569
Quantitative Studies of Transfer In Vivo of
Low Density, Sf 12-60, and Sf 60-400
Lipoproteins Between Plasma and
Arterial Intima in Humans
Munira Shaikh, Richard Wootton, B^rge G. Nordestgaard, Paul Baskerville,
John Stuart Lumley, Agnes E. La Ville, Jeremy Quiney, and Barry Lewis
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To assess the potential of various plasma lipoprotein classes to contribute to the lipid content
of the arterial intima, influx and efflux of these plasma lipoprotein fractions into and from the
intima of human carotid arteries were measured in vivo. While low density lipoprotein (LDL)
is known to transfer from plasma into the arterial wall, there is less information on the
atherogenic potential of lipoproteins of Intermediate density (Sf 12-60) or of very low density
(Sf 60-400). Aliquots of the same lipoprotein (LDL, Sf 12-60 lipoprotein particles, or Sf
60-400 lipoprotein particles) iodinated with iodine-125 and iodine-131 were injected intravenously 18-29 hours and 3-6 hours, respectively, before elective surgical removal of atheromatous arterial tissue, and the intimal clearance of lipoproteins, lipoprotein influx, and fractional
loss of newly entered lipoproteins were calculated. Intimal clearance of Sf 60-400 particles was
not detectable (<03 /ilxhr^xcm" 2 ), whereas the average value for both LDL and Sf 12-60
lipoprotein particles was 0.9 /ilxhr~lxcm~2. Since the fractional loss of newly entered LDL and
Sf 12-60 lipoprotein particles was also similar, the results suggest similar modes of entry and
exit for these two particles. However, due to lower plasma concentrations of Sf 12-60
lipoproteins as compared with LDL, the mass influx of cholesterol in the Sf 12-60 particles was
on the order of one 10th of that in LDL, and that of apolipoprotein B was about one 20th. The
present results suggest that elevated plasma concentrations of Sf 12-60 or "remnant"
lipoproteins share with LDL the potential for causing lipid accumulation in the arterial intima
in humans. (Arteriosclerosis and Thrombosis 1991;ll:569-577)
T
hat elevated plasma levels of low density lipoprotein (LDL) play a causal role in the
development of atherosclerosis is suggested
by epidemiological studies,12 clinical findings,3 controlled trials,4-5 and studies of the cell biology of the
arterial wall.6-7 The role of triglyceride-rich lipoproteins is far less clear. While clinical findings and
observations of cholesterol-fed animals suggest that
From the Department of Chemical Pathology and Metabolic
Disorders (M.S., B.G.N., A.E.L.V., J.Q.), United Medical and
Dental Schools of Guys and St. Thomas' Hospitals, the Department
of Medical Physics (R.W.), Hammersmith Hospital, the Department of Surgery (P.B., J.S.L.), St. Bartholomew's Hospital, and the
Rayne Institute (B.L.), St. Thomas' Hospital, London, U.K.
B.G.N. was supported by the Danish Heart Foundation and the
Danish Medical Research Council during these studies. The
authors acknowledge a research grant provided by Warner-Lambert/Parke-Davis, U.K.
Address for reprints: Dr. B.G. Nordestgaard, Department of
Clinical Chemistry, KK 3011, Rigshospitalet, Blegdamsvej 9, 2100,
Copenhagen, Denmark.
Received June 16, 1989; revision accepted November 30, 1990.
lipoproteins of intermediate density (IDL; remnant
particles; Sf 12-60 lipoproteins) have atherogenic
potential,89 the status of plasma triglyceride as a
causally important independent risk factor for atherosclerotic heart disease and, hence, the role of very
low density lipoprotein (VLDL) are controversial.
If the relative rates of transfer of these lipoproteins
between plasma and the arterial wall are a measure
of their potential for contributing to lipid deposition
and atherogenesis, then their measurement would
help to clarify the pathogenic significance of hypertriglyceridemia due to elevated levels of some or all
classes of triglyceride-rich lipoproteins. We have
used a newly described procedure10 to address these
issues by measuring influx in vivo of LDL, Sf 12-60
lipoprotein particles, and Sf 60-400 lipoprotein particles from plasma into the human atherosclerotic
intima and the fractional loss in vivo from the arterial
intima of newly entered lipoproteins belonging to
these lipoprotein classes.
