Exchange of retinyl and cholesteryl esters between lipoproteins of rabbit plasma

88

Biochimica et Biophysics Actrr, 7 12 (1982) 88-93 Elsevier Biomedical Press

BBA 51131

EXCHANGE OF RETINYL AND CHOLESTERYL OF RABBIT PLASMA DONALD

B. ZILVERSMIT,

RICHARD

E. MORTON,

Divisron of Nutritional Scrences and Section Unmersity, Ithaca, NY 14853 (U.S.A.) (Received

December

ESTERS BETWEEN

L. BARRY

of Biochemistry,

HUGHES

Molecular

LIPOPROTEINS

and KATHRYN

and Ceil Bmlogy,

HAEFLEIN

Divisron

THOMPSON

of Biologicul

Sciences,

* Cornell

30th, 1981)

Key words: Retinyl ester; Cholesterol ester; Lipid exchange;

Transfer protein; Chylomicron

Normal or hypercholesterolemic rabbit plasma stimulates the transfer of retinyl ester as well as cholesteryl ester from rabbit lymph chylomicrons, chylomicron remnants or from cholesteryl ester-rich plasma VLDL to the d > 1.019 lipoprotein fractions. The presence of p-chloromercuriphenylsulfonate does not inhibit the transfer of these esters. Partially purified lipid transfer protein from rabbit or from human plasma also accelerates the transfer of the above esters. Whereas the rabbit plasma transfer protein preferentially accelerates the transfer of retinyl ester, the human plasma transfer protein appears to have a somewhat greater stimulating effect on the transfer of cholesteryl ester from low- to high-density lipoproteins.

Introduction

rabbit fed [ “C]cholesterol, [ 3H]retinyl acetate and oil, were incubated for 60 min with normal or with hypercholesterolemic rabbit plasma and the extent of labeled lipid transfer to the S, < 20 fraction was determined. Under these conditions, the percent transfer of labeled retinyl ester was less than onefourth that of labeled cholesteryl ester. The present investigation extends this observation over longer periods of time and with different lipoprotein fractions.

It has been reported previously that a cholesteryl ester exchange or transfer protein is found in the d > 1.21 fraction of rabbit plasma [ 1,2]. Similar transfer activity was observed in human plasma [3-81, but not in plasma of several other mammalian species [2]. Apparently, the acceleration of lipid transfer is not specific for cholesteryl ester, since triacylglycerol transfer also appears to be accelerated by the same, or a similar, factor [9,10]. It has not been established that other, relatively nonpolar, components of lipoproteins are subject to exchange. In a previous publication from this laboratory the exchangeability of retinyl esters in chylomicrons was compared to that for cholesteryl esters [ll]. Lymph chylomicrons, obtained from a

* Present address: University of New England, College Osteopathic Medicine, Biddeford, ME 04005, U.S.A.

of

Abbreviations: VLDL. very low density 1.006); PCMPS, p-chloromercuriphenylsulfonate.

<

OOOS-2760/82/0000-00000/$02.75

lipoprotein(s)(d

0 1982 Elsevier Biomedical

Materials and Methods

Donor particle preparation Chylomicrons. The thoracic duct of a female New Zealand White rabbit (Dutchland Laboratories, Denver PA) fed Purina Laboratory Rabbit Chow was cannulated. After recovery from surgery, the animal was infused through a duodenal cannula with a 50 mM cholesterol emulsion containing 19.35 g of cholesterol, 45 g of Wesson Oil (Hunt-Wesson, Fullerton CA), 36 g of lecithin, 3 g Press

