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Article history:
AB S T R A C T
Objective. We investigated if daily egg feeding, along with carbohydrate restriction, would
alter lipoprotein metabolism and influence atherogenic lipoprotein profiles and insulin
Keywords:
whole eggs/day (EGG, n = 20) or the equivalent amount of yolk-free egg substitute (SUB, n =
Lipoproteins
Plasma lipids, apolipoproteins (apos), oxidized LDL (oxLDL), cholesteryl ester transfer
Metabolic syndrome
Carbohydrate restriction
baseline and week 12. Lipoprotein particle concentrations and sizes were measured by
NMR spectroscopy
Abbreviations: Apo, apolipoprotein; CETP, cholesteryl ester transfer protein; CHD, coronary heart disease; CRD, carbohydrate-restricted
diet; CVD, cardiovascular disease; EGG, 3 whole eggs per day; HDL-C, HDL-cholesterol; LCAT, lecithin-cholesterol acyltransferase; LDL-C,
LDL-cholesterol; LPL, lipoprotein lipase; MetS, metabolic syndrome; NCEP: ATP III, National Cholesterol Education Program Adult
Treatment Panel III; NMR, nuclear magnetic resonance; oxLDL, oxidized LDL; SUB, egg substitute; TC, total cholesterol; TG, triglycerides.
Corresponding author. Tel.: + 1 860 486 5547; fax: + 1 860 486 3674.
E-mail address: maria-luz.fernandez@uconn.edu (M.L. Fernandez).
0026-0495/$ see front matter 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.metabol.2012.08.014
M ET ABOL I SM CL IN I CA L A N D EX PE RI ME N TA L 6 2 ( 2 0 13 ) 40 04 1 0
1.
Introduction
401
2.
Methods
2.1.
Study design
Forty men and women aged 3070 years and classified with
MetS were recruited and enrolled in a 12-week diet intervention. Participants were included in the study if they met the
NCEP:ATP III revised criteria [1] for MetS at screening.
Exclusion criteria included self-reported diabetes, coronary
heart disease, history of stroke, renal problems, liver disease,
cancer, current pregnancy or lactation, and allergy to eggs. All
participants were asked to follow a moderately carbohydraterestricted diet (CRD) for the entire 12 weeks (25%30% of
energy from carbohydrate, 25%30% of energy from protein,
and 45%50% of energy from fat). The diet was ad libitum, as
there were no specific recommendations or restrictions for
energy intake. Participants were given comprehensive dietary
guidelines and instruction on how to follow a CRD from
trained graduate students. In addition to the CRD, participants
were randomized into two groups at baseline and asked to
consume either 3 whole eggs per day (EGG), or the equivalent
amount of yolk-free egg substitute (SUB) for the entire 12week study period (Sysco Corporation, Houston, TX). Each
daily serving (1/2 cup) of EGG contained approximately 534 mg
cholesterol, 0 g carbohydrate, 16 g protein, 12 g fat, and
equivalent to 186 kcal. A daily serving of the cholesterol/fatfree SUB contained approximately 2 g carbohydrate, 14 g
protein, and equivalent to 60 kcal. Egg products were given
as a liquid and they were equal in both consistency and color.
Participants were blinded to their group assignment and were
given egg products biweekly. Participants were asked to avoid
eating eggs outside of the study allocation. Compliance was
monitored by use of weekly questionnaires and collection of
empty product containers. Participants were asked to maintain their normal physical activity, medications, and dietary
supplement usage upon starting the 12-week study.
A total of 37 participants (n = 37) completed the 12-week
study (25 women and 12 men) and their data were used for
the subsequent analyses. The study was approved by the
University of Connecticut-Storrs Institutional Review Board.
All participants signed the written, informed consent prior
to screening.
2.2.
402
2.3.
M ET ABOL I SM CL IN I CA L A N D E XP E RI ME N TAL 6 2 ( 2 0 13 ) 40 04 10
2.4.
Plasma lipids
2.5.
2.6.
Lipoprotein particle concentrations and average
particle diameter
immobilized fluorescent dye-labeled microspheres to simultaneously quantify apolipoproteins in plasma. The intra-assay
variability was less than 2% for plasma apolipoproteins.
Plasma oxLDL was measured using a sandwich ELISA kit
(Mercodia AB, Uppsala, Sweden) according to the manufacturer's instructions. This kit utilizes the 4E6 murine monoclonal
antibody which binds to oxidized apo B-100. Reactions were
carried out in 96-well plates and quantified using a microplate
reader (BioTek Instruments, Inc., Winooski, VT). The intra-assay
variability was less than 4.5% for plasma oxLDL.
