A Walnut Diet Improves Endothelial Function in
A Randomized Crossover Trial
Emilio Ros, MD; Isabel Núñez, MD; Ana Pérez-Heras, RD; Mercè Serra, RD; Rosa Gilabert, MD;
Elena Casals, MD; Ramón Deulofeu, MD
Background—Epidemiological studies suggest that nut intake decreases coronary artery disease (CAD) risk. Nuts have a
cholesterol-lowering effect that partly explains this benefit. Endothelial dysfunction is associated with CAD and its risk
factors and is reversed by antioxidants and marine n-3 fatty acids. Walnuts are a rich source of both antioxidants and
?-linolenic acid, a plant n-3 fatty acid.
Methods and Results—To test the hypothesis that walnut intake will reverse endothelial dysfunction, we randomized in
a crossover design 21 hypercholesterolemic men and women to a cholesterol-lowering Mediterranean diet and a diet of
similar energy and fat content in which walnuts replaced ?32% of the energy from monounsaturated fat. Participants
followed each diet for 4 weeks. After each intervention, we obtained fasting blood and performed ultrasound
measurements of brachial artery vasomotor function. Eighteen subjects completing the protocol had suitable ultrasound
studies. Compared with the Mediterranean diet, the walnut diet improved endothelium-dependent vasodilation and
reduced levels of vascular cell adhesion molecule-1 (P?0.05 for both). Endothelium-independent vasodilation and
levels of intercellular adhesion molecule-1, C-reactive protein, homocysteine, and oxidation biomarkers were similar
after each diet. The walnut diet significantly reduced total cholesterol (?4.4?7.4%) and LDL cholesterol
(?6.4?10.0%) (P?0.05 for both). Cholesterol reductions correlated with increases of both dietary ?-linolenic acid and
LDL ?-tocopherol content, and changes of endothelium-dependent vasodilation correlated with those of cholesterol-
to-HDL ratios (P?0.05 for all).
Conclusions—Substituting walnuts for monounsaturated fat in a Mediterranean diet improves endothelium-dependent
vasodilation in hypercholesterolemic subjects. This finding might explain the cardioprotective effect of nut intake
beyond cholesterol lowering. (Circulation. 2004;109:GGG-GGG.)
Key Words: antioxidants ? diet ? endothelium ? hypercholesterolemia ? lipoproteins
consumption decreases the risk of coronary artery disease
(CAD), with adjusted relative risk reductions approaching
50% for nut intakes of ?4 to 5 servings per week compared
with little or no intake.2–4Feeding trials have demonstrated
that healthy diets enriched with a variety of nuts consistently
reduce total and LDL cholesterol by 5% to 15%.1,4–8The
lipid effects of nut intake only explain in part the CAD risk
reduction observed in prospective studies, suggesting that
nuts might have antiatherosclerotic effects beyond cholesterol
Besides having a favorable fatty acid profile, nuts are a rich
source of bioactive compounds with potential benefit on
CAD risk such as dietary fiber, folic acid, and antioxidants.1
Nuts also contain sizeable amounts of L-arginine, the precur-
uts are fatty foods rich in unsaturated fatty acids.1
Epidemiological studies have shown that frequent nut
sor amino acid of the endogenous vasodilator nitric oxide
(NO).9Walnuts differ from all other nuts by a high content of
?-linolenic acid (ALA), a vegetable n-3 fatty acid,10which
might confer them additional antiatherogenic properties.11
Endothelial dysfunction, an early event in the development
of vascular disease, is associated with atherosclerosis and its
risk factors, including hypercholesterolemia.12Recent studies
indicate that coronary endothelial dysfunction predicts future
CAD events.13,14Endothelial function can be assessed non-
invasively in the peripheral circulation,15and brachial artery
ultrasound measures correlate with those of coronary endo-
thelial function.16Vascular reactivity may be improved by
dietary factors such as marine n-3 fatty acids, antioxidants,
and L-arginine,17,18but whole foods rich in these compounds
have not been investigated. To test the hypothesis that walnut
intake would improve endothelial function in subjects with
Received May 12, 2003; de novo received October 10, 2003; revision received January 6, 2004; accepted January 12, 2004.