89

of taurocholate, 20 g of albumin and 9 g of NaCl per 1. After absorption was established (as evidenced by milky lymph), 96 PCi of [4-‘4C]cholesterol from Amersham (Arlington Heights, IL) and 406 p.Ci of [3H]retinyl acetate (a gift from Hoffman La Roche, Nutley, NJ), solubilized in ethanol and mixed with 5 ml of the emulsion, were injected into the duodenal cannula over a period of 3 min. The infusion of emulsion was continued at a rate of 3 ml/h throughout the collection period. Lymph was collected in the presence of 0.2 ml of 0.4 M EDTA per 5 ml of lymph. Labeled chylomicrons were isolated by ultracentrifuging lymph in a SW 27 rotor at 4°C for 1.5 h (8.67 . lo6 X g/mm). The chylomicrons were removed from the top of the tube, resuspended in 0.15 M NaCI, and recentrifuged as before. Chylomicron remnants. Remnants were prepared from the chylomicrons by incubation with postheparin rabbit plasma. Postheparin blood was collected 4-5 min after injection of 100 I.U. heparin/kg into a male rabbit maintained on Purina Laboratory Rabbit Chow. Plasma was prepared and frozen until use. Chylomicrons, containing 4.5 mg of cholesterol, were incubated with 5 ml of post-heparin plasma and 5 ml of 50% albumin (fatty acid-free, Pentex, Miles Laboratories, Elkhart, IN) in a 0.2 M TrisHCl buffer,, pH 8.0, for 210 min at 37°C in 1 X 3.5 inch polyallomer centrifuge tubes. More than 50% of the chylomicron triacylglycerol was hydrolyzed. At the end of the incubation the suspension was overlayered with 0.15 M NaCl and centrifuged as described for chylomicrons. The top 5 mm of the tube was sliced to collect large remnants. The infranatant was centrifuged in a 60 Ti rotor at 60000 rpm for 17 h (2.61 . lo* X g/min) at 4°C. The tubes were sliced and the top fractions were collected. These fractions contained the small remnants and possibly a small amount (less than 7.5%) of VLDL derived from the post-heparin normal rabbit plasma. Other plasma lipoproteins. Labeled plasma was collected from two 3 - 4 kg female New Zealand White rabbits that had been fed daily 1OOg of Purina Laboratory Chow supplemented with 0.5 g of cholesterol and 2.5 g of Wesson Oil for 4 days. Shortly after the fourth meal, the rabbits were intubated with a mixture containing 96 PCi of

[‘4C]cholesterol dissolved in ethanol and 5 ml of the same 50 mM cholesterol emulsion used before. The rabbits were fed the 0.5% cholesterol diet again 15 h after the intubation. 18 h after the [‘4C]cholesterol dose the animals were dosed with 5 ml of the emulsion containing 406 PCi of [3H]retinyl acetate dissolved in ethanol. 5 h later, the rabbits were exsanguinated. The blood was collected in tubes containing 0.01 ml of 0.4 M EDTA/ml blood as the anticoagulant. Plasma was centrifuged for 23 h at 4°C in a (Beckman) 60 Ti rotor (3.53. lo8 X g/min). The VLDL was removed by slicing and washed by resuspending in d = 1.006 and centrifuging as before. Intermediate density lipoproteins were removed by ultracentrifugation under similar conditions at a density of 1.019. They were discarded, after which low density lipoprotein was prepared by centrifugation under similar conditions at a density of 1.063. The average specific activity of retinyl ester in the various lipoprotein fractions was 84 2 12 (S.D.) cpm/pmol, whereas that of cholesteryl ester was 11 * 1.4 cpm/nmol in chylomicrons, large and small remnants, and 22.4 in plasma VLDL. Retinyl- and cholesteryl-ester transfer All labeled donor particles were filtered through 0.45 pm Millipore filters (Millipore Corporation, Bedford MA) and stored at 4°C until use. The exchange of labeled lipids from chylomicrons, remnants and VLDL was measured by incubating each of these substrates with normal or hypercholesterolemic plasma for 0, 2 or 6 h at 37°C (details in Table I). 2 mM PCMPS, a lecithin: cholesterol acyltransferase inhibitor [ 121, was added to some incubations. After the incubations, 2 mM PCMPS was added to all samples and lipoprotein fractions were separated by consecutive ultracentrifugation at d = 1.006, 1.O19 and 1.063 in a Beckman 40.3 rotor for 24 h at 4°C for 1.65 . 10’ X g/mm. The recovery of labeled retinol and cholesterol in the lipoprotein fractions was 93 -C 6%. In one experiment the simultaneous transfer of retinyl ester and cholesteryl ester between in vivo labeled rabbit low density lipoprotein and bovine high density lipoprotein (d = 1.08-1.21) was determined in the presence and absence of partially purified lipid transfer protein. This lipid transfer

