Biochirnica et Biophysics Acta, 1166 (1993) 135-137 Elsevier Science Publishers B.V.

BBALIP 54118 Rapid Report

135

Relationship between the size and phospholipid content of low-density lipoproteins

P.J. Barter, O.V. Rajaram, H-Q. Liang and K-A. Rye Lipid Research Laboratory, Hanson Centre, Institute of Medical and Veterinary Science, Adelaide (Australia)

(Received 6 October 19921

Key words: LDL; Particle size; Phospholipid; Acipimox; Lecithin:-cholesterol acyltransferase; Phospholipase A,

In studies performed in vivo and in vitro, it has been found that the Stokes’ diameter of human low-density lipoproteins (LDL) correlates positively and significantly with the molar ratio of phospholipid/apo B in LDL but not with the LDL molar ratios of either cholesterol/ape B or triacylglycerol/apo B. It has been concluded that the phospholipid content of LDL is an important determinant of LDL size.

The low-density lipoproteins (LDL) in human plasma include a number of distinct subpopulations of parti- cles which vary in terms of size [l], density [2], composi- tion [2] and metabolism [3]. While the LDL fraction in any given subject tends to be relatively homogeneous, there is considerable individual variation in terms of which subpopulation predominates. The physiological significance of this LDL heterogeneity is uncertain, although it is of potentially considerable pathological importance in that subjects in whom the LDL are small and dense tend to be at increased risk of developing coronary heart disease [4]. Indeed, small, dense LDL particles are frequently found in patients with com- bined hyperlipidaemia [5], a condition known to be associated with increased coronary risk [61.

The explanation for a predominance of small, dense LDL in such subjects is not known. One suggestion is that it is secondary to an interaction of LDL with a high concentration of very-low-density lipoproteins (VLDL) in a series of reactions which involve the cholesteryl ester transfer protein (CETP) and hepatic lipase. According to this view, CETP catalyses the transfer of cholesteryl esters from LDL to VLDL in exchange for triacylglycerol to form cholesteryl ester- depleted andtriacylglycerol-enriched LDL particles [7].

Correspondence to: P.J. Barter, Department of Medicine, University

of Adelaide, PO Box 498, Adelaide, South Australia, Australia 5000.

Abbreviations: Apo B, apolipoprotein B; CETP, cholesteryl ester

transfer protein; LCAT, lecithin:cholesterol acyltransferase; LDL,

low-density lipoproteins; VLDL, very-low-density lipoproteins.

Subsequent lipase activity hydrolyses much of the newly acquired triacylglycerol, leaving LDL particles which are depleted of core lipids [8]. Such particles are also reduced in size.

The potential importance of core lipid content in determining the particle size of LDL was highlighted recently by the observation in hypertriglyceridaemic patients that successful reduction of the elevated plasma triacylglycerol levels with the nicotinic acid analogue, acipimox, was accompanied by an increase in both the cholesterol content and the particle size of LDL particles [9]. We have since confirmed the capac- ity of acipimox (2.50 mg three times daily) to increase the particle size of LDL in a group of 18 patients (15 males and 3 females) with combined hyperlipidaemia [lo]. However, in our group of subjects, the acipimox- induced increase in LDL size was not accompanied by an increase in the particle content of cholesterol. It was, on the other hand, associated with a significant increase in LDL phospholipid [lo].

In this report, we extend the analysis of the LDL results in these patients with combined hyperlipi- daemia by determining whether the size of LDL parti- cles correlates significantly with any parameter of LDL composition. The size distribution of LDL was deter- mined by non-denaturing gradient gel electrophoresis on 2-16% polyacrylamide gels (Pharmacia-LKB, Upp- sala, Sweden) or 3-13% gels (Gradipore, Sydney, Aus- tralia) [l,ll]. While there is often more than one LDL subpopulation present, invariably one population pre- dominates. The LDL sizes presented in Fig. 1 and Table I refer the Stokes’ diameter (nm) of the predom-

136

28 ]

700 800 900 1000 1100 1200

MOLAR RATIO PL/APO B

Fig. 1. Relationship between the diameter of LDL and the molar

ratio of LDL phospholipid/apo B. Two separate plasma samples

were obtained from 18 subjects with combined hyperlipidaemia; one

was collected prior to commencing therapy with acipimox (closed

symbols) and the other after 3 months of treatment with acipimox

(open symbols). The size distribution of LDL was determined by

non-denaturing polyacrylamide gradient gel electrophoresis. The di-

ameter presented is that of the predominant LDL subpopulation.

inant population. The concentrations of LDL con- stituents were assayed as described [12,13]. The com-

position of LDL has been expressed in terms of the

molar ratios of lipid constituents/ape B, assuming the molecular weights of apo B and phospholipids to be

550000 and 775 Da, respectively.

