Pharmacological Research 61 (2010) 200207

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Pharmacological Research
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Review
Health effects of oleic acid and long chain omega-3 fatty acids (EPA and DHA)
enriched milks. A review of intervention studies
Eduardo Lopez-Huertas

Estacin Experimental del Zaidn, Consejo Superior de Investigaciones Cientcas (CSIC), Profesor Albareda 1, Granada 18008, Spain
a r t i c l e i n f o
Article history:
Received 2 October 2009
Received in revised form 29 October 2009
Accepted 29 October 2009
Keywords:
Review
Functional foods
Oleic acid
Eicosapentaenoic acid (EPA)
Docosahexaenoid acid (DHA)
Enriched milk
Cholesterol
Cardiovascular disease
a b s t r a c t
Substitution of dietary saturated fat by oleic acid and/or polyunsaturated fatty acids (PUFA) has been
described to reduce the cardiovascular risk by reducing blood lipids, mainly cholesterol. Additional ben-
ets have beendescribedfor longchainomega-3PUFA(eicosapentaenoic acidEPAanddocosahexaenoic
acidDHA) from sh oils. In recent years, food technology has been used to produce dairy drinks with
a reduced content of saturated fat in favour of those fatty acids, most of them claiming cardiovascular
benets. This review summarises all the scientic evidence regarding the effects of milks enriched with
long chain omega-3 PUFA (EPA+DHA) and/or oleic acid on cardiovascular health. Nine controlled inter-
vention studies with enriched milks have reported effects on healthy volunteers, subjects with increased
risk factors andcardiovascular patients. The maineffects observedwere reductions of bloodlipids, mainly
cholesterol, LDL-cholesterol and triglycerides.
2009 Elsevier Ltd. All rights reserved.
Contents
1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
2. Health effects of oleic acid, EPA and DHA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
3. Current intakes and recommendations of LC-n3-PUFA and oleic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
4. Summary of intervention studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
4.1. A study in children . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
4.2. Studies with healthy volunteers and hyperlipidemic subjects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
4.3. Studies with patients: cardiovascular disease and metabolic syndrome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
5. The daily dose in the context of the diet. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
6. Bioavailability of EPA+DHA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
7. The magnitude of the effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
8. Concluding remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
Conict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
Abbreviations: CV, cardiovascular; CVD, cardiovascular disease; CHD, coronary heart diseaase; DHA, docosahexaenoic acid; EFSA, European Food Safety Agency; EPA,
eicosapentaenoic acid; HDL, high-density lipoprotein; LDL, low-density lipoprotein; MI, myocardial infarction; MS, metabolic syndrome; MUFA, mono-unsaturated fatty
acids; PUFA, poly-unsaturated fatty acids; PVD, peripheral vascular disease; SFA, saturated fatty acids; TG, triglycerides; RCT, randomised controlled trial; RDI, recommended
daily intake.

Tel.: +34 958 181600x181; fax: +34 958 129600.

E-mail address: eduardo.lopezhuertas@eez.csic.es.
1043-6618/$ see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phrs.2009.10.007
E. Lopez-Huertas / Pharmacological Research 61 (2010) 200207 201
1. Introduction
In the last decade we have witnessed an extraordinary increase
in the number of functional foods targeted to the ever-growing
health conscious population. Several categories among these foods
can be found according to their health target, including the car-
diovascular (CV) system. These foods, when they are consumed
regularly and in the context of a healthy diet and lifestyle, intend
to maintain or improve the CV health by reducing the levels of risk
factors such as cholesterol.
Milk and dairy products are every day consumed foods and con-
stitute a good and popular source of bioavailable calcium. Whilst
milk and dairy foods in general are perceived as healthy foods,
milk fat contains about 70% of saturated fatty acids (SFA). These
fatty acids (mainly myristic and palmitic acids) may raise total and
low-density lipoprotein (LDL) cholesterol, increasing the risk of CV
disease (CVD), hence the consumption of milk fat should be limited
[1].
Since the most effective replacement for saturated fatty acids
in terms of coronary heart disease (CHD) outcome are polyunsat-
urated fatty acids (PUFA) and oleic acid (the main fatty acid from
olive oil) [2], a number of dairy companies have used food technol-
ogy to substitute milk fat with PUFA, oleic acid, or combinations of
both to produce healthier milks. Whilst this substitution should
produce nutritional benets derived from the overall reduced
intake of saturated fat, many of the nal products obtained claim
beneticial effects on blood lipids, i.e. reductions on total and/or
LDL-cholesterol, triglycerides or increases of HDL-cholesterol in
blood.
