Apolipoprotein B-48 and B-100
Very Low Density Lipoproteins
Comparison in Dysbetalipoproteinemia (Type III)
and Familial Hypertriglyceridemia (Type IV)
Francois Terce, Ross W. Milne, Philip K. Weech, Jean Davignon,
and Yves L. Marcel
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A protein band having the same migration as apolipoprotein (apo) B-48 was observed by SDS electrophoresls in the plasma very low density llpoproteln (VLDL) from
14 Type IV and three Type III hyperlipoproteinemlc subjects and from six normal
fasting subjects. The VLDL from five Type IV, three Type III, and one normal subject
were separated Into two subfractlons, retained and nonretained, by Immunoafflnlty
chromatography on monoclonal antl-apo B-100 Sepharose. Based on results of electrophoresls and radloimmunoassay, we have concluded that these two fractions represent apo B-48 and apo B-100 lipoproteins that we have named apo B-48 and apo B100 VLDL. When compared to their respective apo B-100 VLDL, the apo B-48 VLDL
from either Type III or Type IV was principally enriched In total llpids, in apo E, and had
an electrophoretlc migration similar to chylomlcrons. This suggests that apo B-48
VLDL has the same origin (I.e., intestinal) in the two disorders. Both apo B-48 and apo
B-100 VLDL were enriched in cholesteryl ester (CE) and depleted In trlglyceride (TG) in
Type III; however, both fractions were rich In TG and poor In CE In Type IV and In
normal subjects. In addition, compared to their respective apo B-100 VLDL, the apo B48 fraction was enriched In CE In Type III and In TG in Type IV. We conclude that,
despite a possible similar origin for apo B-48 VLDL In Type III and In Type IV subjects,
the composition of apo B-48 VLDL Is variable and the CE/TG ratio is more characteristic of the type of hyperllpldemia than of the particular VLDL subtractions.
(Arteriosclerosis 5:201-211, March/April 1985)
olecular weight,1 immunological,2^1 and metabolic heterogeneity5 of apolipoprotein (apo) B
has been reported, and the literature has been recently reviewed by Kane.6 Using a series of specific
anti-apo B-100 and anti-apo B-100/apo B-48 crossreacting monoclonal antibodies produced in our laboratory,23 we have demonstrated that an immunological heterogeneity was present in the very low
density lipoproteins (VLDL) from familial dysbetalipoproteinemia (Type III hyperlipoproteinemia).7
Thus, we could separate total VLDL into apo B-48
VLDL and apo B-100 VLDL having different apolipoprotein and lipid composition. In addition, we observed immunological heterogeneity in apo B-100
particles.
The principal characteristic of lipoproteins in Type
III hyperlipoproteinemia, which has been recently reviewed,7' 8 is the elevated triglyceride and cholesterol
content in the plasma associated with the presence
of an abnormal VLDL. In part, this is the result of an
accumulation of cholesterol-rich remnant particles
due to a defect in catabolism of both chylomicrons
and VLDL.9-12 In contrast to Type III hyperlipoproteinemia, Type IV hyperlipoproteinemia is characterized
by elevated plasma triglyceride without elevated low
density lipoprotein (LDL) cholesterol, a condition that
has been attributed to an overproduction of
VLDL.13-u Increased triglyceride synthesis with normal apo B production would result in the hypersecretion of large triglyceride-rich VLDL.15-18 In addition, a
saturated or defective catabolism of the VLDL would
be related to the increased triglyceride and VLDL
content in the plasma. 151619
M
From the Clinical Research Institute of Montreal, Montreal,
Quebec, Canada.
Dr. Milne is a scholar of the Fondation de la Recherche en
Sante du Quebec. This study was supported by grants from the
Medical Research Council of Canada (PG-27 AND MT-5427) and
from the Quebec Heart Foundation.
Address for reprints: Dr. Francois Terce, Clinical Research Institute of Montrea), 110 Pine Avenue West, Montreal, Quebec,
H2W 1R7, Canada.
Received August 13, 1984; revision accepted December 5,
1984.
201
202
ARTERIOSCLEROSIS VOL 5, No 2, MARCH/APRIL 1985
Based on the differences described in Type III and
Type IV lipoprotein metabolism and on our recent
results7 on VLDL heterogeneity in Type III, we asked
three questions: 1) Is there apo B heterogeneity in
Type IV VLDL and can apo B-48 VLDL be separated?
2) Is the composition of this fraction similar to that
of apo B-48 VLDL from Type III subjects? 3) Are
the apo B-100 VLDL similar or different in these
two disorders? In this paper, we have used immunoaffinity chromatography to isolate two separate subfractions of VLDL in Type III and Type IV hyperlipoproteinemia and in one normal subject; we refer to
these two fractions as apo B-48 VLDL and apo B-100
VLDL. In each case, the two fractions were characterized with respect to their lipid and apoiipoprotein
composition.
Methods
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Selection of Patients
Patients were selected for their lipoprotein phenotype at the lipid clinic of the Clinical Research Institute of Montreal. The diagnosis of the lipid transport
disorder had been established at a prior visit. The
clinical data are summarized in Table 1.
Diagnosis of Familial Dysbetallpoproteinemia,
Type III
We based our diagnosis on the presence of elevated plasma cholesterol (>250 mg/dl) with or without elevated plasma triglycerides (TG) (>200 mg/dl),
the presence of a (3-VLDL (comparison of electrophoreses of the < 1.006 and > 1.006 g/ml fractions
from ultracentrifugation), and the E 2/2 apo E phenotype as determined by the technique of Bouthillier et
al.20 These characteristics were associated with one
or more of the following features: typical skin xanthomas or palmar creases pigmentation, equally high
levels of plasma cholesterol and TG, and a VLDL
cholesterol/triglyceride ratio of 0.30 or more. Three
patients (two men, one woman) were selected for
the Type III phenotype; one had atherosclerotic vas-
cular disease and two had a VLDL cholesterol/TG
ratio >0.3.
Diagnosis of Hypertrlglyceridemia, Type IV
We based this diagnosis on elevated plasma triglycerides (>200 mg/dl) and VLDL cholesterol (>35
mg/dl) in the presence of normal LDL cholesterol
(<190 mg/dl). The 14 patients (nine men and five
women) had arcus cornaea and/or xanthelasma.
The apo B-LDL level was <119 mg/dl indirectly excluding the diagnosis of a combined hyperlipidemia.
Eight patients had previous atherosclerotic vascular
disease. Three had secondary causes of hyperlipidemia (one with diabetes, one with hypothyroidism,
and one with alcoholism). Their apo E phenotypes20
were distributed as follows: six had apo E 3/3, two
had apo E 3/4, five had apo E 3/2, and one had apo E
4/2. Although a familial history was obtained for each
case, no systematic sampling of first-degree relatives was carried out. Thus, the primary nature of the
disease is only putative. The diagnosis of combined
hyperlipidemia was excluded in all instances, and 11
patients were free of secondary causes of hyperlipidemia. Six of the 11 showed a positive familial context (cardiovascular disease or hypertriglyceridemia
in one of the immediate family). The plasma samples
were obtained while the patients were maintained on
a normal North American diet with no drugs. Six normolipidemic volunteers were used to evaluate for the
presence of apo B-48 in the VLDL fraction. Their
characteristics are reported in Table 1.
