Apolipoprotein A-I inhibits transformation of high density lipoprotein subpopulations during incubation of human plasma

73

Atherosclerosis, IS (1989) 73-82 Elsevier Scientific Publishers Ireland, Ltd.

ATH 04248

Apolipoprotein A-I inhibits transformation of high density lipoprotein subpopulations during incubation of human plasma Garry J. Hopkins and Philip J. Barter Baker Medical Research Institute, Commercial Road, Pmhran, Melbourne,

Victoria 3181 (Australia)

(Received 13 June, 1988) (Revised, received 1 August, 1988) (Accepted 29 August, 1988)

Summary We have examined the effect of added apolipoprotein A-l (apoA-I) on the changes in high density lipoprotein (HDL) particle size that occur when human plasma is incubated in vitro. In the absence of added apoA-I, incubation of plasma at 37 ’ C resulted in a dramatic increase in HDL particle size. When these incubations contained an inhibitor of LCAT, an additional population of smaller HDL particles was formed. These changes in particle size were even more pronounced when the incubations were supplemented with an artificial triglyceride emulsion, Intralipid. All these changes in HDL particle size were markedly inhibited when incubations were supplemented with apoA-I. Even when the amount of added apoA-I was as little as 4.5% of the endogenous apolipoprotein there was an obvious inhibition of the changes in HDL particle size. The presence of added apoA-I sufficient to increase the plasma concentration by 18% virtually abolished the changes in HDL particle size. This effect did not relate to an inhibition of cholesterol esterification, nor did it appear to depend on an incorporation of the added apoA-I into the HDL.

Key words: Apolipoprotein

A-I; HDL; Subpopulation;

Correspondence to: Dr. G.J. Hopkins, Baker Medical Research Institute, P.O. Box 348, Prahran, Victoria 3181, Australia. Abbreuiations: apoA-I, apolipoprotein A-I; HDL, high density lipoproteins; HDL,, HDL subfraction-2; HDL,, HDL subfraction-3; VLDL, very low density lipoproteins; LCAT, lecithin : cholesterol acyltransferase; PCMPS, p-chloro-

0021-9150/89/$03.50

Particle size

Introduction The high density lipoproteins (HDL) in human plasma are heterogeneous consisting of a number of discrete subpopulations of particles of varying size and composition [1,2]. The most common subpopulations have mean particle radii of 5.3,

0 1989 Elsevier Scientific Publishers Ireland, Ltd.

74 4.6, 4.2, 4.0 and 3.8 nm [2]. Studies performed in

vitro have implicated several factors which interact to produce changes in the distribution of HDL subpopulations. These include lecithin : cholesterol acyltransferase (LCAT) [3,4], lipid transfers [5], activities of lipoprotein lipase [6] and hepatic lipase [7,8] and a putative HDL conversion factor [9,10]. The observation that HDL particle size is also changed following enrichment with the acute phase reactant, serum amyloid A protein [ll] indicates that apolipoproteins may also influence the subpopulation distribution of HDL. HDL are also heterogeneous in terms of their capacity to interact with LCAT, the enzyme which catalyses the esterification of cholesterol in plasma [12]. This esterification is a key step in reverse cholesterol transport, the pathway by which cholesterol in peripheral tissues is delivered to the liver for excretion in the bile. Since small HDL are preferred over large HDL as substrates for LCAT [13-151, it follows that the regulation of HDL particle size has important implications in terms of reverse cholesterol transport and the putative role of HDL in protecting against the development of coronary heart disease [16]. This combined with the observation of a marked intersubject variability in the relative proportions of different HDL subpopulations [2,17] has led to a major interest in factors which regulate the distribution of HDL subpopulations. Our previous observation that enrichment of HDL with serum amyloid A protein resulted in changes in HDL particle size [ll] prompted us to investigate the effects of changing one of the more typical HDL apolipoproteins. Specifically, we have examined a possible role of the major protein component of HDL, apolipoprotein A-I (apoA-I). The concentration of apoA-I is known to vary widely between different subjects [18-201. Furthermore, as one of the surface constituents of chylomicrons which is released during lipolysis [21], there exists the potential for acute changes in the concentration of apoA-I in plasma in vivo. To investigate whether such changes in apoA-I are capable of modulating HDL particle size, we have incubated human plasma in vitro in the presence and absence of added apoA-I. It has been found that, while added apoA-I did not promote changes in the HDL, its presence in incubations had a

profound inhibitory effect on the changes promoted by other factors. Materials and methods Isolation of lipoproteins

