Apolipoprotein E gene promoter ?219G3T polymorphism increases
LDL-cholesterol concentrations and susceptibility to oxidation in
response to a diet rich in saturated fat1–3
Juan Antonio Moreno, Francisco Pérez-Jiménez, Carmen Marín, Purificacio ´n Go ´mez, Pablo Pérez-Martínez,
Rafael Moreno, Cecilia Bellido, Francisco Fuentes, and José Lo ´pez-Miranda
Background: The apolipoprotein E (APOE) gene promoter poly-
morphism (?219G3T) has been associated with increased risk of
myocardial infarction, premature coronary artery disease, and de-
creased plasma apolipoprotein E concentrations.
Objective: We aimed to determine in healthy subjects whether this
polymorphism modifies the susceptibility of LDL to oxidation and
the lipid response to the content and quality of dietary fat.
38 GT, and 10 TT) completed 3 dietary periods, each lasting 4 wk.
The first was a saturated fatty acid (SFA)-rich diet [38% fat—20%
SFA and 12% monounsaturated fatty acid (MUFA)—and 47% car-
bohydrates (CHO)], which was followed by a CHO-rich diet (30%
fat—?10% SFA and 12% MUFA—and 55% CHO) or a MUFA-
rich diet (38% fat—?10% SFA and 22% MUFA—and 47% CHO)
LDL oxidation susceptibility, lipids, and lipoproteins were mea-
Results: Compared with carriers of the G allele, TT subjects had a
significantly (P ? 0.05) shorter lag time after the SFA diet. The
replacement of the SFA diet by the CHO or MUFA diet induced a
greater increase (P ? 0.05) in lag time in the TT subjects than in the
GG or GT subjects. Carriers of the T allele had higher LDL-
cholesterol (P ? 0.05) and apolipoprotein B (P ? 0.05) plasma
concentrations after the SFA diet than did GG subjects. Compared
with GG subjects, carriers of the T allele had a significantly (P ?
0.05) greater decrease in LDL cholesterol and apolipoprotein B
when they changed from the SFA to the CHO diet.
Conclusion: The ?219G3T polymorphism may partially explain
phism, ?219G3T, APOE, dietary intervention, LDL oxidation,
LDL cholesterol, cardiovascular disease risk
Apolipoprotein E gene promoter polymor-
Apolipoprotein E (apo E) is a structural component of several
lipoproteins and serves as a ligand for the LDL receptor and the
LDL-receptor-related protein (1, 2). Therefore, apo E plays an
important role in lipid metabolism both by promoting efficient
uptake of triacylglycerol-rich lipoproteins from the circulation
and by taking part in cellular cholesterol efflux and reverse cho-
effective because apo E is present in the population in 3 main
isoforms (apo E2, apo E3, and apo E4) that determine apo E
concentrations and differ in their affinity to bind to the specific
Apo E has antioxidant activity, and this activity differs in extent
knockout mice are highly susceptible to developing atheroscle-
oxidation (8) and also because they lose their normal resistance
to cholesterol feeding. This effect is reversible by antioxidant
supplementation of their diet (9).
In recent years, the interaction between lipoprotein respon-
siveness to dietary intervention and APOE genotypes has been
analyzed; however, the results are controversial (10). Whereas
APOE genotypes to changes in dietary fat or cholesterol content
(11–14). This fact suggests that other genetic or environmental
factors are likely to be responsible for the association of the
APOE gene with individual response to diet.
In accordance with this hypothesis, polymorphisms in the
proximal promoter region of the APOE gene were recently de-
scribed at positions ?491A3T, ?427T3C, and ?219G3T
(15, 16). In particular, there is experimental evidence, both in
vitro and in vivo, that the APOE gene promoter ?219G3T
1From the Lipids and Atherosclerosis Research Unit, Reina Sofía Uni-
versity Hospital, Co ´rdoba, Spain.
FIS 99/0949); Fundacio ´n Cultural “Hospital Reina Sofía-Cajasur,” Conse-
jería de Salud, Servicio Andaluz de Salud (99/116, 00/212, 01/243, 99/165,
00/39, and 01/239); and Consejería de Educacio ´n, Plan Andaluz de Investi-
gacio ´n, Universidad de Co ´rdoba.