This review summarises all the scientic evidence regard-
ing the effects of milks enriched with long chain omega-3 PUFA
(EPA+DHA) and/or oleic acid on CV health. A critical discussion
and a few concluding remarks are also presented.
2. Health effects of oleic acid, EPA and DHA
There is ample scientic evidence showing that modulation of
dietary fat composition affects blood-lipid concentrations. Regard-
ing oleic acid, or monounsaturated fatty acids (MUFA), the most
noticeable effects come fromstudies where the substitution of sat-
urated fat with oleic acid has been tested. Isocaloric replacement
of about 5% of energy from saturated fatty acids by oleic acid (or
PUFA) has been estimated to reduce coronary heart disease risk by
2040% mainly via LDL-cholesterol reduction [3]. Other benecial
effects on risk factors for CVD such as factors related to thrombo-
genesis, in vitro LDL oxidative susceptibility and insulin sensitivity
have also been reported [36]. As intakes of saturated fat in many
countries are higher that the recommended levels [7], an increase
in the intake of oleic acid may be benecial as it limits the intake
of saturated fat. This can be achieved by changing dietary patterns
like using olive oil instead of butter, or by using food technology
to modify the fatty acid prole of foods naturally rich in saturated
fatty acids in favour of oleic acid.
The most important long chain omega-3 PUFA are EPA and DHA
found in sh oils from marine products. The benets derived from
these fatty acids are based on the convincing inverse relationship
observed between its consumption and a decreased risk of CVD,
particularlythecoronaryhearthdisease[2]. Thebiological effects of
EPA and DHA are wide ranging, involving lipoproteins, blood pres-
sure, cardiac function, endothelial function, vascular reactivity and
cardiac electrophysiology, as well as potent antiplatelet and anti-
inammatory effects [810]. These effects depend on dose, time
and baseline characteristics of the subjects. In addition, EPA and
DHA specically reduce plasma triglyceride levels and thereby the
risk of CVD [11].
3. Current intakes and recommendations of LC-n3-PUFA
and oleic acid
Intakes of EPA+DHA are based on food-consumption data and
chemical analyses of diets. Unfortunately, due to the lack of reli-
able food survey data in many European countries, estimates of
EPA+DHA intake are rather scarce. Mean dietary intakes of these
fatty acids among adults have recently reported a daily intake of
265mg in Austria, 380mg in France, 250 in Germany and 90mg
in The Netherlands [12]. These gures are high compared with the
EPA+DHA calculated intake in the US (100200mg/day), but low
compared to the values reported for Japan (up to 2g/day) where
sh consumption is very high [13].
Recommendations of EPA+DHA intake fromnational and inter-
national authorities range200650mgper dayandarebasedonthe
convincing inverse relationshipobservedbetweenits consumption
and a decreased risk of CVD [2,14]. However, intake recommenda-
tions for the general population established by national authorities
are not uniform and reect the different criteria on which they are
based. A selection of these recommendations is given as follows:
- The World Health Organisation (WHO) recommendations for the
general population established that regular sh consumption
(12 servings per week) providing 200500mg of EPA+DHA per
servingis protectiveagainst coronaryheart diseaseandischaemic
stroke [2]. Therefore their recommendations range from 200 to
1000mg. WHO also indicates that people who are vegetarians
and do not take sh are recommended to ensure adequate intake
of plant sources of alpha-linolenic acid, as some of it (0.520%
depending on various factors) is metabolised to EPA [15,16].
- The American Heart Association (AHA), also recommends for the
general population to consume sh, especially oily sh, at least
twiceaweek[11]. Estimates basedonconsumptionof oneportion
(125g) of oily sh (2g EPA+DHA per 100g on average) and one
portion of lean sh (0.2g/100g) result in an approximate intake
of 3g of DHA+EPA per week or 430mg per day. This association
also established intakes of 1g of EPA+DHA from sh or sh oils
for subjects with clinical history of CVD and a 24g supplement
for subjects with high blood triglycerides which produces typical
2040% reductions [17].
- The Foods Standards Agency of the UK, based on work
from the Joint Health Claims Initiative group of experts
(http://www.jhci.org.uk/approv/omega.htm), established Rec-
ommended Daily Intakes (RDI) of EPA+DHA of 3g per week or
450mg per day [18].