570
Arteriosclerosis and Thrombosis Vol 11, No 3 May/June 1991
Methods
Patients
The participants were 18 patients undergoing elective carotid endarterectomy for atherosclerotic
stenoses who presented with transient ischemic attacks or amaurosis fugax. None had clinical evidence
of major primary or acquired disorders of lipoprotein
metabolism, although one (patient H.E.) possibly
had heterozygous familial hypercholesterolemia. Patients were entered into the various studies on a
consecutive basis, without selection according to
their lipoprotein distribution. Each patient gave written consent after being fully informed of the purpose
and protocol of the study. The protocol was approved
by the hospital's ethical committee.
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Radioiodination of Lipoproteins
The entire labeling procedure was performed under sterile conditions. Blood (100 ml) was drawn and
transferred into tubes containing 0.01% EDTA after
each patient had fasted overnight for 12 hours.
Plasma was promptly separated by centrifugation at
800g for 10 minutes at 4°C. Lipoprotein fractions of
Sf 60-400 and Sf 12-60 and LDL were isolated by
sequential preparative ultracentrifugation.1112 Particles with an Sf greater than 400 were removed by
centrifuging aliquots of 3 ml plasma overlayered with
2 ml of d=1.006 g/ml solution at 20,0O0g for 10
minutes at 4°C. The top fractions were aspirated and
discarded, and 3-ml aliquots of the bottom fractions
were overlayered with 2 ml of d=1.006 g/ml solution
and centrifuged at 105,000g for 110 minutes at 4°C to
isolate the Sf 60-400 lipoproteins in the top fraction.
To obtain Sf 12-60 lipoproteins, the bottom fractions, after isolation of lipoproteins with Sf greater
than 60, were adjusted to a final density of 1.019 g/ml
and centrifuged at 105,000g for 18 hours at 4°C. The
top fractions containing Sf 12-60 lipoproteins were
aspirated. LDL was isolated after flotation and removal of lipoproteins of d< 1.019 g/ml; the bottom
fractions were adjusted to d=1.063 g/ml and centrifuged at 105,0O0g for 20 hours at 4°C. The top
fractions containing LDL were aspirated. All lipoprotein fractions were washed by reultracentrifugation at their upper density limits to minimize contamination. After ultracentrifugation, greater than 90%
of the apolipoprotein B of the various lipoprotein
fractions remained intact as assessed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE).
The lipoprotein under investigation was separated
into two 2-ml aliquots; one was labeled with carrierfree [ ia I]NaI and the other with [131I]NaI (Amersham International, Buckinghamshire, U.K.) by the
iodine monochloride method of McFarlane13 as modified by Langer et al.14 Two hundred microcuries of
radiolabeled iodine was added to 0.5 ml lipoprotein
(protein concentration, 4-6 mg/ml), and a specific
activity of about 80 cpm/ng protein was obtained.
After passing the two radiolabeled lipoproteins
through Sephadex G-10 columns (20x1 cm; Pharmacia, Uppsala, Sweden), 2.5 ml of 10% pyrogen-free
sterile human albumin (Blood Products Laboratory,
Elstree, Hertfordshire, U.K.) was added to each dose
to minimize autoirradiation. The doses were subsequently dialyzed extensively against sterile saline containing 0.01% EDTA. In previous studies employing
the same labeling and purification procedures used in
this laboratory, it has been shown that apolipoprotein
B-100 in labeled LDL is homogeneous as assessed by
SDS-PAGE (4-10% gradient gels) and autoradiography.15-17 Immediately before intravenous injection,
the doses were passed through a 0.22-^m filter (Millipore U.K Ltd., Harrow, Middlesex, U.K).
Protocol
Approximately 25 ^.Ci of the autologous iodine125 dose was injected intravenously 18-29 hours
before the patient underwent surgery, and approximately 25 jtCi of the autologous iodine-131 dose was
injected intravenously 3-6 hours before surgery. The
exact dose injected was determined by weighing the
syringe before and after injection. Serial timed blood
samples (10 ml in tubes containing 0.1% EDTA)
were obtained at 10 minutes, 30 minutes, 1 hour, and
thereafter every 2-4 hours after administration of
the 125I dose, and at 10 minutes, 30 minutes, and
thereafter every hour after the administration of the
I31
I dose until the time of operation. The amounts of
radiolabeled lipoproteins remaining in the plasma 10
minutes after injection were 89.5±0.9%, 76±1.5%,
and 73.2±6.7% (mean±SEM) for LDL, Sf 12-60,
and Sf 60-400 lipoproteins, respectively.