3Address reprint requests to J Lo ´pez-Miranda, Unidad de Lípidos y Ar-
teriosclerosis, Hospital Universitario Reina Sofía, Avda Menéndez Pidal,
s/n, 14004 Co ´rdoba, Spain. E-mail: firstname.lastname@example.org.
Received January 9, 2004.
Accepted for publication May 14, 2004.
Am J Clin Nutr 2004;80:1404–9. Printed in USA. © 2004 American Society for Clinical Nutrition
by guest on June 6, 2013
of the gene. Specifically, the ?219G allele shows a higher tran-
European population including control individuals and multiin-
farct patients showed that the ?219G3T polymorphism is also
associated with differential plasma apo E concentrations (17),
illustrating that this polymorphism influences APOE expression
in vivo. Furthermore, the ?219T allele is associated with an
increased risk of myocardial infarction (17) and with premature
coronary artery disease (18).
enhances atherothrombosis remain to be elucidated. In a previ-
ous study, we observed that the presence of this polymorphism
determines serum apo E concentrations and influences the me-
dial period (19). In other study, the ?219T allele was associated
al (17), the ?219G3T polymorphism was not associated with
plasma lipid or lipoprotein concentrations. Thus, the aim of the
oxidation and the lipid response to the quantity and quality of
dietary fat in healthy men with the APOE3/E3 genotype.
SUBJECTS AND METHODS
Because APOE genotypes have been implicated in a variable
lipid response to dietary changes, we studied the effect of the
?219G3T polymorphism in APOE3/E3 subjects, to the exclu-
sion of other apo E isoforms. A group of 55 men (7 with the GG
were recruited from among students at the University of Cor-
doba. The subjects had a mean (?SD) age of 21 ? 0.8 y. All
subjects underwent a comprehensive medical history, physical
examination, and clinical chemistry analysis before enrollment.
levels of physical activity (eg, sports training). None of the sub-
used to calculate individual energy requirements. Mean body
The subjects were encouraged to maintain their regular physical
in smoking habits or alcohol consumption, and intake of foods
not included in the experimental design. The study protocol was
approved by the Human Investigation Review Committee at the
Reina Sofia University Hospital, and informed consent was ob-
tained from all participants.
12% monounsaturated fatty acid (MUFA), and 6% polyunsatu-
rated fatty acid (PUFA)]. After this period, the volunteers were
randomly assigned to 1 of 2 diet sequences. Twenty-eight sub-
jects received a MUFA-rich diet containing 15% protein, 47%
carbohydrates, and 38% fat (?10% SFA, 6% PUFA, and 22%
MUFA) for 28 d. This diet was followed for 28 d by a carbohy-
drate (CHO)-rich diet containing 15% protein, 55% carbohy-
diet. The cholesterol content remained constant (?300 mg/d)
during the 3 periods. Eighty percent of the MUFA diet was
provided by virgin olive oil, which was used for cooking, salad
dressing, and as a spread. The carbohydrate intake of the CHO
diet was based on the consumption of biscuits, jam, and bread.
Butter and palm oil were used during the SFA dietary period.
The composition of the experimental diets was calculated by
using the US Department of Agriculture (20) food tables and
Spanish food-composition tables for local foodstuffs (21). All
by a dietitian. Lunch and dinner were consumed in the hospital
dining room, whereas breakfast and an afternoon snack were
eaten in the medical school cafeteria. Fourteen menus were pre-
pared with regular solid foods and were rotated during the ex-
carbohydrate contents of the diets were analyzed by standard
fatty acids in LDL cholesterol esters at the end of each dietary
minimize seasonal effects and academic stress.
Analysis of fatty acids in LDL cholesterol esters
ids were transmethylated as previously described (23). The re-
II gas chromatograph (Hewlett-Packard, Avondale, PA)
0.25-?m film) obtained from Supelco (Bellefonte, PA) with he-
lium as the carrier gas.
Lipid analysis and biochemical determinations
Venous blood samples were collected at the beginning of the
(1 g/L) tubes from all subjects after they had fasted for 12 h
overnight. Plasma was obtained by low-speed centrifugation
(1500 ? g) for 15 min at 4 °C within 1 h of venipuncture. To
reduce interassay variation, plasma was stored at ?80°C and was
erol concentrations were measured by enzymatic techniques (24,
25). HDL cholesterol was measured after precipitation with phos-
photungstic acid (26). Apo A-I and B were determined by immu-
noturbidimetry (27). LDL-cholesterol concentrations were calcu-
lated by using the Friedewald formula (28).
