absorption of PPC in the rat, Hoppe-Seyler’s Z. Physiol. Chem., 367 (1976) 1321. 23 Beil, F.U. and Gnmdy. S.M., Studies on plasma lipoproteinsduring absorption of exogenous lecithin in man, J. Lipid Res.. 21 (1980) 626. 24 Adams, C.W.M. and Abdulla, Y.H., The action of human HDL on cholesterol crystals, Atherosclerosis, 31 (1978) 466.