An effect of very low density lipoproteins on the rate of cholesterol esterification in human plasma

Biochimica et Blophysrca Acta, 112 ( 1982) I522 160 Elsevier Biomedical Press

152

BBA 51149

AN EFFECT OF VERY LOW DENSITY LIPOPROTEINS ESTERIFICATION IN HUMAN PLASMA G.J. HOPKINS

and P.J. BARTER

Clinical Biochemstry (Received

ON THE RATE OF CHOLESTEROL

January

Kqv words: VLDL;

Unit, School of Medicine,

Flinders University of South Australia,

Bedford Park, South Australia

5042 (Australia)

25th, 1982)

Cholesterol ester; Lecithin:cholesteroI

acyltransferase

Rates of esterification of plasma cholesterol have been compared in two groups of human subjects with widely differing concentrations of very low density lipoproteins (VLDL). Group 1 consisted of subjects with fasting plasma triacylglycerol concentrations over 2.0 mM and group 2 consisted of subjects with concentrations under 1.6 mM. The rate of esterified cholesterol production in incubations of whole plasma from group 1 subjects was much greater than that from group 2 subjects. A clear difference between the rates of esterification was also evident when either total lipoproteins, total high density lipoproteins (HDL) or the HDL subfraction, HDL,, from the two types of subjects were incubated in the presence of a common source of 1ecithin:cholesterol acyltransferase. Since these findings appeared to reflect fundamental differences within the HDL, subfractions, which may have been modified by prior exposure to the high concentrations of VLDL in group 1 subjects, VLDL-deficient plasma and the plasma fraction of d > 1.125 g/ml (containing HDL,) from hypotriglyceridaemic subjects were preincubated at 37°C with an excess of added VLDL in the presence of a reversible inhibitor of lecithin:cholesterol acyltransferase. Following removal of the VLDL and reversal of the inhibition of lecithin:cholesterol acyltransferase, the capacity of the original fractions to esterify cholesterol had been markedly increased. These studies, therefore, show that the lecithin:cholesterol acyltransferase substrate capacity of particles within the HDL, subfraction is enhanced by exposure to VLDL and that this enhancement is not dependent on the continued presence of VLDL during the actual esterification reaction.

Introduction

tions of very low density lipoproteins (VLDL) [2,3]. The mechanism of this enhancement is not obvious since VLDL are unsuitable substrates for 1ecithin:cholesterol acyltransferase [4,5], which is known to act preferentially on high density lipoproteins (HDL) or, more specifically, on particles which reside in the HDL, subfraction of HDL [5]. There are a number of possible explanations for this apparent stimulatory effect of VLDL. (a) Subjects with elevated concentrations of VLDL may also have increased concentrations of 1ecithin:cholesterol acyltransferase. (b) Such subjects are known to have decreased concentrations of HDL, [6,7], a subfraction of HDL reported to

In human plasma the rate of formation of esterified cholesterol in the reaction catalysed by 1ecithin:cholesterol acyltransferase (EC 2.3.1.43) [l] is increased in subjects with elevated concentraAbbreviations: VLDL, very low density lipoproteins (density< 1.006 g/ml); LDL, low density lipoproteins (density= 1.0191.063 g/ml); HDL, high density lipoproteins (density = 1.07high density lipoproteins, subfraction 2 1.21 g/ml); HDL,, (density= 1.063-1.125 g/ml); HDL,, high density lipoproteins, subfraction 3 (density= 1.125.-1.21 g/ml); PCMPS. pchloromercuriphenyl sulphonate; DTNB, 5,5’-dithiobis-2nitrobenzoic acid.

00052760/82/0000-0000/$02.75

0 1982 Elsevier Biomedical

Press

153

inhibit the 1ecithin:cholesterol acyltransferase reaction [8-IO]. (c) VLDL are known to donate free cholesterol for the 1ecithin:cholesterol acyltransferase reaction [ 1l- 131; an increased concentration of VLDL may, therefore, enhance the rate of esterification by providing additional substrate. However, support for this view is undermined by the observation that the availability of free cholesterol is not normally rate-limiting [13]. (d) VLDL are known to be recipients of a proportion of the esterified cholesterol formed in the 1ecithin:cholesterol acyltransferase reaction [ 11,121. Recently, it has been suggested that the rate of esterification is markedly influenced by the capacity of recipient lipoproteins to accomodate the newly formed esterified cholesterol. [ 131; according to this view an increased concentration of recipient VLDL would enhance the rate of esterified

cholesterol formation. (e) Finally, exposure to a high concentration of VLDL may modify the 1ecithin:cholesterol acyltransferase substrate lipoproteins so as to enhance their capacity to act as substrates for the reaction. This final, previously unexplored, possibility is investigated in this report. Materials and Methods Human subjects Subjects were selected on the basis of their 16h-fasted plasma triacylglycerol concentration which was measured as previously described [14]. Only subjects with plasma triacylglycerol concentrations of over 2.0 mM or under 1.6 mM were studied; those with concentrations over 2.0 mM were classified as hypertriglyceridaemic and those

