(Arteriosclerosis, Thrombosis, and Vascular Biology. 1998;18:655-664.)
© 1998 American Heart Association, Inc.
Familial HDL Deficiency Characterized by Hypercatabolism of Mature ApoA-I but Not ProApoA-I
Rami Batal;
Michel Tremblay;
Larbi Krimbou;
Orval Mamer;
Jean Davignon;
Jacques Genest, Jr;
; Jeffrey S. Cohn
From the Hyperlipidemia and Atherosclerosis Research Group and the
Cardiovascular Genetics Laboratory (J.G.), Clinical Research Institute of
Montréal (R.B., M.T., L.K., J.D., J.S.C.); and the McGill University
Biomedical Mass Spectrometry Unit (O.M.), Montréal, Québec,
Canada.
Correspondence to Dr Jeffrey S. Cohn, Hyperlipidemia and Atherosclerosis Research Group, Clinical Research Institute of Montréal, 110 Pine Ave W, Québec, Canada, H2W 1R7.
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Abstract
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AbstractWe have previously
described patients with familial high density lipoprotein (HDL)
deficiency (FHD) having a marked reduction in the plasma concentration
of HDL cholesterol and apolipoprotein (apo) A-I but lacking
clinical manifestations of Tangier disease or evidence of other known
causes of HDL deficiency. To determine whether FHD in these individuals
was associated with impaired HDL production or increased HDL
catabolism, we investigated the kinetics of plasma apoA-I and apoA-II
in two related FHD patients (plasma apoA-I, 17 and 37 mg/dL) and four
control subjects (apoA-I, 126±18 mg/dL, mean±SD) by using a primed
constant infusion of deuterated leucine. Kinetic analysis of
plasma apolipoprotein enrichment curves demonstrated that mature plasma
apoA-I production rates (PRs) were similar in patients and
control subjects (7.9 and 9.1 versus 10.5±1.7 mg ·
kg-1 · d-1). Residence times (RTs) of
mature apoA-I were, however, significantly less in FHD patients (0.79
and 1.66 days) compared with controls (5.32±1.05 days). Essentially
normal levels of plasma proapoA-I (the precursor protein of apoA-I) in
FHD patients were associated with normal plasma proapoA-I PRs (7.8 and
10.4 versus 10.9±2.6 mg · kg-1 ·
d-1) and proapoA-I RTs (0.18 and 0.15 versus 0.16±0.03
day). The RTs of apoA-II were, however, less in patients (3.17 and 2.92
days) than control subjects (7.24±0.71 days), whereas the PRs of
apoA-II were similar (1.8 and 1.9 versus 1.7±0.2 mg ·
kg-1 · d-1). Increased plasma
catabolism of apoA-II in FHD patients was associated with the presence
in plasma of abnormal apoA-IIHDL (without apoA-I). These results
demonstrate that FHD in our patients is characterized, like Tangier
disease, by hypercatabolism of mature apoA-I and apoA-II, but unlike
Tangier disease, by essentially normal plasma catabolism and
concentration of proapoA-I.
Key Words: cholesterol kinetics atherosclerosis reverse cholesterol transport hypoalphalipoproteinemia
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Introduction
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Epidemiological
studies have consistently demonstrated that low plasma HDL
levels are associated with the presence of coronary artery
disease.1 2 This association has been attributed
to a role of HDL in mediating reverse cholesterol
transport.3 HDL has other potentially
antiatherogenic functions, however, such as inhibiting LDL
oxidation,4 reducing the expression of
endothelial cell adhesion
molecules,5 and inhibiting platelet
aggregation.6
ApoA-I and apoA-II are the major structural proteins of
HDL.3 ApoA-I (28 kDa) is synthesized in the liver
and intestine as a pre-propeptide.7 It is
processed cotranslationally to form proapoA-I, which is secreted into
the circulation. A metalloenzyme rapidly cleaves six amino acids from
its amino terminus, leading to the formation of mature apoA-I (243
amino acids),8 which represents the
majority (>95%) of total plasma apoA-I. ApoA-I is believed to be
cleared from the circulation in an almost lipid-free form by
glomerular filtration and tubular reabsorption in the
kidney.9 ApoA-II (17 kDa) is synthesized
predominantly by the liver10 and is secreted in
plasma either as a propeptide, which is readily cleaved, or directly as
mature apoA-II.11 ApoA-II is found in association
with apoA-I in the plasma of normolipidemic subjects (on particles
designated LpA-I:A-II), whereas apoA-I is also present in particles
not containing apoA-II (LpA-I).12 Kinetic studies
in humans have shown that the variation in plasma apoA-I level is
determined by the FCR of apoA-I,13 14 whereas the
variation in plasma apoA-II levels, as well as the distribution of
apoA-I between LpA-I and LpA-I:A-II particles,15
is determined by the rate of production of apoA-II. At the same
time, FCRs of both apoA-I and apoA-II have been found to be inversely
correlated with HDL cholesterol
levels.16
Human HDL deficiency (hypoalphalipoproteinemia) defines a group of
dyslipidemias characterized by an HDL
cholesterol level below the 10th percentile for age- and
sex-matched subjects.17 HDL deficiency has been
shown to be the result of: (1) apoA-I gene abnormalities caused by
deletion, inversion, insertion, nonsense, or missense mutations; (2)
apoA-II deficiency; (3) complete or partial absence of LCAT activity,
as seen in classic LCAT deficiency or fish-eye disease; (4) increase in
cholesteryl ester transfer protein activity; or (5) severe
hypertriglyceridemia (reviewed in
References 18 and 1918 19 ). HDL deficiency is also a major characteristic of
Tangier disease, wherein an abnormality in either HDL-mediated
cholesterol and phospholipid efflux from
peripheral cells20 21 and/or a defect
in HDL particle interconversion22 have been
suggested as possible metabolic causes.