Complete analysis of VLDL (separation on immunoaffinity columns, lipid and apoiipoprotein analysis)
were done for five males of the 14 Type IV subjects
(without secondary causes, with a positive familial
context, and with an apo B-LDL <105 mg/dl), the
three Type III subjects and one normal subject. As
indicated in Table 1, the five Type IV subjects studied
in detail were representative of the Type IV selected.
The study was approved by the Ethics Committee of
the Institute and the informed consent of the patients
was obtained.
Table 1. Physical Characteristics and Plasma Lipid Levels of the Subjects
Plasma lipid concentration
Phenotype
No. and
gender
Age
(yrs)
Weight
(kg)
Triglyceride
(mg/dl)
Cholesterol
(mg/dl)
IV
9M, 5F
49±9
(37-67)
77.9±15
(49.3-99.3)
721 ±372
(202-1231)
277 ± 75
(162-368)
IV*
5M
49±4
(41-57)
79.0±15
(57.4-94.8)
692 ±308
(493-1202)
263 ± 59
(218-362)
III*
2M, 1F
49±8
(41-57)
74.9 ±15
(57.4-94.8)
510±238
(363-785)
466 + 206
(284-690)
Normal
5M, 1F
42+12
(25-62)
72.3 ±13
(53.8-88.8)
118.4±21
(86-143)
217±23
(181-236)
Normal*
1M
25
53.8
Values are means ± SD; the range is given in parentheses.
'These patients were submitted to complete analysis.
143
181
APO B-48 AND B-100 VLDL IN DYSLIPOPROTEINEMIA
Preparation of Llpoprotelns
Blood was collected after a 14-hour fast into EDTA
(1 mg/ml) and the red blood cells were removed by
centrifugation (3000 rpm for 15 minutes). NaN3
(0.02%) and phenyl-methyl sulfonyl fluoride (PMSF)
(0.5 mM) were added to the plasma. Plasma lipoproteins were separated by floatation in a Beckman L565 ultracentrifuge (Beckman Instrument Incorporated, Spinco Division, Palo Alto, California) with a 50.2
Ti rotor at 5° C, 45,000 rmp for 20 hours. The VLDL
chylomicron fraction was isolated at a density of
<1.006 g/ml. LDL from a normal subject was isolated by sequential ultracentrifugation21 at 5° C
between densities of 1.030 and 1.050 g/ml. All of the
lipoprotein subfractions were dialyzed against phosphate-buffered saline (PBS) (0.015 M phosphate,
0.15 M NaCI, pH 7.2) containing 1 mM EDTA and
0.02% NaN3 and stored at 4° C.
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Monoclonal Antibodies and Antlsera
We have previously described the production and
characterization of monoclonal antibodies against
human apo B 2 3 and apo E.22 Affinity-purified rabbit
antimouse IgG was purchased from Kirkegaard and
Perry Laboratories, Incorporated (Gaithersburg,
Maryland).
Immunoadsorbants and Affinity
Chromatography
The preparation of the anti-apo B-100 monoclonal
antibodies immunoadsorbants and the affinity chromatography of the VLDL have been reported.7 The
anti-apo B-100 monoclonal antibodies 4G3 and
5E11 were partially purified from ascitic fluid of
hybridoma-bearing mice, by precipitation with 40%
saturated ammonium sulfate. The precipitated proteins were redissolved and dialyzed against 0.1 M
sodium bicarbonate (pH 8) and were then covalently
coupled to Sepharose 4B.7
VLDL (5 mg protein) of each of three Type III, five
Type IV, or one normal subject were applied on two
columns connected in series containing respectively
15 ml of 5E11 Sepharose 4B and 10 ml of 4G3 Sepharose 4B. The nonretained fractions were collected
after a first washing with 0.015 M PBS (pH 7), EDTA
1 mM. The bound lipoproteins were eluted with 0.05
M citric acid containing 1 M NaCI (pH 2.5). The eluted
fractions were immediately dialyzed against 0.015 M
PBS containing 0.02% NaN3 and 1 mM EDTA.7
Radlolmmunoassay of Apo B
This technique has been described previously.7
The radioimmunoassay (RIA) is based on competition between normal LDL immobilized on removawells (Dynatech Laboratories, Incorporated,
Alexandria, Virginia) and different dilutions of the
sample to be tested for each antibody at its predeter-
Terce et al.
203
mined optimal dilution. Nonspecific binding was determined in wells in which the sample was replaced
by LDL diluted to 80 /xg/ml. All sample dilutions were
made in 0.015 M PBS 1% bovine serum albumin.
Each point is the mean of two values with a coefficient of variation less than 10%. Dilutions of LDL
between 8 /xg/ml and 0.016 /xg/ml were included in
each assay to prepare a standard curve.
Radlolmmunoassay of Apo E
The quantitative determination of apo E in the different VLDL fractions was carried out by previously
described procedures.22 Apo E (10 /xg) purified by
the technique of Weisgraber et al.23 was kindly provided by Daniel Bouthillier and was labeled with 2
mCi of 125I as described by Bolton and Hunter.24 An
apo E-pretitrated plasma stored at - 20° C was used
as the standard curve. All assays were performed
using the monoclonal antibody 6C5.22
Lipld Analysis
Lipids were analyzed in VLDL, apo B-100 VLDL,
and apo B-48 VLDL fractions (100 pig protein) from
each of the three Type III, five Type IV, and one
normal subject, following the procedure of Kuksis et
al.25 The lyophilized samples were digested with 0.3
mg phospholipase C (Sigma Chemical Company, St
Louis, Missouri); then 200 /xg tridecanoyl glycerol
(Serdary Research Laboratories, London, Ontario,
Canada) was added as internal standard, and the
lipids were extracted. The dried extract was redissolved in 80 fi\ pyridine and derivatized with 20 fi\ N,
O-bis- (trimethylsilyl) - trifluoro - acetamide (BSTFA,
Pierce Chemical Company, Rockford, Illinois). The
lipid-silyl ethers were analyzed by gas liquid chromatography (GLC) on 3% OV-1 in a single glass column
(600 x 2 mm) using a Hewlett-Packard model 5880
gas chromatograph (Hewlett-Packard Company,
Palo Alto, California), with a level-4 terminal, in the
single-column compensation mode.