TABLE I CHOLESTEROL ESTERIFICATION IN INCUBATIONS GLYCERIDAEMIC AND A HYPERTRIGLYCERIDAEMIC

OF PLASMA OR LIPOPROTEIN SUBJECT

FRACTIONS

FROM

A HYPOTRI-

Whole plasma (2.5 ml) or lipoprotein fractions (isolated from 2-3.5 ml plasma) were incubated at 37°C in the presence of the d > 1.21 g/ml fraction isolated from 3-5 ml of plasma either pooled from subjectes 1 and 2, or obtained from a separate donor. Rates of esterification were measured as described in Materials and Methods. Each value for initial free cholesterol concentration represents the meantS.E. of (n) determinations. Each value for esterification rate represents the rate of esterification*S.E. calculated by least-squares linear regression analysis on (n) determinations of free cholesterol concentration. Subject 1 is the hypertriglyceridaemic subject; fasting plasma triacylglycerql concentration, 3.5 mM. Subject 2 is the hypotriglyceridaemic subject; fasting plasma triacylglycerol concentration, 0.2 mM. Incubated

fraction

Whole plasma

Subject

Initial free cholesterol concentration (nmol/incubation)

Rate of esterification (nmol/incubation per h)

1 2

4026t-6 2699k6

(6) (6)

272’115 (18)” 712 9 (24)

6.8 2.9

1807*5 1531*5

(6) (6)

86*10 (18)’ 482 6 (22)

4.8 3.1

dc1.21 g/ml+ common lipoprotein-free

plasma

1 2

HDL+ common

lipoprotein-free

plasma

1 2

30022 (6) 723 5 2 (6)

43% 242

2(24)” 2 (24)

14.3 3.3

HDL, + common

lipoprotein-free

plasma

1 2

259‘1 (9) 285” 1 (6)

18-t 7-t

1 (34)a

1 (27)

6.9 2.5

1 2

3412 1 (8) 40622 (8)

108-’ 73’

HDL, +common lipoprotein-free plasma+ common LCAT concentrate

’ Comparison b Comparison ' Concentrate

between subjects between subjects of lipoprotein-free

c

1 and 2 (r-test), P
precipitation

3 (33) a 3 (32)

[ 161) isolated from 5 ml pooled plasma.

31.7 18.0

154

with concentrations under 1.6 mM were classified as normotriglyceridaemic. Subject 1 (Table I) was a 43-year-old male weighing 89 kg (fasting plasma triacylglycerol, 3.5 mM) and subject 2 (TableI) was a 26year-old female weighing 47 kg (fasting plasma triacylglycerol, 0.2 mM). All other subjects were males. The mean age and body weight of group 1 (Table II) were 38.8 * 1.7 years (mean * SE.) and 86.0 * 3.7 kg, and for group 2 (Table II) were 24.5 + 3.2 years and 64.2 +- 3.8 kg.

for 16 h) of plasma at its own density. The recovered fraction of d > 1.006 g/ml was washed at the same density by further ultracentrifugation for 16 h. All other lipoprotein fractions were isolated at 4°C by ultracentrifugation (105000 X g) of plasma samples which had been adjusted to appropriate densities with solid KBr [ 151 and all were washed by a second ultracentrifugal run at the same density. In this manner, the plasma 1.21 g/ml supernatant (containing VLDL, low density lipoproteins (LDL) and HDL) was isolated by ultracentrifugation of plasma for 48 h. HDL were isolated in the density interval 1.07- 1.21 g/ml by ultracentrifugation of plasma at 1.07 g/ml for 24 h followed by ultracentrifugation of the 1.07 g/ml infranatant (adjusted to 1.21 g/ml) for 48 h. HDL, were isolated either in the density interval 1.1255

Preparation of lipoprotein fractions Blood was collected from 16h-fasted subjects into tubes containing EDTA and placed immediately on ice. Plasma was then separated by centrifugation at 4°C. VLDL-deficient plasma was isolated at 4°C by ultracentrifugation (105000 X g