2.8.
Lecithin-cholesterol acyltransferase (LCAT) and
cholesteryl ester transfer protein (CETP)
Plasma LCAT activity was measured with a commercial
fluorometric assay kit (Roar Biomedical Inc., New York, NY).
The plasma samples were incubated with a fluorescently
labeled substrate for 5 h at 37 C. Substrate hydrolysis by LCAT
resulted in an increase in emission at 390 nm and a decrease in
emission at 470 nm. The ratio of the two emission intensities
(390 nm / 470 nm) was used to express LCAT activity.
Plasma CETP activity was determined by a CETP activity
assay kit (Roar Biomedical Inc., New York, NY). CETP activity
was quantified by measuring the transfer of a fluorescent
neutral lipid from a donor molecule to an acceptor molecule
after incubation with plasma for 3 h at 37 C. The CETPmediated transfer of the fluorescent neutral lipid resulted in
an increase in fluorescence (excitation = 465 nm, emission =
535 nm) which was measured by a microplate reader (BioTek
Instruments, Inc., Winooski, VT). The intra-assay variability
for LCAT and CETP assays was each below 4%.
2.9.
Statistical analysis
2.7.
3.
Results
3.1.
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M ET ABOL I SM CL IN I CA L A N D EX PE RI ME N TA L 6 2 ( 2 0 13 ) 40 04 1 0
P < 0.0001) and the SUB group (85.6 16.2 vs. 82.2 14.9 kg,
P < 0.0001).
3.3.
3.2.
Plasma lipids
Plasma total cholesterol (TC) and LDL-C levels were not altered
after 12 weeks, regardless of group (P > 0.1) (Table 1). Plasma TC
and LDL-C for the EGG group did not change over time, even in
the presence of consuming additional cholesterol provided by
the eggs. In contrast, the dietary intervention significantly
improved markers of atherogenic dyslipidemia associated
with MetS (Table 1). Plasma HDL-C (P < 0.0001) increased by
13.6%, while plasma TG (P < 0.0001) was reduced by 24.0% in all
participants over time. Consequently, the TG/HDL-C ratio
decreased significantly from baseline to week 12 for both the
EGG group (3.2 2.1 vs. 1.9 1.4, P < 0.0001) and the SUB group
(3.4 2.6 vs. 2.4 1.5, P < 0.05). The EGG group had a larger HDLC response than the SUB group, displaying greater percent
increases from baseline for the EGG group (19.1%) compared to
the SUB group (9.9%) (P < 0.05). The LDL-C/HDL-C ratio, a
commonly used marker of atherosclerosis risk, improved in
all participants mostly due to increases in HDL-C (Table 1). The
LDL-C/HDL-C ratio decreased by 9.5% in the SUB group and
13.5% in the EGG group from baseline. Interestingly, only
participants in the EGG group had a significant decrease in the
LDL-C/HDL-C ratio over time (P < 0.01).
3.4.
Fasting plasma glucose, insulin, and homeostasis
model assessment (HOMA-IR)
There were no significant effects of the intervention on fasting
plasma glucose (data not shown). In contrast, plasma insulin
Table 1 Plasma lipids, insulin, and insulin resistance as determined by HOMA of participants at baseline, 6 weeks, and
12 weeks of the diet intervention.
Variable
Baseline
Week 6
Week 12
Change
P-value (time)
190.3 33.0
197.4 42.0
192.0 28.1
197.8 40.2
0.4
2.7
> 0.1
NS
113.6 30.4
121.7 37.1
113.2 27.6
119.8 37.5
1.0
2.2
> 0.1
NS
+ 8.6*
+ 4.7
< 0.0001
NS
< 0.0001
NS
55.0 15.0b
51.65 12.8ab
58.5 14.8c
54.8 14.9b
108.6 56.2b
120.0 43.8
100.3 54.0b
116.2 50.5
40.6
26.2
2.2 0.8a
2.5 0.9
2.1 0.7b
2.4 1.1
0.4
0.2
< 0.05
NS
ND
ND
44.3 35.9b
36.4 15.3
11.5
6.0
< 0.01
NS
ND
ND
1.8 1.7b
1.4 0.6
0.5
0.3
< 0.01
NS
Values are mean SD. n = 20 (EGG), n = 17 (SUB). P-values are for time effects and time x group interactions for all subjects analyzed by repeated
measures ANOVA. Values with different superscripts in a row are significantly different using post-hoc multiple comparisons with Bonferroni
correction, P < 0.017. *Indicates significant differences in EGG vs. SUB using independent samples t test, P < 0.05. 1ND = not determined.
