Nutrition, Metabolism & Cardiovascular Diseases (2011) 21, 39e45

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/nmcd

Consumption of diets with different type of fat

influences triacylglycerols-rich lipoproteins particle
number and size during the postprandial state*
P. Perez-Martinez a,*, J.M. Ordovas b, A. Garcia-Rios a, J. Delgado-Lista a,
N. Delgado-Casado a, C. Cruz-Teno a, A. Camargo a, E.M. Yubero-Serrano a,
F. Rodriguez c, F. Perez-Jimenez a, J. Lopez-Miranda a

a
Reina Sofia University Hospital, Lipids and Atherosclerosis Research Unit, Instituto Maimonides de Investigacion
Biomedica Cordoba (IMIBIC), University of Cordoba, Ciber Fisiopatologia Obesidad y Nutricion, Instituto Salud
Carlos III, Spain
b
Nutrition and Genomics Laboratory, Jean Mayer-USDA Human Nutrition Research Center on Aging at Tufts University,
Boston, MA, USA
c
Reina Sofia University Hospital, Clinical Analyses Service, Cordoba, Spain

Received 20 March 2009; received in revised form 8 July 2009; accepted 22 July 2009

KEYWORDS Abstract Background and aims: Previous evidence suggests that dietary fat could influence
Intervention study; the composition and size of triacylglycerols-rich lipoproteins (TRL). In a controlled interven-
Olive oil; tion study on healthy subjects, we evaluated the influence of 3 dietary interventions, with
Postprandial lipemia; different types of fat on postprandial TRL particle size and number.
Nuclear magnetic Methods and results: Volunteers followed three different diets for four weeks each, according to
resonance; a randomized crossover design. Western diet: 15% protein, 47% carbohydrates (CHO), 38% fat (22%
Particle size; saturated fatty acid (SFA)); Mediterranean diet: 15% protein, 47% CHO, 38% fat (24% monounsat-
Dietary fat urated fatty acid (MUFA)); high CHO enriched with ALNA diet: 15% protein, 55% CHO, <30% fat (8%
polyunsaturated fatty acid (PUFA)). After a 12-h fast, volunteers consumed a breakfast with 1 g
fat and 7 mg cholesterol per kg body weight and a fat composition similar to that consumed in
each of the diets: Butter meal: 35% SFA; Olive oil meal: 36% MUFA; Walnut meal: 16% PUFA, 4%
a-linolenic acid. Tryglicerides (TG) in TRL (large and small TRL) were determined by ultracentri-
fugation and size and number of lipoprotein particles were measured with Nuclear Magnetic

*
Support for research: This study was supported by the CIBER CBO/6/03, Instituto de Salud Carlos III; Ministerio de Ciencia e Innovación
(AGL 2004/07907, AGL2006-01979/ALI to JL-M and SAF2003-05770, SAF2007-62005 to FP-J), the Spanish Ministry of Health (FIS PI041619 to
CM)), Consejerı́a de Innovación, Ciencia y Empresa, Proyectos de Investigación de Excelencia Junta de Andalucı́a (P06-CTS- 01425 to JL-M);
Consejerı́a de Salud, Junta de Andalucı́a (06/128, 07/43 to JL-M, 06/129 to FP-J, 06/127 to CM, ), the Diputación Provincial de Córdoba (to
FP-J) and by contract 53-K06-5-10 from NIH and 58-1950-9-001 from the US Department of Agriculture Research Service.
* Corresponding author. Reina Sofia University Hospital, Lipids and Atherosclerosis Research Unit, Avda. Menéndez Pidal s/n, 14004
Córdoba, Spain. Tel.: þ34 957010947; fax: þ34 957218250.
E-mail address: [email protected] (P. Perez-Martinez).

0939-4753/$ - see front matter ª 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.numecd.2009.07.008
40 P. Perez-Martinez et al.