Severe familial FHD has recently been described in three
French-Canadian kindreds as a trait with autosomal codominant
inheritance.23 Two members of one particular
kindred, who were extensively investigated, had normal fasting
triglyceride concentrations with HDL
cholesterol levels below the 5th percentile. One of them
had evidence of coronary artery disease. Both patients had a
50% to 80% reduction in plasma apoA-I concentration, a decrease in
average HDL particle size, and a relative increase in plasma proapoA-I
levels. No evidence was obtained for the presence of an apoA-I or
apoA-II gene abnormality, and the FHD patients had none of the
lipoprotein lipase gene mutations commonly found in French
Canadians.24 LCAT activity was normal in the FHD
patients. Finally, none of the patients had clinical manifestations of
Tangier disease,23 eg, cholesteryl ester
deposition in reticuloendothelial tissues, hyperplastic
orange tonsils, splenomegaly, or relapsing neuropathy.
To investigate the plasma kinetics of HDL apolipoproteins (proapoA-I,
mature apoA-I, and apoA-II) in the aforementioned FHD patients, we
carried out a stable-isotope kinetic study in two FHD and four
normolipidemic control subjects. Our aim was to determine whether
catabolism of apoA-I was greatly increased in these individuals, as
previously documented for Tangier disease
patients25 and other HDL deficiency
states,26 27 28 29 30 31 32 or whether production of
apoA-I was impaired.
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Methods
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Subjects
Six male subjects were investigated in the present study:
two brothers with FHD and four healthy control subjects (Table 1
). The medical history of the two
affected brothers has been described
previously.23 In brief, patient 1 (proband
24430301) was diagnosed with coronary artery disease at the
age of 42. When diagnosed, he had a history of high blood pressure and
he had been a smoker. His HDL cholesterol concentration had
been known to be low, but he had no signs of abnormal liver function or
clinical manifestations of Tangier disease. He underwent
percutaneous transluminal coronary angioplasty
at the age of 42 years and subsequently had coronary artery
bypass surgery at the age of 48. Patient 2 (24430313) did not have
evidence of coronary artery disease, nor did he show clinical
signs of Tangier disease. HDL cholesterol concentrations in
the two patients, measured during routine visits to the lipid clinic of
the Clinical Research Institute of Montreal, were consistently
found to be <0.25 mmol/L. Both patients were not taking any
medications known to affect plasma lipid levels, and their HDL
deficiency was not due to known causes.23 Four
healthy males acted as controls subjects. They had no evidence or
history of dyslipidemia, diabetes mellitus, or any other
metabolic disorder and were not taking any medications
known to affect plasma lipid levels. Only one of them, subject 4, was a
smoker (<1 pack/d). All six subjects gave informed consent to the
study protocol, which was approved by the ethics committee of the
Clinical Research Institute of Montreal.
Infusion Protocol
The in vivo measurement of plasma apolipoprotein kinetics was
carried out as described previously.33 After a
12-hour overnight fast, subjects were given an injection of 10
µmol/kg of body weight of
[D3]L-leucine
([D3]L-leucine 98%, Cambridge
Isotope Laboratories) dissolved in physiological
saline (0.9% NaCl) via an intravenous line attached to a
left forearm vein. After the bolus injection, subjects were infused for
12 hours with 10 µmol · kg-1
· h-1 of
[D3]L-leucine dissolved in
physiological saline. The infusion was carried out
using a volumetric pump (Life Care Pump model 3, Abbott) set to deliver
48 mL of infusate per hour. Subjects were not given food during the
time course of the infusion but had free access to drinking water. They
were encouraged to move around to maintain good blood circulation.
Blood samples (20 mL) were collected from an antecubital vein at
regular intervals (0, 15, 30, and 45 minutes and 1, 1.5, 2, 2.5, 3, 4,
5, 6, 7, 8, 9, 10, 11, and 12 hours) in tubes containing EDTA to a
final concentration of 0.1%. Samples were kept on ice, and plasma was
immediately separated by centrifugation at 3500 rpm for
15 minutes at 4°C. An antimicrobial agent
(NaN3) and a protease inhibitor
(aprotinin) were added to plasma to give a final concentration of
0.02% and 1.67 µg/mL, respectively.
Isolation of Lipoproteins and Apolipoproteins
VLDL, IDL+LDL, and HDL were isolated from 5 mL plasma by
sequential ultracentrifugation in an XL-90
ultracentrifuge using a 50.4 Ti rotor (Beckman) at 50 000 rpm
for 10 hours at densities (d) of 1.006, 1.019, and 1.21
g/mL, respectively. Total lipoproteins were isolated from plasma by
ultracentrifugation (50 000 rpm, 10 hours) of 1 mL of
plasma, adjusted to d=1.25 g/mL with KBr. Lipoproteins
were recovered in the supernatant by tube slicing. VLDL apoB-100 was
isolated by preparative SDSpolyacrylamide gel electrophoresis
on a 4% to 22.5% gradient gel.33 Plasma apoA-I
and apoA-II were isolated by preparative IEF on 7.5%
polyacrylamideurea (8 mol/L) gels (pH gradient, 4 to 7) of
apolipoproteins in total plasma lipoproteins (d<1.25
g/mL).34 Fractions were dialyzed against 10
mmol/L ammonium bicarbonate, preincubated with cysteamine
(ß-mercaptoethylamine, Sigma-Aldrich) in a ratio of 6 mg for
every 1 mg of protein for 4 hours at 37°C, and then delipidated.