From the Lipid Clinic at the Endocrinology and Nutrition Service (E.R., A.P.-H., M.S.), Centre de Diagnòstic per l’Imatge (I.N., R.G.), and Centre
de Diagnòstic Biològic (R.D., E.C.), Institut d’Investigacions Biomèdiques August Pi i Sunyer, Hospital Clínic, Barcelona, Spain.
Correspondence to Emilio Ros, MD, Clínica de Lípids, Servei d’Endocrinologia & Nutrició, Hospital Clínic, Villarroel 170, Barcelona, Spain E-08036.
© 2004 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.orgDOI: 10.1161/01.CIR.0000124477.91474.FF
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<ARTICLEDOCTOPIC??Clinical Investigationand Reports?
DATE??April 6, 2004?
hypercholesterolemia, we performed a randomized, crossover
feeding trial by substituting a walnut-enriched diet for a
healthy Mediterranean-type diet for effects on brachial artery
vasomotor function and circulating markers of endothelial
Twenty-one nonsmoking, asymptomatic men and women with mod-
erate hypercholesterolemia attending the Lipid Clinic at the Hospital
Clínic of Barcelona (Spain) and following a cholesterol-lowering
diet were recruited into a protocol approved by the institutional
review board, and all gave informed consent. Eligibility criteria were
age 25 to 75 years (after menopause in women), serum LDL
?2.82 mmol/L, absence of chronic illnesses or secondary hypercho-
lesterolemia, and no known allergy to nuts. None of the participants
took vitamin supplements, hormone replacement therapy, or medi-
cations known to affect lipid metabolism. They were offered free
walnuts but no monetary compensation.
Before the study, the general recommendations of a Mediterranean-
type, cholesterol-lowering diet were reinforced in all eligible sub-
jects, and baseline data were collected after 4 weeks. The self-
reported nutrient contents of the baseline diet showed good
adherence to dietary advice (Table 1). The 4-week dietary equilibra-
tion period produced a mean change of -?.4?7.2% in total choles-
terol level (P?0.031). Smaller, nonsignificant changes were ob-
served for LDL cholesterol, HDL cholesterol, and triglycerides.
After this period, participants were individually randomized in a
crossover design between 2 diet sequences for 4-week periods: a
control, Mediterranean-type diet and an isoenergetic diet enriched
with walnuts. Twelve participants followed the control diet first for
4 weeks and then switched to the walnut diet for the ensuing 4
weeks; 9 subjects followed the same diets in reverse order. Because
diet-induced lipoprotein changes stabilize in ?4 weeks,19we did not
incorporate a washout period between diets. Participants ate on their
own, a reason why detailed information was provided to them and,
if appropriate, to their partners. Table 1 shows the nutrient content of
the prescribed diets. The diets were composed of natural foodstuffs.
Vegetable products and fish were emphasized, and red and processed
meats, whole-fat dairy products, and eggs were limited. The walnut
diet was similar to the control diet, but walnuts partially replaced
olive oil and other monounsaturated fatty acid (MUFA)–rich foods
such as olives and avocados (no nuts other than walnuts in the walnut
diet were allowed during the study). Prepackaged daily allowances
of raw, shelled walnuts were provided daily in amounts varying from
40 to 65 g (equivalent to 8 to 13 walnuts), according to the
participants’ total energy intake. Walnuts were consumed as snacks
or with meals in desserts or salads. In the walnut diet, walnuts
contributed ?18% of the total energy and replaced 32% of the
energy obtained from MUFA in the control diet.
The walnuts used in the study were analyzed by standard methods
in a reference laboratory (IRTA, Generalitat de Catalunya). The
composition (per 100 g) of walnuts was 14 g protein (of which 18%
arginine), 14 g carbohydrate, and 69 g fat. The fatty acid composition
was 10.1% saturated fatty acids (SFA), 16.9% MUFA, 59.6% n-6
polyunsaturated fatty acid (PUFA; linoleic acid), and 13.4% n-3
PUFA (ALA). Vitamin E components (per 100 g) were 1.8 mg
?-tocopherol and 155 mg ?-tocopherol.
Compliance was assessed from 7-day diet recalls. The diets were
analyzed with the Food Processor, version 8.44, software (ESHA
Research) adapted to nutrient databases of specific Mediterranean
foods when appropriate. Because walnuts are a particularly rich
source of ?-tocopherol, its serum level was measured as a biological
marker of adherence to the walnut diet.
