Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3542-3556
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3542-3556.)
© 1997 American Heart Association, Inc.
Lipoprotein Heterogeneity and Apolipoprotein B Metabolism
Chris J. Packard;
;
James Shepherd
From the Institute of Biochemistry, Glasgow Royal Infirmary, Glasgow, G4
OSF, UK.
Correspondence to Professor Chris J. Packard, Department of Pathological Biochemistry, 4th Floor, Queen Elizabeth Building, Alexandra Parade, Glasgow G31 2ER UK.
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Abstract
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Abstract The apolipoprotein B containing lipoproteins VLDL,
IDL,
and LDL exhibit variation in their structure, function, and
metabolism.
These major lipoprotein classes can be
fractionated into apparently
discrete components by density gradient
centrifugation or affinity
chromatography.
Examination of the behavior of
subfractions in vivo reveals
the presence of metabolic
channels within the VLDL-LDL delipidation
cascade so that the pedigree
of a lipoprotein in part determines
its metabolic fate.
Evidence from VLDL and LDL apoB turnovers
together with epidemiological
data allows the construction of
a quantitative model for the generation
of small, dense LDL.
This lipoprotein subspecies is one component of
the dyslipidemic
syndrome known as the atherogenic
lipoprotein phenotype, a common
disorder in those at risk for
coronary heart disease. Understanding
lipoprotein
heterogeneity is an essential step in the further
discovery
of the pathogenesis of atherosclerosis and in
the tailoring
of pharmacologic treatment for subjects at risk.
Key Words: kinetics • VLDL • plasma triglyceride • small, dense LDL • lipoprotein lipase • hepatic lipase
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Introduction
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Classical
studies by Gofman and his colleagues
1 in the
1950s
and 1960s using the analytical ultracentrifuge, revealed
for
the first time the heterogeneous nature of the
complexes in
plasma that were responsible for lipid transport. Peaks
and
troughs in the Schlieren pattern were clearly observed in the
S
f 0 to 400 range and the same was true of high
density lipoproteins.
However, lipoproteins cannot be isolated from an
analytical
ultracentrifuge and the
publication
2 of a convenient method
for preparing
total lipoprotein classes focused attention on
the broad properties of
very low, low, and high density lipoproteins.
The structure, function,
and metabolism of these lipoprotein
classes has been
intensively investigated over the last three
decades but until recently
little work had been done to discover
if the underlying
heterogeneity is important in determining
the
functional properties of a lipoprotein, in particular its
propensity to
promote or inhibit the processes that lead to
atherosclerosis.
Recognition that subfractions of
lipoproteins may have individual
significance in terms of association
with disease came first
for HDL. Division into
HDL
2 and HDL
3 revealed that
the former
exhibited a strong, negative relationship with risk of CHD
and
was the component of the spectrum that generated most of the
variation
in total HDL plasma concentration.
3
Within the S
f 0 to 400
range of apolipoprotein B
containing particles it was established
that IDL of density 1.006 g/ml
to 1.019 g/ml should be considered
a separate major class with distinct
metabolic properties, but
apart from sporadic reports there
was no comprehensive evaluation
of the role of subfractions within the
VLDL-LDL range. The view
of the majority of workers in the field in the
1970s and early
1980s (the authors of this review included) was that
within
a density interval, the spectrum of lipoprotein particles
differed
only slightly from one another in lipid content or apoprotein
composition
and there were no step changes or discrete entities, ie,
lipoprotein
classes were polydisperse. However, in later years when we
or
others isolated subfractions of VLDL or LDL and studied their
properties
in vivo or in vitro it became increasingly obvious that this
paradigm
was incorrect; the behavior of one component could differ
markedly
from that of another.
4 5 In a similar
vein, in a series of
seminal studies it was demonstrated that
lipoprotein components
of discrete size were present within LDL and
one of these, the
smallest species of 240 Å to 250 Å diameter, was
associated
with CHD risk in a way that particles of larger size were
not.
6 7 Thus, it is now widely accepted that
lipoprotein classes
are paucidisperse, ie, composed of a limited number
of components
with distinct properties and we are only just beginning
to uncover
the basis for this heterogeneity and the
consequences for disease.
Parenthetically, it should be noted that some
investigators,
notably Alaupovic et al,
8 have
continually made the case for
regarding lipoprotein classes as mixtures
of discrete components.
Heterogeneity within lipoprotein classes can be the
result of differing lipid content, different apoprotein composition,
altered protein conformation or as yet unidentified structural
variation (eg, carbohydrate content). Since VLDL, IDL, and LDL are
linked in a continuous metabolic cascade in which lipid
(ie, mainly triglyceride) is lost in a series of small
lipolytic steps, the delipidation process itself cannot give rise to
discrete subfractions. They must arise either by the insertion of new
material within a narrow density interval or by the formation of an
initial stable product that must undergo a step change to the next
state. Immunological methods are used to isolate fractions of differing
apoprotein content from within a lipoprotein class and a sometimes
bewildering array of subfractions containing various combinations of
apoproteins can be prepared by immunoaffinity
chromatography.8 9 It is unclear
as to how many of these species represent entities that could
be considered discrete subfractions, since all apoproteins except apoB
are known to exchange between particles. Evidence is emerging, however,
that specific fractions of different apoprotein composition may retain
their identity long enough in plasma for the properties of the
lipoprotein to be modified. Again, the most convincing data in this
regard have come from studies of HDL particles that contain either
apo-AI and no apo-AII(Lp-AI) or both main apo-A proteins (Lp-AI/AII).
Kinetic investigations have revealed that these species have individual
turnover rates.10
The following review focuses on heterogeneity in
the apoB-containing lipoprotein classes and interprets kinetic studies
of apoB metabolism in light of underlying structural and
functional variation. This is not meant to be an esoteric exercise,
rather it arises from the conviction that only a limited amount of
information about the plasma lipid transport system can be gained from
examination of a whole lipoprotein class, if indeed the true building
blocks of the system are its component subfractions. Furthermore, at a
time when the use of lipid lowering agents in both primary and
secondary prevention of CHD is now accepted medical practice, it is
essential that drugs are targeted towards those at highest risk for
both ethical and financial reasons. Understanding in detail the
aberrations in lipid metabolism and lipoprotein
substructure that give rise to a propensity to develop
atherosclerosis will help identify those measurements
that can be made to assist in prognosis beyond the assay of total
plasma cholesterol or the LDL to HDL ratio.