Resonance Spectroscopy at different time points. The olive oil meal reduced the number of total
TRL postprandial particles compared with the other meals (P Z 0.002). Moreover, the olive oil
meal also increased the TRL particle size compared with the walnut meal (P Z 0.001).
Conclusion: Our data showed that short-term intake of the Mediterranean diet and the acute
intake of an olive oil meal lead to the formation of a reduced number and higher-size TRL particle
compared with other fat sources. These novel findings have implications for understanding the
postprandial lipoprotein mechanisms, and could favour the lower cardiovascular risk in Mediter-
ranean countries.
ª 2009 Elsevier B.V. All rights reserved.

Introduction inconsistencies found so far, this subject is in need of more in

depth investigation. Therefore, we conducted an interven-
Much of our knowledge of the relationship between lipids, tion study on healthy Caucasian subjects in order to define
lipoprotein metabolism and the development of athero- whether the short-term consumption of three diets con-
sclerosis and cardiovascular disease (CVD) is based on taining different types of fat have differential effects on TRL
measurements made in the fasting state. Although such number and particle size during the postprandial state.
measurements remain the foundation of clinical assessment
and are an important basis for therapeutic decisions, it
should be noted that we spend most of the time in a non- Methods
fasting state, with continuous fluctuation in the degree of
lipemia throughout the day. Several studies support the Subjects
concept that the levels of circulating triacylglycerols-rich
lipoproteins (TRL) after meals are significantly associated Twenty men were included in this study, all of whom gave
with the development of atherosclerosis [1]. Moreover the informed consent and underwent a comprehensive medical
access of lipoproteins to the subendothelial space in the history, physical examination, and clinical chemistry anal-
arterial wall appears to be inversely related to the size of ysis before enrolment. They had a mean (S.D.) age of
the particles. For this reason, it was thought for many years 22  1.8 years and a body mass index (BMI) of 24.5  2.7.
that large TRL were not as atherogenic because they were Subjects showed no signs of any chronic disease or obesity,
too large to traverse the endothelial barrier [2], whereas and none had unusually high levels of physical activity.
small TRL formed after the hydrolytic activity of lipoprotein They were selected on the basis of having the apo E3/E3
lipase (LPL) could go across this barrier and thus be more genotype, in order to avoid potential confounding from the
atherogenic than their nascent precursors [3,4]. allele effects of this locus on postprandial lipemia [15].
Postprandial lipemia is influenced by multiple factors such None of the subjects were taking medications or vitamins
as the amount of fat and type of dietary fat present in the known to affect plasma lipids. The study protocol was
meal, as well as other dietary components [5] and genetic approved by the Human Investigation Review Committee of
factors [6]. When high-monounsaturated-fat diets replace the Reina Sofia University Hospital, according to Institu-
high-carbohydrate diets, plasma tryglicerides (TG) concen- tional and Good Clinical Practice guidelines.
trations decrease, both in healthy subjects and type 2 dia-
betics [7,8]. Previous work in healthy, normolipemic subjects Study design
suggest that the adverse effects of low-fat, high-carbohy-
drate diets on blood lipids can be attenuated by long chain Each volunteer in the trial was subjected to three diet
(LC) n-3 PUFA [9]. A recent study showed different patterns intervention periods of 4 weeks duration, in a randomized
of postprandial lipemia after standard fat containing meals in crossover design. The administration of each fat meal was
northern and southern European healthy males [10], sug- conducted according to a Latin-squares design so that all
gesting that background dietary MUFA consumption may subjects received, in random sequence, the 3 diets at 3
influence the nature and extent of postprandial lipemia. different times. The composition of the experimental diets
Moreover, dietary fat can influence the composition and size was calculated using the United States Department of Agri-
of TRL [11], which then modulates their atherogenicity. In culture [16] food tables and Spanish food composition tables
comparison with a reference diet (high in SFA), two diets with for local food [17]. All meals were prepared in the hospital
different levels of MUFA induced marked reductions in apo B- kitchen and were supervised by a dietitian. Lunch and dinner
48 production, suggesting that chylomicrons particle size were eaten in the hospital dining room, whereas breakfast
could increase following moderate to high-MUFA intakes and an afternoon snack were eaten in the medical school
[12]. However, these findings were derived from indirect cafeteria. Fourteen menus were prepared with regular solid
measurement of synthesis and should be interpreted with foods and rotated during the experimental period. The
caution. Less information is available about the effect of composition of the three dietary interventions and the fat
other dietary fats (i.e. omega 3) on TRL particle size and load meals has been previously published [18]. Briefly,
number. Given the widespread interest in the use of omega-3 Western diet: 15% protein, 47% carbohydrates (CHO), 38% fat
fatty acids in the prevention and therapy of a number of (22% saturated fatty acid (SFA)); Mediterranean diet: 15%
diseases including cardiovascular diseases [13,14], and the protein, 47% CHO, 38% fat (24% monounsaturated fatty acid
Dietary fat influences postprandial TG size and number 41