Fasting blood samples were obtained at baseline and at the end of
each diet period. Except for immediate lipoprotein determinations,
serum and EDTA plasma samples were stored at ?80°C and
analyzed at the end of the study. Cholesterol and triglycerides were
measured with enzymatic procedures. HDL cholesterol was quanti-
fied after precipitation with phosphotungstic acid and magnesium
chloride. Apolipoproteins (Apo) Al and B and lipoprotein(a) were
determined by use of turbidimetry. The cholesterol content of VLDL
and LDL was measured after separation of lipoproteins by density
gradient ultracentrifugation as described.6Frozen LDL aliquots,
preserved in a 100-g/L sucrose solution, were stored at ?80°C for
use in copper-induced oxidizability studies and measurement of
vitamin E content at the end of the study.6Analytes determined by
subject in frozen samples of whole serum or plasma as appropriate
were ?-tocopherol and malondialdehyde (MDA) by standard high-
performance liquid chromatography methods, oxidized LDL by a
monoclonal antibody-based immunoassay (Mercodia AB), folic acid
by enzyme immunoassay, homocysteine by fluorescence polariza-
tion immunoassay, soluble intercellular adhesion molecule-1
(ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) by
standard ELISA from DRG Diagnostica (Palex Cormedica), and
TABLE 1.Composition of the Baseline Diet and the Prescribed and Actually Observed Study Diets
Control Diet Walnut Diet
VariableBaseline Diet, Actual* Prescribed Actual*PrescribedActual*P†
Fat, % energy
Marine n-3 PUFA
Protein, % energy
Carbohydrates, % energy
Soluble fiber, g/d
*Mean?SD values estimated from 7-day food records during each diet period.
†Statistical significance for comparison between study diets by paired t test.
April 6, 2004
high-sensitivity C-reactive protein (hsCRP) by particle-enhanced
immunonephelometry. All analyses were done in duplicate.
The noninvasive method of Celermajer et al15was used to evaluate
endothelial function. Studies were performed at the end of each diet
period. On the day of testing, participants ate a standard breakfast at
7 AM and then fasted until 1 PM, when a test meal was given. The
meal consisted of a 100-g bread sandwich with 50 g lean pork ham,
an apple, and tap water. For tests conducted during the control diet,
the bread was soaked with 15 to 20 mL olive oil, whereas 20 to
32.5 g walnuts (half the daily dose) was incorporated into the test
meal during the walnut diet. The varying doses of fat were adapted
to individual energy requirements so that the test meal reproduce
dietary conditions during the trial while keeping a constant amount
of energy (550 to 650 kcal) and fat (19 to 27 g) across the 2 studies
in each participant. Vascular ultrasound studies were performed 4
hours after ingestion of the test meal.
The studies were performed with a color Doppler Toshiba Pow-
erVision ultrasound apparatus using multifrequency transducers
(B-mode, 7.5 to 10 MHz; Doppler, 3.75 MHz). The brachial artery
was imaged longitudinally, 2 to 5 cm above the antecubital crease, by
an experienced ultrasound radiologist (I.N.) who was unaware of the
stage of the experiment. An occluding cuff placed proximally on the
forearm was inflated to a pressure of 300 mm Hg for 4.5 minutes and
rapidly deflated to induce reactive hyperemia. Brachial artery scans
were obtained continuously for 30 seconds before cuff inflation (first
baseline), at 60 to 90 seconds after cuff deflation to assess endothe-
lium-dependent vasodilation (EDV), for another 30 seconds after a
10-minute rest (second baseline), and 3 minutes after 0.4 mg
sublingual glyceryl trinitrate to evaluate endothelium-independent
vasodilation (EIDV). Arterial diameter was measured with ultrasonic
calipers at end diastole, incident with the R wave on the ECG. Four
cardiac cycles were analyzed for each scan, and measurements were
averaged. Reproducibility of measurements over 1 month was
documented in 15 healthy volunteers. Intraobserver reliability for
measurement of EDV was 0.74 (P?0.003). The repeatability coef-
ficient was 5.16, and the mean? SD difference between EDV values
For a crossover design, power calculations indicated that, to detect a
mean absolute difference in EDV of 2% (SD, 2.66), 19 subjects
would need to complete the study (? statistic, 0.05; power ?0.9).