Phosphollpld Analysis
Phospholipids were analyzed by thin layer chromatography (TLC) on silica gel G plates (Analtech
Incorported, Neward, DE) on the same fractions analyzed by GLC. Fractions (50 /xg protein) were extracted following the procedure of Bligh and Dyer26
by three volumes of chloroform/methanol (1 -2, vol/
vol). After removal of the protein precipitate by centrifugation, the chloroform phase was isolated by the
addition of 1 volume of H2O and 1 volume of chloroform, and the methanol/water phase was washed
by 2 volumes of chloroform. After concentration of
the chloroformic phases, the phospholipids were
separated on TLC in the usual solvent system
(CHCLg/MeOH/acetic acid/water, 50:257:3 by volume), and revealed by iodine vapor. The TLC spots
were scraped, and phosphorus analysis was made
by the perchloric acid method as described.27
204
ARTERIOSCLEROSIS VOL 5, No 2, MARCH/APRIL 1985
Apollpoproteln Analysis
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The apolipoprotein composition of the VLDL subfractions was assessed by sodium dodecyl sulfate
(SDS)28 and alkaline urea29 polyacrylamide gel electrophoresis. To identify apo B-100 and apo B-48 in
the VLDL and its subfractions, SDS polyacrylamide
gel electrophoresis was carried out according to the
method of Kane et al.1
To check for the presence of apo B-48 in the different Type IV VLDL, the samples were analyzed by
SDS polyacrylamide electrophoresis followed by
electrophoretic transfer to nitrocellulose paper according to the method of Towbin et al.30 Electrophoresis was performed on 1.5 mm thick slab gels containing 3% acrylamide. After migration, the
separated proteins were electrophoretically transferred to nitrocellulose paper (0.45 fj.m pore size,
Millipore). The nitrocellulose paper was then treated
for immunologicai detection with the anti-apo B-48/
apo B-100 cross-reacting antibody 2D8, following
the technique of Marcel et al.31 and the identification
of apo B was performed after autoradiography on
XAR-5 Kodak films with intensifier screen (Cronex,
B-100
B-48
1 2
3
4
B
B-100
IIM
B-48
1 2
3
4
Figure 1. SDS, 3% polyacrylamide gel electrophoresis of
VLDL from Type IV (A, Lanes 1 to 4), Type III (A, Lane 5; B,
Lane 1), and normal subjects, (B, Lanes 2 to 4). 100 /*g
protein VLDL was applied on each gel except in A, Lane 5
where 50 ju.g protein was applied. Apo E phenotype was
determined for Type IV VLDL as following: A, Lane 1:
E3/E3; A, Lane 2: E4/E3; A, Lanes 3 and 4 E2/E3. Gels are
stained with Coomassie blue.
Dupont). Paper electrophoresis in an albumin containing buffer was performed according to the method of Lees and Hatch.32 Protein concentration was
measured by the Lowry technique.33
Results
VLDL proteins from 14 patients with Type IV hyperlipoproteinemia, from three patients with Type III
hyperlipoproteinemia, and from six normolipemic
subjects were separated by SDS electrophoresis on
3% polyacrylamide gels. Typical patterns for the
three groups are shown in Figure 1. As expected,
apo B-48 was observed in the VLDL of all Type III
subjects examined (Figure 1 A, lane 5,50 ^g protein;
Figure 1 B, Lane 1, 100 ^q protein). In addition, a
protein having an apparent molecular weight identical to Type III apo B-48 was also observed in all Type
IV VLDL samples (Figure 1A, Lanes 1-4) and in
normal VLDL samples (Figure 1 B, Lanes 2-4). The
staining intensity of the apo B-48 band appeared
greatest with Type III VLDL, intermediate with Type
IV VLDL, and least with normal VLDL.
The typical Type III profile, including the accumulation of apo B-48 in the plasma, is associated with
the presence of the e2 allele in a homozygous form
(apo E 2/2 phenotype). To see if the presence of apo
B-48 in Type IV VLDL was associated with the e2
allelle, we examined the apo E isoforms of all subjects studied. Of the 14 subjects, six were heterozygotes for the e2 allele. In Type IV hyperlipidemics,
there was no correlation between the apparent apo
B-48/apo B-100 ratio based on staining intensity and
the presence of e2 allele in the heterozygous form. In
Figure 1 A, the VLDL in Lanes 1 and 2 were prepared
from subjects heterozygous for the e2 allele, whereas the VLDL in Lanes 3 and 4 were from subjects
who did not have the apo E2 isoform. This observation was confirmed on the VLDL of the 14 Type IV
subjects that had been separated by SDS electrophoresis and transferred electrophoretically to nitrocellulose paper, and for which the apo B had been
detected by sequential incubations with a monoclonal antibody that cross-reacts with apo B-48 and
apo B-100 (2D8), and 125l-antimouse IgG (results not
shown). Again, the differences in the apparent apo
B-48/apo B-100 ratio could not be associated with
the presence of E2 allele. We did not find a significant
difference in the level of triglyceride between the
patients having the E2 allele (740 ± 422 mg/dl) and
those without e2 allele (585 ± 380 mg/dl).
Separation of Apo B-48 VLDL and Apo B-100
VLDL
VLDL was separated into nonretained and retained fractions by immunoaffinity chromatography
on anti-apo B-100 sepharose, a technique that has
been previously described and validated.7 The separation of the VLDL and the analysis of the resulting
fractions were performed on five Type IV subjects,
three Type III subjects, and one normolipemic sub-
204
ARTERIOSCLEROSIS VOL 5, No 2, MARCH/APRIL 1985
Apollpoproteln Analysis
Downloaded from http://atvb.ahajournals.org/ by guest on July 31, 2017
The apolipoprotein composition of the VLDL subfractions was assessed by sodium dodecyl sulfate
(SDS)28 and alkaline urea29 polyacrylamide gel electrophoresis. To identify apo B-100 and apo B-48 in
the VLDL and its subfractions, SDS polyacrylamide
gel electrophoresis was carried out according to the
method of Kane et al.1
To check for the presence of apo B-48 in the different Type IV VLDL, the samples were analyzed by
SDS polyacrylamide electrophoresis followed by
electrophoretic transfer to nitrocellulose paper according to the method of Towbin et al.30 Electrophoresis was performed on 1.5 mm thick slab gels containing 3% acrylamide. After migration, the
separated proteins were electrophoretically transferred to nitrocellulose paper (0.45 fj.m pore size,
Millipore). The nitrocellulose paper was then treated
for immunologicai detection with the anti-apo B-48/
apo B-100 cross-reacting antibody 2D8, following
the technique of Marcel et al.31 and the identification
of apo B was performed after autoradiography on
XAR-5 Kodak films with intensifier screen (Cronex,
B-100
B-48
1 2
3
4
5
B
B-100
B-48
1 2
3
4
Figure 1. SDS, 3% polyacrylamide gel electrophoresis of
VLDL from Type IV (A, Lanes 1 to 4), Type III (A, Lane 5; B,
Lane 1), and normal subjects, (B, Lanes 2 to 4). 100 /*g
protein VLDL was applied on each gel except in A, Lane 5
where 50 ju.g protein was applied. Apo E phenotype was
determined for Type IV VLDL as following: A, Lane 1:
E3/E3; A, Lane 2: E4/E3; A, Lanes 3 and 4 E2/E3. Gels are
stained with Coomassie blue.