Blood from pormal human subjects fasted for 12 h was collected into tubes containing EDTA (1 mg/ml) and immediately placed on ice. The plasma was separated by centrifugation at 4 o C. In some experiments VLDL-deficient plasma was obtained as the infranatant after ultracentrifugation of plasma at 173000 X g for 16 h. All ultracentrifugal separations were performed at 4°C. Isolation of apoA-I

ApoA-I was purified from HDL which were isolated from human plasma by sequential ultracentrifugation. HDL (d= 1.085-1.21 g/ml) were isolated using a single spin at 1.085 g/ml (158000 X g for 30 h) and two spins at 1.21 g/ml (158000 X g for 40 h and a wash at 247000 x g for 16 h). Densities were adjusted using solid KBr [22]. The HDL were dialysed exhaustively against 50 mM NH,HCO,, pH 8, freeze dried and delipidated with chloroform, methanol and ether [23]. The apo-HDL was dissolved in and dialysed against 0.1 M Tris-HCl, pH 8.2 containing 6 M urea for 24 h. The apoA-I was then isolated by gel filtration chromatography on a column (100 X 5 cm) containing Sephadex G-150 (Pharmacia-LKB, Uppsala, Sweden) run at 20 ml/h [24]. Column fractions were monitored by SDS-polyacrylamide gel electrophoresis (Phastsystem, Pharmacia-LKB, Uppsala, Sweden); fractions containing only apoA-I were pooled, dialysed against 50 mM NH,HCO,, pH 8 and freeze dried. The apoA-I was then redissolved in 0.02 M phosphate buffered saline and stored at - 20 o C. Incubations

Plasma or VLDL-deficient plasma, either alone or supplemented with graded amounts of apoA-I, was either kept at 4 o C or incubated at 37 o C in a shaking waterbath. Some of these incubations also contained 2 mM p-chloromercuriphenyl sulphonate (PCMPS) to inhibit LCAT [25]. Other incubations were supplemented with an artificial triglyceride emulsion (15%, v/v) (Intralipid 20%,

75 KabiVitrum, Stockholm, Sweden): in this case LCAT was inhibited with 1.4 mM dithiobisnitrobenzoic acid [26] because PCMPS has been reported to inhibit the transfer of triglyceride between triglyceride-rich lipoproteins and HDL [27]. Following incubation, any added Intralipid was removed by ultracentrifugation at 178000 X g for 16 h. Incubation mixtures were then adjusted to 1.25 g/ml with KBr [22] and the 1.25 g/ml supernatants and infranatants were recovered after ultracentrifugation at 178000 X g for 24 h.

Gel-filtration chromatography In some experiments gel-filtration chromatography was used to isolate HDL from the 1.25 g/ml supematants recovered after incubation. 0.2 ml of sample was applied to a Superose 6 column (Pharmacia-LKB, Uppsala, Sweden) equilibrated with 50 mM Tris-HCl, pH 7.5 containing 0.15 M NaCl.

Analyses The particle size distribution of HDL was determined by gradient-gel electrophoresis on 4-30s polyacrylamide gradient gels (Pharmacia-LKB, Uppsala, Sweden) as previously described [28]. Cholesterol esterification was measured as the reduction in free cholesterol concentration during incubation. Free cholesterol, total cholesterol and protein were measured as previously described [28]. Phospholipids and triglyceride (without free glycerol) were measured enzymatically (Boehringer Mannheim, West Germany); all assays were adapted for use on a Cobas-Bio centrifugal analyzer (Roche Diagnostics, Basel, Switzerland). Concentrations of apoA-I were measured using an immunoturbidimetric assay performed on the Cobas Bio centrifugal analyzer. The assay mixture consisted of 5 ~1 sample, 40 ~1 diluent and 250 ~1 of a solution containing 6% (w/v) polyethylene glycol 6000 and 2.5% (v/v) buffer concentrate (OUEC 40/41, Behringwerke, Marburg, F.R.G.). After incubation for 2 min at 37O C, 20 ~1 sheep anti-human apoA-I antiserum (Boehringer-Mannheim, F.R.G.) was added and the incubation continued for a further 20 min. Absorbances were read at a wavelength of 340 nm. The assay was standardized using appropriate dilutions of

apolipoprotein calibration ringer-Mannheim.