The carotid endarterectomy specimen was received and processed immediately after resection. If
any media was attached, it was removed by gentle
cleavage from the intima and discarded. The intima
was then washed with 25 ml ice-cold saline 10 times
to remove adhering plasma. Most labile tissue radioactivity, presumably due to adhering plasma, was
removed by the first four to six washes (Figure 1).
Most of the remaining radioactivity was subsequently
extracted from the minced tissue as described below.
If appropriate, the specimen of intima was separated
into regions with macroscopically elevated atherosclerotic regions and the remaining area that comprised fatty streaks or macroscopically normal intimal tissue. Each region was weighed and its area
mapped by planimetry, after which the tissue was
washed and then finely minced with scissors. The
minced specimen was extracted overnight on a tube
rotator (Denley Spiramix 10, Denley Tech Ltd.,
Sussex, U.K.) at 4°C. Arterial tissue of individuals
given radiolabeled lipoproteins of Sf greater than 12
was extracted in a solution of d=1.006 g/ml and of
those given LDL, in a solution of d=1.019 g/ml.
Analyses
Plasma and arterial Sf 60-400 and Sf 12-60 lipoproteins and LDL were isolated by ultracentrifugation as described above using a tube slicer. Apolipo-
Shaikh et al
Lipoprotein Transfer Between Plasma and Arterial Wall
571
600
500
125-1 Ratfioactivity
400
E
a
u
131-1 Radioactivity
300
FIGURE 1. Bar graph showing removal of plasma contamination of human carotid intima by serial washing
(1-10). Radioactivity (cpm) represents the total counts per
minute in wash/extract of a single arterial sample.
200
o
o
m
E
•o
100
WuhM
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protein B in lipoprotein fractions from plasma and
arterial tissue and in the doses (to which cold LDL of
0.6 mg protein content was added) was precipitated
with redistilled 1,1,3,3-tetramethyl urea (Sigma
Chemical Co., Poole, Dorset, U.K.) by the method of
Kane et al.18 More than 90% of the radioactivity was
tetramethyl-urea precipitable. To remove lipids, the
precipitates were washed overnight at -20°C in
ethanol/ether (3:2, vol/vol) followed by three washes
with ether at 4°C. Since low counts were anticipated,
radioactivity in the washed precipitates was determined by counting each sample for 3x20 minutes in
a double-channel gamma counter (LKB Wallac
model 1280 Ultrogamma, Bromma, Sweden) to a
precision of 2.5% or better. Protein was determined
by the method of Lowry et al19 using bovine serum
albumin as a calibrator. Cholesterol and triglyceride
concentrations in plasma and lipoprotein fractions
were estimated by automated enzymatic methods
using Boehringer Mannheim Kit No. 237574 and
Wako Chemical Kit number 991-00405, respectively
(Boehringer Mannheim, Mannheim, F.R.G., and
Wako Chemicals GmbH, Neuss, F.R.G.).
Equivalency of I25I- and m[-Labeled
Lipoproteins
The calculation of influx and efflux requires that
lipoproteins of a given class (LDL, Sf 12-60, or Sf
60-400 lipoprotein particles) are transported into
and out of the arterial intima at the same rate when
iodinated with 123I or 131I. To test whether a lipoprotein labeled with the two isotopes behaved identically, plasma curves for the two labeled lipoprotein
preparations in each study were superimposed and
were found to be satisfactorily congruent (Figure 2).
The volume of distribution of 131I-labeled lipoprotein
was 97 ±2% (mean±SEM, N=18) of that obtained
with the 125I-labeled lipoprotein. Equivalency regarding arterial uptake of '"I-LDL and 131I-LDL has
previously been tested in cholesterol-fed rabbits.20 In
these studies, when albumin was added to the labeled
lipoproteins as in the present study, the arterial influx
of '%LDL was 98% of that of 125I-LDL.