Oxidation of LDL
LDL was isolated from fresh plasma samples by sequential
with a type NVT65 rotor (Beckman, Palo Alto, CA) for 2 h at
405 000 ? g and 4 °C. The formation of conjugated dienes was
measured by incubating 100 ?g LDL protein with 5 ?mol
INTERACTION OF DIET AND APOE POLYMORPHISM
by guest on June 6, 2013
CuSO4/L in 1.0 mL phosphate-buffered saline medium. Absor-
bance at 234 nm was measured continuously every 5 min for 4 h
at 37 °C in a spectrophotometer as previously described (29).
Results are expressed as the duration of the lag time before
propagation of the LDL oxidation reaction, which was deter-
mined by the absolute increase in absorbance above the initial
DNA amplification and genotyping
Genomic DNA extraction and APOE E2, E3, E4 (30) and
?219G3T (12–14) genotypes were determined as previously
8% nondenaturing polyacrylamide gel at 150 V for 2 h. Bands
were visualized by silver staining. Samples containing the T
time to verify the genotype.
effects of the APOE ?219G3T polymorphism on plasma total
cholesterol, LDL-cholesterol, HDL-cholesterol, triacylglycerol,
apo A-I, and apo B concentrations in each dietary stage. When
statistical significance was found, Tukey’s post hoc comparison
test was used to identify between-group differences. Statistical
analyses were carried out by using SPSS statistical software,
version 8.0 (SPSS Inc, Chicago).
studied when we compared the baseline characteristics of the
the subjects heterozygous for the T allele (GT; n ? 38) and the
composition of the participants’ mean daily intake is shown in
Table 2. Analysis of LDL cholesterol esters obtained after each
dietary period showed good adherence during the different in-
tervention stages. After the SFA diet period, we observed a sig-
nificantly greater (P ? 0.005) increase in palmitic acid in the
LDL cholesterol esters than were observed after the CHO and
MUFA diets: 27.3 ? 1.4% compared with 19.8 ? 3.9% and
15.2 ? 0.4%, respectively. A significantly greater (P ? 0.05)
the MUFA diet (50.3 ? 4.7%) than after the CHO diet (38.8 ?
9.0%) but not after the SFA diet (47.2 ? 4.4%).
and concentrations of total cholesterol, LDL cholesterol, HDL
cholesterol, apo A-I, and apo B after the 3 diets are shown in
0.05) longer lag times and significantly lower concentrations of
total (P ? 0.001), LDL (P ? 0.001), and HDL (P ? 0.05)
diet, the CHO diet was associated with significantly shorter lag
times (P ? 0.01) and significantly lower plasma concentrations
of HDL cholesterol (P ? 0.05) and apo A-I (P ? 0.01). No
served after the different diets (P ? 0.695).
A significant diet-by-genotype interaction effect was ob-
LDL-cholesterol (P ? 0.048) concentrations (Table 3). Carriers
homozygous for the G allele. Thus, in TT and GT subjects, the
decrease in apo B plasma concentrations was significantly (P ?
0.049) greater than that in the GG subjects when the subjects
switched from the SFA diet to the CHO one (TT: ?14%; GT:
?16%; GG, ?5%). In carriers of the T allele, the decrease in
LDL-cholesterol concentrations was significantly (P ? 0.048)
the SFA diet to the CHO diet (TT, ?21%; GT, ?17%; GG,
?5%). However, when the SFA diet was compared with the
MUFA diet, no significant differences in apo B or LDL-
cholesterol concentrations were observed between genotypes.
Baseline anthropometric characteristics and plasma lipid and
apolipoprotein (apo) concentrations according to ?219G3T APOE
GG (n ? 7)
GT (n ? 38)
TT (n ? 10)
Apo A-1 (g/L)
Apo B (g/L)
20.6 ? 1.6
24.3 ? 2.4
21.4 ? 1.8
23.4 ? 2.7
20.8 ? 2.2
23.8 ? 2.9
4.1 ? 0.5
2.4 ? 0.3
1.2 ? 0.2
1.2 ? 0.2
0.5 ? 0.1
1.0 ? 0.4
4.1 ? 0.7
2.5 ? 0.6
1.18 ? 0.26
1.2 ? 0.2
0.6 ? 0.1
0.8 ? 0.5
4.1 ? 0.6
2.4 ? 0.5
1.3 ? 0.4
1.2 ? 0.2
0.6 ? 0.2
0.9 ? 0.4
1All values are x ? ? SD. There were no significant differences between
genotype groups by ANOVA.