- The French authority for food safety also established recom-
mendations for adult men (500mg/day) and adult women
(400mg/day) [19] whereas The Netherlands proposed450mgper
day [20].
- The European Food Safety Agency (EFSA) has recently reviewed
and proposed labelling reference intakes values of 250mg per
day of EPA+DHA [12]. These values intend to represent the Euro-
pean recommended daily intakes (RDI) for adults in the future
(as currently RDI values in Europe are not harmonised across
countries). Interestingly, although the above mentioned RDI cur-
rentlyusedinEuropearewell abovethis value(400450mg/day),
EFSA argues that the most recent evidence shows that, when
only healthy subjects are considered, the intake of EPA+DHA
is negatively related to CV risk in a dose-dependent way up to
250mg/day(12servings of oilyshper week) [12]. Theserecom-
mendations shouldinthe future extendto all Europeancountries.
As oleic acidis the quantitatively most important representative
of MUFAin the diet, oleic acid and MUFAintakes and recommenda-
tions are virtually the same. According to the TRANSFAIR study and
recent national surveys, the total intake of oleic acidinadults varies
202 E. Lopez-Huertas / Pharmacological Research 61 (2010) 200207
Table 1
Study design, intervention groups, daily intake of nutrients from dairy and duration of the interventions of the studies with omega 3 and oleic acid enriched milks. RCT is
randomised control trial, CnR is control non-randomised.
Study design/reference Intervention groups Daily intake of nutrients from the food used in
the intervention
Duration intervention
RCT with crossover [21] Control group: whole milk Per 500mL (daily intake): total fat: 16g, of
which SFA: 10.7g, MUFA: 4.8g, PUFAs: 0.48g
14 months
Supplemented group: milk enriched with
oleic acid and PUFA
Per 500mL (daily intake): total fat: 16g, of
which SFA: 2.4g, MUFA: 11.2g, PUFAs: 2.4g
14 months
CnR [22] Control: semi-skimmed milk group 500mL/day of standard semi-skimmed milk
(1.7g dairy fat fat/100mL) providing: total
dairy fat 8.5g
4 weeks
Supplemented group consuming the
enriched dairy drink. Group (S)
500mL/day of a skimmed dairy drink
supplemented with EPA and DHA, -linolenic
acid and vit E (1.7g fat/100mL) providing:
total fat: 8.5g, n3 fatty acids 0.4g, of which,
-linolenic acid: 100mg, DHA: 180mg, EPA:
120mg, Vit E: 15mg
6 weeks
CnR [23] Control: semi-skimmed milk 500mL/day of Standard semi-skimmed milk
(1.9g dairy fat fat/100mL) providing 8.5g of
dairy fat: SFA: 6.7g, oleic acid: 1.82g, PUFAs:
0.22g, no EPA, no DHA
4 weeks
Supplemented group: enriched dairy drink 500mL/day of a skimmed dairy drink
supplemented with Epa and DHA, oleic acid
and vitamins (1.9g fat/100mL) providing SFA:
2.25g, oleic acid 5.12g, PUFAs:1.85g, DHA:
200mg, EPA: 130mg
8 weeks
CnR [24] Control: Semi-skimmed milk 500mL/day as in [21] 4 weeks
Supplemented group: enriched dairy drink. 500mL/day as in [21] 8 weeks
RCT [29] Control: Semi-skimmed milk 500mL/day as in [21] 12 months
Supplemented group: enriched dairy drink. 500mL/day as in [21], but providing EPA:
200mg, DHA: 130mg
12 months
RCT [31] Control: semi-skimmed milk 500mL/day (1.9g dairy fat fat/100mL)
providing SFA: 6.7g, Oleic acid: 1.85g, PUFAs:
0.22g, No EPA or DHA
3 months
Supplemented group consuming the
enriched dairy drink. Group (S)
500mL/day of a skimmed dairy drink
supplemented with sh oils, oleic acid and
vitamins (1.9g fat/100mL) providing SFA:
2.8g, oleic acid 5.7g, EPA: 15mg, DHA: 171mg
3 months
RCT [30] Control: Semi-skimmed milk 500mL/day as in [29] 12 months
Supplemented group: enriched dairy drink 500mL/day as in [29] 12 months
RCT [27] Control 1: semi-skimmed milk Per 500mL (daily intake): SFA: 6.7g, oleic acid:
1.82g, PUFAs: 0.22g, EPA+DHA: 0
1 year
Control 2: skimmed milk Per 500mL of daily intake: SFA: 0.21g, oleic
acid 0.06g, PUFAs: 6.9mg, EPA+DHA: 0
1 year
Enriched dairy drink Per 500mL of daily intake: SFA: 2.25g, oleic
acid 5.12g, PUFAs: 1.85g, EPA: 200mg, DHA:
130mg
1 year
RCT [28] Intervention 1: Control. Maltodextrin
and soy oil enriched dairy drink
Total fat 3gSFA: 1.36g, MUFA: 0.88g, PUFAs:
0.8g, EPA+DHA: 0, insol. bre: 1.16g, sol.