serum

from

Boeh-

Results Changes in HDL particle size during incubation of human plasma and effects of adding apoA-I (Fig. I) Fig. 1 shows the effect of added apoA-I on the HDL particle size in plasma incubated in the absence and presence of an inhibitor of LCAT [21]. The HDL in non-incubated plasma consisted of two major populations of particles with radii 4.3 nm and 4.0 nm and one minor population with a particle radius of 5.3 nm (Fig. 1, Profile A). This distribution was identical to that in non-incubated plasma supplemented with an inhibitor of LCAT (Profile B). When plasma in which LCAT was

1

Fig. 1. Effect of added apoA-I on changes in HDL particle size. Plasma was kept at 4” C in either the absence (Profile A) or presence (Profile R) of an inhibitor of LCAT [21]. Aliquots of these plasma samples were also incubated for 24 h at 37 o C with LCAT active (Profile C) or inactive (Profile D). Prior to incubation, samples of the same plasma were also supplemented with 576 pg of apoA-I which approximately doubled the concentration of apoA-I in the incubations. These samples were subsequently incubated with LCAT active (Profile E) or inactive (Profile F). Following incubation, the fraction of d -c 1.25 g/ml was recovered by ultracentrifugation and the HDL particle size distribution determined by gradient-gel electrophoresis as described in the Materials and Methods. In all figures the vertical lines represent particle radii: Alb represents albumin.

76 active was incubated at 37 o C a prominent population of enlarged particles with a mean radius of 4.7 nm appeared (Profile C). In the presence of an inhibitor of LCAT, incubation resulted in the appearance of two new populations of HDL (Profile D). One, with a mean radius of 4.7 nm, was comparable to that appearing when LCAT was active; the other, with a particle radius of 3.7 nm, was not observed in the incubations in which LCAT was active. Aliquots of the same plasma were supplemented with an amount of apoA-I, which approximately doubled the plasma concentration of this apolipoprotein. In samples kept at 4 o C, the addition of apoA-I had no effect on HDL particle size (results not shown). These samples were then incubated at 37 o C in the presence and absence of LCAT activity. In both cases, the addition of exogenous apoA-I virtually abolished the changes in HDL particle size which had been seen in its absence. In the incubation in which LCAT was active, the HDL now consisted of a major population of particles with a radius of 4.1 nm and two minor populations with radii of 4.3 and 4.7 nm (Profile E). When LCAT was inhibited in incubations containing added apoA-I, the HDL consisted of populations of particles with radii of 4.3 and 4.0 nm (Profile F). These results are representative of experiments performed with 6 separate plasma samples. Although the size distributions of the non-incubated HDL varied slightly from subject to subject, qualitatively similar results were obtained in each experiment. Effects of adding increasing amounts of apoA-I to plasma (Fig. 2) To determine the amount of apoA-I that had to be added to inhibit the changes in HDL particle size, plasma in which LCAT was active was supplemented with increasing amounts of apoA-I and incubated at 37O C. In this plasma sample, the non-incubated HDL was comprised of populations with radii of 5.3, 4.3 and 4.0 nm (Fig. 2, Profile A). Incubation again resulted in the formation of a prominent population of enlarged particles of radius 4.7 nm (Profile B). A population of smaller particles of radius 3.7 nm also appeared. The addition of 33 I-18of apoA-I, which increased the apoA-I concentration by only 4.5%, had an

Particle radius (nm)

5.3

4.340

3.7

Fig. 2. Effect of increasing amounts of added apoA-I on changes in HDL particle size. Plasma was either kept at 4OC (Profile A) or incubated with LCAT active at 37 o C for 7 h in the absence (Profile B) or presence of increasing amounts of apoA-I. The amounts added increased the concentration of apoA-I in plasma by 4.5% (Profile C), 9% (Profile D) and 18% (Profile E). Following incubation, the particle size distribution of the HDL was determined as described in the legend to Fig. 1.