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Structure and Function of VLDL Subfractions
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VLDL comprises the class of lipoproteins present after an
overnight
fast that float when plasma is subjected to a centrifugal
force.
On the basis of this broad definition there is no reason why
this
lipoprotein fraction should be homogeneous and indeed
any investigation
of its structure has revealed marked
heterogeneity in size and
in lipid and apoprotein
composition. VLDL is synthesized and
secreted continuously by the
liver. Lipoprotein assembly is
initiated by the addition of lipid to
the growing apoB chain
in the rough endoplasmic reticulum. More lipid,
principally
triglyceride, is added to nascent VLDL
particles as they pass
along the secretory pathway. Details of the VLDL
assembly process
are beyond the scope of the present review but
have been described
in a number of recent
articles.
11 12 13 However, concepts derived
from
cell culture studies will be revisited following a discussion
of the in
vivo properties of VLDL subfractions and what these
reveal about the
synthetic machinery.
Two methods have been employed most commonly to fractionate VLDL into
structurally and metabolically discrete components. These
are centrifugation that separates mainly on the basis
of size and density (ie, particle lipid content) and affinity
chromatography where differences in apoprotein
composition or conformation are exploited. VLDL vary in size from about
350Å to 700Å diameter, which corresponds to 8-fold range in volume,
ie, from 22x106 Å3 to
180x106 Å3 . Most of the
difference in size is due to the triglyceride core since
the phospholipid/apoprotein coat is of constant thickness in
lipoproteins.14 15 Separation by density is
likely to be a useful approach since the subfractions generated vary in
triglyceride content and will have arisen either because
lipoproteins with a different lipid load have been released by the
liver or because lipolysis has produced a smaller particle from a
larger one. In fact, there is evidence that both processes are
responsible for the heterogeneity observed. Techniques
such as gel chromatography or density gradient
centrifugation have divided VLDL into multiple
fractions14 15 16 17 but a convenient, commonly used
and highly reproducible method is that of cumulative flotation
centrifugation,18 which provides
two or three subfractions in a concentrated form suitable for further
analysis, namely VLDL1 of
Sf 60 to 400 and VLDL2 of
Sf 20 to 60 or alternatively
VLDL1 of Sf 100 to 400,
VLDL2 of Sf 60 to 100 and
VLDL3 of Sf 20 to 60.
Compared to larger VLDL, VLDL of Sf 20 to 60 are
smaller, enriched in cholesteryl ester, deplete in
triglyceride and have a lower ratio of apoE and apoC to
apoB.14 15
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Properties of VLDL Subfractions Separated by Density
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In a series of studies we investigated the metabolism
of apoB
in VLDL subfractions in normal and dyslipidemic
subjects.
5 19 20 21 The results, summarized
diagrammatically in Fig 1
, revealed
not
only differences in the kinetics of the component fractions
but also
the presence of "metabolic" channeling within the
VLDL-LDL
delipidation cascade where apparently parallel (ie,
nonintersecting)
processing pathways generate IDL and LDL products
of differing
metabolic potential from precursors in the
VLDL range. In early
experiments we trace-labeled VLDL of
S
f 100 to 400 with the
idea of following the
various lipolytic and catabolic steps
through to LDL, in line with
current models at the time we envisaged
a single delipidation cascade
from the largest VLDL particles
to LDL. However, on injection of
radioiodinated S
f 100 to 400
VLDL
into subjects we observed that while substantial amounts
of its apoB
appeared in smaller VLDL and IDL following delipidation
of the
particle, less than 10% was converted to LDL.
19
Thus,
large VLDL was postulated not to form LDL to a significant
degree,
a tenet that has been confirmed by
others.
22 Rather, its delipidation
ceases in the
VLDL or IDL density ranges where it is thought
to generate remnants
that persist in the circulation for considerable
periods (Fig 1
).
Intermediate sized VLDL of S
f 60 to 100 has
kinetic
properties similar to those of S
f 100 to
400 VLDL, although
more of the apoB from this interval is delipidated
to LDL.
5 19 20 ApoB containing particles in the
S
f 20 to 60 density
range, in contrast, are
rapidly and efficiently converted to
LDL with >50% of the tracer
being observed in LDL within
24 hours after
injection.
19 As explained in detail
elsewhere,
23 analysis of dual tracer
turnovers using
131I-VLDL S
f 60 to
400
and
125I-VLDL S
f 20 to 60 by
multicompartmental modeling led
to a complex but
physiologically important conceptualization
of
apoB metabolism that placed kinetic
heterogeneity at the
center of an understanding of how
VLDL and LDL synthesis and
catabolism are regulated. Studies in a wide
variety of subjects
indicate that the liver synthesizes and secretes
VLDL particles
that range in size and density across the full
S
f 20 to 400
spectrum. Furthermore, there is
increasing evidence that the
production of
VLDL
1 (S
f 60 to 400) and
VLDL
2 (S
f 20 to 60) are
regulated
independently of one another.
21 24 25
VLDL
1 production was
increased in
subjects with high-normal compared to low-normal
plasma
triglyceride levels [C.J.P. et al, 1997, unpublished],
was
stimulated by estrogen treatment in post-menopausal
females,
24 and specifically inhibited by the
infusion of insulin to normolipidemic
men.
25
VLDL
2 production was not perturbed by the
administration
of these hormones but was found to be elevated in
moderately
hypercholesterolemic
subjects.
21 Increased LDL production has
long
been considered to be a major cause of raised LDL levels in
common
forms of
hypercholesterolemia
26
and our investigations
located enhanced hepatic output into
VLDL
2 as the likely source
of this
metabolic abnormality (Fig 1
). The mechanism by which
the
liver is able to vary the amount of large versus small VLDL
secreted is
unknown. Recent experiments in cell culture
12 13
suggest that VLDL assembly is more complex than was originally
thought.