bre: 0.16g
6 weeks
Intervention 2: omega-3 enriched dairy
drink
Total fat 3.44gSFA: 1.84g, MUFA: 1g, PUFAs:
0.8g, EPA+DHA: 0.4g, insol. bre: 3.24g, sol.
bre: 0.2g
6 weeks
Intervention 3. bre enriched dairy drink Total fat 3.56gSFA: 1.56g, MUFA: 1.04g,
PUFAs: 0.96g, EPA+DHA: 0g, insol. bre:
1.32g, sol. bre: 0.52g
6 weeks
Intervention 4. omega-3+bre enriched
dairy drink
Total fat 3.24gSFA: 1.68g, MUFA: 1g, PUFAs:
0.56g, EPA+DHA: 0.36g, Sol. bre: 0.64g
6 weeks
between 12% and 18% of energy (E%), for most countries around
1012E% [7]. The intakes are higher in Southern European coun-
tries (up to 29%) like Greece, Italy or Spain [3], where the main
source of oleic acid is olive oil, and lower in western and north-
ern countries where oleic acid intake comes from meat and meat
products mainly.
Oleic acidintake recommendations are basedonthe recommen-
dations for intakes of total fat (around 30% of the total energy),
maximum intakes of saturated fatty acids (10% or less), and mini-
mum and maximum intakes of PUFA (610% of the energy) [2,14].
As a consequence, dietary intake of oleic acid should be therefore
in the range of 1015%. Whilst the average intake of fat in the Euro-
pean population is ca. 35% of the total energy, most countries are in
the lowrange of intake (withthe exceptionof the SouthernEurope)
and should probably increase the consumption of high oleic acid
foods like olive oil.
4. Summary of intervention studies
A total of 9 intervention studies reporting CV effects of milks
containing EPA and DHA PUFA and/or oleic acid were found using
PubMed searches. Out of those, 7 were carried out with healthy
subjects or groups with increased CV risk factors, and two studies
with CVD patients. In all the studies, milk fat was substituted by
E. Lopez-Huertas / Pharmacological Research 61 (2010) 200207 203
EPA and DHA PUFA from rened sh oils with or without other
vegetable oils (mainly olive oil and high-oleic sunower oil). Study
design, interventiongroups, daily intake of nutrients fromthe dairy
drinks used and duration of the interventions are summarised in
Table 1.
4.1. A study in children
Therst studythat investigatedtheblood-lipideffects of aliquid
milk with a modied fatty acid prole was a randomised con-
trol crossover trial that compared the consumption of whole milk
with a milk preparation enriched with oleic acid and PUFA from a
vegetable oil mix made up with 60% olive, 20% peanut and 20% sun-
ower [21]. 7-month consumption of 500mL/day of the enriched
dairydrink, containingaquarter of thesaturatedfat present instan-
dard whole milk, produced desirable blood lipid-lowering effects:
7.2%intotal cholesterol, 13.2%intriglycerides and9.5%inLDL-
cholesterol. The authors concluded that to reduce serum levels
of total cholesterol and LDL-cholesterol, without reducing caloric
intake, it could be benecial to substitute the whole milk custom-
arily consumed by children with a milk preparation of fat-free milk
enriched with oleic acid.
4.2. Studies with healthy volunteers and hyperlipidemic subjects
The rst study reporting the effects of milk enriched with sh
oils was published by Visioli et al. [22]. The healthy young sub-
jects for the study (n=8) rstly consumed 500mL/day of standard
semi-skimmed milk for 4 weeks and then change to 500mL of
the enriched dairy drink that was consumed for 6 further weeks.