Dupont). Paper electrophoresis in an albumin containing buffer was performed according to the method of Lees and Hatch.32 Protein concentration was
measured by the Lowry technique.33
Results
VLDL proteins from 14 patients with Type IV hyperlipoproteinemia, from three patients with Type III
hyperlipoproteinemia, and from six normolipemic
subjects were separated by SDS electrophoresis on
3% polyacrylamide gels. Typical patterns for the
three groups are shown in Figure 1. As expected,
apo B-48 was observed in the VLDL of all Type III
subjects examined (Figure 1 A, lane 5,50 ^g protein;
Figure 1 B, Lane 1, 100 ^q protein). In addition, a
protein having an apparent molecular weight identical to Type III apo B-48 was also observed in all Type
IV VLDL samples (Figure 1A, Lanes 1-4) and in
normal VLDL samples (Figure 1 B, Lanes 2-4). The
staining intensity of the apo B-48 band appeared
greatest with Type III VLDL, intermediate with Type
IV VLDL, and least with normal VLDL.
The typical Type III profile, including the accumulation of apo B-48 in the plasma, is associated with
the presence of the e2 allele in a homozygous form
(apo E 2/2 phenotype). To see if the presence of apo
B-48 in Type IV VLDL was associated with the e2
allelle, we examined the apo E isoforms of all subjects studied. Of the 14 subjects, six were heterozygotes for the e2 allele. In Type IV hyperlipidemics,
there was no correlation between the apparent apo
B-48/apo B-100 ratio based on staining intensity and
the presence of e2 allele in the heterozygous form. In
Figure 1 A, the VLDL in Lanes 1 and 2 were prepared
from subjects heterozygous for the e2 allele, whereas the VLDL in Lanes 3 and 4 were from subjects
who did not have the apo E2 isoform. This observation was confirmed on the VLDL of the 14 Type IV
subjects that had been separated by SDS electrophoresis and transferred electrophoretically to nitrocellulose paper, and for which the apo B had been
detected by sequential incubations with a monoclonal antibody that cross-reacts with apo B-48 and
apo B-100 (2D8), and 125l-antimouse IgG (results not
shown). Again, the differences in the apparent apo
B-48/apo B-100 ratio could not be associated with
the presence of E2 allele. We did not find a significant
difference in the level of triglyceride between the
patients having the E2 allele (740 ± 422 mg/dl) and
those without e2 allele (585 ± 380 mg/dl).
Separation of Apo B-48 VLDL and Apo B-100
VLDL
VLDL was separated into nonretained and retained fractions by immunoaffinity chromatography
on anti-apo B-100 sepharose, a technique that has
been previously described and validated.7 The separation of the VLDL and the analysis of the resulting
fractions were performed on five Type IV subjects,
three Type III subjects, and one normolipemic sub-
APO B-48 AND B-100 VLDL IN DYSLIPOPROTEINEMIA
Downloaded from http://atvb.ahajournals.org/ by guest on July 31, 2017
ject. The starting VLDL and the nonretained and retained fractions were systematically tested in RIA
using four apo B-1 OO-specific monoclonal antibodies
(4G3, 5E11, 3A10, and 3F5) and with 1D1 and 2D8;
these are two anti-apo B-100 monoclonal antibodies
that cross-react with the apo B-48 of Type III VLDL
and normal thoracic duct lymph chylomicrons.2-3 As
shown in Figure 2, both the retained and nonretained
fractions, as well as the starting VLDL, competed
with LDL for antibodies 2D8 and 1D1, whereas only
the retained fraction and starting VLDL showed reactivity with the four anti-apo B-1 OO-specific antibodies.
The same pattern of reactivity with the monoclonal
antibodies was seen with the VLDL fractions from
Type IV, Type III, and normal subjects. Therefore the
nonretained fraction of Type IV and normal VLDL
appeared immunochemically identical to apo B-48
VLDL of Type III. Based on these results, we have
defined apo B-48 VLDL as the subfraction of particles isolated from fasting subjects at d < 1.006 g/ml
that are not retained on the anti-apo B-100 immunoadsorbants, and apo B-100 VLDL as the subfraction
0.5
1.0
o
CD
0.5
B-100
B-48
Figure 3. Typical SDS, 3% polyacrylamide gel electrophoresis of the apolipoproteins of Type IV VLDL and subtractions from trie experiment shown in Figure 2. Lane 1. Starting VLDL. Lane 2. Nonretained fraction. Lane 3. Retained
traction. In Lane 1, 100 /^g protein was used; in Lanes 2
and 3, 50 /xg protein was used.
Paper Electrophoresis of Apo B-48 and Apo
B-100 VLDL
1.0
0.5
205
of particles of d < 1.006 g/ml that are retained on the
immunoadsorbants.
The VLDL fractions were examined on 3% polyacrylamide gel electrophoresis (Figure 3). As previously shown with Type III VLDL,7 apo B-48 from
Type IV and normal subjects was present only in the
nonretained fraction. Occasionally, as in Figure 3,
the nonretained fraction obtained after a single passage was contaminated with a trace amount of apo
B-100 that could be removed by rechromatography.
Like the VLDL of Type 111 subjects, the apo B-100 and
apo B-48 were associated with distinct VLDL particles in Type IV and normal subjects. The proportion
of protein associated with apo B-48 VLDL was highest in the three Type III subjects (10.0% ± 1.1),
intermediate in the five Type IV subjects (5.0% ±
0.8), and lowest in the one normal subject (2%).
f.o r
CD
Terc6 et al.
NON-RETAINED
FRACTION
0.1
10
1
PROTEIN (tig/ml)
100
Figure 2. Type IV VLDL (5 mg) was passed on 5E11
Sepharose 4B, then on 4G3 Sepharose 4B. The starting
VLDL (A), retained fraction (B) and nonretained fraction
(C) were tested in a solid phase RIA using the anti-apo
B-100 specific monoclonal antibodies 4G3 (x), 5E11 (o),
3A10 ( + ) and 3F5 (A), or the cross-reacting anti-apo
B-100/anti-apo B-48 monoclonal antibodies 2D8 (•) and
1D1 (a).
Type III and Type IV VLDL and their nonretained
and retained fractions obtained by immunoaffinity
chromatography were analyzed by paper electrophoresis. For Type IV, the apo B-100 VLDL had the
same mobility as the bulk of the unfractionated VLDL
(prebeta), whereas the apo B-48 VLDL remained at
the point of origin (Figure 4). The migrations of the
Type III VLDL were identical to that previously described7 (data not shown), i.e., a broad (3-band for
apo B-100 VLDL and a band that remained at the
origin for apo B-48 VLDL.
Apollpoproteln Composition
For Type III and Type IV, the proteins of starting
VLDL and the two subtractions were separated by
SDS polyacrylamide gel electrophoresis using 10%
acrylamide in the presence of B-mercaptoethanol,
APO B-48 AND B-100 VLDL IN DYSLIPOPROTEINEMIA
Downloaded from http://atvb.ahajournals.org/ by guest on July 31, 2017
ject. The starting VLDL and the nonretained and retained fractions were systematically tested in RIA
using four apo B-1 OO-specific monoclonal antibodies
(4G3, 5E11, 3A10, and 3F5) and with 1D1 and 2D8;
these are two anti-apo B-100 monoclonal antibodies
that cross-react with the apo B-48 of Type III VLDL
and normal thoracic duct lymph chylomicrons.2-3 As
shown in Figure 2, both the retained and nonretained
fractions, as well as the starting VLDL, competed
with LDL for antibodies 2D8 and 1D1, whereas only
the retained fraction and starting VLDL showed reactivity with the four anti-apo B-1 OO-specific antibodies.