A two-step process is envisaged in which a small lipoprotein
particle
containing at first little triglyceride is formed in
the
rough ER and then the bulk of the triglyceride core added
to
this at the junction of the rough and smooth ER. It is possible
that
the release of small VLDL follows the addition of a relatively
small
quantity of triglyceride (or of cholesteryl ester) to
the
nascent particle while large VLDL is formed by the addition
of a
substantial triglyceride core in a second, quantum step.
Enhanced
VLDL
2 secretion in moderate
hypercholesterolemics may result
from factors other
than triglyceride availability, for example,
the rate of
cholesterol synthesis,
13 cholesteryl
ester availability,
11 13 27 or microsomal
transfer protein activity.
28
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Figure 1. ApoB metabolism. ApoB is made
constitutively in the liver. Initiation of lipoprotein assembly
requires the addition of a small amount of neutral lipid to the growing
polypeptide chain. Further lipid is added during particle assembly and
secretion and the nature of the final lipoprotein released (which
ranges from LDL to VLDL1) depends on the extent of
lipidation. ApoB containing lipoproteins on entering the circulation
are subject to lipolysis and remodeling so that the composition of the
coat of the particle, especially its apoproteins, and the core change
during the course of intravascular metabolism.
Production rates for VLDL1 (Sf 60 to
400), VLDL2 (Sf 20 to 60) and IDL plus LDL-apoB
are quoted in mg/d. Subjects with elevated plasma
triglycerides (HTG) produce an excess of VLDL1
apoB (unpublished, 1997), whereas those with raised plasma
cholesterol levels overproduce VLDL2
apoB.21 IDL plus LDL apoB production is
variable over the range shown and is inversely related to the
plasma triglyceride concentration.21
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Cross-sectional surveys revealed that variation in plasma
triglyceride is principally a function of changing
VLDL1 levels.29 Likewise,
it is larger VLDL that accumulate in
hypertriglyceridemics and are lowered by
fibrate therapy.30 31 32 The plasma concentration
of VLDL1 has been found to be controlled by both
the rate of production and the rate of delipidation of the
lipoprotein [References 30 and 3130 31 ; C.J.P. et al, 1997, unpublished
data]. The former appears to be under the influence of
hormones24 25 while the latter has a number of
possible determinants. For example, investigation of the
VLDL1 to VLDL2 conversion
in subjects with genetic dyslipidemia revealed that the
step was critically dependent on LpL, (the fractional transfer rate
from VLDL1 to VLDL2 was
reduced 90% in LpL deficiency22 33 ) but was not
affected by HL deficiency,34 by homozygous
FH35 or by homozygosity for apolipoprotein
E2.20 From studies of VLDL
composition,36 human
mutants,37 and genetically altered
mice,38 it appears that apo-CIII as well as
apo-CII is a modulator of LpL activity39 (Fig 1
).
Recent kinetic experiments showed that VLDL1
lipolysis but not that of VLDL2 was inhibited
profoundly by the presence of chylomicron-like emulsion particles in
the circulation.40 The implication of this
finding is that VLDL1 lipolysis does not
"compete" effectively with chylomicron clearance but is virtually
suspended when the plasma content of chylomicrons is high and then
resumes when lipid absorption is complete. Presumably
VLDL1 is a much poorer substrate for LpL than
dietary particles. It is not yet clear what regulates lipolysis in
vivo, LpL activity as measured in post-heparin plasma showed only a
weak correlation with either the extent of alimentary lipemia or
VLDL1 concentration.29 41
More likely it is the surface composition of VLDL, both its lipid and
apoprotein content (eg, apoC-II/C-III or apoE/C ratios) that is
critically important in most subjects.36 37 38 39
VLDL1 has two distinct
metabolic fates, conversion by LpL to
VLDL2 and direct catabolism. The nature of the
second process is unknown at present but it is quantitatively
significant since up to half the apoB in a VLDL1
tracer was removed directly from the circulation without appearing in
denser fractions20 21 22 (Fig 1). Since the rate of
VLDL1 direct catabolism was similar to normal in
FH homozygotes,35 it is unlikely that the LDL
receptor is involved. However, catabolism of the lipoprotein was
reduced substantially in normolipemic apoE2
homozygotes20 and in subjects lacking
LpL.33 These proteins have been implicated in the
binding of lipoproteins to agents such as the LRP and the VLDL
receptor42 43 and the observations raise the
possibility that these entities mediate the direct removal of large
triglyceride-rich VLDL (Fig 1). The ligand for the putative
receptor is likely to be apoE since apoB appears not to be in a
receptor competent conformation on large triglyceride-rich
VLDL.44 By extrapolation from animal studies it
is also probable that apoCIII plays an inhibitory role by
interacting with or displacing apoE.38 45 46 The
metabolic properties of VLDL1 suggest
that in many ways it acts as a liver-derived "chylomicron
particle"; it is produced in response to the presense of
triglyceride in the cell; and it is cleared rapidly by the
same agents (initially LpL and then receptors) that metabolize dietary
particles. The finding that VLDL1 release is
suppressed by insulin,25 a hormone elevated
during the absorption of fat in the diet, indicates that chylomicron
and VLDL1 secretion may be controlled in a
reciprocal fashion. In this context it is noteworthy that some
mammalian species release B-48 containing large VLDL when faced with
the need to secrete increased quantities of triglyceride
from the liver.47
The VLDL2 density interval
(Sf 20 to 60) contains the products of
VLDL1 delipidation and newly secreted VLDL
particles. The need to postulate synthesis of small VLDL by the liver
was seen in kinetic studies where the amount of apoB generated by
VLDL1 delipidation was insufficient to account
for the total mass seen in the density interval23
and it was only by permitting VLDL2 direct
production that the kinetics of this lipoprotein fraction could
be explained. As shown in Fig 1, 131I-VLDL2 derived from injected
131I-VLDL1 was found to be converted
to IDL and LDL at a slower rate than a tracer of radio-labeled whole
VLDL2. This can only occur if a new lipoprotein
species is introduced into the VLDL2 interval.