Calculations
All calculations are based on radioactivity in apolipoprotein B of plasma and of arterial lipoproteins.
For studies in which iodinated LDL was injected
intravenously, radioactivity in plasma and arterial
LDL fractions was taken into consideration. For
studies on iodinated Sf 12-60 lipoprotein particles,
calculations were performed for Sf 12-60 apolipoprotein B radioactivity in plasma and arterial fractions. For studies on iodinated Sf 60-400 lipoprotein
particles, radioactivity in arterial Sf 60-400 apolipoprotein B was indistinguishable from background
counts. However, estimates of maximal possible intimal clearances were made using the minimum counts
that could have been detected by our method.
The arterial intima is assumed to be a single wellmixed pool for newly entered lipoproteins, that is,
lipoproteins that have entered the intima within the
experimental period. The intima is assumed to receive
plasma lipoproteins in proportion to their concentration in plasma and to be depleted of newly entered
lipoproteins in proportion to their concentration in
intima. No assumptions concerning the route (into
plasma, into media, or degraded or irreversibly attached in intima) or the mechanism by which newly
entered lipoproteins leave intima (as intact or degraded lipoproteins, or irreversibly attached lipoproteins to arterial components) are necessary. Furthermore, as usual in isotope experiments, it is assumed
that the labeled lipoproteins behave like unlabeled
lipoproteins and that the system under investigation is
in a steady state. The above assumptions have previously been validated and/or discussed.10
572
Arteriosclerosis and Thrombosis Vol 11, No 3 May/June 1991
dq a /d t =R i -R e =k i q p -k e q a
125-1 LDL
131-1 LDL
30 -
20 -
10
t
1
0
0
Time after Injection (hours)
20
where q, (counts per minute x centimeter"2) and qp
(counts per minute) are the contents of labeled
lipoproteins in the excised arterial intima and the
plasma volume, respectively.
Since the intimal content of labeled lipoproteins at
time zero is zero, the solution of this first-order
differential equation is
-M) Pexp(k T)q (T)dT
q a (t)=kjexp(-
e
p
Jo
where T is a dummy variable of integration. Since
this equation is valid for both a short-term (3-6
S» 12-60 lipoprotein
hours) exposure and a long-term (18-29 hours)
in
o
30 exposure with 131I- and 125I-labeled lipoproteins, reT3
D
125 I SI 12-60
spectively, two equations with two unknowns k, and k^
131-1 SI 12-60
•
CO
could be derived and solved as follows. Smooth
E
curves were fitted with cubic spline functions21-22 to
20 m
the observations of both 125I and 131I apolipoprotein B
a.
c
radioactivity of the plasma lipoproteins under inves<n
^ \
tigation. These curves were the functions qp(T).
c
10 'o
Based on initial estimates of the fractional uptake
o
and loss, k, and k,., respectively, the equations for 131I
Q.
o
and I25I radioactivity were integrated numerically
a.
0 with an adaptive integration routine.21-22 This gener0
1 0
20
•o
Time after Injection (hours)
ated two values for the content of 131I- and 12iI01
labeled lipoproteins in the intima, which were compared with those actually observed. An objective
CO
Sf 60-400 lipoprotein
function, the sum of the squared differences between
calculated and observed 131I and I25I radioactivity in
intima, was then minimized by adjusting k, and k^ by
the modified simplex method of Nelder and Mead.23
Intimal clearance of lipoproteins (microlitersx
hour"1 x centimeter"2) was calculated as the product of
fractional uptake (kj) and plasma volume (microliters).
Plasma volume was taken as the average volumes of
distribution of 125I- and 131I-labeled lipoproteins; this
was calculated by dividing the injected amounts of
labeled material by the concentrations of labeled material in plasma at time zero, obtained by extrapolation
Time after Injection (hours)
of the plasma curves. The influxes of apolipoprotein B
(micrograms x hour"1 x centimeter"2) and of lipoproFIGURE 2. Line plots showing superimposability of plasma
1
2
radioactivity-time curves in three patients injected with iodine- tein cholesterol (nanomolesx hour" x centimeter" )
125 (a)- and iodine-131 (^-labeled low density lipoprotein was the intimal clearance multiplied by the plasma
concentrations of lipoprotein apolipoprotein B
(upper panel), 5/12-60 lipoprotein (middle panel), or Sf
(microgramsxmilliliters" 1 ) and cholesterol
60-400 lipoprotein (lower panel).