(MUFA)); high CHO enriched with a-linolenic acid (ALNA) chylomicron and large VLDL fraction of TRL (also referred to
diet: 15% protein, 55% CHO, <30% fat (8% polyunsaturated as large TRL), were isolated from 4 ml plasma overlayered
fatty acid (PUFA) (Table 1)). The PUFA enrichment of the with 0.15 m NaCl, 1 mm EDTA (pH 7.4; density, 1.006 g/ml)
high-CHO diet was achieved via the use of natural food by a single ultracentrifugal spin (28,000  g, 30 min, 4C) in
components rich in a-LNA of vegetable origin (based on a 50-type rotor (Beckman Instruments, Fullerton, CA). We
walnuts (Juglands regia L.)). The cholesterol content of the added 200 ml of the following preservative solution (Sodium
diets was <300 mg/day, and it was kept at a constant level Azide 100 mg, Aprotina (4TIU/mg) 2.75 mg, Cloramphenicol
throughout the three dietary intervention periods. At the end 80 mg, Benzamidine Hydrochloride hydrate 160 mg, Genta-
of the dietary intervention period and after a 12-h fast, at micin 80 mg, water 10 mL) to the plasma before TRL sepa-
time 0, the subjects were given a fatty breakfast with a fat ration. Large TRL contained in the top layer were removed by
composition similar to that consumed in each of the diets, aspiration after cutting the tubes. The infranatant was
consisting of 50e66% of the subjects’ daily normal intake of transferred to another tube and it was centrifuged at
calories and composed of 1 g fat, 7 mg cholesterol and 40 a density of 1.019 g/ml for 24 h at 115,000  g in the same
equivalent retinol per kg body weight, with the following rotor. The nonchylomicron fraction of TRL (also referred to as
caloric distribution: 60% fat, 15% protein, 25% CHO. The small TRL) was removed from the top of the tube.
composition of the three meals was: Butter meal: 35% SFA;
Olive oil meal: 36% MUFA; Walnut meal: 16% PUFA, 4% from Nuclear Magnetic Resonance spectroscopy
ALNA and 12% from linoleic acid (C18:2, LAA). The butter
meal was based on the consumption of butter, wholemeal The TRL particle number and size were measured by proton
bread, a hard-boiled egg and whole milk. The olive oil meal NMR spectroscopy on nonfractionated plasma (Lip-
was administered in the form of a typical Mediterranean meal oscience). This method quantifies particle concentration by
with extra virgin olive oil, bread and tomato, accompanied converting the characteristic signal amplitude generated by
by skimmed milk and a hard-boiled egg. The walnut meal the methyl group NMR signal of each lipoprotein particle
consisted of walnuts, wholemeal bread, jam and skimmed [24]. The process begins with automated measurement of
milk. The amount of each ingredient was calculated as the proton NMR spectrum of the patient’s plasma using
a function of individual body weight so that all subjects a dedicated 400-MHz NMR analyzer. The digitized spectrum
consumed the same type of food at different amounts. is stored in a computer memory and the analysis software
then extracts the amplitudes of the individual subclass NMR
Lipid analysis signals, converts them to concentration units (typically
nmol/L for particles number and nm for particle size), and
Blood was collected in tubes containing EDTA to give a final outputs the data in the NMR LipoProfile report format [25].
concentration of 0.1% EDTA. Plasma was separated from
red cells by centrifugation at 1500  g for 15 min at 4  C.
Statistical analysis
Cholesterol and TG in plasma and lipoprotein fractions
were assayed by enzymatic procedures [19,20]. Apo A-I and
Statistical analysis was carried out using SPSS statistical
Apo B were determined by turbidimetry [21]. HDL-choles-
software, version 15.0 (SPSS Inc, Chicago, IL) after log
terol was quantified by analyzing the supernatant obtained
transformation appropriate for TRL particle numbers. ANOVA
after precipitation of a plasma aliquot with dextran sulfate-
for repeated measures was used to analyse the differences in
Mg2þ, as described by Warnick et al. [22]. LDL-cholesterol
plasma lipid and lipoprotein concentrations. In these anal-
level was calculated from the total cholesterol, TG and
yses, we studied the statistical effects of the time alone or
HDL-cholesterol values using the Friedewald formula [23].
the change in the variable after ingesting fatty food over the
entire lipemic period (represented as P1: time effect) and
Lipoprotein separations the effect of the meal (represented as P2: meal effect),
independently of the time in the postprandial study. We also
TG in triacylglycerol-rich lipoproteins (TRL) (large and small studied the effect of the interaction of both factors, meal
TRL) were determined by ultracentrifugation. Thus, the and time, which is indicative of the magnitude of the