obvious inhibitory effect on these changes in HDL particle size (Fig. 2, Profile C). The addition of 130 pg of apoA-I, which increased the apoA-I concentration by 18%, virtually abolished the changes in HDL particle size (Profile E). Effects of adding apoA-I at serial times during incubations of plasma (Figs. 3 and 4) The effect of adding varying amounts of apoA-I was further examined in a study of VLDL-deficient plasma to which up to 5 equal aliquots, each containing 123 pg of apoA-I, were added at 2 h intervals during a 10 h incubation. Fig. 3 shows

77

radius (nm)

5.3

4.3 4.0 3.7

incubated plasma consisted of populations of particles with radii of 5.3, 4.3 and 4.0 nm, as seen in the previous experiments, as well as a minor population with a particle radius of 4.5 nm (Figs. 3 and 4, Profiles A and H show the same sample). Incubation with LCAT active in the absence of added apoA-I resulted in changes to the HDL

Particle radius (nm)

5.3

4.34.0 3.7

Fig. 3. Effect of apoA-I added at serial times during incubation in the presence of LCAT activity. VLDL-deficient plasma was either kept at 4” C (Profiles A and H) or incubated at 37O C for 10 h in the absence (Profile B) or presence (Profiles C to G) of aliquots of apoA-I added at 2 h intervals during incubation. Aliquots containing 123 pg of apoA-I were added at 0 h (Profile C); 0 and 2 h (Profile D); 0, 2 and 4 h (Profile E); 0, 2, 4 and 6 h (Profile F) and 0, 2, 4, 6 and 8 h (Profile G). Following incubation, the particle size distribution of the HDL was determined as described in the legend to Fig. 1.

the results of incubations in which LCAT was active, while Fig. 4 shows experiments in which LCAT was inhibited. The HDL in the non-

Fig. 4. Effect of apoA-I added at serial times during incubation in the presence of an inhibitor of LCAT [23]. The descriptions of Profiles A to H are given in the legend to Fig. 3. Following incubation, the particle size distribution of the HDL was determined as described in the legend to Fig. 1.

78 comparable to those described above; the original predominant population of particles of radius 4.3 nm were converted into particles of radius 4.7 nm (Fig. 3, compare Profiles A and B). The addition at the beginning of the incubation of only a single aliquot of apoA-I (to increase the concentration of apoA-I in the incubation mixture by 8%) was sufficient to reduce this change in HDL particle size (Fig. 3, Profile C). This reduction was even more apparent when a second aliquot of apoA-I was added 2 h after commencing the incubation (Fig. 3, Profile D). Addition of a third aliquot after 4 h of incubation almost completely abolished the changes observed in the control incubation (Fig. 3, Profile F). Additional aliquots added after 6 and 8 h of incubation were without further effect (Fig. 3, Profiles F and G). The presence of added apoA-I had no influence on the rate of cholesterol esterification; in the absence of apoA-I, 30% of the free cholesterol was esterified during 10 h of incubation while in the incubation containing the highest concentration of the apolipoprotein 31% was esterified. The effects of two-hourly additions of aliquots of apoA-I were also studied when VLDL-deficient plasma was incubated in the presence of an inhibitor of LCAT (Fig. 4). In the absence of added apoA-I, there was an appearance of new populations of particles with radii ranging from 3.7 to 4.7 nm (Fig. 4, Profile B). With serial additions of apoA-I these changes became progressively less obvious and were completely abolished in mixtures to which four or more aliquots of apoA-I were added (Fig. 4, Profiles C to G). Incorporation of added apoA-I incubations of plasma (Table 1)

into HDL

during

To gain insight into how much of the added apoA-I remained unassociated with lipoproteins and how much was incorporated into the HDL, the incubation mixtures in the experiment described in Figs. 3 and 4 were separated into fractions of d < 1.25 g/ml (lipoprotein fraction) and d > 1.25 g/ml (lipoprotein-free fraction). The concentration of apoA-I was then measured in these fractions. Gel-filtration chromatography of the lipoprotein fraction established that more than 99% of the apoA-I co-eluted with HDL. For simplicity of presentation, Table 1 shows only the

TABLE

1

INCOBPORATION OF APO INCUBATION OF PLASMA

A-I

INTO

HDL

DURING

VLDL-deficient plasma was incubated at 37“C for 8 h in the presence and absence of PCMPS to inhibit LCAT. Incubations were supplemented with apoA-I as described in the legend to Fig. 3. In the absence of added apoA-I, samples contained 1495 pg of apoA-I in an incubation volume of 1.1 ml. Following incubation, the fractions of d cl.25 g/ml (lipoprotein fraction) and d > 1.25 g/ml (lipoprotein-free fraction) were recovered as described in Materials and Methods. Amount of apoA-I added to incubations (cg)