404
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3.5.
3.6.
54
50
48
46
Baseline
Wk 12
44
42
52
40
EGG
SUB
21.3
21.2
21.1
21
Baseline
20.9
Wk 12
20.8
20.7
21.5
21.4
20.6
20.5
EGG
SUB
Fig. 1 (A) Changes in VLDL particle size (diameter) from baseline to week 12 for both the EGG and SUB groups. Values are
means SEM. *Indicates significantly different from baseline at P < 0.05. (B) Pearson correlation between the changes in plasma
triglycerides (TG) and VLDL particle size from baseline to week 12. (C) Changes in LDL particle size (diameter) from baseline to
week 12 for both the EGG and SUB groups. Values are means SEM. *Indicates significantly different from baseline at P < 0.01. (D)
Pearson correlation between the changes in plasma TG/HDL-C ratio and changes in LDL particle size from baseline to week 12.
405
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9.2
9.15
9.05
9
8.95
Baseline
8.9
Wk 12
8.85
8.8
9.1
8.75
8.7
8.65
EGG
SUB
Fig. 2 (A) Changes in HDL particle size (diameter) from baseline to week 12 for both the EGG and SUB groups. Values are
means SEM. *Indicates significantly different from baseline at P < 0.01. (B) Pearson correlation between the changes in HDL-C
and changes in HDL particle size from baseline to week 12.
3.7.
3.8.
Table 2 Total number of IDL, VLDL, large, medium, and small VLDL particles for participants at baseline and after 12 weeks
of the diet intervention.
Variable (nmol/L)
Total VLDL
EGG
SUB
Large VLDL
EGG
SUB
Medium VLDL
EGG
SUB
Small VLDL
EGG
SUB
Total IDL
EGG
SUB
Baseline
Week 12
Change
P-value (time)
82.7 25.3a
77.6 29.5
66.8 33.2b
77.2 34.3
15.9*
0.3
< 0.05
< 0.05
3.6 2.6a
3.7 3.7a
1.9 2.2b
2.0 2.1b
1.8
1.7
< 0.001
NS
29.9 13.5a
25.8 13.1
19.9 20.3b
29.8 18.9
9.3*
+ 4.0
NS
< 0.05
49.9 18.5
47.8 22.7
45.0 18.2
45.4 19.9
4.8
2.5
NS
NS
53.6 36.9
62.7 47.9a
47.5 44.8
39.3 43.0b
6.1
23.4
< 0.05
NS
Values are mean SD. n = 20 (EGG), n = 17 (SUB). P-values are for time effects and time x group interactions for all subjects analyzed by repeated
measures ANOVA. Values with different superscripts in a row are significantly different, P < 0.05. *Indicates significant differences in EGG vs.
SUB using independent samples t test, P < 0.05. NS, not significant (P > 0.05).
406
M ET ABOL I SM CL IN I CA L A N D E XP E RI ME N TAL 6 2 ( 2 0 13 ) 40 04 10
Table 3 Total number of LDL, large, medium, and small LDL particles and plasma oxidized LDL for participants at baseline
and after 12 weeks of the diet intervention.
Variable (nmol/L)
Total LDL
EGG
SUB
Large LDL
EGG
SUB
Medium LDL
EGG
SUB
Small LDL
EGG
SUB
OxLDL1
EGG
SUB
Baseline
Week 12
Change
P-value (time)
1266.6 341.8
1353.0 393.3
1157.8 303.6
1313.0 387.9
108.9
39.9
NS
NS
371.7 149.1a
401.9 217.2
456.2 170.6b
446.6 192.4
+ 84.5
+ 44.6
< 0.01
NS
173.4 78.8a
175.3 86.4
132.4 73.8b
166.0 86.6
41.0
9.3
< 0.05
NS
668.1 301.9a
712.9 351.7
521.1 283.7b
661.2 327.7
147.0
51.7
< 0.05
NS
50.7 11.6
51.9 16.3a
48.2 12.9
47.3 13.6b
2.6
4.6
< 0.05
NS
Values are mean SD. n = 20 (EGG), n = 17 (SUB). P-values are for time effects and time x group interactions for all subjects analyzed by repeated
measures ANOVA. Values with different superscripts in a row are significantly different, P < 0.05. NS, not significant (P > 0.05). 1Expressed as
arbitrary units/L.
by 30.4% in the EGG group (P < 0.01) and 10.3% in the SUB group
(P < 0.05). In addition, there was a significantly greater increase
in large HDL particles for the EGG group compared to the SUB
group (P < 0.05). Increases in large HDL particles from baseline
to week 12 were associated with increases in HDL-C for both
the EGG group (r = 0.923, P < 0.001) and the SUB group (r = 0.493,
P < 0.05). Regarding other HDL subclasses, medium HDL
particles decreased significantly by 38.8% for all participants
(P < 0.01), while there were no changes in the number of small
HDL particles for either group.