Furthermore, in a continuous separation medium such as a density
gradient, a distinct peak was seen in the VLDL2
range that strongly suggested the insertion of newly secreted
lipoprotein.17 The possibility of
metabolic channeling within the VLDL-LDL delipidation
cascade was first mooted by Fisher,48 and its
confirmed presence23 49 indicates that the
properties of a circulating lipoprotein depend heavily on its pedigree
and that exchange of key lipid and protein components between particles
is relatively slow compared to the processes of lipolysis and
catabolism. VLDL2 delipidation proceeded
efficiently in subjects with LpL deficiency, evidence that the enzyme
was not essential for the processing of this
lipoprotein.33 Likewise, in HL deficiency
VLDL2 was converted to IDL at about half the
normal rate.34 It is likely that both lipases
contribute to this step (Fig 1). LDL receptors are likely to be
responsible for direct catabolism of VLDL2;
smaller VLDL are more effective ligands than their larger counterparts
for this receptor49 and in vitro studies support
the view that binding occurs via the apoB rather than
apoE.50 In line with this suggestion we found
that VLDL2 direct catabolism was similar to
normal in apoE2 homozygotes.20
Further, while cyclohexanedione modification of apoB in
Sf 60 to 400 VLDL that abolishes receptor
mediated clearance did not affect its clearance rate (presumably
because apoE mediates the receptor-lipoprotein interaction in this
density interval43 ), treatment of
Sf 12 to 60 lipoproteins with this agent
substantially retarded their removal from the
circulation.51
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Properties of VLDL Subfractions Separated by Affinity
Chromatography
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Cell surfaces are coated with heparan sulfate and other
similar
compounds believed to facilitate the proximation of
lipoproteins
containing apoB and apoE to lipases and receptors. These
apoproteins
have specific glycosaminoglycan binding
sites and so adhere
to columns containing Sepharose-bound heparin. The
affinity
of VLDL fractions for heparin, however, is variable and
dependent
on the apoE content of particles and the conformation of
apoB.
52 53 54 In the case of the latter protein,
expression of heparin
binding domains, like the receptor binding
site,
50 appears
to be influenced by the size of
the lipoprotein and its lipid
composition. VLDL when applied to
heparin-Sepharose is separated
into bound and unbound fractions.
Retained particles accounted
for over half the VLDL mass in
normolipemics, had a higher apoE:
apoB ratio, were relatively rich in
cholesteryl esters, and
were smaller than unretained
particles.
52 53 54 55 The two
fractions are
present in variable proportions throughout the
S
f 20 to 400 density interval, although
VLDL
2 has been shown to
have a substantially
higher content of retained particles.
56 Early
studies
57 of unretained VLDL showed that it was
particularly
rich in phosphotidylethanolamine(PE), a major phospholipid
found
in abundance in cell membranes but not in the majority of plasma
lipoproteins
and that it was a poor ligand for the LDL receptor,
possibly
because it was lacking in apoE but also due to the
conformation
of its apoB being inappropriate for binding. Fielding and
Fielding
on the basis of these properties suggested that unretained
particles
were newly secreted.
57 Co-incubation of
apoE poor (ie, unretained)
VLDL with retained apoE rich VLDL did not
lead to significant
inter-particle transfer of PE or apoE, and the same
workers
in a later investigation
58 reported that
unretained VLDL required
to be lipolysed to be able to bind apoE to its
surface. This
modification of VLDL was associated with, and was
possibly dependent
on, a perturbation in the structure of apoB. It was
postulated
58 that unretained VLDL was the
metabolic precursor to retained
VLDL and that lipolysis was
necessary to change apoB conformation
and so reveal heparin binding
sites and permit apoE to adhere
to the particle (Fig 2
). The hypothesis is consistent
with data
showing that in
hypertriglyceridemic subjects, who are
known
generally to overproduce VLDL (Fig 1
),
59
unretained VLDL accounted
for 40% of total VLDL compared to 25% in
normals.
55 56 Treatment
with diet or a fibrate
led to a decrease in the proportion of
unretained
VLDL.
55 The requirement of a nascent particle to
undergo
lipolysis before it can, through a perturbation in the apoB
conformation
or through the acquisition of apoE, bind to receptors may
be
a mechanism by which futile cycling of VLDL is prevented in
the
liver. This organ secretes VLDL that could in theory be
immediately
removed by cell surface receptors especially since
apoE is present
in abundance on the surface of
hepatocytes.
60
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Figure 2. Metabolic scheme for VLDL subfractions
isolated by heparin-Sepharose chromatography. VLDL can
be fractionated by affinity chromatography on solid
phase heparin into bound and unbound fractions. The former are retained
either by virtue of the apoE on the particle or by the expression of a
heparin binding site on apoB. Unbound VLDL show characteristics of
nascent particles and when injected in vivo are converted to a bound
species. In vitro evidence indicates that unbound VLDL must undergo
lipolysis before apoE will adhere to the particle.58 The
proportion of unbound VLDL is higher in the VLDL1 than in
the VLDL2 flotation range.56
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Metabolic studies reveal that when unretained VLDL was
radiolabeled and injected into normal or
hypertriglyceridemic subjects, it was
converted into a VLDL that was able to bind to heparin and thereafter
was delipidated to IDL.52 However, it was found
that retained VLDL was not simply the product of lipolysis of
unretained VLDL. Barrett et al56 provided
evidence for the direct input of both triglyceride and apoB
into the bound and unbound fractions. Furthermore, their results were
consistent with direct catabolism of unbound VLDL as well as
lipolytic conversion to bound lipoproteins. Turnover experiments in
rabbits and mini-pigs revealed that apoE-rich VLDL was catabolized at a
greater rate than apoE-poor VLDL, principally by direct clearance from
the circulation,53 56 and apoE-rich VLDL were
less likely to be converted to LDL in rabbits than apoE-poor
particles.61 These findings were
consistent with the in vitro observations regarding the
relative abilities of these lipoprotein fractions to interact with the
LDL receptor.54
Aware that VLDL particles may bind to heparin through both apoB and
apoE, Campos et al54 have recently examined the
distribution of VLDL subfractions separated first on anti-apoE affinity
columns and then on heparin-Sepharose. VLDL from subjects with a range
of plasma triglyceride concentrations were first passed
through the anti-apoE column; about 30 to 50% were unretained and
hence were deficient in apoE. Chromatography of these
"B" particles on heparin-Sepharose generated both bound (1% to
35% of total VLDL) and unbound (14% to 36% of total VLDL) fractions.