In the baseline plasma samples collected before commencing therapy with acipimox, the diameter of

LDL in these combined hyperlipidaemia subjects did

not correlate significantly with the ratios of either LDL cholesterol/ape B (r = 0.24, ns) or LDL triacyl- glycerol/ape B (r = 0.13 ns>. The LDL diameter did,

however, correlate positively and significantly with the ratio of LDL phospholipid/apo B (Y = 0.55, P < 0.05, Fig. 1).

As reported,previously [lo], after these subjects had

been treated for 3 months with acipimox, there was a significant increase in the diameter of LDL from 24.6

nm to 25.2 nm (P < 0.051 but there were no significant changes in the LDL molar ratios of either cholesterol/

apo B or triacylglycerol/apo B. There was, however, a significant increase in the ratio of phospholipid/apo B

from a pretreatment value of 831 f 68 (mean f S.D.>

to a treatment value of 955 f 101 (P < 0.001). When the pre-treatment data was pooled with that obtained

after 3 months of treatment with acipimox, to provide 36 data points in the 18 subjects, the correlation be-

tween LDL diameter and the molar ratio of LDL phospholipid/apo B remained positive and significant

(Y = 0.55, P < 0.001) (Fig. l), whereas the correlations between LDL size and the ratios of LDL cholesterol/ape B and LDL triacylglycerol/apo B re- mained non-significant.

To investigate further the relationship between the lipid composition and the particle size of LDL, two

groups of experiments were performed in vitro. In the first group, mixtures of LDL and HDL isolated from

normal human subjects were incubated with purified 1ecithin:cholesterol acyltransferase (LCAT) which had been isolated and assayed precisely as described previ-

ously [14]. Details of the experimental conditions are

given in the legend to Table I. Incubation in the absence of LCAT had no measurable effects on either the particle size or the composition of LDL (results not

TABLE 1

Effects of LCAT and phospholipase A, on the composition and particle size of LDL

Experiment A. The combined LDL+ HDL fractions (d 1.019-1.21 g/ml) was isolated as described [I6], mixed with human serum albumin

(Sigma Chemical, St. Louis, MO, USA) and incubated for 8 h at 37°C in the absence (control) or in the presence of LCAT. The final incubation

volume was 700 ~1. The total cholesterol concentration in the incubation mixture was 1.67 mmol/L (range 1.56-1.78); the concentration of

albumin was 39 mg/ml and of LCAT 10.3 units/ml. In the mixtures containing LCAT, a mean of 114 mmol/ml of unesterified cholesterol (29%

of that initially present) became esterified during the 8 h of incubation. Following incubation, the LDL were isolated ultracentrifugally as the

fraction of d < 1.063 g/ml and assayed for lipids, apo B and particle size. Values represent the means and S.D. of 5 separate experiments.

Experiment B. LDL (d 1.019-1.055 g/ml) were isolated from normal human plasma, mixed with either human or bovine serum albumin (Sigma)

and incubated for 3 h at 37°C in the absence (control) or in the presence of phospholipase A, (PLA,). The final incubation volume ranged from

300-500 ~1. The total cholesterol concentration ranged from 1.4-2.5 mmol/L; the concentration of albumin was 39 mg/ml and of PLA, 400

units/ml. Foliowing incubation, the LDL were reisolated as the fraction of d < 1.063 g/ml, and assayed for composition and particle size. Values

represent the mean + SD. of 5 separate experiments for the PL/apo B ratios and LDL diameters and of 3 experiments for the ratios of CE/apo

B, TG/apo B and UC/ape B.

Molar ratios in LDL LDL diameter

CE/Apo B a TG/Apo B UC/APO B PL/Apo B (run)

Experiment A

Control 3010&230 221* 47 873 _t 132 1080& 130 27.0 + 1 .O

LCAT 3360f292 *** 262* 58 635+ 29 ** 952* 93 ** 26.61_ 1.0 ** Experiment

Control 2 930 + 320 346 k 129 990 + 193 1150 + 280 28.9 i_ 1.3 PLA, 2900+330 355 f 132 969 + 147 800+315 *** 27.4kO.7 **

* * P < 0.01, * * * P < 0.001, indicates the significance of the difference from the control value as determined by paired t-test.

a Abbreviations: CE. cholesteryl ester; TG, triacylglycerol; UC, unesterified cholesterol; PL, phospholipids; PLA,, phospholipase A,.

137

shown). Incubation in the presence of LCAT, on the other hand, resulted in a significant increase in the LDL molar ratio of cholesteryl ester/ape B and signif- icant decreases in the LDL molar ratios of both unes- terified cholesteroljapo B and phospholipid/apo B. Despite the increase in cholesteryl ester content of the LDL, there was a small but significant decrease in the diameter of LDL (Table IA).