3.9.
over time for the SUB group (Fig. 3B). Plasma CETP activity did
not change over time, and there were no differences between
the EGG and SUB groups (P > 0.5, data not shown).
3.10.
Plasma apolipoproteins
1.18
1.16
390 nm / 470 nm
1.14
1.12
1.1
Baseline
1.08
Wk 12
1.06
1.04
1.02
1
EGG
SUB
Fig. 3 (A) Pearson correlation between the changes in the number of small + medium LDL particles and changes in oxidized
LDL (oxLDL) from baseline to week 12. (B) Changes in LCAT activity from baseline to week 12 for both the EGG and SUB groups.
Values are means SEM. *Indicates significantly different from baseline at P < 0.05.
407
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Table 4 Total number of HDL, large, medium, and small HDL particles for participants at baseline and after 12 weeks of the
diet intervention.
Variable (mol/L)
Total HDL
EGG
SUB
Large HDL
EGG
SUB
Medium HDL
EGG
SUB
Small HDL
EGG
SUB
Baseline
Week 12
Change
P-value (time)
33.0 5.3
31.0 4.9
33.2 5.7
30.2 5.8
+ 0.2
0.8
NS
NS
5.6 2.7a
5.8 3.0a
7.3 3.2b
6.5 3.4b
+ 1.7*
+ 0.6
< 0.001
< 0.05
5.2 3.4
5.0 4.7a
3.4 3.4
2.8 2.8b
1.8
2.2
< 0.01
NS
+ 0.3
+ 1.4
NS
NS
22.1 4.2
20.1 6.2
22.5 5.7
21.6 6.8
Values are mean SD. n = 20 (EGG), n = 17 (SUB). P-values are for time effects and time x group interactions for all subjects analyzed by repeated
measures ANOVA. Values with different superscripts in a row are significantly different, P < 0.05. *Indicates significant differences in EGG vs.
SUB using independent samples t test, P < 0.05. NS, not significant (P > 0.05).
4.
Discussion
Table 5 Plasma apolipoproteins for participants at baseline and after 12 weeks of the diet intervention.
Variable (mg/L)
ApoA-I
EGG
SUB
ApoA-II
EGG
SUB
ApoB
EGG
SUB
ApoC-II
EGG
SUB
ApoC-III
EGG
SUB
ApoE
EGG
SUB
Baseline
Week 12
Change
P-value (time)
1249.0 196.8
1162.2 204.2
1241.1 181.5
1136.0 162.1
7.9
26.2
NS
NS
288.9 62.4
256.5 45.8
267.5 48.4
243.2 50.3
21.4
13.4
< 0.05
NS
1308.9 536.7
1243.0 468.2
1224.9 386.5
1229.0 404.5
84.0
14.0
NS
NS
70.0 19.9
68.2 26.9
63.1 23.3
61.5 25.9
7.0
6.8
< 0.05
NS
182.6 53.0
169.0 57.9
165.2 54.5
151.0 59.7
17.5
18.0
< 0.05
NS
51.5 10.2
53.9 28.2
48.2 13.9
47.7 25.7
3.3
6.2
< 0.05
NS
Values are mean SD. n = 20 (EGG), n = 17 (SUB). P-values are for time effects and time x group interactions for all subjects analyzed by repeated
measures ANOVA. NS, not significant (P > 0.05).
408
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M ET ABOL I SM CL IN I CA L A N D EX PE RI ME N TA L 6 2 ( 2 0 13 ) 40 04 1 0
Author contributions
CNB conducted the research, analyzed data and wrote the
paper; CJA conducted the research and provided input for the
paper; JB participated in data collection and interpretation;
JSV participated in interpretation and provided input; MLF
designed research, analyzed data and had primary responsibility for final content. All authors read and approved the
final manuscript.
Funding
This study was funded by the American Egg Board/Egg
Nutrition Center.
409
Conflict of interest
MLF received funds to conduct the study. No other conflicts of
interest exist for the remaining authors.
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