The latter form of "B" particle was larger and more
triglyceride-rich than either the bound "B" particles
or the B/E particles whose lipid composition resembled each other. This
was clear evidence that in earlier studies the retained
heparin-Sepharose fraction was a mixture of "B"–and
"B/E"–VLDL with the former binding by virtue of the apoB
conformation (Fig 2). The apoC:apoB ratio was similar in all fractions
isolated. Heparin-Sepharose unbound "B" particles failed completely
to bind to the LDL receptor in vitro, while heparin bound "B" VLDL
interacted with the receptor at an affinity approaching that of B/E
VLDL. Indeed the affinity of VLDL for the LDL receptor was not related
to the particles' apoB/apoE ratio. Campos et
al62 have also shown that B/E VLDL can be
subdivided by affinity chromatography on an antibody
column that recognizes an epitope near the mid-portion of apoB
polypeptide. This antibody appears to be selective for remnant
particles and consideration must now be given to the possibility that
the "signal" that a VLDL particle has become a remnant may reside
in the conformation of apoB. Thus, there is now evidence that a minimum
of four VLDL subfractions can be isolated on the basis of their apoE
content and apoB conformation, so ample heterogeneity
exists within VLDL to support the phenomenon of metabolic
channeling described above.
It is difficult to relate unambiguously the structural and
metabolic heterogeneity in VLDL revealed by
centrifugation based fractionation to that based on
affinity chromatography. VLDL1
(Sf 60 to 400) has been shown to have a high
content of apoE-poor particles that do not bind to heparin-Sepharose.
This is consistent with the high triglyceride
content and other attributes expected of new secreted VLDL.
Heparin-Sepharose bound VLDL1 may be remnants
already formed within the density interval. Lipolysis of "B"
particles to VLDL2 leads to a change in the
conformation of apoB and the acquisition of some apoE, thus rendering
the VLDL able to bind to heparin. Thus, within the
VLDL2 density interval the relatively slowly
metabolized remnants of VLDL1 delipidation (Figs 1, 2) are likely to be heparin-Sepharose bound "B" or "B/E"
particles. The nature of newly secreted VLDL within the
Sf 20 to 60 is less clear. It is conceivable that
heparin-Sepharose unbound VLDL2 comprises, as in
VLDL1, nascent particles. These are reported to
represent 10% to 50% of the VLDL2 apoB
mass,56 a figure in line with the amount of the
lipoprotein estimated from kinetic studies to be present in newly
secreted material.20 21 Hence, it is tempting to
speculate that heparin unbound VLDL of both density intervals is the
form in which particles are released from the liver (Fig 2). However,
Barrett et al56 required newly synthesized
lipoprotein to enter both heparin-bound and heparin-unbound
VLDL2 to fit the model to the observed data and
so the form of nascent particle remains an open question. The
metabolic fate of heparin-bound versus heparin-unbound VLDL
has not been followed through to LDL in man, and so it is not yet
obvious which particles act as precursors to the latter lipoprotein.
Analogy with rabbit studies suggests that those particles that acquire
sufficient apoE will be efficiently removed by receptors and this
presumably occurs throughout the Sf 0 to 400
range. Others will progress down the delipidation cascade to form
LDL.
|
Heterogeneity of Intermediate Density
Lipoproteins
|
|---|
Kinetic studies in the mid-1970s identified IDL as a class of
lipoprotein
with distinct metabolic properties that acted
as a transient
intermediate in the delipidation cascade from VLDL to
LDL. Subsequently
it was shown that not all IDL were derived from VLDL
and not
all IDL were converted to LDL, a portion of the lipoprotein
was
catabolized directly from plasma probably by the LDL receptor
since the
rate of this process was dramatically reduced in FH
homozygotes.
35 63 Turnovers conducted in subjects
with genetic dyslipidemia
indicate that hepatic lipase was
essential for the IDL to LDL
conversion.
34
Similarly, infusion of antibodies to HL in animals
gave rise to an
accumulation of S
f 12 to 20 lipoprotein and
a
decrease in LDL.
64 The IDL to LDL conversion was
also found
to be dependent on the apoE phenotype of a subject.
In a study
of normolipidemic apoE
2 homozygotes we
observed a 60% reduction
in the rate of transfer of IDL to LDL while
direct catabolism
of the fraction, presumably mediated by its apoB
component,
was normal.
20 The diminished
circulating LDL mass in apoE
2 homozygotes was the
result of this failure to delipidate IDL
efficiently. Unexpectedly the
IDL to LDL lipolytic step was
also greatly impaired in FH homozygotes.
Up to 5 days was required
to complete the delipidation of VLDL to LDL
in such subjects
35 compared to less than 1 day in
normals. Thus, the mechanism
of conversion appeared to be independent
of LpL
33 but involved
the possibly concerted
action of HL, apoE
3, and the LDL receptor.
It is
conceivable that on the liver sinusoidal cells when HL
is sited, an
interaction between apoE and the LDL receptor facilitates
the
delipidation step. About half of the apoB entering the IDL
density
interval was transmitted to LDL in normals and half
is removed by
catabolism.
20 What determines the
metabolic fate
of the particles is unknown. It could be due
to structural heterogeneity
(the basis of which is as
yet unrecognized) or to the relative
likelihood of IDL particle binding
to HL and being lipolysed
to LDL or binding to the LDL receptor located
on a hepatic parenchymal
cell and being internalized and degraded.