To determine whether the reduction in LDL size resulting from incubation in the presence of LCAT was related to the reduction in phospholipid content of the particles, further experiments were conducted in which preparations of LDL isolated from normal human sub- jects were incubated in vitro with phospholipase A, (Boehringer-Mannheim, Germany) (Table I, Experi- ment B). Experimental details are given in the legend to the table. In these incubations, a significant reduc- tion in the molar ratio of phospholipid/apo B 1150 + 280 to 800 + 315 (P < 0.001) was again accompanied by a significant reduction in LDL diameter from 28.9 -t 1.3 to 27.4 + 0.7 (P < 0.01) (Table IB). There were no changes in the ratios of cholesteryl ester/ape B, tri- acylglycerol/apo B or unesterified cholesterol/ape B after incubation with phospholipase A,.

These results do not support the previous conclu- sions that the size of LDL particles is determined mainly by the core lipid content of the particle. Al- though the cholesteryl ester content of LDL was not determined in the study described above, the fact that there was no change in the LDL particle content of total cholesterol (of which about 70% is esterified) after treatment with acipimox, indicates that any change in LDL cholesteryl ester content would, at most, have been minor. This, combined with the fact that treat- ment with acipimox produced a small (but not signifi- cant) reduction in the LDL content of triacylglycerol indicates that treatment with acipimox in the present study did not substantially increase the core lipid con- tent of LDL. The reasons for the discrepancy between the present results and those of Franceschini et al. [9] are unclear.

In vivo studies cannot address the mechanism un- derlying a positive correlation between LDL size and phospholipid content. For example, it is possible that larger LDL particles have a greater surface area and can therefore accommodate a larger number of phos- pholipid molecules, in which case the increase in the LDL particle content of phospholipid may have been secondary to the increase in LDL size. On the other hand, an increase in the phospholipid content of the LDL may be the primary determinant of the increase in particle size. This latter suggestion is strongly sup- ported by the results of the in vitro studies presented in this report.

The small but significant decrease in LDL particle size which occurred during incubation with LCAT was

unexpected, since the measured increase in cholesteryl ester content of the LDL would have been predicted to lead to an increase in the core volume and a conse- quent increase in particle size. The fact that an in- crease in the cholesteryl ester content of LDL particles was accompanied by a decrease in size indicates that there must have been a change to the packing of core constituents such that a larger number of cholesteryl ester and triacylglycerol molecules were accommo- dated in a smaller volume. It is possible that this change was secondary to the reduction in surface phos- pholipid and unesterified cholesterol. The results of the studies with phospholipase A, provide additional support for this proposition and suggest that phospho- lipid content of LDL may be an important determinant of LDL particle size. This raises obvious questions about factors which may regulate LDL phospholipid physiologically and how such factors relate to the regu- lation of the subpopulation distribution of LDL in vivo. What, if any, is the role played by the recently reported phospholipase A, activity of apo B [15] remains to be determined.

Acknowledgements

This work was supported by a grant from the Na- tional Health and Medical Research Council of Aus- tralia.

References

1 Krauss, R.M. and Burke, D.J. (1982) J. Lipid Res. 23, 97-104.

2 Hammond, M.G. and Fisher, W.R. (1971) J. Biol. Chem. 246,

5454-5465.

3 Fisher, W.R., Zech, L.A., Bardalaye, P., Warmke, G. and Berman,

M. (1980) J. Lipid Res. 21, 760-774.

4 Austin, M.A., Breslow, J.L., Hennekens, C.H., Buring, J.E., Wil-

let, W.C. and Krauss, R.M. (1988) JAMA 260, 1917-1921.

5 Austin, M.A., Brunzell, J.D., Fitch, W.L. and Krauss, R.M.(1990)

Arteriosclerosis 10, 520-530.

6 Brunzell, J.D., Schrott, H.G., Motulsky, A.G. and Bierman, E.L.

(1976) Metabolism 25, 313-320.

7 Barter, P.J., Gorjatscho, L. and Calvert, G.D. (1980) Biochim.

Biophys. Acta 619, 436-439.

8 Deckelbaum, R.J., Eisenberg, S., Oschry, Y., Butbul, E., Sharon,

I. and Olivecrona, T. (1982) 257, 6509-6517.

9 Franceschini, G., Bernini, F., Michelagnoli, S., Bellosta, S., Vac-

carino, V., Fumagalli, R. and Sirtori, C.R. (1990) Atherosclerosis

81, 41-49.

10 Barter, P.J., Nelson, L., Devlin, S. and Jenkins, P. (1991) Ather-

sclerosis Rev. 22, 213-216.

11 Meyer, B.J., Ha, Y.C. and Barter, P.J. (1989) Biochim. Biophys.

Acta 1004, 73-79.

12 Hopkins, G.J. and Barter, P.J. (1986) J. Lipid Res. 27, 1265-1277.

13 Hopkins, G.J. and Barter, P.J. (1989) Atherosclerosis 75, 73-82. 14 Rajaram, O.V. and Barter, P.J. (1985) Biochim. Biophys. Acta

835, 41-49.

15 Pathasarathy, S. and Barnett, J. (1990) Proc. Natl. Acad. Sci. USA 87, 9741-9745.

16 Barter, P.J., Chang, L.B.F. and Rajaram, O.V. (1990) Athero- sclerosis 84, 13-24.