90

protein fraction was prepared from lipoprotein-deficient plasma obtained by dextran sulfate/MnCl, precipitation as described previously [4]. Purification of the lipid transfer protein in both rabbit and human lipoprotein-deficient plasma was performed by sequential chromatography on phenyl Sepharose and CM 52 cellulose (Whatman) [ 131. Lipid transfer was assayed as previously described [4], except that the assay volume was increased 4-fold and the donor and acceptor lipoprotein concentrations were 400 pg total cholesterol each. Lipid analysis All lipoprotein fractions and substrates were extracted by partitioning into hexane from 43% ethanol in water [ 141. Butylated hydroxytoluene (50 pg/ml) was added to the ethanol as an antioxidant. Aliquots of the hexane phase were separated into free and esterified cholesterol by thin-layer chromatography on pre-coated silica gel type 60 TLC plates (Merck, E.M., Darmstadt, F.R.G.) in 60 : 40 : 1 hexane/diethyl ether/acetic acid (v/v). The triacylglycerol and free and esterified cholesterol were eluted from the silica gel with chloroform/methanol, 9 : 1 (v/v). After removal of the solvent, the samples were saponified [15] and extracted with hexane, and aliquots were taken for liquid scintillation counting and cholesterol determination [ 161. Triacylglycerol in chylomicrons and chylomicron remnants was determined by the method of Sardesai and Manning [ 171. Protein was determined by the method of Lowry et al. [ 181. Another aliquot of the initial hexane phase was dried under N, and the residue redissolved in 0.1-0.3 ml of absolute ethanol. Retinol and retinyl ester fractions in the ethanol solution were separated by HPLC on a Waters Bondapak C-18 column [19] with absolute ethanol as the eluting solvent. Retinol mass was determined from the absorbance at 340 nm and was quantitated by comparison with retinol and retinyl palmitate standards (Eastman). The retinol and retinyl ester peaks were collected separately from the column. They were dried under N, and radioactivity was determined in a toluene scintillator. Recovery of injected label was over 95%.

Results During the course of several experiments in which labeled retinol was fed to rabbits or in which retinol-labeled chylomicrons were injected into hypercholesterolemic rabbits, significant amounts of labeled retinyl ester were found in low density lipoprotein and high density lipoprotein fractions. In order to investigate the possibility that retinyl ester might exchange between lipoprotein fractions, VLDL and low density lipoprotein from a hypercholesterolemic rabbit, which had been fed [ ‘HIretiny acetate and [ l4 Clcholesterol previously, were added to separate flasks containing unlabeled hypercholesterolemic plasma. In a 6 h incubation at 37°C and after re-isolation of the lipoprotein fractions, VLDL had lost approximately 9% of its labeled retinyl ester and 11% of its labeled cholesteryl ester. These preliminary results led us to investigate the relative exchangeability of retinyl and cholesteryl esters from several lipoprotein fractions which might resemble the intermediate lipoprotein fractions in chylomicron degradation. In one experiment the in vitro transfer of retinyl ester is compared to that of cholesteryl ester in normal and in hypercholesterolemic rabbit plasma. The latter was obtained from a rabbit fed 0.5% cholesterol, 2.7% oil added to Purina Laboratory Rabbit Chow (Ralston Purina, St. Louis, MO) (100 g/day for 4 days). Four types of labelled donor particles were used: chylomicrons. large and small remnants, and hypercholesterolemic plasma very low density lipoprotein. Incubations were arranged to measure the loss of radioactive labels from the donor particles at 2 and 6 h. Table I shows the results of incubating retinyl ester- and cholesteryl ester-labeled chylomicrons, chylomicron remnants and hypercholesterolemic rabbit VLDL with either normal or with hypercholesterolemic rabbit plasma. As observed previously [ll], the labeled retinyl ester from chylomicrons was transferred to the d 1 1.019 lipoproteins more slowly than the chylomicron cholesteryl ester. In 6 h 13% of radioactive retinyl ester had been lost from chylomicrons to normal d > 1.019 plasma lipoproteins, compared to a loss of 30% for cholesteryl ester. Similar results were observed for the incubation of the same chylomicrons with hy-

_.._,I

I

OF LABELED

RETINYL-

AND

CHOLESTERYL-ESTERS

FROM

LOWER

DENSITY

LIPOPROTEINS

TO d > 1.019 LIPOPROTEINS

14 7

--PI-

a 56-~6’% of [ ‘HIretiny b 90%4’% of [3H]retinyl

$

20 32

rabbit

0 18 30

--_

ester and 46~4% ester and 8314%

in hypercholesterolemic

plasma

Incubated

rabbit 0 7 13

in normal

RE

CE

lipoprotein

Chylomicron

Donor

Incubated 0 2” 6”

(h)

Time

23 16

__~,

of [ “C]cholesteryl of [‘4C]cholesteryl

plasma

3 17 29

RE

_

CE

25 14

4 15 24

ester in the d > 1.019 fraction ester in the d > 1.019 fraction

21 12

0 11 23

RE

CE

was present was present

16 8

1 7 15

in LDL. in LDL.