Krauss and co-workers in a series of
investigations65 66 have examined the structural
and metabolic heterogeneity of IDL. They
reported the presence on gradient gel electrophoresis of two major
subfractions that overlapped in size and density and hence cannot be
readily isolated. IDL-1 were larger (280 Å to 300 Å in diameter),
less dense and seemed to form a component of a spectrum of particles
that ranged from Sf 14 to 60, ie, into the
VLDL2 density range. This species was relatively
triglyceride-rich whereas IDL-2 was smaller (270 Å to 280
Å in diameter) and contained relatively more cholesterol.
Given the fact that both VLDL and LDL exhibit a high degree of
heterogeneity it is entirely likely that the IDL class
also contains discrete species with individual metabolic
properties. These have been examined in a rat model system by Musliner
et al.66 IDL-1 appeared to give rise to
intermediate-sized LDL particles while conversely IDL-2 was the
precursor to the largest LDL species, LDL-1. These nonintersecting
pathways of IDL and LDL interconversion have been formulated into a
metabolic scheme by Krauss.67 The
importance of IDL lies not only in the fact that it is the immediate
precursor to LDL but that it appears to be particularly atherogenic. A
number of studies have linked raised IDL levels to increased risk of
CHD as recently summarized by Superko.68
|
LDL Structural and Metabolic Heterogeneity
|
|---|
LDL as the major cholesterol-carrying lipoprotein in
plasma
is the fraction most strongly implicated in atherogenesis. When
examined
in the analytical ultracentrifuge "shoulders" were
apparent
on the LDL peak suggestive of the presence of subfractions of
differing
flotation rate and early reports from
Fisher
4 highlighted the
polydispersity of LDL in
hypertriglyceridemia compared to the
"monodisperse"
distribution seen in normals or subjects with
elevated cholesterol
levels. It was considered for many
years that LDL comprised
a population of particles with continuously
variable size and
density across the range of 200 Å to 270 Å and
d
1.019 g/ml to 1.063 g/ml. However, Krauss
69
using the high
resolution technique of gge provided convincing evidence
that
in virtually all subjects LDL was made up of a small number
of
subtypes of particles with relatively discrete size and density,
ie, it
was "paucidisperse." A seminal observation was that patients
with a
preponderance of small-sized LDL detected by gge (pattern
"B" LDL)
had a three-fold increased risk of having a myocardial
infarction
independent of the total concentration of LDL
present.
70 This finding triggered the current
widespread interest in LDL
size as a predictor of CHD risk and the
association has been
confirmed many times.
71 72 73
Since small, dense LDL is clinically
important it is essential to
understand its origins and the
basis for its increased atherogenic
potential compared to larger
species of the lipoprotein. At present
opinions are divided
as to whether a high concentration of small, dense
LDL in the
plasma is the result of a specific inherited
trait
68 or if
the particle subtype is generated
in anyone given the appropriate
conditions. In the discussion that
follows, evidence is presented
to support mainly the latter
view, although in some individuals
it is conceivable that an overriding
genetic factor may be present
to cause the generation of small
LDL.
|
LDL Particle Size and Plasma Triglyceride
|
|---|
From the earliest studies of LDL heterogeneity,
whether assessed
by size on gradient gels or by density in a
centrifuge rotor,
it was noted that subjects with smaller and
denser LDL had higher
plasma triglyceride levels than those
with lighter particles.
Austin et al in a classic
paper
70 found that pattern B was
associated with
a two-fold increase in plasma triglyceride,
higher plasma
apoB and IDL levels and reduced HDL cholesterol
and apoA-I
concentration, ie, small, dense LDL did not appear
in isolation from
other plasma lipid abnormalities. Campos et
al
73
reported a highly significant correlation between LDL
size score (a
continuous index of LDL diameter rather than the
dichotomous
classification of Austin and Krauss
70 ) and plasma
triglyceride.
Further, they showed that once
triglyceride was taken into account,
LDL size was not an
independent discriminator of CHD risk. Methods
based on
centrifugation of LDL in a density gradient were
developed
by Swinkels et al,
71 Chapman et
al,
74 and ourselves
72 in
an
attempt to quantify the amount of various LDL subfractions
present.
Although some workers divided LDL into multiple
fractions,
74 it was clear that if plasma (rather
than prepared LDL) was
applied to a gradient and the subfractions
generated rapidly
that three discrete fractions were present in
most normal or
moderately hyperlipidemic subjects
[LDL-I, d=1.025 g/ml to 1.034
g/ml, LDL-II, d=1.034 g/ml to 1.044 g/ml
and LDL-III, d=1.044
g/ml to 1.060 g/ml (nb, these ranges vary slightly
between publications)]
while in severe
hypertriglyceridemics very small and dense
species
were observed (LDL-IV
69 ). The link
between plasma triglyceride
and LDL size phenotype
was first explored by Austin et al.
7 They found
that pattern A (large LDL predominant) was universally
present at
low plasma triglyceride levels (<0.5 mmol/l) whereas
pattern
B was found in most individuals whose triglyceride
exceeded
2.0 mmol/l. Similarly, McNamara et
al
75 showed that change
in LDL size was related
to change in plasma triglyceride level.
However, it is
necessary to turn to a continuous, quantitative
assessment of LDL
subfraction concentrations in order to uncover
the precise nature of
the relationship between plasma triglyceride
and LDL size.
In our laboratory LDL subfraction concentrations
were measured in a
large number of normal and CHD affected
subjects.
29 72 In the study of Griffin et
al,
72 it was found that LDL-I
and LDL-II had
similar plasma concentrations in those with and
without CHD but LDL-III
was 2-fold higher in affected subjects.
An LDL-III level of 100 mg/dl
was the best discriminant between
the two groups and was associated
with a 7-fold increase in
risk.
72 Close
examination of the relationships between LDL
subfraction concentrations
and plasma triglyceride revealed
that LDL-I decreased as
plasma triglyceride rose across the
normal range. LDL-II
showed a biphasic association with a positive
correlation below a value
of 1.5 mmol/l for the plasma lipid
and a negative one above that
(Fig 3A
). LDL-III remained below
100
mg/dl until the plasma triglyceride exceeded 1.5
mmol/l
and then the level of the subfraction increased dramatically.