TABLE

11

CHOLESTEROL ESTERIFICATION ACYLGLYCEROLCONCENTRATIONS

IN

TWO

GROUPS

OF

HUMAN

SUBJECTS

WITH

DIFFERING

PLASMA

TRI-

Whole plasma (3 ml) or HDL, (isolated from 6.5 ml plasma) were incubated at 37°C in the presence of lipoprotein-free plasma isolated from 11.5- 13 ml of pooled plasma. Rates of esterification were measured as described in Materials and Methods. Human subjects with fasting plasma triacylglycerol concentrations over 2 mM or under 1.6 mM were studied: six subjects with levels over 2 mM were allocated to group 1 and six subjects with under 1.6 mM were allocated to group 2. Each value represents the mean? S.E. of determinations on six subjects. Rates of esterification in incubations of HDL, were compared by randomly pairing each subject in group 1 with a subject in group 2 and, in six separate experiments, determining rates of esterification when HDL, from each pair of subjects were incubated in the presence of a common 1ecithin:cholesterol acyltransferase source (pooled d> 1.21 g/ml fraction). This experimental design was chosen because of the technical difficulty of measuring rates of esterification in incubations of HDL, (performed in the presence of a common 1ecithin:cholesterol acyltransferase source) from all 12 subjects in a single experiment. n.s.. not significant. Incubated

fraction

Comparison by f-test

Group 1

Triacylglycerol concentration (~mol/mI plasma) Rate of esterification (nmol/ml plasma per h) Initial free cholesterol concentration (nmol/ml plasma) Rate of esterification (nmol/incubation per h) Initial free cholesterol concentration (nmol/incubation)

’ Independent r-test. ’ Paired /-test.

Whole plasma

2

2.6-t

Whole plasma

116

Whole plasma

1504

-t

0.2 4

-‘IO0

HDL, +common lipoprotein-free plasma

128

i-

HDL, + common lipoprotein-free plasma

881

iI23

8

0.9*

0.2

59

*

5

P
1056

i

4X

P
94

-

8

P
876

klO7

ns. h

155

1.21 g/ml or with other plasma proteins in the fraction of d> 1.125 g/ml by ultracentrifugation for either 36 h at 1.125 g/ml or 48 h at 1.21 g/ml. Lipoprotein-free plasma was isolated as the infranatant after ultracentrifugation of plasma at 1.21 g/ml for 48 h. This fraction was used as a source of 1ecithin:cholesterol acyltransferase in incubations of various lipoprotein fractions. To reduce incubation volumes, the lipoprotein-free plasma and the fraction of d > 1.125 g/ml were concentrated 2-4-fold by dialysis against 60% sucrose. In certain experiments the lipoprotein-free plasma was subjected to (NH,)*SO, precipitation [16] to obtain a more concentrated source of 1ecithin:cholesterol acyltransferase. Following isolation, all fractions were dialysed against 0.02 M

phosphate buffer (pH 7.4) containing 0.01% EDTA and 0.02% NaN,.

0.15 M NaCl,

Incubations In vitro rates of cholesterol esterification were determined in whole plasma or in mixtures of lipoprotein fractions and lipoprotein-free plasma incubated for 50-60 min at 37°C in a shaking water-bath. At four different time-points aliquots of the incubation mixtures were taken into tubes containing p-chloromercuriphenyl sulphonate (PCMPS), an inhibitor of 1ecithin:cholesterol acyltransferase [17], and placed immediately on ice. Multiple (6-8) determinations were made of the free cholesterol concentration in each aliquot using an enzymatic method (Boehringer Man-

5oL,

I,

I 60

40

20

0

“.*

d

100

5

I

15

30

50

Minutes Fig. 1. Decline in free cholesterol concentration in incubations containing (a) whole plasma, (b) the d < 1.2 1 g/ml fraction of plasma, (c) HDL or (d) HDL, from either a hypotriglyceridaemic subject (0) or a hypertriglyceridaemic subject (0). Lipoprotein fractions (isolated from 2-3.5 ml plasma) were incubated at 37°C in the presence of lipoprotein-free plasma isolated from 3-4.7 ml of plasma either pooled from both subjects or obtained from a separate donor. Incubations in Fig. Id also contained an amount of an 1ecithin:cholesterol acyltransferase concentrate (see Materials and Methods) derived from 5 ml pooled plasma. Aliquots of the incubation mixtures were taken for free cholesterol determination at various times up to 60 min. Each point and error bar represents the decline in mean free cholesterol concentration and summed S.E.