Protein bands were identified on gels by Coomassie blue staining. The
aim of cysteamine treatment was to separate proapoA-I from apoE3 and
apoA-II from asialylated apoC-III (apoC-III0),
which normally comigrate to the same positions on IEF gels. Cysteamine
introduces an amino group to the single cysteine residues of
apoE335 and apoA-II but does not affect proapoA-I
and apoC-III0, since these proteins do not
contain cysteine. Cysteamine-modified apoE3 and apoA-II thus migrated
to a higher position on the gel due to their increased positive charge
(Fig 1
).

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Figure 1. Separation by IEF gel electrophoresis of
apolipoproteins in cysteamine-treated (+) and nontreated (-) VLDL
(d<1.006 g/mL) and total plasma lipoprotein
(d<1.25 g/mL) fractions. Chemical modification by
cysteamine treatment (Reference 34) caused amino groups to be added to
cysteine residues in apoE3 and apoA-II, resulting in a single, positive
charge increase and migration of these proteins to a higher position in
IEF gels. Cysteamine-treatment of apoE3 and apoA-II in
d<1.25 g/mL fractions allowed these proteins to be
separated from proapoA-I and apoC-III, as indicated in the right-hand
gels. The major isoform of mature apoA-I (apoA-Io), which
was excised for stable-isotope analysis, is also
indicated.
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Separation of Lipoproteins by Two-Dimensional Gel
Electrophoresis
Plasma lipoproteins were separated by two-dimensional
nondenaturing gel electrophoresis, essentially as described
previously.36 Agarose strips (
7.5 cm long)
containing electrophoretically separated lipoproteins were positioned
at the top of 3% to 24% nondenaturing concave gradient
polyacrylamide gels and sealed with agarose. Molecular-size
markers (Pharmacia) labeled with radioiodine were also separated on
each gel. They were equilibrated by a prerun for 20 minutes at a
constant voltage of 125 V. Samples were pre-electrophoresed at 70 V for
1 hour and then separated for 24 hours at 125 V. After electrophoresis,
lipoproteins were transferred (180 mA, 24 hours) to a nitrocellulose
membrane (0.4-µm pore size). Fixing, blocking, and immunolocalization
steps of apoA-Iand apoA-IIcontaining lipoproteins were performed as
described36 by using immunopurified polyclonal
anti-human apoA-I antibody and monoclonal anti-apoA-II antibody
(125I labeled). Plasma without apoA-Icontaining
lipoproteins was prepared by immunoaffinity
chromatography by using antiapoA-I latex (Genzyme).
Plasma (200 µL) was added to 1 mL of anti-apoA-I latex suspension,
gently mixed for 15 minutes at room temperature, and then
centrifuged at 12 000 rpm for 10 minutes. The infranatant,
which contained nonlatex-bound plasma now devoid of apoA-I, was
concentrated by using Centricon-10 concentrators (Amicon) before
separation by electrophoresis.
Plasma Lipids and Apolipoproteins
Plasma and lipoprotein fractions were assayed for total (free
and esterified) cholesterol and triglyceride
with a Cobas Mira-S automated analyzer (HoffmanLa Roche)
using enzymatic reagents. Plasma apoB concentration was measured by
noncompetitive ELISA using an immunopurified goat anti-human apoB
antibody and horseradish peroxidaseconjugated monoclonal
antibody.37 Plasma apoA-I concentration was
measured by nephelometry on a Behring nephelometer 100 (Behring) using
the Behring protocol and reagents. Plasma apoA-II was measured by
nephelometry in the laboratory of Dr Linda Bausserman (Meriam Hospital,
Brown University, Providence, RI).38 Plasma apoE
and apoC-III concentrations were measured by ELISAs developed in our
laboratory.39 40 ApoE phenotype was
determined by IEF of delipidated VLDL.34
Quantification of ProApoA-I
Mature apoA-I and proapoA-I concentrations in plasma of
normolipidemic and FHD subjects were derived from total plasma apoA-I
concentrations measured by nephelometry. The proportion of each apoA-I
isoform contributing to total plasma apoA-I was determined by GC-MS and
densitometric scanning of IEF gels. The amount of leucine associated
with the major mature apoA-I IEF band (apoA-I0)
and the major proapoA-I band (isoform
apoA-I+2)41 was determined
by comparing the areas under the peaks of leucine and norleucine (an
internal standard; see the following section) separated by GC-MS. The
amount of apoA-I protein present in each band was then calculated
as
The amount of protein in minor mature apoA-I bands (isoforms
apoA-I-1 and apoA-I-2) and
the minor proapoA-I band (isoform
apoA-I+1)41 was then
estimated by measuring the relative amounts of these isoforms by IEF
gel scanning densitometry. Plasma concentration of proapoA-I was then
calculated as
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Determination of Isotopic Enrichment in Isolated
Apolipoproteins
Apolipoprotein bands as well as blank (nonprotein-containing)
gel slices were excised from polyacrylamide gels (VLDL apoB-100
from SDS-polyacrylamide and apoA-I and apoA-II from
IEF).33 Each slice was added to a borosilicate
sample vial containing 600 µL of 6N HCl and an internal standard of
250 ng norleucine (Sigma-Aldrich) dissolved in 50 µL
double-distilled water. Gel slices were hydrolyzed at 110°C for 24
hours, cooled to -20°C for 20 minutes, and centrifuged at
3500 rpm for 5 minutes. Free amino acids in the hydrolysate were
separated from precipitated polyacrylamide, purified by
cation-exchange chromatography using AG 50 W-X8 resin
(Bio-Rad), and derivatized by treatment with 200 µL of acetyl
chlorideacidified 1-propanol (1:5, vol/vol) for 1 hour at 100°C and
50 µL of heptafluorobutyric anhydride (Supelco) for 20 minutes at
60°C.33 Enrichment with deuterated leucine was
determined by GC-MS (Hewlett-Packard, 5988 GC-MS) using negative
chemical ionization and methane as the reagent gas. Selective ion
monitoring at m/z=352 and 349 (ionic species
corresponding to derivatized deuterated and derivatized nondeuterated
leucine, respectively) was performed, and the tracer to tracee ratio
was calculated from the isotopic ratio in each sample according to the
formula derived by Cobelli et al42
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where z(t) is the tracer to tracee ratio
at time t, r(t) is the isotopic ratio
at time t, and rN and rI are the
naturally occurring and infusate isotopic ratios, respectively.