The enriched milk contained 400mg of total n3 fatty acids
(300mg of EPA+DHA) and a total fat of 1.7g/100mL, equivalent to
semi-skimmed milk. The relatively low amounts of EPA and DHA
supplemented in the milk resulted in unexpected but clear incre-
ments inplasma EPA(>40%) and DHA(>30%). The triglycerides (TG)
concentration decreased by 19% and the high-density lipoprotein
(HDL) cholesterol concentration increased by 19% at the end of the
enriched milk period; plasma vitamin E rose by 21% whilst the sus-
ceptibility of plasma to oxidation was unaffected. In addition, a
negative correlation between plasma EPA or DHA and TG levels,
and a positive correlation between HDL-C and DHA were found,
suggesting the importance of plasma DHA and EPA for blood lipid
control.
Two controlled non-randomised studies were later carried out
comparingsemi-skimmedmilkwithaskimmedmilkenrichedwith
an oil blend made up with rened sh oils, olive oil and high-
oleic sunower oil. Thenal supplementeddairyproduct contained
equal amounts of the total fat present in standard semi-skimmed
milk (1.9g dairy fat/100mL) but with a different fatty acid prole:
66%less saturatedfat, twice the amount of oleic acid, and8-times of
the amount of PUFA, including 60mg/100mL of EPA+DHA, which
are not present in cows milk. The dairy drink was also supple-
mented with vitamins A, D, E, B6 and folic acid.
The rst study [23] included young (2545 years) healthy sub-
jects (n=30). Firstly, 500mL/day of standard semi-skimmed milk
was consumed for 4 weeks and then changed to 500mL of the
enricheddairy drink for 8 further weeks. The amounts of EPA+DHA
supplementedinthemilkresultedinsignicant plasma increments
of EPA(48%) and DHA(50%) at the end of the study. LDL-cholesterol
signicantly decreased by 20% and total cholesterol by 7%, whilst
maintaining the normal levels of TG and HDL-cholesterol. The lack
of effect in plasma TG levels was unexpected although mean TG
values at the beginning of the study were within the range of
normality. The enriched dairy drink of the study also reduced
plasma levels of total homocysteine, another independent CVDrisk
factor, known to be reduced by vitamins B6, B12 and folic acid,
present in the enriched milk. A reduction in the levels of the sol-
uble form of VCAM-1, a molecule that plays an important role in
early atherosclerosis, was also reported.
In the second study [24], middle age (4565 years) subjects
(n=30) withmildhyperlipidemiabut withnoadditional riskfactors
were included in the study to investigate the effects of the enriched
dairydrinkina baseline context of highbloodlipids. Usingthe same
study design, consumption of the enriched milk also increased
plasma concentrations of EPA by 33% and DHA by 20% and reduc-
tions were observed for cholesterol (9%), LDL-cholesterol (13%), TG
(24%), total homocysteine (20%) and VCAM-1. The lipid-lowering
effect was more prominent during the rst 4-week consumption of
the enriched milk and the subjects restored their normal values at
the end of the 8-week study period, suggesting the effect was more
pronounced in the context of high blood lipids. Interestingly, simi-
lar TG-lowering effects have been reported over a 5-week period of
daily administration of pharmacologic amounts of EPA+DHA(4.5g
administered in capsules) [25], showing that the vehicle of admin-
istration (milk) also plays a role in the effects produced (discussed
below). A meta-analysis of 36 randomised control crossover tri-
als using 34g of EPA+DHA on average for more than 2 weeks
also showed more prominent effects on TG reduction when the TG
baselinevalues of thesubjects wereabove2.0mmol/L(>177mg/dL)
[26].
Recently, the effects of the same enriched dairy drink have been
tested in a large randomised control trial (RCT) including 297 sub-
jects (2565 years) with moderate CV risk that consumed 500mL
of the test milks for 1 year [27]. The subjects were randomised in
3 groups: (1) Skimmed milk (0.3g of total fat/100mL), (2) semi-
skimmed milk (2.2g/100mL), and (3) the enriched dairy drink
(2.2g/100mL). The inclusion of a skimmed milk group was inter-
esting as regarding the intake of dairy, nutritional medical advice
to subjects with borderline high or high blood lipids is to consume
skimmed milk or low-fat dairy in general. Besides, results fromthis
interventiongroups helpedto differentiate the effects of just the fat
present in the other milks. Consumption of the enriched milk for 1
year produced signicant increases in serumHDL-cholesterol (4%).
Plasma TG (10%), total cholesterol (4%), and LDL-cholesterol (6%)
were reduced only in the enriched drink group whilst no changes
weredetectedintheskimmedmilkor semi-skimmedgroups. These
results indicate greater benets of EPA+DHAand oleic acid admin-
istered in dairy compared with low-fat dairy and deserves further
investigation.