20 10

2 9 22

RE

~ PCMPS

Small

Large

Plasma

Remnants

VLDL

11 4

0 4 11

CE

_

2 7 21

RE

+ PCMPS

_ _

0 2 10

CE

Normal rabbit plasma and plasma from a rabbit fed 0.5 g of cholesterol/day for 4 days contained 71.4 and 447 mg of cholesterol/d1 plasma. 4 ml of each plasma was incubated at 37°C with chylomicrons, remnants or VLDL containing 0.58 mg total cholesterol and 9.5-16.8 nCi of [ “C]choIesterol and 21-58 nCi of [‘Hlretinol. Some incubations also contained 2 mM PCMPS which inhibits 1ecithin:cholesterol acyltransferase. Values arc % transfer. RE, retinyl ester; CE. cholesteryl ester.

TRANSFER

TABLE

92

percholesterolemic plasma. For small chylomicron remnants (Table I), prepared in vitro with post-heparin plasma, the loss of labeled retinyl ester and cholesteryl ester were more comparable. In 6 h 23-29% of retinyl ester had been transferred out of the donor particles, compared to 21-23% for cholesteryl ester. For large chylomicron remnants the retinyl ester transfer exceeded that for cholesteryl ester (Table I), but further experiments will be needed to generalize this conclusion. The transfer of labeled retinyl ester from hypercholesterolemic VLDL to other lipoprotein fractions appears to be rapid compared to that for cholesteryl ester. After 2 and 6 h of incubation the percent of labeled retinyl ester transferred was about twice that for the cholesteryl ester fraction (Table I). Neither the transfer of retinyl ester nor that of cholesteryl ester was affected by the sulfhydryl reagent PCMPS. Because of the similarity in the transfer of retinyl- and cholesteryl-esters in either normal or hypercholesterolemic rabbit plasma, we investigated the effect of partially purified cholesteryl ester transfer protein from rabbit plasma on the transfer of both lipids. Table II shows that addi-

TABLE

tion of increasing amounts of partially purified transfer protein or of a d> 1.21 fraction increased not only the cholesteryl ester transfer from hypercholesterolemic rabbit low density lipoprotein to bovine high density lipoprotein, but had a similar effect on the transfer of retinyl ester. The addition of the rabbit transfer protein stimulated transfer of retinyl ester relatively more than the transfer of cholesteryl ester, particularly at the lower levels of transfer protein. For partially purified human transfer protein (Table II) the relative magnitudes are reversed, i.e., cholesteryl ester transfer was consistently greater than the transfer of retinyl ester. Apparently the specificity of the transfer protein for the transfer of different lipids differs between animal species. Discussion In a previous study from this laboratory [ll], rabbit lymph chylomicrons, containing [ ‘HIretiny ester and [ “C]cholesteryl ester, were incubated with either normal or with hypercholesterolemic rabbit plasma for 60 min. Exchange of retinyl ester between rabbit lymph chylomicrons and the S,C 20 fraction was found to be considerably

II

ACCELERATED TEINS.

TRANSFER

OF RETINYL-

AND CHOLESTERYL-ESTERS

FROM

LOW- TO HIGH-DENSITY

LIPOPRO-

Labeled rabbit low density lipoproteins (1.063 1 d > 1.O19) and unlabeled bovine high density lipoproteins ( 1.2 I> d > 1.08) (400 ).tg total cholesterol each) were incubated with partially purified lipid transfer protein (CM-cellulose fraction) or d > 1.21 from rabbit or human plasma for 3 h at 37°C. The low density lipoprotein fraction contained 4601 cpm [3H]retinyI ester and 8404 cpm [‘4C]cholesteryl ester. The relative transfer quotient was calculated from the increments due to transfer protein. Transfer

% Transfer

protein

Relative (A/B)

Fraction

_ Rabbit Rabbit Rabbit Rabbit

mg protein

CM CM d > 1.2 1

d z 1.21

0 0.12 0.24 11.5 23.0 0

Human Human Human

CM CM CM

0.075 0.15 0.225

Retinyl

ester

Cholesteryl

(A)

(B)