r(t) was obtained from the ratio of the areas
under the peak of leucine ionic species m/z=352 and 349. The
ratios of the areas under the peak of the ionic species
m/z=349 of leucine and the internal standard (norleucine)
detected in blank samples were used to correct for background
(method-introduced) leucine, which was 0.44±0.27%, 6.04±2.06%, and
4.12±2.96% (mean±SD) of total leucine in mature apoA-I, proapoA-I,
and apoA-II samples, respectively.
Kinetic Analysis
Tracer to tracee ratios of VLDL apoB-100, mature plasma apoA-I,
proapoA-I, and apoA-II were fitted to a monoexponential
function using SAAM II computer software (SAAM II
Institute). The function was defined as
z(t)=Zp{1-e[-k(t-d)]},
where z(t) was the tracer to tracee ratio at time
t, Zp was the tracer to tracee ratio of the
tissue precursor amino acid pool from which the protein in question was
derived (estimated from VLDL apoB-100 and proapoA-I enrichment curves
at plateau; see below), d was the delay time in hours, and
k was the FPR (pools per hour). RT was calculated as the
reciprocal of FPR (1/FPR), and the absolute PR in (in milligrams per
kilogram per day) was calculated as
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where pool size=plasma concentration (mg/dL)xplasma volume
(0.045 L/kg).
The tracer to tracee ratios (Zp) of the intestinal and
hepatic precursor amino acid pools from which proapoA-I and hence
apoA-I were derived were taken to be the enrichment of proapoA-I at
plateau. Since apoA-II, like apoB-100, is predominantly of hepatic
origin, the enrichment of the apoA-II precursor pool was taken to be
the enrichment of VLDL apoB-100 at plateau.
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Results
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Plasma Lipids and Apolipoproteins
Plasma concentrations of lipids and apolipoproteins in
subjects on the day of the infusion are shown in Table 1
. The two brothers with FHD had plasma
HDL cholesterol concentrations of 0.19 and 0.27
mmol/L, which were, on average, 18% and 26% of HDL
cholesterol concentrations in control subjects. Their
plasma concentrations of apoA-I and apoA-II were 14% and 29% (for
apoA-I) and 46% and 44% (for apoA-II) of control values,
respectively. Total plasma triglyceride, VLDL
triglyceride, and apoB concentrations were slightly higher
in FHD patients than in control subjects, whereas plasma apoE levels
were slightly lower. Total plasma cholesterol and apoC-III
levels were within the normal range.
The separation by IEF gel electrophoresis of proapoA-I isoforms,
mature apoA-I isoforms, and apoA-II from total plasma lipoproteins
(d<1.25 g/mL) of two control subjects and two FHD
patients is shown in Fig 2
.
ApoA-I+2 and apoA-I+1
correspond to proapoA-I, whereas apoA-I0,
apoA-I-1, and apoA-I-2
correspond to mature apoA-I.41 The increased
positive charge on the two proapoA-I isoforms is due to the additional
six-amino-acid amino terminal tail of proapoA-I. The minor mature
apoA-I isoform (apoA-I-2) is a deamidation
product of apoA-I0 and
apoA-I-1.41 The same
amount of protein (600 µg) was loaded onto each gel. In relative
terms, samples from FHD patients contained considerably less mature
apoA-I but similar amounts of proapoA-I (apoA-I+2
isoform). As is evident from Fig 2
, the minor proapoA-I isoform
(apoA-I+1 isoform) was somewhat reduced in FHD
patients compared with that in control subjects; however,
quantification of total plasma mature apoA-I and total proapoA-I
revealed that the decrease in plasma apoA-I concentration in patients
was due to a decrease in mature apoA-I and not proapoA-I concentration
(Table 2
). The relative contribution of
proapoA-I to total plasma apoA-I was thus higher in FHD patients
(18.2% and 9.5% for patients 1 and 2, respectively) compared with
control subjects (2.9±0.3%, mean±SD for four subjects).