Amount of apoA-I recovered in lipoprotent-free fraction ( pg)

S of added apoA-I recovered in Iipoprotein-free fraction

(A) LCAT active

0 123 246 369 492 615

45 44 52 98 132 211

0 2.8 14.4 17.7 27.0

(B) LCAT inhibited ’

0 123 246 369 492 615

82 120 218 309 430 439

30.9 55.3 61.5 70.7 58.0

’ LCAT was inhibited

by the addition

of 2 mM PCMPS [21].

amount of apoA-I recovered in the lipoprotein-free fraction; in each case the balance was in the HDL. When plasma in which LCAT was active was incubated in the absence of added apoA-I, only 3% of the apoA-I was recovered in the lipoproteinfree fraction. When these incubations were supplemented with apoA-I, most of the added apolipoprotein was incorporated into the HDL with only a small proportion recovered in the lipoprotein-free fraction. Addition of up to 246 pg of exogenous apoA-I (corresponding to a 16.5% increase in apoA-I) had no effect on the amount recovered in the lipoprotein-free fraction. Indeed, even when as much as 615 pg of apoA-I was added, only about one third remained unassociated with HDL (Table 1). By contrast, in the incubations in which LCAT was inhibited a much greater proportion of the

79

added apoA-I was recovered in the lipoprotein-free fraction (Table 1). Even when only 123 pg of apoA-I was added, 31% remained unassociated with the HDL. When 246 pg or more was added, the major proportion remained unassociated with HDL.

Particle ratio (rd

5:3

4.3 4.0 3.7

,

Effect of apoA-I added only at the termination of a period of incubation of plasma (Fig. 5)

To determine whether changes in HDL particle size would be inhibited (or reversed) by the addition of apoA-I at the end of incubations, plasma was incubated at 37’ C before adding apoA-I for the final hour of incubation. Under these circumstances, the addition of 1728 pg of apoA-I, which approximately doubled the plasma concentration of the apolipoprotein, had no effect on the incubation-induced changes in HDL particle size (Fig. 5, Profile C).

Fig. 6. HDL particle size in incubations supplemented with Intralipid: effect of added apoA-I. Plasma containing an inhibitor of LCAT [22] was kept at 4OC (Profile A), or incubated at 37’C for 16 h in the presence of either Intralipid (15% v/v) (Profile B) or both Intralipid and 650 ng of added apoA-I (Profile C). This amount of apoA-I was sufficient to approximately double the concentration in the plasma. Following incubation, the particle size distribution of the HDL was determined as described in the legend to Fig. 1.

Incubation of plasma supplemented with Intralipid: effects of adding apoA-I (Fig. 6)

Fig. 5. Effect of apoA-I added 1 h before termination of incubation. Plasma was either kept at 4OC (Profile A) or incubated at 37 ’ C for 17 h in the absence of added apoA-I (Profile B). A third aliquot of plasma was incubated for 16 h in the absence of added apoA-I and then for a further 1 h after being supplemented with 1728 cg of apoA-I (Profile C). This amount of apoA-I was sufficient to approximately double the concentration in the plasma. Following incubation, the particle size distribution of the HDL was determined as described in the legend to Fig. 1.

We have previously shown that incubation of HDL, in the presence of Intralipid and a source of lipid transfer activity results in the appearance of new populations of particles, some larger and some smaller than the original HDL [5]. Figure 6 shows the effect of incubating whole plasma supplemented with Intralipid (15%, v/v). These incubation mixtures also contained an inhibitor of LCAT. The HDL in the non-incubated plasma consisted of two populations of particles with radii of 5.3 and 4.3 nm (Profile A). Following incubation in the presence of Intralipid the original major population of HDL, with a particle radius of 4.3 nm, had disappeared to be replaced by populations of both larger and smaller particles (Profile B). When however, 650 pg of apoA-I was added to approximately double the concentration