Table 1 Dietary interventions and meal compositions (% of energy intake).

Western diet Mediterranean diet High CHO enriched Butter meal Olive oil meal Walnut meal
with ALNA
Protein 15 15 15 15 15 15
CHO 47 47 55 25 25 25
Fat 38 38 <30 60 60 60
SFA 22 <10 <10 35 20 20
MUFA 12 24 12 22 36 24
PUFA 4 4 8 4 4 16
ALNA 0.4 0.4 2 0.7 0.7 4
CHO: Carbohydrates; SFA: Saturated fatty acid; MUFA: Monounsaturated fatty acid; PUFA: polyunsaturated fatty acid; ALNA: a-linolenic
acid.
42 P. Perez-Martinez et al.

postprandial response in each meal (represented as P3: compared to subjects who ate the other two meals (butter
time  meal interaction). When statistically significant and walnuts).
effects were found we used ANOVA to identify group differ-
ences in each time. The Tukey’s post-hoc comparison test NMR spectroscopy
was used to identify group differences and the contrast In the postprandial state, subjects who consumed the olive
statistic used when the sphericity assumption was not satis- oil meal had a reduced number of total TRL particles than
fied was Greenhouse-Geisser. A probability of less than 0.05 subjects who ate butter and walnut meals (P2 Z 0.002)
was considered significant. All data presented in the text and (Fig. 2A). On the other hand, we observed that consumption
tables are expressed as mean  SE. of the olive oil meal diet induced an increase in the TRL
particles size as compared with the walnut-enriched meal
Results (P2 Z 0.001) (Fig. 2B).