(nanomolesxmilliliters"1), respectively. The calculation of lipoprotein cholesterol influx assumes that the
intimal clearances measured for apolipoprotein B are
1
2
The fractional uptake, kj (hour" x centimeter ),
also valid for the cholesterol moiety of the lipoproteins.
of plasma lipoproteins into the carotid intima and the
Stender and Zilversmit24 have provided evidence that
1
fractional loss, k,. (hour' ), of newly entered lipoproin cholesterol-fed rabbits, the cholesterol ester and
teins from the same intimal sample were calculated
protein of LDL enter the arterial intima in the same
10
as described by Wootton et al. On the principle of
proportions in which they are present in plasma LDL.
conservation of matter, the rate at which the intimal
Intimal clearance (microlitersxhour"'xcenticontent of labeled lipoproteins changes, dqjdu is the
meter"2) was also calculated by the "sink method," in
difference between the influx, R, (counts per
which the amount of radioactivity in apoliprotein B
minutexhour~'xcentimeter~ 2 ), and efflux, Re
(counts per minute x centimeter"2) in the intima is
(counts per minutexhour"'xcentimeter"2), of ladivided by the area beneath the plasma apolipoprobeled lipoproteins
K
Downloaded from http://atvb.ahajournals.org/ by guest on June 14, 2017
X
Shaikh et al LJpoprotein Transfer Between Plasma and Arterial Wall
573
tein B radioactivity-time curve (hour x counts per
minute x milliliters"1).
tima); that of Sf 12-60 lipoproteins was underestimated by 27±5%.
Statistics
Discussion
Using a recently developed procedure, the validity
of which has been tested previously,10 we have compared the influx into and fractional loss from human
atherosclerotic intima of LDL, Sf 12-60, and Sf
60-400 lipoproteins. To date, few quantitative studies of lipoprotein transfer have been made in humans; such studies have involved the measurement of
influx by the integral or sink method.25-29 To our
knowledge, efflux or fractional loss of lipoproteins
from human arterial intima has not been measured
previously.
The sink method does not take into account efflux
of labeled lipoproteins from the arterial wall during
the uptake period and may thus underestimate the
true influx of lipoproteins from plasma into the
arterial wall. The present investigations show that the
sink method underestimates lipoprotein intimal
clearance (and thus, lipoprotein influx) by 19-27%
after 3-6 hours' exposure of labeled lipoproteins to
the arterial wall when measured by use of iodinated
lipoproteins. Our results may be compared with
those of Bremmelgaard et al,29 in which the sink
method was employed and in which average total
plasma cholesterol influx into the intima of lesioned
human abdominal aortas was 3.6 nmolxhr~ 1 xcnr 2 .
Since high density lipoprotein appears to contribute
about 40% to plasma cholesterol influx into the
human intima,28 the total cholesterol influx from
VLDL plus LDL in the article by Bremmelgaard et
al29 would be 2.2 nmolxhr"'xcm"2. Considering the
large interindividual variation in arterial influx measurements, this compares well with our average LDL
and Sf 12-60 lipoprotein cholesterol influxes of 6.2
and 0.5 nmolxhr"'xcm"2, respectively. An earlier
study from this laboratory employing the sink method
has shown lower mean values for minimum influx
compared with our present estimates of absolute
influx.26 The disparity may reflect the fact that "minimum influx" (the difference between influx and efflux) underestimates true influx even with a mean
3-hour experimental period for VLDL and 25 hours
for LDL. Nevertheless, the results were concordant in
that LDL minimum influx greatly exceeded that of
IDL. Our data suggest that Sf 60-400 lipoproteins do
not contribute importantly to cholesterol influx, since
the intimal clearance was less than 0.3 ^lxhr~'xcm" 2 .
The lipoprotein content of the arterial wall reflects
the balance between the rates at which lipoproteins
enter and leave the arterial wall and are degraded
within it. Ghosh et al30 found an average fractional
loss of LDL from aortic fatty streaks of cynomolgus
monkeys of 0.09 hr"1. This compares with our average fractional loss for LDL of 0.12 hr"1. Interestingly,
Schwenke and Zirversmit31 did not find any loss of
labeled cholesteryl ester from uninjured rabbit aortas
but did find an average fractional loss from injured
Values are given as mean±SEM. Differences
between groups were evaluated by the unpaired
Student's t test.