Daily intake during each experimental diet period1
SFA dietCHO dietMUFA diet
Protein (% of energy)
Fat (% of energy)
Carbohydrates (% of energy)
1SFA diet, saturated fatty acid–rich diet; CHO diet, low-fat, high-
carbohydrate diet; MUFA diet, monounsaturated fatty acid–rich diet.
MORENO ET AL
by guest on June 6, 2013
lag time before propagation of the LDL oxidation reaction (P ?
0.025). Compared with carriers of the G allele, TT subjects had
significantly (P ? 0.05) shorter lag times after the SFA diet. In
addition, there was a significant diet-by-genotype interaction
lag time in TT subjects (14.2 min, or 72%) than in GG subjects
greater increase in lag time (29.25 min, or 154%) was observed
in TT subjects than in carriers of the G allele (GG: 16.25 min, or
30%; GT: 9.33 min, or 34%) when the SFA diet was compared
with the MUFA diet. In addition, we observed a significant cor-
relation (r ? ?0.27, P ? 0.005) between the increase in LDL
cholesterol and the decrease in lag time.
Our results show that the presence of the T allele in the APOE
?219G3T polymorphism increases the susceptibility of
plasma LDL to oxidative modifications and enhances the re-
sponse of apo B and LDL cholesterol to the presence of SFA in
the diet of healthy men. Previous studies suggested that differ-
ences in individual response to diet exist. Therefore, the influ-
ence of the genetic loci of the principal apolipoproteins, such as
The allelic variations of these genes influence the degree of
response by both HDL and LDL cholesterol (31, 32). Thus, the
hyperresponse of LDL-cholesterol concentrations associated
with the E4 allele occurs only when the fat content of the diet is
varied (33). However, the inconsistencies observed in several
tors may interact with the APOE gene in determining the indi-
vidual response to diet. Thus, in our study, we observed that
carriers of the T allele showed a higher response of LDL choles-
B was significantly (P ? 0.05) higher when they changed from
the SFA to the CHO diet.
cardial infarction, premature coronary artery disease, and de-
creased plasma apo E concentrations (17, 18). These facts sug-
gest that the basal ability of cells to synthesize and secrete apo E
expression produces an increase of up to 300% in the uptake of
chylomicron remnants, VLDL, and LDL (34). In addition, the
uptake of apo B–containing lipoproteins was increased by the
LDL receptor, probably through an interaction with the LDL-
APOE expression results in a massive accumulation of
cholesterol-rich VLDL-like remnants and also LDL-like parti-
cles. The higher plasma LDL concentrations and the prolonged
circulation of these atherogenic lipoproteins in the plasma of
ApoE knockout mice (E°) cause an oxidative stress that is asso-
ciated with the increased susceptibility of the lipoprotein to ox-
idation, especially when the animals are stressed by an athero-
genic diet. This phenomenon could explain our results, wherein
plasma LDL to oxidative modifications when consuming an
SFA-rich diet. In accordance with this hypothesis, we observed
a significant correlation between increases in LDL cholesterol
and decreases in lag time. In addition, apo E can protect against
Plasma lipid and apolipoprotein (apo) concentrations at the end of each dietary period and the percentage change in LDL cholesterol (LDL-C), apo B, and
lag time according to APOE genotype and experimental diet1
Genotype and dietLag time TC LDL-CHDL-CTGApo A-IApo B
min (%)mmol/Lmmol/L (%) mmol/L mmol/L g/Lg/L (%)
GG (n ? 7)
GT (n ? 38)
TT (n ? 10)
67.75 ? 40.48a,2
77.25 ? 46.16 (14)3
84.00 ? 37.91 (30)
3.87 ? 0.41
3.61 ? 0.49
3.59 ? 0.48
2.23 ? 0.38a
2.09 ? 0.38 (?5)
2.06 ? 0.44 (?7)
1.18 ? 0.19
1.06 ? 0.22
1.08 ? 0.19
0.95 ? 0.48
0.95 ? 0.24
0.94 ? 0.20
1.22 ? 0.20
1.11 ? 0.17
1.14 ? 0.15
0.54 ? 0.11a
0.52 ? 0.13 (?5)
0.51 ? 0.11 (?5)
45.58 ? 20.88a
47.44 ? 26.08 (12)
54.91 ? 24.05 (34)