Effects of the different diets on fasting lipids Discussion

The biochemical characteristics of participants at the end This study reveals that consumption of an olive oil-rich
of each dietary intervention period and before each fat- meal leads to the formation of a reduced number of TRL
load meal are shown in Table 2. The intake during 4 weeks particles compared with butter and walnut-based meals.
of a Western diet induced a significant increase in plasma Moreover, TRL particle size was greater after the intake of
concentrations of LDL-cholesterol and apoB compared with the olive oil meal compared with the walnut meal.
the ingestion of a Mediterranean and high CHO enriched The use of non-fasting TG concentrations in coronary
with ALNA diet at the end of the intervention period heart disease (CHD) risk assessment appears to be clinically
(Table 2). In addition, the intake of the Western diet relevant as impaired postprandial lipemia is emerging as
induced a significant increment in plasma concentrations of a relevant factor in the pathogenesis of atherosclerosis
total cholesterol compared with the intake of the high CHO [26]. Plasma concentrations of chylomicrons and their
enriched with ALNA diet (Table 2). HDL-C was lower after remnants reflect the workings of postprandial lipid
the high CHO enriched with ALNA diet compared with the metabolism and correlate with both lesion progression and
Mediterranean diet. No significant differences were found cardiovascular events [27]. Furthermore, the size and
between the three diets on fasting TG, TG in TRL (large and number of TRL particle may be better predictors of
small TRL) glucose, and APO A-I (Table 2). atherosclerosis than conventional TG measurements [1].
The major change in lipoprotein particle number during
Effects of the test meals on TRL responses alimentary lipemia relates to the increase of postprandial
large VLDL particles [28]. Large VLDL particles accumulate
Ultracentrifugation during alimentary lipemia secondary to preferential lipol-
We analyzed postprandial responses of plasma and lipo- ysis of chylomicrons by LPL [29] and account for 90% of the
proteins TG following the intake of the different diets. cholesterol increase in the TRL fractions observed during
Postprandial plasma TG, large TRL-TG and small TRL-TG alimentary lipemia [30]. Most studies with n-3 PUFA-rich
concentrations are shown in Fig. 1. Interestingly, post- fats showed a reduced postprandial lipemia when ingested
prandial TG and large TRL-TG concentrations were much in the daily diet or as a single meal compared with other fat
greater during the early postprandial phase after the olive sources [31]. On the other hand, a previous study showed
oil meal and returned to near-fasting concentrations much an exacerbated chylomicron response after a butter meal
earlier than after the intake of the butter and walnuts meal compared with an olive oil one [32]. In contrast, data from
(P3 Z 0.002 and P3 Z 0.012, respectively) (Fig. 1) Mekki et al. showed that single mixed meals containing

Table 2 Biochemical characteristics of participants at the end of each dietary intervention period and before each fat load
meal.
Western diet (n Z 20) High CHO enriched Mediterranean diet (n Z 20) P value
with ALNA diet (n Z 20)
Total cholesterol, mmol/L 3.74 (3.46e4.16)* 3.40 (3.05e3.95)* 3.53 (3.31e4.06) 0.038
LDL-C, mmol/L 2.36 (2.06e2.66)*,y 2.13 (1.73e2.66)* 2.11 (1.83e2.63)y 0.093
HDL-C, mmol/L 1.16 (0.98e1.29) 1.11 (0.98e1.24)* 1.12 (1.06e1.26)* 0.029
TG, mmol/L 0.83 (0.62e1.08) 0.80 (0.58e1.03) 0.75 (0.57e0.95) 0.832
Apo A1, mmol/L 1.26 (1.09e1.34) 1.19 (1.10e1.35) 1.26 (1.13e1.39) 0.318
Apo B, mmol/L 0.68 (0.62e0.78)*,y 0.58 (0.56e0.69)* 0.63 (0.57e0.68)y 0.012
Glucose, mmol/L 4.53 (4.45e4.84) 4.58 (4.4e4.78) 4.83 (4.23e4.89) 0.717
TG large-TRL, mmol/L 0.07 (0.03e0.09) 0.04 (0.02e0.09) 0.05 (0.03e0.09) 0.983
TG small-TRL, mmol/L 0.21 (0.11e0.31) 0.25 (0.12e0.32) 0.20 (0.13e0.30) 0.772
Values are presented as median (interquartile range). ANOVA for repeated measures and post-hoc Tukey test to compare means. Same
superscript indicated P < 0.05 within each group.
Dietary fat influences postprandial TG size and number 43

P1=0.001 P1=0.001
2,1 P2=0.970 4 P2=0.002

Total VLDL & chylomicron

A

particles number (nmol/L)

P3=0.002 P3>0.050
1,9
3,9
1,7
TG(mmol/L)

1,5 3,8
1,3
1,1 3,7
0,9
3,6
0,7
0,5 3,5
0 1 2 3 4 5 6 8.5 11 0 1 2 3 4 5 6
Time (hr) Time (hr)

1 P1=0.001
Large-TRL TG (mmol/L)

60 P1=0.001
P2=0.448 P2=0.001
B

TRL particle size (nm)

0,8 P3=0.012 P3=0.007

0,6 55

0,4
50
0,2

0 45
0 1 2 3 4 5 6 8.5 11 0 1 2 3 4 5 6
Time (hr) Time (hr)