80 of the apolipoprotein, the changes to HDL particle size were virtually eliminated (Profile C). Discussion Several plasma factors are known to mediate changes in HDL particle size. LCAT, for example, promotes the conversion of HDL, into particles of the size and density, if not the composition, of HDL, [29]. Increases in HDL particle size are also induced by activity of lipoprotein lipase which provides additional surface components released during the hydrolysis of triglyceride-rich lipoproteins [6]. An exchange of cholesteryl ester in HDL for triglyceride in VLDL or Intralipid promoted by lipid transfer proteins provides another mechanism by which HDL become enlarged [S]. Subsequent interaction with hepatic lipase, however, converts these enlarged triglyceride-rich HDL into particles even smaller than the original HDL [30]. Changes in HDL particle size have also been attributed to a recently identified HDL conversion factor which converts HDL, into new populations of both larger and smaller particles [9,10]. The present finding that such changes in HDL particle size are markedly inhibited by apoA-I provides evidence that apolipoproteins are also involved in the regulation of HDL particle size and subpopulation distribution. The effect of apoA-I was apparent over a wide range of experimental conditions. For example, changes in HDL particle size were inhibited regardless of whether LCAT was active or inactive, whether VLDL or Intralipid was present or absent, and regardless of the duration of incubation. The amount of added apoA-I required to inhibit changes in HDL particle size in different plasma samples was also variable. In some experiments, changes in HDL particle size were inhibited when the concentration of apoA-I was increased by 10-258. In one experiment an inhibitory effect was apparent when the plasma concentration of apoA-I was increased by only 4.5%. In these studies a significant proportion of the apoA-I added to plasma was incorporated into HDL during incubations in which LCAT was active. By contrast, when LCAT was inhibited most of the added apoA-I remained unassociated with HDL. Nevertheless, the capacity of the

apoA-I to inhibit changes in HDL particle size was as obvious in the absence of LCAT activity as in its presence, suggesting that the inhibitory effect did not depend on an incorporation of the apolipoprotein into HDL particles. This raises the possibility that the inhibition is mediated by a pool of apoA-I which is not associated with any of the major lipoprotein fractions. The existence of a pool of “free” apoA-I in human plasma has been suggested by several investigators, with reports that it may account for as much as 20-30% of the total plasma apoA-I in some subjects [18-201. Since the capacity of apoA-I to inhibit changes in HDL particle size in the present studies was apparent when added in amounts sufficient to increase the endogenous concentration by as little as lo-15%, the possibility of physiological significance must be seriously considered. In the present studies the amount of apoA-I which was not associated with lipoproteins was assessed by measuring the concentration of the lipoprotein in the supernatant and infranatant after a single 24 h ultracentrifugal spin at a density of 1.25 g/ml. Although it has been suggested elsewhere that a proportion of the apoA-I is dissociated from HDL particles during prolonged ultracentrifugation [31], this was unlikely in the present experiments. In the control incubations which were performed in the absence of added apoA-I, only 3-5.5% of the total plasma apoA-I was recovered in the fraction of d > 1.25 g/ml. Comparably low concentrations of “free” apoA-I have been reported elsewhere using techniques other than ultracentrifugation. The proportion of “free” apoA-I measured after immunabsorption and starch block electrophoresis, for example, has been reported to be 5-25% of total apoA-I [18]. Ishida et al. [20] found that “free” apoA-I, separated by agarose gel electrophoresis, accounted for 4-59;; of total apoA-I in normolipaemic subjects, but up to 40-50% in hyperlipidaemic subjects. Other workers [19], using quantitative radial immunodiffusion, reported proportions of free apoA-I ranging from 10 to 30% of total apoA-I in a group of 100 normal subjects. In the present studies, the relatively low proportions of free apoA-I recovered in. the lipoprotein-free fraction after ultracentrifugation is likely to reflect the use of a single 24-h period of ultracentrifugation rather