The same pattern of reactivity with the monoclonal
antibodies was seen with the VLDL fractions from
Type IV, Type III, and normal subjects. Therefore the
nonretained fraction of Type IV and normal VLDL
appeared immunochemically identical to apo B-48
VLDL of Type III. Based on these results, we have
defined apo B-48 VLDL as the subfraction of particles isolated from fasting subjects at d < 1.006 g/ml
that are not retained on the anti-apo B-100 immunoadsorbants, and apo B-100 VLDL as the subfraction
0.5
1.0
o
CD
0.5
B-100
B-48
Figure 3. Typical SDS, 3% polyacrylamide gel electrophoresis of the apolipoproteins of Type IV VLDL and subtractions from the experiment shown in Figure 2. Lane 1. Starting VLDL. Lane 2. Nonretained fraction. Lane 3. Retained
fraction. In Lane 1, 100 /^g protein was used; in Lanes 2
and 3, 50 /xg protein was used.
Paper Electrophoresis of Apo B-48 and Apo
B-100 VLDL
1.0
0.5
205
of particles of d < 1.006 g/ml that are retained on the
immunoadsorbants.
The VLDL fractions were examined on 3% polyacrylamide gel electrophoresis (Figure 3). As previously shown with Type III VLDL,7 apo B-48 from
Type IV and normal subjects was present only in the
nonretained fraction. Occasionally, as in Figure 3,
the nonretained fraction obtained after a single passage was contaminated with a trace amount of apo
B-100 that could be removed by rechromatography.
Like the VLDL of Type 111 subjects, the apo B-100 and
apo B-48 were associated with distinct VLDL particles in Type IV and normal subjects. The proportion
of protein associated with apo B-48 VLDL was highest in the three Type III subjects (10.0% ± 1.1),
intermediate in the five Type IV subjects (5.0% ±
0.8), and lowest in the one normal subject (2%).
f.o r
CD
Terce et al.
NON-RETAINED
FRACTION
0.1
10
1
PROTEIN (tig/ml)
100
Figure 2. Type IV VLDL (5 mg) was passed on 5E11
Sepharose 4B, then on 4G3 Sepharose 4B. The starting
VLDL (A), retained fraction (B) and nonretained fraction
(C) were tested in a solid phase RIA using the anti-apo
B-100 specific monoclonal antibodies 4G3 (x), 5E11 (o),
3A10 ( + ) and 3F5 (A), or the cross-reacting anti-apo
B-100/anti-apo B-48 monoclonal antibodies 2D8 (•) and
1D1 (a).
Type III and Type IV VLDL and their nonretained
and retained fractions obtained by immunoaffinity
chromatography were analyzed by paper electrophoresis. For Type IV, the apo B-100 VLDL had the
same mobility as the bulk of the unfractionated VLDL
(prebeta), whereas the apo B-48 VLDL remained at
the point of origin (Figure 4). The migrations of the
Type III VLDL were identical to that previously described7 (data not shown), i.e., a broad (3-band for
apo B-100 VLDL and a band that remained at the
origin for apo B-48 VLDL.
Apollpoproteln Composition
For Type III and Type IV, the proteins of starting
VLDL and the two subtractions were separated by
SDS polyacrylamide gel electrophoresis using 10%
acrylamide in the presence of B-mercaptoethanol,
206
ARTERIOSCLEROSIS VOL 5, No 2, MARCH/APRIL 1985
ci--
67 43 -
-E
30-
cm
20 14-
1
VLDL
B-100
VLDL
2
-
2
3
Ill
1
3
-,
B-48
VLDL
B
Downloaded from http://atvb.ahajournals.org/ by guest on July 31, 2017
Figure 4. Paper electrophoretogram of Type IV VLDL
showing retained and nonretained fractions isolated by
immunoaffinity chromatography. The arrow indicates the
point of application of the samples. Fractions were detected with oil Red O.
Figure 5. SDS 10% (A) and Tris urea (B) polyacrylamide
gel electrophoresis of Type IV VLDL (Lane 1), nonretained
fraction (Lane 2) and retained fraction (Lane 3). 50 fj.g of
protein was applied to the gels. The position of apoproteins
C-l and E in B are tentatively identified.
and on alkaline urea, polyacrylamide gel. The results
for Type III VLDL (not shown) were the same as
previously reported7 and no qualitative difference
could be seen between apo B-100 and apo B-48
VLDL in their apoprotein composition. The results for
Type IV VLDL are shown in Figure 5. In Figure 5A,
the starting VLDL and the two fractions seemed to be
similar on 10% polyacrylamide gel. Because of its
high molecular weight, apo B was found on the top of
the gels. An intense band was seen with a molecular
weight between the standards 43,000 and 30,000
corresponding to that of apo E (35,000). Near the
bottom of the gels at approximately 10,000 to
20,000, a broad band was present in the region
where apo A-ll and the apo Cs would migrate. The
fractions from Type IV VLDL were qualitatively similar to those from Type III.
On alkaline urea polyacrylamide gels (Figure 5 B),
apo C-ll, C-lll0, C-lll,, and C-lll2 were tentatively
identified as major bands in the middle of the gels as
well as apo C-l and apo E near the top of the gels. A
weakly stained band just above the apo C-ll may be
apo D-2 as described by Chang et al.34 Starting
VLDL and the two subfractions appeared qualitatively similar.
Apo E was measured by RIA in all Type IV and
Type III VLDL and subfractions. The results are
shown in Table 2. In Type IV, as in Type III, the apo
E/protein ratio was significantly increased in the apo
B-48 VLDL fraction, compared to starting VLDL and
apo B-100 VLDL. This ratio was similar in starting
VLDL and apo B-100 VLDL for Type IV, but higher in
starting VLDL than in apo B-100 VLDL in Type III.
Lipld Analysis
Starting VLDL, as well as the nonretained and retained fractions, from the five Type IV subjects, three
Type III subjects, and one normal subject were analyzed for their total lipid composition by gas-liquid
chromatography following the method of Kuksis et
al.25 and for their phospholipid composition by phosphorus assay. These results are summarized in
Tables 3 to 6. Each value represents the mean for
the number of subjects indicated, each analyzed in
triplicate. The range was less than 3% between the
three analyses, (intraassay variability) and all the
samples were analyzed in the same experiment with
the same standards.
Although not all comparisons were statistically significant in Type III (due to having only three subjects), the differences between B-48 VLDL and B100 VLDL were similar to those previously reported
for this type of patient.7 In Type III and Type IV pa-
Table 2. Relative Apo EConcentrations in Type III and Type IV VLDL Subfractions
Type IV(n = 5)
Type III (n = 3)
apo E/protein
VLDL
Apo B-100
VLDL
Apo B-48
VLDL
VLDL
Apo B-100
VLDL
Apo B-48
VLDL
0.43 ±0.09
0.21+0.02
0.50±0.2*
0.23 ±0.06
0.19 ±0.05
0.31 ±0.03*
The apo E concentration was determined by radioimmunoassay. The protein concentration was
determined by Lowry assay.