0,5 Butter meal

Small-TRL TG (mmol/L)

P1=0.001 Walnut meal

0,4 P2=0.970 Olive oil meal
P3=0.001
0,3
Figure 2 A. Line plots of postprandial total VLDL and
0,2 chylomicron particles number normalised by log trans-
0,1
formation (nmol/L) in participants according to the consump-
tion of the butter (continuous line, black diamonds), walnut
0 (discontinuous line, black squares) and olive oil (discontinuous
0 1 2 3 4 5 6 8.5 11
line, black triangles) meals. ANOVA for repeated measures. P1:
Time (hr)
time effect; P2: meal effect; P3: time  meal interaction.
Butter meal Values are mean  SE. B. Line plots of postprandial mean TRL
Walnut meal particles size (nm) in participants according to the consump-
Olive oil meal
tion of the butter (continuous line, black diamonds), walnut
Figure 1 Line plots of postprandial TG, large-TRL TG and (discontinuous line, black squares) and olive oil (discontinuous
small-TRL TG (mmol/L) response in participants according to line, black triangles) meals.
the consumption of the butter (continuous line, black dia-
monds), walnut (discontinuous line, black squares) and olive oil
(discontinuous line, black triangles) meals. ANOVA for Extrapolating these results to humans, Jackson et al.
repeated measures. P1: time effect; P2: meal effect; P3: observed that meals containing olive oil result in a greater
time  meal interaction. Values are mean  SE. apo B-48 response compared with palm oil, safflower oil, and
a mixture of fish and safflower oil, and it stimulated the
formation of both small (S[f] 60e400) and large (S[f] > 400)
butter reduce postprandial lipemia and chylomicron accu- apo B-48-containing lipoproteins [35]. These findings were
mulation in the circulation in healthy young men [33]. derived from indirect but consistent measurements where
However, this does not necessarily apply to other cate- the structural protein apo B-48, was used to determine the
gories of subjects or patients. In our study we tested number of chylomicron particles present in the circulation
whether the chronic consumption of three dietary models after each of the test meals and to estimate their particle
with different types of fat modify TRL number and particle size. In this context, we have shown that consumption of an
size during the postprandial state. Interestingly, the olive oil meal increases TG-rich lipoproteins particle size
consumption of the olive oil meal led to the formation of compared with butter and walnut meals. However, it is
a reduced number of higher-sized TRL particles compared important to note that these previous studies used different
with butter and walnut meals. Previous studies in Caco-2 methodologies and experimental designs and we need to be
cells, a human colon carcinoma cell line, have shown an cautious about extrapolating these results to our population.
increase in the number of particles secreted after incuba- The consistency of our findings using NMRs for quantification
tion with oleic acid as opposed to linoleic and palmitic acid of the number and size of lipoprotein particles is remarkable
[34]. The increase in apo B-containing lipoproteins released [25]. From an analytical perspective, the NMR method of
from the cells was attributed to the specific increase in apo lipoprotein subclass analysis used in this study has several
B-48, suggesting that olive oil may increase the expression advantages over the classic techniques of electrophoresis
of the apoB messenger RNA editing enzyme. and ultracentrifugation in terms of ease and speed of
44 P. Perez-Martinez et al.

measurement [36]. Moreover, NMRs eliminates the sources of conflict of interest; Javier Delgado-Lista, no conflict of
analytical variability inherent in separation procedures. interest; Nieves Delgado-Casado, no conflict of interest;
The mechanisms underlying the observed associations of Cristina Cruz-Teno, no conflict of interest; Antonio
dietary fat and particle size and number of TRL need to be Camargo, no conflict of interest; Elena M Yubero-Serrano,
elucidated. TG-rich lipid droplets formed during the post- no conflict of interest; Fernando Rodrı́guez, no conflict of
prandial state fuse with immature apo B-48-containing lipo- interest; Francisco Pérez-Jiménez, no conflict of interest;
proteins to form heterogeneously sized large-TRL and the size José López-Miranda, no conflict of interest.
of the mature large-TRL is partly determined by lipid droplet
size. Efficient lipidation of nascent apo B-48 requires micro-
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