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Results
Table 1 gives the clinical details and lipoprotein
levels of the patients studied. Mean plasma LDL
apolipoprotein B and cholesterol concentrations
were 13 and nine times higher, respectively, than the
plasma Sf 12-60 apolipoprotein B and cholesterol
concentrations.
The kinetics of transfer in vivo of LDL, Sf 12-60,
and Sf 60-400 particles between the plasma and
carotid arterial intima are shown in Table 2. Details
of lipoprotein transfer in each patient are given in
Table 3. In patients studied for the transfer of LDL,
the fractional uptake, intimal clearance, and influx
(of cholesterol and apolipoprotein B) appeared
greater in elevated than in nonelevated regions of the
carotid intima of these patients although the difference was not formally statistically significant. Fractional loss of newly entered LDL was similar in the
two regions (Table 2).
Influx of LDL apolipoprotein B and cholesterol
into specimens with elevated lesions were 19-fold
and 16-fold greater, respectively, than those of the Sf
12-60 class (p<0.05 andp=0.05, respectively; compare Table 2). However, the intimal clearances of
LDL and Sf 12-60 lipoprotein, which normalized for
differences in plasma concentrations, were similar.
Fractional loss of newly entered lipoproteins from
the carotid intima was also similar for these two
classes of lipoproteins (Table 2).
For the two patients who received labeled Sf
60-400 lipoprotein, the intimal radioactivity was not
significantly different from zero (Table 2). By our
method, in these two patients it would have been
possible to detect intimal clearances of 0.3 and 0.1
^lxhr~'xcm" 2 , respectively (Table 3). Thus, it appears that in human carotid intima, the intimal
clearance of Sf 60-400 lipoprotein particles is less
than 0.3 ^lxhr'xcm" 2 .
LDL apolipoprotein B influx into elevated regions
of the arterial intima showed a positive correlation
with plasma LDL apolipoprotein B concentration
(r=0.89, p<0.01; the positive correlation between
influx and plasma level of LDL cholesterol was also
significant: r=0.93,/><0.001). No significant correlation was observed between apolipoprotein B and
cholesterol influxes of LDL in nonelevated regions or
of the Sf 12-60 lipoprotein particles and their respective plasma concentrations.
When the sink method was used to calculate
intimal clearance using values for the short-term
(3-6 hours) exposure to 13lI-labeled lipoproteins,
intimal clearance of LDL was underestimated by
19±3% (mean±SEM; elevated plus nonelevated in-
54
65
66
59
71
59
55
60
55
68
47
57
62
64
61
61
62
62
59
85
60
5.4
8.8
7.1
6.8
5.6
6.3
6.9
4.2
6.0±0.5
8.7
6.1
5.9
13.1
6.6
7.8
7.7
6.0
55
5.3
6.6
7.2±0.7
Plasma
1.0
1.4
1.2
1.2
1.0
1.9
1.1
1.0
1.2±0.2
1.5
1.0
1.2
1.2
0.9
1.1
1.4
1.7
1.3
1.3
1.9
1.3±0.1
HDL
3.1
4.6
3.9
3.7
3.5
3.8
5.4
1.2
3.5±0.7
6.4
3.6
3.5
10.8
4.6
5.8
5.9
4.0
3.8
3.4
3.9
5.1±0.7
LDL
0.8
1.5
1.3
1.1
0.9
0.9
0.5
0.4
0.4
0.6
0.8
0.8±0.1
VLDL+IDL
0.1
03
0.2
0.4
05
0.2
0.3
1.9
0.5±0.3
Sf 12-60
Total cholesterol (mmoiyi)
2.0
2.0
2.6
0.9
1.9
3.0
2.0
1.6
2.7
1.9
1.9
2.1±0.2
1.2
25
1.9
1.1
3.1
2.2
1.0
1.5
1.5
0.7
1.2
1.7
1.2
0.7
1.6
0.9
0.8
1.2±0.1
Plasma VLDL+IDL
1.4
1.5
2.1
0.5
1.4
0.4
1.2
0.2
1.1
0.1
1.4±0.2
0.7±0.3
Sf >60
0.1
0.2
0.2
0.2
1.5
0.2
0.2
0.2
0.5±0.3
Sf 12-60
Trigrycerides (mmol/1)
h density lipoprotein; LDL, low density lipoprotein; VLDL, very low density lipoprotein; IDL, intermediate density lipoprotein.