*The difference between apo B-48 and apo B-100 VLDL by Student's paired /test was significant at 2a
< 0.05 for Type III and at 2a < 0.01 for Type IV (see underlined values).
APO B-48 AND B-100 VLDL IN DYSLIPOPROTEINEMIA
207
Terc<§ et al.
Table 3. Chemical Composition of Type III, Type IV, and Normal VLDL, Apo B-100, and Apo B-48 VLDL
Type III (n = 3)
Proteins
Normal (n =
D
VLDL
B-48
VLDL
VLDL
B-100
VLDL
B-48
VLDL
VLDL
B-100
VLDL
B-48
VLDL
7.5±1. 1
8.5±1.7
4 .1±0.4*
9 .5 ±3.2
8.7±1.9
3 .9±0. 5*
9.6
10.7
6.5
90 .5 ±3.2
91.3±1.9
90.4
89.3
93.5
92.5±1. 1
Total lipids
Type IV (n = 5)
B-100
VLDL
91.5±1.7 95 .9±0.4
96 .1±0. 5
The percentage composition by weight is expressed as the mean value ± SD of the number of indicated subjects. The
protein content was determined by Lowry assay, and the total lipids by gas liquid chromatography.
The difference between apo B-48 and apo B-100 VLDL by Student's paired Mest was significant at 2a < 0.05 for Type III
and at 2a < 0.005 for Type IV (see underlined values).
Table 4.
Lipld Composition of Type III, Type IV, and Normal VLDL, Apo B-100, and Apo B-48 VLDL
Type IV (n = 5)
Type III (n = 3)
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VLDL
B-100
VLDL
7.3 ±1.3*
7.3 ±1.5
Free
cholesterol
Cholesteryi
esters
32.8 ± 8
31.3±5
Triglycerides
47.3 ± 8
Phospholipids 12.2±1.8
B-48
VLDL
B-100
VLDL
VLDL
4.7 ±1.3
4.98 ±1
38.1 ±0.9
20.9 ±8
46.3 ± 4
39.9 ±3.7
14.7±2.6
B-48
VLDL
Normal (n = 1)
B-100 B-48
VLDL VLDL VLDL
4.7 ±0.7
4.6
4.5
4.2
21.7±7.1
16.1 ±3.1
12.9
10.5
8.4
61.8±3
58.9 ±8
67.9 ±4.7
65.2
69.0
71.3
12.9 ±2.6
12.7 ±0.6
14.0 ±0.8
11.7±0.6
16.0
15.6
15.7
1.97 ±0.5
1.67
1.40
1.19
0.24±0.06§
0.20
0.15
0.12
7.4 ±1
EC/FCt
2.66 ±0.26 2.59 ±0.4
2.94±0.18
2.57 ±0.6
2.6 ±0.7
CE/TG*
0.72 ±0.28 0.68 ±0.17
0.96±0.15§
0.34 ±0.2
0.37 ±0.1
"Values are expressed as the mean percentage ± SD by weight of total lipids as analyzed by gas liquid chromatography.
fMolar ratio of esterified cholesterol to unesterified cholesterol (EC/FC).
^Weight ratio of cholesteryi ester to trigiyceride.
§Difference between apo B-48 and apo B-100 VLDL by Student's paired Mest was significant at 2 < 0.1 for Type III and 2
< 0.05 Type IV (see underlined values).
Table 5. Carbon Number Distribution of Cholesteryi Esters and Trigiyceride In Type III, Type IV, and Normal VLDL
and Subtractions
Type IV (n = 5)
Type III (n = 3)
VLDL
Cholesteryi esters
C16
17.9 ±0.4*
Normal (n = 1)
B-100 B-48
VLDL VLDL VLDL
B-100
VLDL
B-48
VLDL
VLDL
B-100
VLDL
B-48
VLDL
19.5±1.4
19.2±0.6
18.5±1.4
20.1 ±1.7
18.6 ±3.3
21.6
27.4
25.3
C18
70.8 ±1.2
68.4 ±1.2
71.3 ±2.2$
68.4 ±4.8
64.5 ±3.5
71.7±3.1$
62.6
56.8
61.2
C20
11.2±1.1
12.1 ±0.8
9.5 ±2.3
12.9±3.7
15.4±2.4
11.6±0.4
15.7
17.1
13.5
11.7±2.5
9.7 ± 2
8.9 ±2.2
10.1 ±1.7
8.2±2.6
5.4
6.9
8.2
Triglycerides
C48
9.3±0.4t
C50
17.6 ±2.9
18.3 ±2.9
16.0 ±0.9
15.9 ±2.2
16.7±1.8
16.3 ±2.5
12.5
15.5
15.4
C52
43.3 ±2.4
40.8 ±2.3
39.6 ±3.6
43.4 ±5.2
40.9 ± 5
40.2 ±2.8
44.9
47.0
43.7
C54
29.9 ± 5
27.2 ±3.6
34.7±3.2$
31.8±7.5
32.3±3.1
35.8 ±3.0$
37.2
30.6
32.7
'Values are expressed as the mean percentage ± SD of total cholesteryi esters.
tValues are expressed as the mean percentage ± SD of total triglycerides.
JThe difference between apo B-48 and apo B-100 VLDL by Student's Mest was significant at 2a < 0.1 for Type III and 2a
< 0.05 for Type IV (see underlined values).
208
ARTERIOSCLEROSIS VOL 5, No 2, MARCH/APRIL 1985
Table 6. Phosphollpld Composition of VLDL, B-100 VLDL, and B-48 VLDL from Type III and Type IV Patients
Analyzed by their Phosphorus Content
Phospholipid
(mol %)
LPC
Sph
PC
PS-PI
PE
Sph/PC
Type III (n = 3)
VLDL
B-100 VLDL
B-48 VLDL
VLDL
11.9±0.8
19.3±0.9
62.7 ±2.2
3.1 ±0.6
3.3 + 0.1
0.31 ±0.02
10.7±0.5
17.5 + 0.6
64.2 ±1
4.3 ±0.8
3.2 ±0.7
0.27 ±0.01
11.5±0.8
24.4 ±0.6
58.5±0.9
2.9 ±0.4
2.6 ±0.4
0.42 ±0.01*
9.9 ±1
14.7 ±2.2
68.8 ±1.8
2.7 ±0.8
3.8 ±0.6
0.21 ±0.03
Type IV (n = 5)
B-100 VLDL B-48 VLDL
11.3±2
10.3 ±2.0
14.9±2
15.8±2.8
66.6 ±3
69.0 ±3
4.0 ±0.9
3.8 ±0.9
3.2 ±0.9
2.8 ±0.8
0.22 ±0.04
0.23 ±0.06
Normal
VLDLt
2-15
15-20
65-70
—
2-10
0.23-0.29
Values are expressed as the mean molar percentage ± SD of total phospholipids. LPC = lysophosphatidylcholine; Sph
= sphingomyelin; PC = phosphatidylcholine; PS-PI = phosphatidylserine-phosphatidylinositol; PE = phosphatidylethanolamine.
'Significant difference between apo B-48 and apo B-100 VLDL in Type III by Student's paired f test at 2a < 0.001 (see
underlined values).
fData are from reference 36.