Data are presented as mean?SD. Two-tailed t tests or the Wilcoxon
rank-sum test for paired samples as appropriate was used to compare
changes in outcome variables in response to diets. To examine
whether there was any carryover effect from the order of diets for the
2-period crossover design, we performed a repeated-measures
ANOVA with order and treatment as independent variables and
outcome as the dependent variable. Differences between diets were
tested also by ANCOVA using general lineal models, with baseline
values or sex as covariates. Pearson’s correlation coefficients were
used to assess relationships between continuous variables. Analyses
were performed with SPSS software (version 10.0).
Of the 21 participants randomly assigned to dietary interven-
tion, 20 completed both study phases. One subject withdrew
for personal reasons. The 8 men and 12 women who com-
pleted the trial had a mean age of 55 years (range, 26 to 75
years). The nutrient content of the self-reported diets was in
good agreement with the planned diets (Table 1). The fatty
acid composition of the control diet reflected the high MUFA
content of olive oil, and that of the walnut diet mirrored the
PUFA constituents of walnuts. According to participants’
reports and recounts of empty packages, compliance with
walnut ingestion was 100%. This was confirmed by the
increase in the serum ?-tocopherol level during the walnut
diet compared with the control diet (3.46?1.28 versus
1.89?1.05 ?mol/L, P?0.001). Daily walnut consumption
was well tolerated by all subjects.
Suitable brachial artery ultrasound measurements were
available in 18 subjects. Compared with the olive oil–rich
meal during the control diet, the walnut meal during the
walnut diet significantly (P?0.043) improved EDV from
3.6?3.3% to 5.9?3.3%, a relative increase of 64% (the
Figure and Table 2). Of the 18 participants, EDV improved
by ?2% in 9 subjects on the walnut diet. When these 9
diet-responsive subjects are compared with the 9 subjects
who showed smaller changes or no benefit in response to the
walnut diet, the only difference was percent change of the
EDV in brachial arteries of 18 men and women with moderate
hypercholesterolemia after control Mediterranean diet and wal-
nut diet given for 4 weeks each in crossover design. Bold lines
represent mean values. Differences are statistically significant
(P?0.043) by paired t test.
Endothelial Activation at Baseline and at the End of Each Diet Period
Vasomotor Function of the Brachial Artery and Soluble Markers of
Variables Baseline Control Diet Walnut DietP*
Baseline artery diameter, mm
Hyperemic flow, mL/min
Values are mean?SD.
*Statistical significance for comparison between experimental diets by paired t test.
Ros et alWalnuts Improve Endothelial Function
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P?0.070). There was no evidence of a carryover effect
between the periods. Adjustment for sex or baseline EDV did
not change the results. At the end of each diet period, brachial
ultrasound studies showed no significant differences in the
baseline vessel diameter, EIDV, or hyperemic flow (Table 2).
The level of VCAM-1 was significantly (P?0.045) lower
during the walnut diet than during the Mediterranean diet. We
did not find significant differences in ICAM-1 levels between
the diets. There was an inverse correlation between changes
in EDV and changesof
Table 3 shows the actual values at baseline and at the end
of each diet period for other outcomes. Body weight and
blood pressure were stable throughout the study. Compared
with the Mediterranean diet, the walnut diet produced signif-
icant (P?0.05) reductions in total cholesterol (?4.4?7.4%)
and LDL cholesterol (?6.4?10.0%). Parallel nonsignificant
reductions in apoB and the LDL:HDL ratio were also
observed with the walnut diet. The differences of effect
between the 2 diets on the lipid profile did not change
materially when adjusted for baseline values or gender. The
ratios(?5.9?7.9 versus 2.1?9.5;
changes between diets in total cholesterol, LDL cholesterol,
and apoB were inversely correlated with both self-reported
changes in dietary ALA as percent of energy (r??0.561,
P?0.010; r??0.578, P?0.008; and r??0.594, P?0.006,
respectively) and changes in LDL ?-tocopherol (r??0.459,
P?0.044; r?0.446, P?0.048; and r??0.516, P?0.020,
respectively). The 2 diets had opposite effects on the LDL
content of ?-tocopherol and ?-tocopherol, whereas other
biomarkers of oxidative stress and serum folic acid, homo-
cysteine, and hsCRP were unaffected (Table 3).