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Figure 2. Comparison of relative amounts of proapoA-I,
mature apoA-I, and apoA-II in total plasma lipoproteins
(d<1.25 g/mL) of control and FHD subjects separated by
IEF gel electrophoresis. Gels are shown for two control and two FHD
patients, corresponding to control subjects 1 and 2 and FHD patients 1
and 2 in Table 1 . Different isoforms of proapoA-I and mature apoA-I are
indicated. Similar amounts of total protein (600 µg) were separated
on each gel.
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Analysis of ApoA-I and ApoA-IIContaining HDL
Plasma lipoproteins of control and FHD patients were
separated according to their charge and size by two-dimensional
gradient gel electrophoresis. ApoA-Icontaining HDL subfractions for
two control and two FHD patients are shown in Fig 3
. Considerably less apoA-I was detected
in plasma samples from FHD patients, due to a marked reduction in
apoA-I associated with
-migrating HDL (ie,
-LpA-I). The amount of
apoA-I associated with pre-ßmigrating HDL
(preß1-LpA-I and
preß2-LpA-I) was, however, normal. The average
particle size of
-LpA-I in FHD patients tended to be smaller than
that of control subjects.

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Figure 3. Two-dimensional gel electrophoretic separation of
apoA-Icontaining lipoproteins from the plasma of control and FHD
subjects. Gels (A and B) are shown for two control and two FHD
patients, corresponding to control subjects 1 and 2 and FHD patients 1
and 2 in Table 1 . Plasma (200 µL) was separated in the first
dimension (left to right) by agarose gel electrophoresis and in the
second dimension (top to bottom) by 3% to 24% polyacrylamide
gradient gel electrophoresis. Lipoproteins containing apoA-I were
detected with 125I-labeled polyclonal anti-human apoA-I
antibody after electrotransfer to nitrocellulose membranes. Different
apoA-Icontaining HDL subpopulations are indicated with vertical
arrows. Molecular size markers were separated between plasma samples
(in the center of each gel): thyroglobulin (17 nm), ferritin (12.2 nm),
catalase (9.5 nm), lactate dehydrogenase (8.2 nm), and albumin
(7.1 nm), from top to bottom.
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Electrophoretically separated HDL of plasma from FHD patients was also
found to contain less apoA-II compared with controls (Fig 4
), which corresponded to a twofold
reduction in total plasma apoA-II concentration (measured by
nephelometry) (Table 2
). Larger
-migrating HDLs containing apoA-II
were virtually absent from the plasma of FHD patients, which meant that
the average particle size of
-LpA-II in FHD patients was smaller
than that of control. As shown in gels C and D in Fig 4
, these
apoA-IIcontaining HDLs were unique, in that they did not contain
apoA-I, unlike the majority of apoA-IIHDL in control subjects, which
was removed by apoA-I affinity chromatography; the
efficiency of removal of apoA-Icontaining lipoproteins was >97% for
patients and control subjects, as assessed by an essentially complete
absence of apoA-I in electrophoretically separated samples after
affinity chromatography. This result is
consistent with the observation that all HDL apoA-II is (under
normal circumstances) bound to apoA-I in LpA-I:A-II particles in
normolipidemic subjects.12

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Figure 4. Separation of apoA-IIcontaining lipoproteins, in
the presence and absence of apoA-I, from the plasma of control and FHD
subjects. Gels (A and B) show the presence of apoA-IIcontaining
lipoproteins in the plasma (200 µL) of two control and two FHD
patients corresponding to control subjects 1 and 2 and FHD patients 1
and 2 in Table 1 . Lipoproteins containing apoA-II were detected with
125I-labeled polyclonal anti-human apoA-II antibody after
electrotransfer of lipoproteins separated by two-dimensional gel
electrophoresis. Gels (C and D) show the presence of
apoA-IIcontaining lipoproteins in the plasma from the same subjects
after apoA-Icontaining lipoproteins were removed by affinity
chromatography (see "Methods"). ApoA-IIcontaining
HDLs in control plasmas were almost completely removed by this
procedure, in contrast to plasma from FHD patients where a large
proportion of apoA-II was not bound to apoA-I. Molecular size markers
were separated between plasma samples (in the center of each gel):
thyroglobulin (17 nm), ferritin (12.2 nm), catalase (9.5 nm), lactate
dehydrogenase (8.2 nm), and albumin (7.1 nm), from top to
bottom.
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Stable-Isotope Enrichment of Plasma ApoA-I
Newly synthesized mature apoA-I, enriched with deuterated leucine,
was detected in the plasma of all subjects within 2 hours of the start
of the stable-isotope infusion experiment (Fig 5
). The rate of appearance of
deuterium-labeled mature apoA-I measured as percentage tracer
(deuterated leucine) to tracee (nondeuterated leucine) ratio was linear
for the 12-hour duration of the study in all six subjects. The
fractional rate of appearance of labeled mature apoA-I (the slope of
the enrichment curve) was sevenfold and threefold higher in the two FHD
patients compared with control, corresponding to significantly
increased FPRs (measured in pools per day; Table 2
). Because mature
apoA-I pool sizes were proportionately decreased, absolute apoA-I PRs
were similar in patients and control subjects (Table 2
), ranging
between 7.9 and 12.9 mg · kg-1 ·
d-1. The RT of mature apoA-I in plasma was,
however, significantly shorter in patients (0.79 and 1.66 days) than in
control subjects (5.32±1.05 days), demonstrating that mature apoA-I
catabolism was significantly increased in FHD patients. Thus, in the
case of FHD patient 1, each molecule of mature apoA-I remained in the
plasma for a period that was 6.7 times less than that of control
subjects, corresponding to a plasma apoA-I concentration that was 8.8
times lower than that in control. In the case of FHD patient 2, each
molecule of mature apoA-I remained in the plasma 3.2 times less than
that of control subjects, corresponding to a plasma apoA-I
concentration that was 3.7 times lower than control.