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tients, as well as in a normal subject, apo B-48 VLDL
was enriched twofold in total lipids with respect to
proteins, compared to apo B-100 VLDL or starting
VLDL (Table 3). Lipid composition (Table 4) indicated that the usual lipid species are found in all fractions, particularly free cholesterol, cholesteryl esters,
triglycerides, diglycerides, and some monoglycerides (not reported). This latter class is derived from
lysophosphatidylcholine and perhaps by some degradation of diglycerides. This represents only about
0.3 to 0.4% of total lipids. Diglycerides were derived
from phospholipids and sphingomyelins. Lysophosphatidylcholine, sphingomyelin and other phospholipids were analyzed more precisely based on their
phosphorus content (see below). Triglycerides were
the major component in all the samples; cholesteryl
esters were the second most important class, followed by phospholipids and finally free cholesterol.
All Type III VLDL and subfractions had a triglyceride
percentage lower, and cholesteryl esters and free
cholesterol percentage higher, than in normal VLDL;
as expected,7 Type III apo B-48 VLDL were richer in
cholesteryl esters and poorer in triglycerides and
phospholipids compared to apo B-100 VLDL; free
cholesterol content was the same in Type III VLDL
subfractions.
The Type IV VLDL composition was closer to that
of the normal VLDL than to that of the Type III VLDL
with, however, more cholesteryl esters and less
phospholipid and triglyceride in Type IV VLDL than in
normal VLDL. In contrast to Type III, Type IV apo B48 VLDL were enriched in triglycerides and depleted
in cholesteryl esters compared to apo B-100 VLDL.
Free cholesterol content was the same in the two
subfractions, whereas phospholipids were slightly
more abundant in apo B-100 VLDL. Apo B-48 VLDL
and apo B-100 VLDL compositions from one normal
subject were comparable to that of Type IV, with
more triglycerides and less cholesteryl esters in the
apo B-48 fraction.
Lipid analysis25 permitted the distinction between
the different carbon number classes for cholesteryl
esters and triglycerides (Table 5). Cholesteryl esters
with chains of 16, 18, and 20 carbons (as well as
triglycerides with total carbon numbers of 48, 50, 52,
and 54) were identified as independent and welldefined peaks on the chromatograms. As reported
for total VLDL,35' x cholesteryl ester with an 18-carbon fatty acid was the most important cholesteryl
ester in all fractions of Type III, Type IV, and normal
VLDL, and was increased in all apo B-48 VLDL compared to apo B-100 VLDL For the triglycerides, 52
and 54 carbon number compounds were the most
important, with a higher percentage for the 52. This
latter class was in the same amount in apo B-48
VLDL and apo B-100 VLDL of Type IV and Type III.
On the other hand, 54 acyl carbon triglycerides were
increased in Type IV and Type III apo B-48 VLDL
compared to apo B-100 VLDL at the expense of 48
acyl carbon triglycerides.
The results for phospholipids analysis are summarized in Table 6. They have not been previously reported for Type III and Type IV; however, in general,
our results are similar to the composition reported for
normal VLDL.37 Phosphatidylcholine was the major
phospholipid constituent in all fractions and seemed
to be present in similar proportions in Type IV subfractions, but decreased in Type III apo B-48 VLDL
compared to apo B-100 VLDL. Sphingomyelin was
the second major component and was increased in
apo B-48 VLDL compared to apo B-100 VLDL, particularly in Type III. No difference could be seen in
lysophosphatidylcholine levels. The minor subclasses, phosphatidylethanolamine, and phosphatidylserine-phosphatidylinositol were present in all the
subfractions.
Discussion
We have previously described7 a method to separate apo B-100 VLDL and apo B-48 VLDL particles
from a pool of plasma of Type III hyperlipemic patients, based on their immunologica! heterogeneity
defined by monoclonal antibodies. In this paper, we
used the same method to separate VLDL from plasma of five Type IV and of a new series of three Type
III subjects into subfractions that contained particles
APO B-48 AND B-100 VLDL IN DYSLIPOPROTEINEMIA
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with either apo B-100 or apo B-48 as the sole apo B
species. We studied some physical and chemical
properties of the starting VLDL and of these two subTractions to make two comparisons: 1) a comparison
between the two groups of patients, and 2) a comparison between the two subtractions of VLDL,
which were present in all individuals of both groups
of patients. In addition, VLDL from one normal subject were submitted to the same analysis.
While the presence of apo B-48 VLDL has been
documented 791038 in the VLDL from the fasting
plasma of Type I, Type V, and Type III hyperlipidemic
subjects, it has not been reported in fasting normal or
Type IV subjects. Recently, Meng et al.38 using
3.25% SDS polyacrylamide gel electrophoresis
showed the presence of apo B-48 in VLDL from Type
III, but not from Type IV, subjects. On the other hand,
Kane6 reported the presence of appreciable
amounts of apo B-48 in VLDL from hypertriglyceridemic subjects without identifying the phenotype of the
hypertriglyceridemia. In our studies, when we have
applied 100 fig of VLDL protein from Type IV and
normal subjects to 3.5% SDS polyacrylamide gels,
we have consistently seen a band migrating at the
same position as apo B-48. Although the reason for
these discrepancies is unknown, it is apparently not
related to the quantity of protein applied on the gels.
In addition to its mobility in SDS electrophoresis,
immunochemical characterization indicated that this
band is indeed identical to apo B-48 from Type III
VLDL or chylomicrons. By passing Type IV VLDL
through anti-apo B-100 immunoaffinity columns, we
separated starting VLDL into a nonretained fraction
that contained only the protein with a mobility of apo
B-48 and a retained fraction that contained only apo
B-100. In RIA, the retained fraction reacted with all of
the monoclonal antibodies, whereas the nonretained
fraction reacted with only those monoclonal antibodies (1D1, 2D8) previously shown to cross-react
with apo B-48.23
These results exclude the possibility that the low
molecular weight species seen in Type IV VLDL corresponds to the previously described apo B-50, an
apo B species or fragment that has a migration
similar to apo B-48 in SDS polyacrylamide gel electrophoresis12 and that reacts with some of the antiB-100 monoclonal antibodies.2 Therefore, in the
absence of contradicting evidence, we have considered the apo B-48 of Type IV and normal VLDL to be
identical to that of Type III VLDL or chylomicrons. As
in Type III VLDL, apo B-48 and apo B-100 are present on different particles in VLDL from Type IV and in
normal subjects. While apo B-48 VLDL represents a
relatively minor subpopulation of the total VLDL (5%
for Type IV and 2% for normal subjects), it was present in all the subjects studied. In Type IV subjects,
there was no obvious correlation in the apparent apo
B-48/apo B-100 ratio and the e2 allele.
The comparison between the total VLDL from
Type III and Type IV subjects indicated that the composition of apolipoproteins was qualitatively similar
Terce et al.