In
both surveys
29 72 the LDL-III-plasma
triglyceride curve
gave the impression of a breakpoint at
the level of about 1.5
mmol/l, at least in male subjects. This
concurred with the earlier
observation of Austin et
al
7 that pattern B only becomes prevalent
above
this value. The fact that the total LDL concentration
remained
relatively constant in subjects with plasma triglyceride
in
the range 1.0 mmol/l to 3.0 mmol/l suggested to us that
1.5
mmol/l was a "threshold" above which either LDL-II was
increasingly
converted to LDL-III in the circulation or LDL-III rather
than
LDL-II was the preferred product of VLDL delipidation. The
postulated
existence of a threshold concentration of plasma
triglyceride
rich lipoproteins for the formation of small,
dense LDL was
intriguing and helped explain the observation of Superko
and
Krauss
76 that nicotinic acid treatment of
moderately hypercholesterolemic
subjects led to a
change in LDL size from pattern B to A only
if the plasma
triglyceride fell below 1.6 mmol/l. Similarly
in a
number of later drug studies, reduction in plasma
triglyceride
led to a diminution of LDL-III and an increase
in LDL-II even
if total LDL mass was unaltered on therapy [Reference
77
77 and M.J. Caslake et al, 1997, unpublished data (Fig 3B
)]. Further
evidence
for a threshold effect in the conversion of LDL-II to LDL-III
has
been observed during gestation.
78 Plasma
triglyceride climbed
from 1.0 mmol/l to 2.5
mmol/l from the first to third trimester
and the LDL concentration rose
70%. At first LDL-II accumulated
but as plasma
triglyceride exceeded about 1.8 mmol/l in the
group of
women studied, there was an abrupt change in the relative
concentration
of LDL-II and LDL-III and at term most subjects
exhibited a high
LDL-III level.
78 The appearance of pattern
B at
the end of pregnancy and the reversion of LDL to pattern
A postpartum
has been reported by Silliman et al.
79 These
investigations
reveal the malleability of LDL subfraction profile and
also
raise the question as to the mechanism that underlies a step
change
in LDL size.
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|
Figure 3. Influence of plasma triglyceride on the
concentration of intermediate sized (LDL-II) and small dense LDL
(LDL-III) in normal and hyperlipidemic subjects.
|
|
In our and others' experience80 few individuals
display small, dense LDL at low normal plasma triglyceride.
The vast majority of the population who express pattern B-LDL or a
LDL-III >100 mg/dl do so against a background of disturbances
in lipoprotein metabolism linked to high normal or
moderately elevated plasma triglyceride levels with
attendant changes in IDL and HDL. The term atherogenic lipoprotein
phenotype(ALP) was coined by Austin et
al7 to describe a syndrome of small, dense LDL,
moderately elevated VLDL and low HDL. It is increasingly recognized as
a significant, and possibly in population terms the most important,
lipid-associated risk factor for CHD. As described in following
sections, elevation in plasma triglyceride due to increased
VLDL1 levels may be the key metabolic
disturbance that gives rise to the syndrome.
|
LDL Particle Size and Lipases
|
|---|
Generation of small, dense LDL requires the removal of lipid
from
the particle while, of course, apoB is retained. LDL is
cholesteryl
ester rich and since plasma is deficient in esterases
that hydrolyze
this lipid species, it is predictable that particles
in which the core
is predominantly cholesteryl ester are resistant
to shrinkage.
However, the action of CETP mediates the exchange
of neutral lipid
between lipoproteins with the result that when
circulating levels of
triglyceride-rich lipoprotein (VLDL, chylomicrons)
are
high, a portion of their core triglyceride is transferred
into
LDL (and HDL) and replaced with cholesteryl ester from the denser
species
(Fig 4
).
Triglyceride-enriched LDL is then a substrate for
endothelial-bound
lipases and their action leads to the
formation of smaller,
lipid-depleted particles. Cycles of neutral lipid
transfer and
lipolysis have been shown to promote the formation of
HDL
3 from
HDL
281 and there is reason to
believe that the same phenomenon occurs
in the LDL density
range.
67 82 Either lipoprotein lipase or
hepatic
lipase(HL) could, in theory, hydrolyze triglyceride
in LDL.
In vitro and in vivo evidence strongly suggests that
the latter enzyme
is the more likely agent. It has a much higher
affinity for LDL than
lipoprotein lipase and has the capability
to act as a phospholipase as
well, hence removing both core
and surface components from the
particle.
83 84
The possibility that LpL was involved in the regulation of LDL size was
investigated by Karpe et al,85 who found a
positive association between LpL activity and the amount of LDL in a
light (d <1.040 g/ml) fraction. Furthermore, obligate heterozygotes
for LpL deficiency show multiple lipoprotein abnormalities including
pattern B LDL.86 However, these associations are
likely to have been mediated through the effect of LpL variation on
chylomicron and VLDL concentrations (Fig 4). Hepatic lipase activity,
on the other hand, was shown to be positively related to the amount of
small, dense LDL87 although this association was
not confirmed in the subsequent study of Jansen et
al.88 We explored the relationship between
lipases and LDL subfraction concentration in normolipemic men and women
and in an initial study89 were able to show a
strong correlation between HL activity and the plasma concentration of
LDL-III. Moreover, there was a strong suggestion that common factors
regulated the subfraction distribution in LDL and HDL. In a larger
number of subjects,29 it was observed that women
at plasma triglyceride levels above the indicated threshold
for the formation of small, dense LDL (ie, about 1.5 mmol/l) had
less LDL-III than men (Fig 3A). Multivariate
analysis showed that in men the LDL-III concentration was
explained mainly by plasma triglyceride level but in women
HL activity and plasma triglyceride were both determinants.
Apparently for LDL-III to rise above 100mg/dl in females it was
necessary for HL to be in the male range. Normally the HL level in
women is half that seen in men, a result that is readily explained by
the regulatory influence of sex hormones on the
enzyme.29 The discrepant results in the study of
Jansen et al88 can be explained by the fact that
they examined only men. Further support for a key role of HL was seen
in the condition of inherited deficiency of the enzyme; affected
subjects had large, buoyant LDL subfractions in
plasma.90 These findings led us to postulate a
model in which the formation of a significant amount of LDL-III in
plasma (ie, above 100 mg/dl) required the combination of a plasma
triglyceride >1.5 mmol/l and a HL >15 u/l (Fig 4).