Castro et al. [28] published a RCT of a 6-week duration with
70 healthy subjects comparing the blood-lipid effects produced by
the consumptionof 4different enricheddairy drinks supplemented
with (1) maltodextrin and soy oil (control), (2) EPA+DHA, (3) bre
(guar gumand oat our containing beta-glucan), and (4) EPA+DHA
plus the above bre, all containing similar amounts of total fat.
Unexpectedly, no signicant effects were reported on blood lipids
between the study groups. The only within-group effects detected
were a TG increase in the placebo and in the omega-3 enriched
drinks. Acloser analysis of the study population, the randomisation
of the subjects, the placebo drink and the dietary control shows a
number of confounding factors, as follows. Before the intervention,
the authors classied the 70 subjects into 3 clusters: (I) younger,
normal lipids/lowrisk, (II) middle age/moderate risk, and(III) older,
high lipids/high risk. From these clusters, the volunteers were ran-
domised into the 4 study groups. Using this approach, the groups
obtained were highly heterogeneous, as included volunteers from
16 to 69 years of age in the same cluster, mixing high and low
baseline blood lipids and makes it very difcult to obtain signi-
cant effects, as shown for instance by the high standard deviation
of the TG values in the omega-3 group (Baseline 102.369.3, after
6 weeks 155.6141.2). Using this approach, also the bre drinks of
the study that contained beta-glucan (reported to reduce choles-
204 E. Lopez-Huertas / Pharmacological Research 61 (2010) 200207
terol) did not show any effect on cholesterol and LDL-cholesterol,
in fact, increasing trends were observed. Besides, the control drink
of the study contained soy oil providing the same daily intake of
PUFA as the omega-3 enriched drinks (0.8g). Finally, it is difcult
to explain why in the context of an increased omega-3 intake, the
plasma linoleic acid(omega 6) increased(P=0.009) at the endof the
study period in the omega 3 and bre drinks compared with base-
line. Since the PUFA intake from the drinks of the study was very
similar, we conclude that the dietary intake of nutrients among the
study groups was poorly controlled. This study has been included
as it describes blood-lipid effects produced regular consumption
of a dairy drink enriched with PUFA, EPA and DHA but the design
used by the authors, the composition of the placebo drink and the
follow up of the study does not allow us to withdraw any clear
conclusion.
4.3. Studies with patients: cardiovascular disease and metabolic
syndrome
The effects of long-term consumption (1 year) of the enriched
dairydrinkdescribedabove have beeninvestigatedinpatients with
peripheral vascular disease (PVD) [29] and also in patients with
clinical history of myocardial infarction (MI) [30] that followed a
cardiac rehabilitation program. The same RCT design was used for
both studies and the patients were divided in two study arms con-
suming 500mL/day of semi-skimmed milk or the same amounts of
the enriched dairy drink.
PVDis amanifestationof atherosclerosis causedbytheocclusion
of the arteries tothe legs that typically produces painwhenwalking
as a clinical symptom. The functionality in this type of patients is
tested by measuring the distance that the patient can walk without
pain and also by measuring the blood pressure ratio taken in the
ankle and in the arm (ankle-brachial pressure index). The patients
in the enriched dairy drink group reduced total cholesterol, mainly
in the group of patients with high cholesterol values at baseline
(9%), but maintained the normal values of LDL-cholesterol and
TG. Signicant reductions of Apolipoprotein B, a marker for all the
potential atherogenic particles, were also reported. In addition, the
enriched drink group increased the walking distance before the
onset of pain by ca. 3-fold, together with the ankle-brachial pres-
sure index, showing outstanding benets in the clinical symptoms
of the patients.
Results fromthe MI patients inthe enricheddrinkgroupshowed
increased in plasma oleic acid, DHA and EPA by 10%, 16%, and
53%, respectively. Plasma total cholesterol, and ApoB concentra-
tions decreased by 11% and 13%, and LDL-cholesterol decreased by
13%, whilst maintaining healthy levels of TG. Besides, the plasma
concentration of high sensitive C-reactive protein, a marker of sys-
temic inammation and a strong predictor of the risk of future CV
events, signicantly decreased by ca. 50% at the end of the study.
These two studies with CV patients indicate that the inclusion
of specic nutrients (EPA, DHA, oleic acid, folic acid, and vitamins
B6 and E) in their everyday diet may have the ability of control-
ling a variety of risk factors and even improve clinical outcomes,
providing new evidence for the potential role of nutrition in the
management of these patients that deserves future research.