209
in the two syndromes and similar to the normal VLDL
composition. The characteristic enrichment in apo E
in the Type III plasma39 and VLDL40 could be observed from the data of the RIA (Table 2). The lipid
composition was characteristic of the syndrome with
a CE/TG ratio elevated in Type III and low in Type IV,
consistent with previous findings.735 Similar to Type
IV VLDL, normal VLDL were also triglyceride-rich
particles, according to reported data. 7 3 8 3 7
Apo B-100 VLDL is the major fraction of total VLDL
in Type III, as it is in Type IV or in normal subjects.
Thus, the differences described above between total
VLDL from these two groups of patients were also
present in the apo B-100 subtractions, and we noted
particularly the difference in the electrophoretic migration (prebeta in the Type IV and a broad 13 band in
the Type III), and the CE/TG ratio, which was much
higher in Type III than in Type IV or normal VLDL.
Characterization of Type III VLDL subfractions
from the three new patients confirms the differences
between apo B-48 and apo B-100 VLDL previously
reported for a pool of patients.7 Differences between
the apo B-48 and apo B-100 VLDL from Type IV
subjects became apparent when they were similarly
analyzed. In addition, certain similarities between
apo B-48 from Type III and Type IV subjects were
seen when the two apo B-48 subfractions were compared with their respective apo B-100 VLDL; and
these similarities include: 1) an increase in the ratio
of total lipid-to-protein; 2) an enrichment in apo E;
3) an enrichment in cholesteryl ester with 18-carbonacyl chains and in triglycerides with a 54-carbons
number; and 4) a similar electrophoretic mobility
charcteristic of chylomicrons. Thus, the apo B-48
VLDL may have a common origin, probably the intestine in Type III and Type IV hyperlipoproteinemia.
On the other hand, we cannot exclude the possibility
that an increased particle size of apo B-48 VLDL, as
indicated by the increased lipid/protein ratio, rather
than a common site of synthesis, is responsible for
the similarities between the apo B-48 VLDL subfractions of Type III and Type IV hyperlipidemia.
In the case of Type III hyperlipoproteinemia, the
presence of apo B-48 VLDL in fasting plasma can be
rationalized by a defect in the chylomicron clearance10-12 due to the presence of a defective apo E
isoform.41-*3 Because the disappearance of chylomicrons is extremely rapid in individuals with normal
lipoprotein lipase activity,44 it is more difficult to explain the persistence of apo B-48 VLDL in fasting
plasma of normal and Type IV subjects, but several
possibilities may be considered: 1) The synthesis,
assembly, and secretion of chylomicrons or intestinal VLDL may occur in the fasting stage, 2) The apo
B-48 VLDL may represent chylomicron remnants
that have escaped hepatic capture, and 3) Hepatic
apo B-48 synthesis may occur in the human as has
been described in the rat.45-46 These three possibilities will be considered in order.
Endogenous intestinal lipoprotein synthesis has
been previously reported.47'« Thus, lipoproteins of
210
ARTERIOSCLEROSIS VOL 5, No 2, MARCH/APRIL 1985
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the size of VLDL have been found in the fasting
human jejunal mucosa,49 and intestinal VLDL can be
isolated from fasting chylous urine,50 thoracic duct,51
and mesenteric lymph.52 The apo B species (apo B48 or apo B-100) that is present in such endogenous
intestinal VLDL has not been reported. After incubation with plasma, intestinal VLDL acquires the characteristics of plasma VLDL.47 However, unlike the
apo B-48 VLDL described here that has an electrophoretic mobility of chylomicrons, the intestinal
VLDL has a pre-beta migration.48 Therefore, in at
least one physical property, the Type IV apo B-48
VLDL differs from previously described endogenous
intestinal VLDL.
Type IV apo B-48 VLDL could also represent chylomicron remnants that have escaped hepatic capture, in spite of this fraction's enrichment in apo E,
which presumably can be recognized by the hepatic
receptor.5354 Such chylomicron remnants accumulate in Type III hyperlipidemia because of the defective apo E. However, the difference in the CE/TG
ratio between Type III and Type IV VLDL (noted earlier for apo B-100 VLDL) was even more pronounced
on comparing Type III with Type IV apo B-48 VLDL.
Type III apo B-48 VLDL had not only a higher CE/TG
ratio than Type IV apo B-48 VLDL, but also the highest CE/TG ratio of any VLDL fraction, whereas Type
IV apo B-48 VLDL had the lowest CE/TG ratio of any
VLDL fraction. The difference in the CE content may
reflect the time the lipoproteins have been circulating
because they can be enriched by the combined actions of lecithin-cholesterol acyltransferase and cholesteryl ester transfer protein.55 In addition, the higher TG content of Type IV apo B-48 VLDL also reflects
the saturation of the lipoprotein lipase as well as the
composition of a newly secreted lipoprotein. The
Type IV apo B-48 VLDL thus presents a CE/TG ratio
characteristic of a recently secreted particle. This
could suggest that despite a possible common origin
(intestinal), the apo B-48 VLDL may be metabolized
quite differently in Type III and in Type IV subjects.
Therefore, the CE/TG ratio appears more characteristic of the type of hyperlipidemia than of the apo
B-48 or apo B-100 VLDL subfraction. Although only
one normal subject was studied, normal apo B-48
VLDL resembled the apo B-48 VLDL from Type IV
subjects much more than that of Type III subjects, in
CE/TG ratio.
In rats, hepatic apo B-48 synthesis has been
shown4546 but this has not been seen in humans.
However, until it can be demonstrated directly that
hepatic apo B-48 VLDL secretion does not occur in
humans, there remains the possibility that the apo
B-48 VLDL in fasting plasma is of hepatic origin.
In summary, we have isolated and characterized a
subpopulation of VLDL from Type IV and Type III
subjects that had as its sole apo B species a protein
with a molecular weight and immunological properties indistinguishable from apo B-48. Some physical
and chemical properties of the apo B-48 VLDL were
similar in Type III or in Type IV, suggesting a com-
mon origin. The most striking difference between the
apo B-48 VLDL fractions isolated from Type III and
Type IV subjects was that the enrichment in cholesterol ester seen in Type III apo B-48 VLDL was not
found in Type IV apo B-48 VLDL. The origin of apo
B-48 VLDL in fasting Type IV subjects and the metabolic processes responsible for its characteristic
properties remain speculative.
Acknowledgments
We thank Daniel Bouthillier for providing apo E. We thank Lucie
Boulet for help with the paper electrophoresis, and Denise Brossard, Helene Milloux, and Suzanne Quidoz for taking blood samples. We are also indebted to Louise Lalonde for impeccable
secretarial assistance.
References
1. Kane JP, Hardman DA, Paulus HE. Heterogeneity of apolipoprotein B: isolation of a new species from human chylomicrons. Proc Natl Acad Sci USA 1980;77:2365-2369
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Index Terms: apolipoprotein B • dysbetalipoproteinemia
very low density lipoprotein • monoclonal antibody
• familial hypertriglyceridemia
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Apolipoprotein B-48 and B-100 very low density lipoproteins. Comparison in
dysbetalipoproteinemia (type III) and familial hypertriglyceridemia (type IV).
F Tercé, R W Milne, P K Weech, J Davignon and Y L Marcel
Arterioscler Thromb Vasc Biol. 1985;5:201-211
doi: 10.1161/01.ATV.5.2.201
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