In this 8-week crossover feeding trial in subjects with
moderate hypercholesterolemia, we found that substituting
walnuts for ?32% of the energy from MUFA in a cholester-
ol-lowering Mediterranean diet improves vascular endothelial
function. Consistent with prior reports,1,4–7the walnut diet
decreased total cholesterol and LDL cholesterol levels. Cho-
lesterol reductions with the walnut diet were inversely related
to self-reported dietary ALA increases. This was not unex-
pected, because cell culture experiments have shown that
LDL enrichment with ALA following a walnut diet facilitates
Oxidative Stress at Baseline and at the End of Each Diet Period
Body Weight, Blood Pressure, Serum Lipids, and Biomarkers of
Variables BaselineControl Diet Walnut DietP*
Body weight, kg
Blood pressure, mm Hg
LDL ?-tocopherol, nmol/mg protein
LDL ?-tocopherol, nmol/mg protein
Lag time of LDL CD production, min
Oxidized LDL, U/L
Folic acid, ng/mL
CD indicated conjugated dienes. Values are mean?SD or medians (interquartile ranges). To
convert cholesterol and triglycerides to mg/dL, multiply by 38.67 and 88.57, respectively.
*Statistical significance for comparison between the experimental diets by paired t test or the
Wilcoxon rank-sum test.
April 6, 2004
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receptor-mediated LDL clearance.20Our results provide di-
rect evidence that regular walnut intake may reduce cardio-
vascular risk by a dual mechanism in subjects at risk for
One limitation of the study is the use of outpatient
intervention diets as opposed to a controlled feeding trial with
meals prepared at a metabolic kitchen. However, compliance
was very good, and the actual diets consumed closely
matched the prescribed diets (Table 1). In fact, the results
show that motivated, free-living persons who have been given
appropriate dietary advice may closely follow designed diets
and incorporate substantial quantities of walnuts into their
meals. Another limitation is that we evaluated postprandial
endothelial function but did not obtain fasting measurements.
Vascular reactivity may be impaired after a fatty meal, and
this effect may be counteracted by n-3 fatty acids or antioxi-
dants in the meals.18Nevertheless, the test meals were
designed to reproduce the feeding conditions during the 2
dietary periods, and no other sources of variation of vascular
reactivity were introduced. From this study, we cannot know
whether, compared with an olive oil-rich meal and as a result
of acute effects of dietary components, a walnut meal lessens
the deterioration of EDV that presumably follows a fatty
meal. However, because humans spend a good part of the day
in a postprandial state, when the effects of foods on endothe-
lial function are more likely to be relevant, it is arguable
whether the chronic effect of the walnut diet can be separated
from the repeated acute effects of walnut meals.
The walnut meal in a background walnut diet was associ-
ated with significant improvement in brachial artery EDV.
The walnut diet also attenuated endothelial activation, as
suggested by the reduction in VCAM-1 levels. The mecha-
nism by which walnut intake may improve endothelial
function remains uncertain. The inverse correlation between
changes in EDV and those in cholesterol-to-HDL ratios
suggests that the effect of the walnut diet may be mediated in
part through an improved lipid profile. It is well established
that hypercholesterolemia impairs EDV and that endothelial
dysfunction can be reversed by aggressive cholesterol-
lowering treatment.12Although significant, the average 6.4%
decrease in LDL cholesterol observed with the walnut diet is
modest compared with the profound LDL cholesterol reduc-
tions induced by hypolipidemic drugs or LDL apheresis that
have been associated with improved EDV in clinical trials.12
This suggests that other factors may play a role in the
beneficial vascular effects of the walnut diet.
One component of walnuts that might favorably influence
endothelial function is the plant n-3 fatty acid ALA. The
walnut allowances used in the study provided daily amounts
of ALA ranging from 3.7 to 6.0 g. These doses of ALA are
nearly double those used in the intervention groups of 3
secondary prevention trials showing marked reductions in
cardiac end points that have been attributed in part to
supplemental ALA.21–23Intake of 20 g/d ALA from flaxseed
oil for 1 month has been reported to improve arterial
compliance despite increased LDL oxidation,24but no formal
endothelial function studies have been performed after ALA
supplementation. On the other hand, fish oil supplements at
doses ranging from 3 to 10 g/d consistently ameliorate EDV
in clinical studies.18,25,26This beneficial effect of marine n-3
fatty acids might be mediated by increased membrane fluidity
of endothelial cells promoting enhanced synthesis and/or
release of NO.27There is also in vitro evidence of reduced
endothelial expression of VCAM-1 by marine n-3 fatty
acids,28but fish oil intake has been associated with increased
VCAM-1 levels, perhaps because of an enhanced oxidative
Despite its high PUFA content, the walnut diet had no
deleterious effects on biomarkers of oxidative stress. Both the
resistance of LDL to in vitro oxidation and the level of
oxidized LDL were similar after the 2 diets, whereas the
plasma level of MDA, an end product of the peroxidation
process, decreased nonsignificantly after the walnut diet.