nheim GmbH, CHOD-PAP method) adapted for use in a centrifugal analyzer (Centrifichem System 400, Union Carbide Corp.). The rate of decline in free cholesterol concentration (i.e., the rate of esterification) and the standard error of the rate were calculated by least-squares linear regression [ 181 on data obtained from these multiple determinations of free cholesterol concentration. The coefficient of variation of the rates determined using this method generally ranged from 3 to 10%. In most incubations the rates of esterification were linear up to the fourth time-point. Less commonly (see Fig. la, b), linearity was maintained only until the third time-point (40 min); in these cases the fourth time-point was excluded from the calculations. Rates of esterification were then compared using the slopes obtained from linear regression: the slopes are normally distributed, and the difference between slopes can be examined using Student’s t-test [18]. Since the number of degrees of freedom always exceeded 30, the normal approximation to the t distribution was appropriate. In some experiments VLDL-deficient plasma or the plasma fraction of d > 1.125 g/ml were pre-incubated with added VLDL at either 4 or 37°C for 6 h in the presence of 1.4 mM 5,5’-dithiobis-2nitrobenzoic acid (DTNB), a reversible inhibitor of 1ecithin:cholesterol acyltransferase [ 191. The VLDL were then removed by ultracentrifugation (105000 X R) for 16 h at 4°C at a density of 1.006 g/ml. The re-isolated VLDL-deficient fractions were subsequently re-incubated at 37°C in the presence of 3.5 mM dithiothreitol to reverse the DTNB-induced inhibition of 1ecithin:cholesterol acyltransferase [20] and rates of esterification were measured as described above. Initial concentrations of esterified cholesterol in these incubations were measured as previously described [21]. Results Cholesterol esterification in incubations of whole plasma or lipoprotein fractions isolated from a hypertriglyceridaemic and a hypotriglyceridaemic subject The rate of cholesterol esterification in incubations of plasma from a hypertriglyceridaemic subject (fasting plasma triacylglycerol, 3.5 mM) was much greater than in incubations of plasma from a

hypotriglyceridaemic subject (fasting triacylglycerol concentration, 0.2 mM) (Fig. la, Table I). When lipoproteins (the d < 1.21 g/ml plasma fraction containing VLDL, LDL and HDL) from each subject were incubated with aliquots of a common pool of lipoprotein-free plasma (providing a common source of 1ecithin:cholesterol acyltransferase) the rate of cholesterol esterification in the incubations of lipoproteins from the hypertriglyceridaemic subject was again greater than in those containing lipoproteins from the hypotriglyceridaemic subject (Fig. 1b, Table I). This difference between the rates of esterification was still evident when either the total HDL fractions or the HDL, subfractions from the two subjects were incubated with aliquots of the same pool of lipoprotein-free plasma (Fig. lc, TableI). When the lipoprotein-free plasma was omitted from these incubations of isolated lipoproteins there was no measurable esterification of cholesterol. Since rates of esterification in the incubations containing HDL, were rather low, this experiment was repeated in the presence of additional 1ecithin:cholesterol acyltransferase, provided in the form of a concentrate of lipoprotein-free plasma obtained by (NH4)zS04 precipitation (see Materials and Methods). Again, the rate of cholesterol esterification was greater in the incubations containing HDL, from the hypertriglyceridaemic subject (Fig. Id, Table I). The initial concentrations of free cholesterol in the various incubations are also shown in Table I. The free cholesterol concentration in incubations containing either whole plasma or the total lipoprotein fraction from the hypertriglyceridaemic subject was greater than in those from the hypotriglyceridaemic subject. However, in the incubations containing total HDL the free cholesterol concentration was greater in those from the hypotriglyceridaemic subject. In the case of the HDL, incubations, the concentration of free cholesterol was similar for both subjects. Cholesterol esterification in incubations of whole plasma and HDL, from two groups of human subjects To determine whether the results of the previous experiments were unique to the two subjects

III OF VLDL-DEFICIENT

PLASMA

AND

THE

PLASMA

FRACTION

OF d>1.125

g/ml.

of

B. Plasma d>1.125

a Comparison

_

_ VLDL VLDL

_ VLDL VLDL

Temperature

4 37 4 37

37 4 37

4

(“C)

by r-test with each other value in the same experiment:

fraction g/ml

plasma

A. VLDL-deficient

Addition

Pre-incubation

incubation

1

969 956 936

983

Expt. 2

1

each difference

445 _ 500

Expt.

was statistically

430 422 393 442

Expt. 2

Initial free cholesterol concentration

1038 981 1049

1001

Expt.