The time course of enrichment of plasma proapoA-I for the four
control subjects (A) and two FHD patients (B) is shown in Fig 6
. The tracer to tracee ratio of plasma
proapoA-I increased monoexponentially and reached a
plateau level during the time course of the infusion. The mean level of
proapoA-I enrichment at plateau for the control subjects was 6.7±2.4%
compared with 8.2±2.2% for VLDL apoB-100. Because the proapoA-I
plateau varied somewhat from one individual to another (being a
function of the amount of deuterated leucine reaching the proapoA-I
precursor amino acid pool and the level of unlabeled leucine in this
pool), curves in Fig 6
have been normalized to allow the data from
individual subjects to be visually compared (this measure was
unnecessary for slower-turning-over mature apoA-I). ProapoA-I ratios
have thus been expressed as a percentage of the tracer to tracee ratio
of proapoA-I at plateau in each individual. As shown in Fig 6B
, the
time course of enrichment of proapoA-I for the two FHD patients was not
different from that of the control subjects; mean data (±SD) for the
four control subjects are shown with open diamond symbols. Kinetic
analyses of these curves demonstrated that neither the RT nor
the PR of proapoA-I was different in FHD patients compared with control
subjects (Table 2
).
Stable-Isotope Enrichment of Plasma ApoA-II
The time course of plasma apoA-II enrichment with deuterated
leucine for the four control subjects and two FHD patients is shown in
Fig 7
. As was the case for mature apoA-I,
the tracer to tracee ratio of apoA-II increased linearly, and the
fractional rate of appearance of newly synthesized apoA-II was about
twofold higher in patients than in control subjects. Because plasma
apoA-II concentration (and hence apoA-II pool size) was approximately
two times less, apoA-II PRs in FHD patients (1.8 and 1.9 mg ·
kg-1 · d-1) were
not significantly different from those of control (1.7±0.2 mg ·
kg-1 · d-1).
ApoA-II RTs, on the other hand (3.17 and 2.92 days), were significantly
less compared with controls (7.24±0.71 days), demonstrating that the
FCR of plasma apoA-II was significantly increased in FHD patients.
 |
Discussion
|
|---|
Our results have demonstrated that the FHD patients in the
present study (with no clinical symptoms of Tangier disease or
evidence of other known causes of HDL deficiency) had significantly
reduced levels of plasma apoA-I and apoA-II, which were not caused by
reduced apoA-I or apoA-II production. Reduced HDL levels were
instead associated with increased plasma catabolism of mature apoA-I
and, to a lesser extent, of apoA-II but not increased catabolism of
proapoA-I. We have therefore concluded that hypercatabolism of mature
plasma apoA-I, but not proapoA-I, was responsible for the HDL
deficiency in these patients.
Two previous studies have investigated the plasma kinetics of HDL
apolipoproteins in patients with severe genetic HDL deficiency of
unknown origin. Emmerich et al31 described a
46-year-old man with coronary artery disease who had severely
reduced levels of plasma HDL cholesterol (5.0 mg/dL) and
total plasma apoA-I (4.5 mg/dL). His brother and two children had
reduced HDL levels, suggesting codominant inheritance of the
abnormality. His apoA-I was structurally normal, he had no clinical
features of Tangier disease, and he was found to have marked
hypercatabolism of apoA-I. Rader et al29
similarly identified two male and three female probands with very low
HDL levels (of apparently familial origin), who had no evidence of
premature coronary heart disease. They had no clinical or
biochemical characteristics typical of known HDL deficiency states, and
all five individuals had increased catabolism of HDL apoA-I and
apoA-II. These reported cases of severe HDL deficiency of unknown
etiology resemble those described in the present study, in that an
increase in plasma apoA-I catabolism, rather than a reduction in apoA-I
production, was responsible for reduced HDL levels. This
situation is analogous to the one in patients with less severe
reductions in HDL, in whom the FCR of plasma apoA-I has
consistently been shown to be the primary metabolic
predictor of intersubject variability in plasma apoA-I and HDL
cholesterol concentrations.13 14 16
It has been hypothesized that increased fractional catabolism of apoA-I
in these individuals is caused by triglyceride enrichment
and cholesterol depletion of HDL particles, the formation
of smaller HDL, increased interaction of HDL with lipoprotein and
hepatic lipases, greater dissociation of apoA-I from HDL, and
subsequent clearance of "free" apoA-I by the
kidney.9 Increased renal clearance of apoA-I in a
lipid-free or greatly lipid-depleted form may also be responsible for
apoA-I hypercatabolism in our more severely affected FHD patients. This
may be the result of impaired HDL3- and
apoA-Imediated cellular cholesterol and phospholipid
efflux, which we have found to be a characteristic of fibroblasts from
FHD patients (data not shown), similar to that of fibroblasts from
Tangier patients.20 21 A decrease in availability
of tissue-derived cholesterol in precursor HDL particles
could reduce cholesteryl ester formation in HDL (catalyzed by LCAT),
resulting in impaired maturation of these particles into larger,
cholesteryl esterrich HDLs and formation of relatively small,
lipid-depleted apoA-I particles, which are rapidly cleared. Support for
this concept is provided by our two-dimensional electrophoretic
analysis of apoA-Iand apoA-IIcontaining HDL-size
lipoproteins (Figs 3
and 4
), showing that larger
-migrating HDLs,
particularly those containing apoA-II, were greatly reduced in FHD
patients, whereas small pre-ß1LpA-I,
representing lipid-poor43 or
lipid-free22 apoA-I particles, were present
in similar amounts compared with controls and at significantly elevated
levels relative to other apoA-I subfractions in FHD plasma.