0.9 15.6 18.5 20.8 26.9

0.3 7.0 11.3 15.3 26.4

1.8

2.7

9.2 15.9 17.5

20.0 33.4 45.9

ester

_ 2.2 1.6 1.3 1.0 _ 0.43 0.46 0.36

transfer

93

slower than that observed for cholesteryl ester. In the present study we studied this process for a longer period of time and for different lipoprotein fractions. For all lipoprotein fractions, simultaneously labeled with retinyl- and cholesteryl-esters, the transfer of retinyl esters was significant. In the case of chylomicrons, the retinyl ester transfer appeared to be slower than that for cholesteryl ester, but for low density lipoprotein, VLDL and for chylomicron remnants prepared in vitro, the retinyl ester transfer was as rapid or more rapid than that for cholesteryl ester. The transfer of both esters appears, however, to be mediated by the same transfer protein in plasma. In a recent study on the metabolism of chylomicrons in hypercholesterolemic dogs [20], the authors observed a significant transfer of retinyl ester from chylomicrons to the higher density lipoprotein fractions. They concluded that in vivo exchange of retinyl ester could have been responsible for this finding, but they were not able to exclude that transfer of retinyl ester was the result of progressive lipolysis and the transfer of apolipoproteins from chylomicrons to other plasma fractions. The present results cannot furnish an answer to this question, since the retinyl ester transfer in our studies appears to require the presence of lipid transfer protein which does not appear to be present in dog plasma (unpublished observations). Acknowledgments We wish to thank Judi Dougherty for her assistance in preparation of the manuscript. This investigation was supported by NIH Research Grant HL 10933 from the National Heart, Lung and Blood Institute of the U.S. Public Health Service. D.B.Z. is a Career Investigator of the American Heart Association. R.E.M. is a Postdoctoral Fel-

low of the National Heart, Lung and Blood Institute of the U.S. Public Health Service. K.H.T. was a Predoctoral Trainee supported by National Research Service Award HL 07245 from the National Heart, Lung and Blood Institute of the U.S. Public Health Service. References 1 Zilversmit, D.B., Hughes, L.B. and Balmer, J. (1975) Biochim. Biophys. Acta 409, 393-398 2 Barter, P.J. and Lally, J.I. (1979) Metabolism 28, 230-236 3 Pattnaik, N.M., Montes, A., Hughes, L.B. and Zilversmit, D.B. (1978) B&him. Biophys. Acta 530, 428-438 4 Morton, R.E. and Zilversmit, D.B. (1981) B&him. Biophys. Acta 663, 350-355 5 Barter, P.J. and Jones, M.E. (1980) J. Lipid Res. 21,238-249 6 Chajek, T., Aron, L. and Fielding, C.J. (1980) Biochemistry 19, 3673-3677 I Ihm, J., Harmony, J.A.K., Ellsworth, J. and Jackson, R.L. (1980) Biochem. Biophys. Res. Comm. 93, 1114- 1120 8 Marcel, Y.L., Vezina, C., Teng, B. and Sniderman, A. (1980) Atherosclerosis 35, 127-133 9 Rajaram, O.V., White, G.H. and Barter, P.J. (1980) Biochim. Biophys. Acta 617, 383-392 10 Morton, R.E. and Zilversmit, D.B. (1981) Fed. Proc. 40, 1694 11 Ross, A.C. and Zilversmit, D.B. (1977) J. Lipid Res. 18, 169-181 12 Glomset, J.A., Norum, K.R. and King, W. (1970) J. Clin. Invest. 49, 1827-1837 13 Morton, R.E. and Zilversmit, D.B. (1981) J. Biol. Chem. 256, 11992- 11995 14 Thompson, J.N., Erdody, P., Brien, R. and Murray, T.K. (1971) B&hem. Med. 5, 67-89 15 Abell, L.L., Levy, B.B., Brodie, B.B. and Kendall, F.E. (1952) J. Biol. Chem. 195, 357-366 16 Zak, B., Moss, N., Boyle, A.J. and Zlatkis, A. (1954) Anal. Chem. 26, 776-177 17 Sardesai, V.M., and Manning, J.A. (1968) Clin. Chem. 14, 156-161 18 Lowry, O.H.. Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 19 Bieri, J.G., Tolliver, T.J. and Catignani, G.L. (1979) Am. J. Clin. Nutr. 32, 2143-2149 20 Melchior, G.W., Mahley, R.W. and Buckhold, D.K. (1981) J. Lipid Res. 22, 598-609