81 than the prolonged and multiple spins used elsewhere [31]. The mechanism by which apoA-I inhibited the changes in HDL particle size cannot be determined from these studies. It did not, however, relate to an inhibition of cholesterol esterification. Nor did it appear to depend on an incorporation of the apolipoprotein into HDL. It is possible that the effect of apoA-I on the changes in HDL particle size may have been secondary to some other function of the apolipoprotein. It is known, for example, that addition of apoA-I will displace apoA-IV from HDL [32]; it is possible that the consequent increase in a pool of “free” apoA-IV may have been inhibitory. ApoA-I has also been reported to inhibit transfers of cholesteryl ester and triglyceride between lipoproteins [33]. This would be expected to reduce the changes in HDL particle size induced by lipid transfers [?I]. ApoA-I may also inhibit changes in HDL particle size by preventing fusion of lipoprotein particles [34]. Studies of model lipoproteins have shown that the formation of HDL from precursor populations incubated with LCAT is apparently achieved by a process involving fusion of particles [35]. In conclusion, it is apparent from these studies that relatively small increases in the concentration of apoA-I are associated with a profound inhibition of the changes in HDL particle size promoted by other factors operating in plasma. Furthermore, the inhibitory effect is apparent when apoA-I is added at concentrations comparable to those reported for “free” apoA-I in vivo in human subjects. Further studies will be required to elucidate both the mechanism of the inhibition and its precise physiological significance. Acknowledgements This work was supported by grants from the National Health and Medical Research Council of Australia and the National Heart Foundation of Australia. The excellent technical assistance of Shona Devlin is gratefully acknowledged. References 1 Kostner, G.M., Isolation, subfractionation,

and characteri-

2

3

4

5

6

7

8

9

zation of human serum high-density lipoproteins. In: C.E. Day (Ed.), High Density Lipoproteins, Marcel-Dekker, New York, 1981 p. 1. Blanche, P.J., Gong, E.L., Forte, T.M. and Nichols, A.V., Characterization of human high-density lipoproteins by gradient gel electrophoresis, Biochim. Biophys. Acta, 665 (1981) 408. Daerr, W.H. and Greten, H., In vitro modulation of the distribution of normal human plasma high density lipoprotein subfractions through the lecithin : cholesterol acyltransferase reaction, Biochim. Biophys. Acta, 710 (1981) 128. Schmitz, G., Assmann, G. and Melnik, B.. The role of lecithin : cholesterol acyltransferase in high density lipoproteins/high density lipoprotein, interconversion, Clin. Chim. Acta, 119 (1981) 225. Hopkins, G.J., Chang, L.B. and Barter, P.J., Role of lipid transfers in the formation of a subpopulation of small high density lipoproteins, J. Lipid Res., 26 (1985) 218. Patsch, J.R., Gotto, A.M., Jr., Olivecrona, T. and Eisenberg, S., Formation of high density lipoprotein,-like particles during lipolysis of very low density lipoproteins in vitro, Proc. Natl. Acad. Sci. USA, 75 (1978) 4519. Patsch, J.R., Prasad, S., Gotto, A.M., Jr. and BengtssonOlivecrona, G., Postprandial lipemia. A key for the conversion of high density lipoprotein, into high density lipoprotein, by hepatic lipase, J. Clin. Invest., 74 (1984) 2017. Hopkins, G.J. and Barter, P.J., Role of triglyceride-rich lipoproteins and hepatic lipase in determining the particle size and composition of high density lipoproteins, J. Lipid Res., 27 (1986) 1265. Gambert, P., Lallemant, C., Athias, A. and Padieu, P., Alterations of HDL cholesterol distribution induced by incubation of human serum, Biochim. Biophys. Acta, 713 (1982) 1.

10 Rye, K.-A. and Barter, P.J., Changes in the size and density of human high-density lipoproteins promoted by a plasmaconversion factor, B&him. Biophys. Acta, 875 (1986) 429. 11 Clifton, P.M., Mackinnon, A.M. and Barter, P.J., Effects of serum amyloid A protein (SAA) on composition, size, and density of high density lipoproteins in subjects with myocardial infarction, J. Lipid Res., 26 (1985) 1389. 12 Glomset, J.A., The plasma lecithin : cholesterol acyltransferase reaction, J. Lipid Res., 9 (1968) 155. 13 Fielding, C.J. and Fielding, P.E., Purification and substrate reactivity of lecithin-cholesterol acyltransferase from human plasma, FEBS Lett., 15 (1971) 355. L. and Jones, 14 Barter, P.J., Hopkins, G.J., Gorjatschko, M.E., Competitive inhibition of plasma cholesterol esterification by human high-density lipoprotein subfraction-2, Biochim Biophys. Acta, 793 (1984) 260. 15 Barter, P.J., Hopkins, G.J. and Gorjatschko, L., Lipoprotein substrates for plasma cholesterol esterification, Atherosclerosis, 58 (1985) 97. 16 Miller, G.J. and Miller, N.E., Plasma-high-density-lipoprotein concentration and development of ischaemic heart disease, Lancet, i (1975) 16. 17 Nichols, A.V., Human serum lipoproteins and their interrelationships, Adv. Biol. Med. Phys., 11 (1967) 109. 18 Kunitake, S.T., La Sala, K.J. and Kane, J.P., Apoli-