A distinguishing feature of the present investigation is the
measurement (for the first time) of the plasma kinetics of proapoA-I
with an endogenous, stable-isotope labeling technique and
the finding that the plasma proapoA-I PR and RT were essentially normal
in FHD patients compared with control subjects (Table 2
). The in vivo
kinetics of plasma proapoA-I has been previously studied in two
normolipidemic and two Tangier disease patients by Bojanovski et
al,44 45 using purified and radioactively labeled
proapoA-I. The two normolipidemic subjects with plasma proapoA-I
concentrations of 5.5 and 6.0 mg/dL had proapoA-I RTs of 0.19 and 0.27
day and proapoA-I PRs of 11.6 and 8.8 mg ·
kg-1 · d-1,
respectively.45 These values are similar to those
obtained in the present study for control subjects (RT, 0.16±0.03
day; PR, 10.9±2.6 mg · kg-1 ·
d-1; Table 2
), providing evidence that exogenous
and endogenous tracer techniques give comparable
results.
In view of this similarity, we have made a direct comparison between
kinetic parameters obtained previously for Tangier
patients45 and those obtained for our control and
FHD patients (Fig 8
) (average results for
two subjects are shown in the case of FHD and Tangier patients). As
depicted by the relative sizes of shaded circles in Fig 8
, FHD patients
had mean plasma mature apoA-I concentrations of 24 mg/dL compared with
1.0 mg/dL in Tangier disease patients and 123 mg/dL in control
subjects. Plasma pools of mature apoA-I were thus five times smaller,
on average, in FHD patients than in control subjects and were >100
times smaller in Tangier disease patients (reflecting the greater
severity of HDL deficiency in Tangier disease). In relative terms,
plasma concentrations of proapoA-I were increased in FHD and Tangier
disease patients (ie, proapoA-I represented 14% of total
apoA-I in FHD patients and 60% in Tangier disease patients compared
with 3% in control subjects). In absolute terms, however, average
plasma concentrations of proapoA-I were 3.3, 1.5, and 3.7 mg/dL,
respectively, and proapoA-I pool sizes were thus reduced in Tangier
disease patients though not in patients with FHD (Fig 8
). It is
significant that these relatively normal levels of proapoA-I in FHD
patients were associated with essentially normal rates of proapoA-I
production and fractional catabolism unlike Tangier disease
patients, who were characterized by slightly reduced rates of proapoA-I
production and significantly increased rates of proapoA-I
fractional clearance from plasma (9.3 pools/d versus 0.3 and 0.3 pool/d
in FHD and control subjects, respectively). Thus, as depicted by the
relative widths of arrows in Fig 8
, 3% to 7% of proapoA-I is cleared
from plasma and escapes conversion to mature apoA-I in control and FHD
patients, whereas a significant proportion (
70%) of proapoA-I in
Tangier disease patients is removed in unconverted form from plasma.
Mature apoA-I is subsequently catabolized at an increased fractional
rate in both FHD and Tangier disease, with hypercatabolism being six
times greater in Tangier than FHD patients. ApoA-II fractional
catabolism is also increased in FHD patients (Table 2
), though not to
the same extent as in Tangier disease patients (plasma apoA-II RT in
FHD versus Tangier, 3.0 versus 0.8 days).25 These
data taken together demonstrate that from an HDL kinetic perspective,
FHD patients are clearly dissimilar from control subjects. Furthermore,
they are distinguishable from patients with Tangier disease, since (1)
reduction in plasma apoA-I and hypercatabolism of plasma apoA-I are
significantly more severe in Tangier disease, (2) increase in
fractional catabolism of plasma apoA-II is also more severe in Tangier
disease, and (3) plasma catabolism and concentration of proapoA-I are
essentially normal in FHD but not in Tangier disease. The reason for
this latter difference in proapoA-I metabolism and its
pathophysiological significance deserve further
investigation.