Initial free cholesterol concentration (nmol/ml)

Esterification

1

significant,

_ _

_ _

Expt.

P
8

1

41*11 38k 9 106* ga

33-c

Expt.

634

1334 1215 1334

Expt. 2

Initial esterified cholesterol concentration

1078 1091 1027

Expt. 3

Rate of cholesterol esterification (nmol/ml per h)

a

132*5

_ 81&5

Expt.

a

1

Rate of cholesterol esterification

45-t5 58*4 106-‘5

53*4

Expt. 2

88*4 90*3 77*3 122*4

Expt. 2

56+6 62*5 11Ok6”

_

Expt. 3

a

VLDL-deficient plasma (isolated from 7 ml plasma) and the plasma fraction of d > 1.125 g/ml (isolated from 5-7 ml plasma) were obtained from hypotriglyceridaemic subjects (fasting plasma triacylglycerol, < 1 mM) and pm-incubated at 37 and 4°C with or without added VLDL in the presence of 1.4 mM 5,5’-dithiobis-2-nitrobenzoic acid, a reversible inhibitor of 1ecithin:cholesterol acyltransferase [ 191. The VLDL-deficient fractions were then m-isolated at 4°C by ultracentrifugation at 105000X g for 16 h. Rates of cholesterol esterification were determined in 37°C incubations of these fractions performed in the presence of 3.5 mM dithiothreitol to reverse the inhibition of lecithin:cholesterol acyltransferase. To provide an additional common source of 1ecithin:cholesterol acyltransferase, the esterification incubations in B also contained added amounts of a concentrate of lipoprotein-free plasma (obtained by (NH,),SO, precipitation [16]) isolated from 5-7 ml plasma. Each value is the mean* S.E. Values in B are nmol/incubation. In A, the following amounts of VLDL triacylglycerol were added/ml of final pre-incubation mixture: Expt. 1, 6142 nmol; Expt. 2, 1825 nmol; Expt. 3, 3379 nmol. In B, the following amounts of VLDL triacylglycerol were added/ml of final pre-incubation mixture: Expt. 1. 3003 nmol; Expt. 2, 2666 nmol.

RATE OF CHOLESTEROL ESTERIFICATION IN INCUBATIONS INFLUENCE OF PRE-INCUBATION WITH VLDL

TABLE

158

studied, rates of cholesterol esterification were also measured in incubations of plasma and lipoprotein fractions from two groups of subjects selected on the basis of their fasting plasma triacylglycerol concentrations. Group 1 comprised six subjects with fasting plasma triacylglycerol concentrations over 2 mM and group 2 comprised six subjects with plasma triacylglycerol concentrations under 1.6 mM (Table II). The mean rate of cholesterol esterification in incubations of whole plasma from subjects in group 1 was significantly greater than in incubations of plasma from subjects in group 2 (Table II). Subjects in group 1 also had higher plasma concentrations of free cholesterol than subjects in group 2 (Table II). To compare rates of esterification in incubations of HDL,, each subject in group 1 was paired randomly with a subject in group 2 and, in six separate experiments, rates of esterification were measured when HDL, from each pair of subjects were incubated with aliquots of a common pool of lipoprotein-free plasma. Thus, in each experiment HDL, from one subject in group 1 and HDL, from one subject in group 2 were each incubated in the presence of a common source of 1ecithin:cholesterol acyltransferase. This experimental design was chosen to overcome the technical difficulty of measuring rates of esterification in incubations of HDL, (performed in the presence of a common source of 1ecithin:cholesterol acyltransferase) from all 12 subjects in a single experiment. Paired analysis showed that the mean rate of cholesterol esterification in incubations of HDL, from group 1 subjects was significantly greater (by 40 * lo%, mean +- SE.) than in incubations of HDL, from group 2 subjects (Table II). There was no significant difference between the mean initial concentrations of free cholesterol in the incubations of HDL, from the two groups. Cholesterol esterification in incubations of VLDLdeficient plasma or the plasma fraction of d > I. 125 g/ml. Influence of pre-incubation with VLDL VLDL-deficient plasma and the plasma fraction of d > 1.125 g/ml (containing HDL,) were obtained from hypotriglyceridaemic subjects (fasting plasma triacylglycerol, under 1 mM) and preincubated for 6 h at 37” or 4°C in the presence or absence of added VLDL. Rates of cholesterol