Although there were predictable,29reciprocal changes in the
LDL content of ?- and ?-tocopherol between the 2 diets, the
data suggest that endogenous antioxidants were spared during
the walnut diet. Phenolic compounds in walnuts probably
counteracted the pro-oxidant effects of PUFA on LDL.30
These results provide further evidence that walnut diets are
not associated with lipid peroxidation.6,7A recent report
indicates that, among edible plants, walnuts have one of the
highest contents of total antioxidants.31Oxidation markers
were not worsened by the walnut diet, but neither were they
improved; hence, our study does not support the antioxidant
potency of walnuts as an alternative explanation for the
observed beneficial effects on vascular reactivity.
?-Tocopherol is another strong antioxidant that is particu-
larly abundant in walnuts. The walnut diet provided 90 to 135
mg/d of this vitamin E component and increased its serum
level ?2-fold. Although ?-tocopherol has been investigated
much less than ?-tocopherol, it is increasingly recognized as
a relevant antiatherogenic molecule.32The lack of clinical
trials using ?-tocopherol for effects on markers of cardiovas-
cular risk precludes any conclusions on its eventual vasomo-
tor effects. Interestingly, serum cholesterol reductions were
related to increases in the ?-tocopherol content of LDL. A
likely explanation is that LDL ?-tocopherol is a marker of the
bioavailability of the walnut constituents responsible for
As the substrate for endothelium-derived NO, the amino
acid L-arginine, still another important constituent of nuts,9
also might have a positive effect on EDV. Walnut intake
increased dietary L-arginine by 0.9 to 1.4 g/d. Endothelial
function improves after treatment with L-arginine supple-
ments starting at doses of 2 g/d, particularly in subjects with
impaired NO synthesis such as patients with hypercholester-
olemia or prior congestive heart disease.17,18,33
Endothelial dysfunction associated with cardiovascular
risk factors or established congestive heart disease may be
restored by various food components in the absence of any
cholesterol lowering. Thus, supplements of marine n-3 fatty
acids, L-arginine, folic acid, and antioxidant vitamins C and E
and beverages rich in phenolic compounds such as red wine,
fruit juice, and tea have demonstrated a beneficial vasomotor
effect in various arterial beds.18Replacement of a diet rich in
SFA with 2 cholesterol-lowering diets, a low-fat diet or an
olive oil–rich, Mediterranean-type diet, has also been shown
to improve brachial artery EDV in hypercholesterolemic
Ros et alWalnuts Improve Endothelial Function
balt2/zhc-ci/zhc-ci/zhc01304/zhc9723-04ataylormpS?6 3/5/0416:17 Art: 145119
men.34This argues against the partial replacement of olive oil Download full-text
and other MUFA-rich foods in the walnut diet as being
relevant to improved EDV. The unique composition of
walnuts, a whole food rich in several nutrients and phyto-
chemicals with the capacity to improve vascular reactivity,
probably explains why substituting walnuts for part of the
MUFA in a Mediterranean diet was associated with improved
endothelial function in men and women with hypercholester-
olemia. This finding might explain the cardioprotective effect
of regular nut intake beyond cholesterol lowering. It also
provides further support for the inclusion of walnuts in
Financial support was provided by grants from the California Walnut
Commission, Spanish Ministry of Health (FIS 00/0992 and ISCIII
Red G03/140), and Fundació Privada Catalana de Nutrició i Lípids.
We sincerely thank the patients participating in the study; Montserrat
Portas, RN, for careful extraction and handling of blood samples;
María J. Ortega, RN, for expert assistance with ultrasound studies;
Emili Corbella for expert statistical advice; and Catherine Bauchet
and Christine Bauchet, who provided excellent assistance with
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