The model helped to explain the reduced penetrance of pattern B LDL in
females (low HL activity) and in younger
subjects7 91 (plasma triglyceride
climbs dramatically in the population from about 0.5 mmol to
1.0 mmol at age 20 to 1.5 mmol/l to 2.0 mmol/l at age
40).92 Alteration in HL activity may alter the
"threshold" seen in the plasma triglyceride - LDL-III
association. Thus, a high HL in a man may result in the generation of
small, dense LDL at plasma triglyceride less than 1.5
mmol/l. Conversely, an elevated plasma triglyceride may
cause LDL-III to form even in women with normal HL levels, eg, as in
pregnancy. If the model is correct, then the mechanistic implication is
that the formation of small-sized LDL depends on the rate of transfer
of triglyceride molecules into LDL (a function principally
of the concentration of triglyceride-rich lipoproteins in
plasma since CETP activity is not normally
rate-limiting93 ) and the rate of their hydrolysis
by HL. The susceptibility of LDL to cycles of triglyceride
enrichment and hydrolysis has been demonstrated in vitro by Lagrost et
al.82 The suggested existence of a threshold
indicates that these rates must achieve certain values before
significant quantities of normal-sized LDL are converted to their
smaller counterparts. Also, the symmetry of the mechanisms for
formation of small, dense species in LDL and
HDL81 89 94 helps to explain why low HDL levels
are a component of an ALP and why HDL cholesterol is
strongly inversely correlated with LDL
size.29 73 75 80
|
LDL Heterogeneity and CETP
|
|---|
CETP deficiency is an extremely rare inherited condition mainly
found
so far in the Japanese population. It is associated with high
levels
of HDL cholesterol (3 mmol/l to 4 mmol/l)
but relatively normal
levels of other plasma
lipoproteins.
95 Investigation of LDL
structure in
affected subjects revealed the presence of two
major species, one of
near normal size and the other much smaller
than
normal.
96 From the gradient gel electrophoresis
it appeared
that both co-existed throughout almost the entire LDL
density
range and IDL also showed two components on gradient gel
electrophoresis.
LDL were found to be cholesteryl ester poor and
triglyceride
rich and Sakai et al
96
proposed from these observations that
VLDL was secreted by the liver as
two discrete species, which
were delipidated through nonintersecting
pathways through IDL
to LDL. The metabolic model described
in Fig 1
accords with
this suggestion. They further postulated that the
small LDL
in their patients normally received cholesteryl ester from
HDL
via CETP and so maintained (or attained) a size comparable to
that
of the larger LDL species (Fig 4
). CETP may, therefore,
have a dual
action on the LDL subfraction distribution. That
is, it facilitates the
transfer of cholesteryl esters from HDL
to LDL and this may be the
major fate of this lipid in subjects
with low levels of
triglyceride-rich lipoproteins (the other
potential
acceptor for cholesteryl esters) and in the fasting
state. This action
favors the formation of larger LDL and as
noted above the
maintenance of such species in the circulation.
If, however,
plasma triglyceride levels rise CETP mediates other
transfers
that lead to a reduction in LDL size (Fig 4
). The intriguing
observation
that not all LDL is affected uniformly by CETP deficiency
may
be explained by the presence of metabolically distinct
species
in this density range.
Excess alcohol intake leads to a secondary, partial CETP deficiency and
has been shown to be associated with the appearance of multiple LDL
species on gge. Heavy drinkers with <25% of normal CETP activity had
small-sized as well as normal-sized LDL and on abstention from alcohol
the pattern normalized.97 The authors of this
report also noted that in obligate heterozygotes for CETP deficiency
where CETP levels were half normal the LDL pattern was not disturbed,
thus a marked decrease in activity was required before very small LDL
species were seen. It is of interest to note that the only other
patient group where LDL are so small is severe
hypertriglyceridemics69 98
and it is tempting to speculate that in this situation also there is a
markedly reduced transfer of HDL cholesteryl ester into LDL since VLDL
and chylomicrons are present in large quantities to act as
preferential acceptors. The role of CETP as a determinant of LDL size
has been explored in detail by Guerin et al99 and
Lagrost et al.100 Guerin et
al101 identified in experiments in vitro that it
was larger triglyceride-rich LDL subfractions that were the
preferential acceptors of CETP-mediated transfer of HDL cholesteryl
ester in plasma from normal subjects. Absence of transfer activity in
CETP deficiency presumably led to this LDL species in particular
becoming delipidated by the action of HL to small sized particles.
|
Genetic Influences on LDL Heterogeneity
|
|---|
LDL size pattern B appears to be an inherited trait. Austin
and
Krauss who have conducted many studies on this
topic
7 91 102 103 104 showed that about 30% of men
exhibit this pattern
and that in families its appearance was due to a
major gene
effect with a dominant or additive mode of inheritance.
There
was reduced penetrance in men under 20 years old and women under
50
years,
80 91 possibly explained by the model
postulated above.
Examination of families with familial combined
hyperlipidemia
revealed that transmission of pattern B
(with an allele frequency
of 0.25) was independent of plasma apoB
levels but closely linked
to the presence of elevated plasma
triglyceride.
102 Twin studies
indicated
that about a third to a half of the variation seen in the LDL
subfraction
profile was due to genetic factors with the remainder being
attributed
to hormonal, nutritional, and environmental
influences.
80 104 From the proposed model for the
generation of small, dense
LDL it is obvious that there are a
substantial number of candidate
genes that could be responsible for
inheritance of pattern B,
eg, variation in LpL or apoCIII may elevate
plasma triglyceride
above the threshold whereas variation
in HL or CETP may have
a more direct effect on LDL structure. Linkage
studies have
associated pattern B with polymorphisms in or near the
LDL receptor,
Apo AI-CIII-AIV, CETP, and manganese superoxide dismutase
loci[reviewed
in 8
Top
Abstract
Introduction
and ß) arise from different sources. Pool