esterification were subsequently determined in 37”C-incubations of the re-isolated VLDL-deficient plasma or the d > 1.125 g/ml fraction. In three separate experiments, in which VLDLdeficient plasma was pre-incubated with an excess of added VLDL (Table IIIA), the rate of esterification in incubations of the re-isolatled VLDL-deficient plasma was enhanced 2-3-fold compared to that which had not been preincubated or had been pre-incubated at 37°C in the absence of added VLDL. Rates of esterification were also enhanced in incubations of the re-isolated plasma fraction of d > 1.125 g/ml which had been pre-incubated with added VLDL at 37°C (Table IIIB). These increases in the rates of esterification were not associated with greater initial concentrations of free cholesterol, but were associated with a 50% reduction in the concentration of esterified cholesterol in the d> 1.125 g/ml fraction (Table IIIB). Discussion The results presented in this report confirm previous observations that the rate of cholesterol esterification in whole plasma [2,3] or VLDL-depleted plasma [22] of hypertriglyceridaemic subjects is enhanced compared to those with lower triacylglycerol levels. This difference was found to persist when either total lipoproteins, total HDL or the HDL, subfraction from either type of subject were incubated in vitro. The greater rate of esterification in incubations containing HDL, isolated from hypertriglyceridaemic as compared to hypotriglyceridaemic subjects was apparent even when the incubations contained the same amount of 1ecithin:cholesterol acyltransferase, when the potentially inhibitory HDL, subfraction [8-lo] had been removed and when the concentration of substrate free cholesterol in the incubations were virtually identical. In simple terms, it was apparent that the HDL, subfraction isolated from hypertriglyceridaemic subjects was more effective as a substrate for 1ecithin:cholesterol acyltransferase than was the corresponding subfraction from hypotriglyceridaemic subjects. These findings, therefore, establish that the enhanced rate of cholesterol esterification in the plasma of subjects with elevated concentrations of VLDL is at least partly the consequence of a

159

fundamental difference within the HDL, subfraction of such subjects (mechanism (e) outlined in the Introduction). However, they do not exclude other mechanisms, such as those suggested in (a)(d), as possible contributors to the differences in whole plasma rates of esterification. Such factors may have contributed to the lower molar rates of esterification in incubations of HDL,, compared to those containing whole plasma. One possible explanation for these results is that during prior exposure to a high concentration of VLDL, the 1ecithin:cholesterol acyltransferase substrate lipoproteins within the HDL, subfraction had been altered so as to enhance their substrate capacity. It is known from in vitro studies that there is a net mass transfer of esterified cholesterol from HDL to VLDL which is increased with inceasing concentrations of VLDL [ 11,121. If esterified cholesterol is also transferred from 1ecithin:cholesterol acyltransferase substrate particles to VLDL and this net mass transfer is capable of relieving a product inhibition of 1ecithin:cholesterol acyltransferase, it follows that an elevated concentration of VLDL would result in an enhanced rate of esterified cholesterol production. This enhancement should also be evident when the VLDL have been removed from the incubation mixture. This proposal was tested by exposing HDL,and VLDL-deficient plasma from hypotriglyceridaemic subjects to high concentrations of VLDL prior to measuring the rate of esterified cholesterol formation. Pre-incubation with VLDL was found to enhance markedly the rate of esterified cholesterol formation even though the VLDL were removed prior to the actual measurement of the rate of esterification. The effect of VLDL in these studies cannot be explained in terms of donating free cholesterol or accepting newly formed esterified cholesterol during the esterification reaction. Rather, the pre-incubation appeared to modify the substrate lipoprotein particles in some way which enabled them to be more effective substrates for 1ecithin:cholesterol acyltransferase. The precise mechanism of this effect clearly requires further investigation, although it is possible that the substantial decline in the concentration of esterified cholesterol in the d> 1.125 g/ml fraction pre-incubated with VLDL may have resulted

of product inhibition in in a release 1ecithin:cholesterol acyltransferase substrate lipoproteins. Such a transfer to VLDL is compatible with observations by others [23,24] that a more dense subpopulation of HDL, is prominent in some subjects with hypertriglyceridaemia. It can be concluded, however, that one mechanism by which elevated concentrations of VLDL enhance esterified cholesterol production involves a modification of particles within the HDL, subfraction and that this enhancement of esterification is not dependent on the continued presence of VLDL during the actual esterification reaction. Acknowledgements We wish to thank Dr. Michael Jones for advice on statistical evaluation, and Anita Stibbs, Joanne Berry and Alison Dooley for technical assistance.

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