4.30 ? 0.67
3.70 ? 0.60
3.75 ? 0.63
2.68 ? 0.66b
2.20 ? 0.55 (?17)
2.22 ? 0.61 (?17)
1.20 ? 0.31
1.08 ? 0.21
1.14 ? 0.27
0.88 ? 0.32
0.86 ? 0.37
0.81 ? 0.26
1.25 ? 0.22
1.16 ? 0.20
1.20 ? 0.23
0.67 ? 0.17b
0.56 ? 0.14 (?16)
0.57 ? 0.14 (?14)
25.25 ? 14.22b
39.37 ? 18.58 (72)
54.50 ? 15.94 (154)
4.35 ? 0.45
3.77 ? 0.46
3.95 ? 0.35
2.80 ? 0.47b
2.23 ? 0.57 (?21)
2.33 ? 0.42 (?16)
1.13 ? 0.26
1.13 ? 0.36
1.23 ? 0.37
0.87 ? 0.40
0.84 ? 0.38
0.82 ? 0.34
1.19 ? 0.18
1.15 ? 0.18
1.22 ? 0.22
0.67 ? 0.15b
0.59 ? 0.19 (?14)
0.60 ? 0.18 (?11)
diet group, P ? 0.05 (repeated-measures ANOVA).
2x ? ? SD (all such values).
3Percentage change from the SFA diet in parentheses (all such values).
INTERACTION OF DIET AND APOE POLYMORPHISM
by guest on June 6, 2013
greater resistance to in vitro oxidation, and the extent of athero-
sclerosis was significantly lower than that in untreated mice. E°
mice fed high-fat, low-cholesterol diets enriched with olive oil
showed a reduction in atherosclerotic lesions and a decrease in
hepatic lipid peroxidation (37).
We recently observed that the presence of the ?219G3T
lipoproteins during the postprandial period, thus prolonging
teins. Thus, the increased plasma triacylglycerol-rich lipoprotein
concentration during the postprandial phase causes cholesterol ex-
change between triacylglycerol-rich lipoproteins and LDL and
dial state, it is postulated that the extent of exchange may be deter-
an enhanced exchange in subjects with prolonged postprandial li-
pemia, as in TT subjects. The resultant triacylglycerol-enriched
LDL and HDL particles are subject to lipolysis by hepatic lipase,
APOE gene enhances the susceptibility of LDL to oxidation.
LDL susceptibility to oxidation is determined by antioxidant
content, fatty acid composition, and the size of the particle. As
resistance of LDL particles to oxidation (40). MUFA-rich diets
tochemicals, which may beneficially increase LDL oxidation
resistance beyond that due to fatty acid composition (41). The
mechanism of the increased oxidative stress of LDL when sub-
jects consume an SFA diet, given that these fatty acids are not
prone to oxidation, probably involves other factors, such as the
LDL concentration and the particle residence time in the circu-
lation. Thus, the SFA diet was associated with significantly
higher plasma LDL-cholesterol concentrations than was either
hypolipidemic diets. Furthermore, we observed a correlation
between increases in LDL cholesterol and decreases in lag time.
Our data seem to indicate that the ?219G3T polymorphism
influences lipoprotein concentrations and LDL susceptibility to
oxidation according to the presence of SFAs in the diet of
healthy, young, normolipemic men. We included only healthy,
young, normolipemic men to avoid the effect of other factors
(age, sex, BMI, etc) on lipid response to the content and quality
of dietary fat. Studies conducted with conditions representing
impaired metabolism, such as in dyslipoproteinemic subjects,
will generally be more successful in finding differential effects
across APOE genotypes, and such studies may be helpful in
present study suggest the use of genotyping of the APOE
counseling and intervention and more efficacious primary and
secondary coronary artery disease prevention.
diet because of a greater increase in apo B, LDL-cholesterol
concentrations, and oxidative modifications in LDL. The allelic
variability in the APOE gene promoter polymorphism may par-
tially explain the differences in individual response to diet.
JAM was responsible for data collection, data analysis, and writing of the
and RM contributed to the collection of data and the writing of the manuscript.
JL-M were responsible for the conception and design of the study, analysis of
data, and writing of the manuscript. None of the authors had any conflicts of
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