poprotein studies
F
58
63
M
54
80
F
F
F
M
M
70
63
63
82
80
61
75
69
46
43
60
weight (kg)
oprotein studies
M
M
M
M
M
F
M
M
F
F
F
Age
(yr)
linical and LJpoprotein Findings in Study Participants
ex
\At
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1.5
2.4
0.6
0.8
0.3
0.7
0.4
0.4
0.5±0.1
Sf >60
691 ±73
547
486
684
472
685
914
691
1,270
430
619
798
LDL
53±S
56
48
28
55
79
Sf 12-60
Sf
Apolipoprotein B (
Shaikh et al Lipoprotein Transfer Between Plasma and Arterial Wall
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aortas of 0.06 hr '. The fractional loss measured by
our method is assumed to be a combination of 1)
efflux of lipoprotein from the intima into plasma, the
lumen, or the vasa vorum, 2) the metabolism of
apolipoprotein B by the cells of the arterial wall, and
3) lipoprotein that becomes nonexchangeable, that is,
by forming complexes with glycosaminoglycans of the
arterial wall. The above processes may have different
implications for atherogenesis, with the second and
possibly the third processes contributing to cholesterol delivery to the intima and the first process
decreasing it. These processes are therefore likely to
contribute, to different degrees, to the calculated
fractional loss in the artery. Further studies are
needed to identify the fate of lipoproteins entering
the arterial wall.
The early clearance of about one fourth of Sf
12-60 and Sf 60-400 apolipoprotein B from plasma
may not be attributable to partial denaturation of
labeled apolipoprotein B.15"17 Other explanations for
early clearance of these particles could be the catabolism of these particles by lipolysis, transfer into
extravascular space, or segregation within the circulation, that is, in the liver. The present influx and
efflux calculations were based on the specific activity
of particles isolated in the same flotation range from
arterial tissue and from plasma; rapid lipolytic conversion of injected lipoproteins to denser particles is
therefore unlikely to influence our estimates.
The cholesteryl ester content of the artery has
been considered as a mixture of an inert pool of
cholesterol along with a labile pool of cholesteryl
ester entering the wall after injury to the artery.31
Similarly, the apolipoprotein B content of the intima
may be representative of two such pools, the labile
one comprising "newly entered" lipoproteins. In the
present study, it was not possible to measure the
intimal apolipoprotein B content derived from newly
entered lipoproteins, and therefore efflux in terms of
microgramsx hour"'x centimeter"2 could not be calculated. Hence, as seen from Tables 2 and 3, the
product of fractional loss (k,.) and the total apolipoprotein B content of the intima exceeds the corresponding influxes. However, assuming influx equals
efflux, the pool of newly entered lipoproteins
(microgramsxcentimeter~2) can be calculated as influx (micrograms x hour"'x centimeter"2) divided by
k, (hour"1).
In the present investigations, although the absolute
influx into elevated lesions of LDL cholesterol and
LDL apolipoprotein B was 19-fold and 16-fold
higher than that of the Sf 12-60 lipoproteins, intimal
clearance and fractional loss of the two classes of
lipoproteins were similar. This suggests that LDL
and Sf 12-60 lipoproteins enter and leave the arterial
wall by similar mechanisms. On comparing the relative rates of uptake of VLDL, LDL, and high density
lipoprotein by rabbit aortas, Stender and Zilversmit24
have also suggested similar mechanisms for uptake of
these particles.