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|
Figure 8. Comparison of plasma apoA-I kinetics in
normolipidemic subjects, FHD patients, and patients with Tangier
disease. Shaded circles represent plasma pools of mature apoA-I
and proapoA-I; circle sizes are proportional to the size of in vivo
pools that they represent. Rate of movement of apoA-I in and
out of these pools is described by open and closed arrows,
respectively; sizes of these arrows are proportional to the mass of
protein movement. Fractional transport rates for arrows indicating
irreversible loss of proapoA-I or apoA-I from plasma are indicated by
numbers (with units in pools/day). Kinetic parameters
for normolipidemic and FHD subjects have been taken from the
present data; kinetic parameters for Tangier
disease patients were obtained by taking the average of data for two
patients as reported by Bojanovski et al.44 Mean plasma
concentrations of mature apoA-I and proapoA-I were, in normolipidemic
subjects, 123±18 and 3.7±0.7 mg/dL; in FHD patients, 24 and 3.3
mg/dL; and in Tangier disease patients, 1.0 and 1.5 mg/dL.
|
|
As shown by the data in Fig 4
and as demonstrated
previously,12 nearly all apoA-II in the plasma of
normolipidemic subjects is associated with HDL containing apoA-I. We
have found, however, that our patients with FHD had abnormal
apoA-IIcontaining HDL, which did not contain apoA-I. It is
significant that similar particles have been detected in the plasma of
patients with Tangier disease.46 47 48 These
lipoproteins, designated Lp(A-II), were found to be equally effective
as Lp(A-I) in promoting cholesterol efflux from
cholesterol-loaded human
fibroblasts,48 and therefore, no evidence has
been obtained suggesting that these lipoproteins are of particular
atherogenic potential. Nevertheless, they represent a
characteristic feature of HDL deficiency states, and rapid turnover of
plasma apoA-I is perhaps responsible for abnormal conversion of
LpA-I:A-II to LpA-II or simply prevents the normal formation of
LpA-I:A-II particles. Alternatively, impaired
HDL3- and apoA-Imediated cellular
cholesterol and phospholipid efflux, shown to be a
characteristic of our FHD patients (as mentioned previously), leads to
the formation of abnormal apoA-IIonlycontaining HDL. A third
possibility is that the absence or deficiency (as shown for Tangier
disease patients22) of a plasma factor
responsible for the formation of mature HDL (by converting
pre-ß1LpA-I to
-LpA-I) results in the
abnormal formation of LpA-II. The reason for the existence of LpA-II
thus remains speculative, although we have assumed that these
lipoproteins represent a consequence rather than a cause of HDL
deficiency.
We have found in the present study that the tracer to tracee ratio
of plasma proapoA-I reaches a plateau during the time course of a
12-hour infusion experiment (Fig 6
). This is of methodological
significance, since the tracer to tracer ratio of proapoA-I at plateau
can be assumed to represent the enrichment of the intestinal
and hepatic precursor amino acid pools from which proapoA-I is derived,
in the same way that plateau enrichment of VLDL apoB-100
represents the enrichment of hepatic precursor amino acid
pools.33 We have found that the level of plasma
proapoA-I enrichment at plateau was less than that of VLDL apoB-100 in
FHD patients as well as all four control subjects (proapoA-I plateau
enrichment, 6.7±2.4% versus VLDL apoB-100, 8.2±2.2%), suggesting
that intestinal precursor leucine pools were enriched with deuterated
leucine to a lesser extent than were hepatic leucine pools. This idea
is consistent with previous data showing that VLDL apoB-48 of
intestinal origin plateaus at lower enrichment than does VLDL apoB-100
of hepatic origin.49 50 We therefore contend that
using proapoA-I enrichment at plateau rather than VLDL apoB-100
enrichment at plateau to calculate apoA-I kinetic
parameters is more accurate and results in an average RT
for mature apoA-I in plasma of 5.3 days for control subjects compared
with values of 3.412 and 4.5
days13 for total apoA-I and 6.5 days for mature
apoA-I44 that have been obtained in exogenous
apoA-I radioiodination studies. A somewhat higher RT is expected for
mature apoA-I than for total apoA-I, since total apoA-I includes both
mature apoA-I and proapoA-I (the latter having a faster rate of
turnover, ie, shorter RT).
In conclusion, although a specific gene defect has not yet been
identified as the cause of HDL deficiency in our FHD patients, the
present results demonstrate that these patients are kinetically
distinguishable from control subjects and from patients with Tangier
disease. The gene responsible for low HDL levels and associated
hypercatabolism of HDL in FHD patients may therefore be different from
that of Tangier disease, or it may represent a less severe
abnormality of the same gene. Further investigation of this kindred is
thus warranted, since it has the potential to provide new insight into
genetic factors affecting HDL metabolism.
 |
Selected Abbreviations and Acronyms
|
|---|
| FCR |
= |
fractional catabolic rate |
| FHD |
= |
familial HDL deficiency |
| FPR |
= |
fractional production rate |
| GC-MS |
= |
gas chromatographymass spectrometry |
| IEF |
= |
isoelectric focusing |
| LCAT |
= |
lecithin cholesterol acyltransferase |
| Lp |
= |
lipoprotein |
| PR |
= |
production rate |
| RT |
= |
residence time |
|
 |
Acknowledgments
|
|---|
This study was supported by a joint university-industry grant
from the Medical Research Council (MRC) of Canada and Parke-Davis
(PA-14006) and by an MRC operating grant (MT 12884) to J.G., Jr. R.B.
received a scholarship and J.S.C. was supported by a grant-in-aid from
the Heart and Stroke Foundation of Québec. We would particularly
like to acknowledge the help of Denise Dubreuil and the other nurses of
the Lipid Clinic of the Clinical Research Institute of Montréal
and the excellent technical assistance of Hélène Jacques
and Nancy Doyle. The generous gift of anti-apoE antibody and
antiapoA-I latex gel from Tom Pasani of Genzyme Corp was also very
much appreciated.
Received August 7, 1997;
accepted December 11, 1997.
 |
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