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25

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poprotcin A-I-containing lipoproteins with pm-beta elcctrophorctic mobility, J. Lipid Rcs., 26 (1985) 549. Borrcsen, A.-L. and Berg, K., Presence of “free” apoA-I in sorum: implications for immunological quantification of HDL and its apoproteins, Artery, 7 (1980) 139. Ishida, B.Y., Frolich, J. and Fielding, C.J., Prebeta-migrating high density lipoprotein: quantitation in normal and hyperlipidcmic plasma by solid phase radioimmunoassay following clcctrophoretic transfer, J. Lipid Res., 28 (1987) 778. Eisenbcrg, !I., High density lipoprotein metabolism, J. Lipid Res., 25 (1984) 1017. Hatch, F.T. and Lees, R.S., Practical methods for plasma lipoprotein analysis, Adv. Lipid Rcs., 6 (1%8) 1. Huff, M.W., Fidgc, N.H., Nest& P.J., Billington, T. and Watson, B., Metabolism of C-apolipoproteins: kinetics of C-II, C-III, and C-III,, and VLDL-apolipoprotcin B in normal and hypcrlipoproteinemic subjects, J. Lipid Res., 22 (1981) 1235. Scanu. A., Totb, J., Edelstein, C., Koga, !I. and Stiller, E., Fractionation of human serum high density lipoprotein in urea solutions. Evidence for polypeptide heterogeneity, Biochemistry, 8 (1969) 3309. Glomset, J.A., Norum, K.R. and King, W., Plasma lipoproteins in familial lecithin: cholesterol acyltransferase deficiency: lipid composition and reactivity in vitro, J. Clin. Invest., 49 (1970) 1827. Stokke, K.T. and Norum, K.R., Determination of lecithin : cholesterol acyltransfcrase in human blood plasma, Stand. J. Clin. Lab. Invest., 27 (1971) 21. Hopkins, G.J. and Barter, P.J., Dissociation of the in vitro transfers of estcrificd cholesterol and triglyceride between

human lipoproteins. Metabolism, 31 (1982) 78. 28 Hopkins, G.J. and Barter, P.J., Capacity of lipoproteins to act as substrates for lccithin:cholaterol acyltransferasc. Enhancement by pm-incubation with an artificial triacylglycerol emulsion, Biochim. Biophys. Acta, 794 (1984) 31. 29 Rajaram, O.V. and Barter, P.J., Increases in the particle size of high-density lipoproteins induced by purified lecithin : cholesterol acyltransferasc: effect of low-density lipoproteins, Biochim. Biophys. Acta, 877 (1986) 406. 30 Hopkins, G.J. and Barter, P.J., Role of triglyceriderich lipoproteins and hepatic lipase in determining the particle size and composition of high density lipoproteins, J. Lipid Res., 27 (1986) 1265. 31 Kunitake, S.T. and Kane, J.P., Factors affecting the integrity of high density lipoproteins in the ultracentrifuge, J. Lipid Rcs., 23 (1982) 936. 32 Lcfevre, M., Chuang, M.-Y. and Roheim, P.S., ApoA-IV metabolism in the rat: role of lipoprotein lipase and apolipoprotein transfer, J. Lipid Res., 27 (1986) 1163. 33 Son, Y.-SC. and Zilvcrsmit, D.B., Purification and characterization of human plasma proteins that inhibit lipid transfer activities, B&him. Biophys. Acta, 795 (1984) 473. 34 Parks, J.S., Martin, J.A., Johnson, F.L. and Rude& L.L., Fusion of low density lipoproteins with cholesterol esterphospholipid microemulsions. Prevention of particle fusion by apolipoprotein A-I, J. Biol. Chem., 260 (1985) 3155. 35 Nichols, A.V., Gong, E.L., Blanche, P.J., Forte, T.M. and Shore, V.G., Pathways in the formation of human plasma high density lipoprotein subpopulations containing apolipoprotein A-I without apolipoprotein A-II, J. Lipid Res., 28 (1987) 719.