NE
NE
NE
NE
NE
NE
E
E
E
E
E
E
E
E
E
E
pe of
laque
1.16
1.97
2.94
190
610
460
950
580
750
740
420
580
800
4.20
6.20
4.30
300
1,050
190
350
700
82
360
2.40
2.00
2.20
3.30
278
56
(Sf 60-400 apo B)
231
331
222
81
45
45
76
104
(Sf 12-60 apo B)
857
186
234
263
311
138
501
330
899
208
30
180
366
234
104
(LDL apo B)
45
Apolipoprotein B
concentration
(Mg/g)
ND*
<0.32t
<0.14t
<0.34t
<0.09f
0.57
1.48
0.50
ND*
ND*
ND*
2.04
201
401
142
1.28
0.41
439
106
699
217
0.21
0.84
0.59
0.76
0.21
295
303
54
54
0.36
0.52
1.53
1.27
3.99
357
1,278
97
109
459
1.90
0.67
548
322
0.45
0.11
1.79
0.49
136
144
29
622
0.16
0.29
100
71
76
84
67
60
66
59
96
88
88
76
71
65
72
88
89
86
71
0.4
0.9
1.0
0.1
0.3
0.6
0.2
0.04
0.03
0.11
0.02
0.07
0.03
0.1
0.8
0.07
0.02
0.11
2.3
0.40
0.37
2.6
3.1
6.1
2.4
4.5
43.1
1.7
12.2
0.36
0.72
0.55
5.07
0.24
1.63
1.50
0.42
0.5
10.3
0
0
0
0
0
0
0
0
0
0
0
0
0.08
0
4.9
0.57
73
66
96
0
1.7
0.21
0
Fracti
0.6
1.9
Cholesterol influx
(nmolxhr'xcm- 2 )
0.10
0.23
Apolipoprotein
B influx
(/xgxhr-'xcnT 2 )
81
89
Sink assumption
(% of k,x plasma
volume)
Intimal clearance
k,x plasma
volume (/xlx
hr-'xcm- 2 )
77
84
(hr'xcrn"2)
Fractional
uptake, lC|Xl0-«
density lipoprotein; apo, apolipoprotein; NE, nonelevated lesions; E, elevated lesions.
al uptake and loss were not calculated since arterial Sf 60-400 apolipoprotein B radioactivity was not detectable (ND).
clearance that could have been measured by our method.
nts fractional uptake of lipoprotein from the plasma pool; kj represents fractional loss of newly entered lipoprotein from intima. Short-term (3-6 hours) exposure was used to
rance by the sink method.
NE
E
E
poprotein studies
170
NE
300
1,000
2.70
2.20
5.90
4.30
1.50
300
250
NE
NE
NE
E
E
E
E
E
940
540
7.65
1350
proteiri studies
4.38
5.20
3.09
2.68
4.84
2.57
4.26
8.18
2.64
2.26
4.20
3.13
Area
(cm2)
Intima
1,350
200
70
650
230
Wet
weight
(mg)
ransfer In Vivo of Plasma Lipoprotelns Between Plasma and Intima of Carotid Arteries in Each Patient
Downloaded from http://atvb.ahajournals.org/ by guest on June 14, 2017
Shaikh et al
Lipoprotein Transfer Between Plasma and Arterial Wall
LDL influx into the intima is greater due to its
greater concentration in plasma compared with that
of Sf 12-60 lipoprotein. Nevertheless, the present
findings suggest that at elevated plasma concentrations, Sf 12-60 lipoprotein may share with LDL the
potential for mediating cholesterol transfer into the
arterial intima. Thus, in conditions where the latter
class of lipoproteins accumulate in plasma, such as
familial dysbetalipoproteinemia (type III hyperlipidemia), or secondary hyperlipidemic conditions, such
as diabetes mellitus and chronic renal failure,32 the Sf
12-60 lipoprotein class may contribute substantially
to lipid accumulation in the intima. Levels of Sf
12-60 lipoprotein are influenced by dietary cholesterol; hence, persons who are hyperresponsive to
cholesterol may be particularly at risk.
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KEY WORDS • low density lipoproteins • intermediate density
lipoproteins • very low density lipoproteins • arterial
wall-lipoprotein interaction • arterial influx/efflux of lipoproteins
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Quantitative studies of transfer in vivo of low density, Sf 12-60, and Sf 60-400 lipoproteins
between plasma and arterial intima in humans.
M Shaikh, R Wootton, B G Nordestgaard, P Baskerville, J S Lumley, A E La Ville, J Quiney and
B Lewis
Arterioscler Thromb Vasc Biol. 1991;11:569-577
doi: 10.1161/01.ATV.11.3.569
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville
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