Finally, a recent RCT [31] investigated the effects of a differ-
ent EPA+DHA PUFA and oleic acid enriched milk in 72 metabolic
syndrome (MS) patients. MS syndrome is diagnosed when the sub-
ject is presented with 3 or more elevated CV risk factors from
a list of 5, including blood glucose (110126mg/100mL), TG
(>150mg/100mL), low HDL-cholesterol (<40mg/100mL), blood
pressure (85130mm Hg) and body mass index (>30). The MS
patients were randomised into 2 parallel groups that consumed
500mL/day of enriched milk or semi-skimmed milk (control) for
a period of 3 months. The enriched milk of the study was also
obtained by the addition of oleic acid and EPA+DHA (presum-
ably from sh oils) to skimmed milk that provided a daily intake
of 186mg of EPA +DHA, ca. 40% less that the previous omega-3
milks, and 5.7g of oleic acid. At the end of the 3-month study,
the MS patients of the enriched milk reduced the serum levels
of total cholesterol (6.2%), LDL-cholesterol (7.5%) and TG (13.3%).
The reductions were measurable after 2 months. In addition, the
enriched drink improved the blood glucose control as fasting
plasma glucose levels signicantly decreased by 5.3% to reach nor-
mal values. This effect of blood glucose was not reported before but
it was not unexpected as an increase in the intake of oleic acid (but
not EPA and DHA) at the expense of saturated fat often results in
better blood glucose control [6].
5. The daily dose in the context of the diet
The combination of nutrients contained in the enriched dairy
drink and the pattern of consumption of the milks could be rea-
sonably considered as part of a healthy balanced diet. The amounts
of consumption of the milks in nearly all the studies was 500mL
per day (equivalent to 2 average size glasses) for a time period
of between 6 weeks and 14 months. Consumption of 2 glasses of
this dairy, containing an average of 600mg of calcium provides
about 65% of the RDI of calcium established for adults (900mg).
Besides, the consumption did not produce major dietary changes
in the subjects in accordance with food intake surveys that indi-
cate that milk and dairy products generally are the main source
of dietary calcium constituting 66%, 72%, and 45% of the typical
Spanish, US and UK diet, respectively [3234]. The daily dose of
EPA+DHA mostly used in the studies was approximately 300mg
(190330mg range) and represents the consumption of two por-
tions of shper week, one oily [12]. These amounts are very close to
the250mgper dayrecommendedbyEFSAbut still 30%less thanthe
amounts recommended by national agencies like in the UK, France
or The Netherlands (as described above). Regarding oleic acid, the
daily amounts used to substitute saturated fatty acids frommilk fat
in were in the 210g range, mostly 5g, which represent only 14%
of the energy based on a 2500Kcal intake.
6. Bioavailability of EPA+DHA
The average inclusion of 300mg of EPA+DHA in the milks pro-
duced a 2550% enrichment in the plasma levels of the fatty acids
after a minimum period of 6 weeks. The high effectiveness of such
low dose of the fatty acids included in the milks is somehow unex-
pected but has been reported and discussed by several research
groups [22,24,31]. Milk is a very efcient carrier for fat absorption,
because milk fat highly dispersed in micelles providing in a large
surface for absorption: the average diameter of the fat globules
in homogenised bovine milk is 13mm; estimations indicate that
1g of fat in milk is distributed in a surface of approximately 1m
2
.
Besides, milk is a uid that is slowly and repeatedly ingested dur-
ing the day. This may further facilitate the availability and enhance
the biological actions of the omega-3 fatty acids, in turn rendering
them active even at low doses [22].
An important feature of all the enriched milks used in the study
is that their formulation involves a pre-emulsication process of
the supplemented fats using emulsiers. This process can make a
signicant contribution to the absorption of the EPA+DHA fatty
acids as described in a recent randomised crossover trial in healthy
adults indicating improved absorption of omega-3 fatty acids by
pre-emulsication [35]. In this study, pre-emulsication of an oil
mixture (comprising concentrated sh oil 43%, borage oil 31% and
axseed oil 26%) prior to ingestion, compared with the oil admin-
istered as such, increased the absorption EPA+DHA by 2- to 3-fold
E. Lopez-Huertas / Pharmacological Research 61 (2010) 200207 205
calculated by measuring the area under the curve (9h) obtained
after the administration of the oils. All these features are likely to
improve the absorption of EPA+DHA in the enriched milks and
could explain the high bioavailability of the fatty acids in spite of
the low dose administered.
7. The magnitude of the effects
Several meta-analyses and reviews have been carried out to
estimate the impact of SFA on cholesterol levels [36]. These anal-
yses indicate that for every 1% increase in calories from SFA as
a percent of total energy, the serum LDL-cholesterol rises about
2%. Conversely, a 1% reduction in saturated fatty acids will reduce
serum cholesterol by about 2% [37,38]. Blood cholesterol and LDL-
cholesterol are well-established independent risk factors of CVD
and are primary targets of nutritional and pharmacological strate-
gies aimed to reduce the risk of this disease. Also, high fasting TG
concentration and HDL-cholesterol are other markers of increased
CV risk.
The blood-lipid effects of the enriched milks of the studies are
summarised in Table 2. In all the studies identied with healthy
volunteers or subjects with increased blood lipids, consumption
of the dairy drink in which the dairy fat has been substituted
by PUFA, EPA+DHA and oleic acid for more than 6 weeks pro-
duced sustainable reductions of total cholesterol (range 411%)
and LDL-cholesterol (620%), specially when baseline values at
baseline were elevated. Since a higher intake of saturated fat is
associated with higher cholesterol levels, perhaps the substitu-
tion of a percentage of the dietary saturated fat maybe more
effectiveat loweringcholesterol onthesubjects withhigher choles-
terol baseline values. The reductions were consistent across the
studies. The effects on HDL-cholesterol are less clear as only 2 stud-
ies showed signicant increases. However, the total cholesterol
reductions observed did not occurred at the expense of HDL-
cholesterol. With regard to TG, the reduction range observed was
1024%. The only study showing reductions in fasting blood glu-
cose included subjects with borderline high values (MS patients).
A common feature observed in the studies is that the lipid-
lowering effect was more prominent when baseline values were
elevated, suggesting that the supplemental nutrients may have
contributed to blood-lipid stabilisation in the context of a blood-
lipid imbalance. This could perhaps be applicable to the high blood
glucose situation but requires further research more focused on
insulin/glucose response with pre-diabetic subjects or type-2 dia-
betes patients.
The studies carried out with CVD patients showed that 1-year
consumption of the enriched dairy drinks helped the patients
to keep blood-lipid levels under control, without the need of
lipid-lowering drugs. Controlling the blood-lipid values and other
risk factors of these patients by nutritional therapy and/or lipid-
lowering drugs is key to prevent the occurrence of further CV
events; therefore baseline values of blood lipids of the patients
were mostly within the range of normality. In this situation, con-
sumption of the enriched drink was useful as nutritional support
to improve the clinical outcomes (PVD patients) and/or inamma-
tory markers (MI patients) but the effects on blood lipids were
mild to none. There is wide scientic evidence indicating that these
reductions of blood lipids (mainly cholesterol and LDL-cholesterol
levels) produce desirable decreases in the CV disease risk. In this
sense, earlyclinical trials [39,40] foundthat a 1%reductioninserum
total cholesterol level reduced risk for coronary heart disease by
about 2%. Also, using data from a large number of cohort studies,
Law et al. found that a 10% reduction in serum cholesterol level
attained at age 40 yielded a reduction in relative risk for CHD of
50% [41,42].
Table 2
Summary of the effects of the enriched milks on total cholesterol (TC), LDL-cholesterol (LDL-C) and triglycerides (TG) reported in the intervention studies. Normal, borderline
high (B.high) and high baseline values have been dened as reported by the National Cholesterol Education Programme-Adult Treatment Panel III [50].
Reference Subjects, n Fat used to substitute dairy fat Bloodlipids inthesubjects fromtheenrichedgroups
TC LDL-C TG
Benito et al. [31]
Patients, metabolic
syndrome, n=72
Oleic acid, PUFA,
EPA+DHA
Lipid values at baseline B. High B. High B. High
Lipid-lowering effect Yes Yes Yes
% reduction vs. baseline 6.2 7.5 13.3
Carrero et al. [24]
Healthy, mild
hyperlipidemic, n=30
Oleic acid, EPA+DHA
Lipid values at baseline High High High
Reduction at the end Yes Yes Yes
% reduction vs. baseline 9 13 24
Bar et al. [23] Healthy adults, n=30 Oleic acid, EPA+DHA
Lipid values at baseline Normal Normal Normal
Reduction at the end Yes Yes No
% reduction vs. baseline 7 20 0
Carrero et al. [30]
Myocardial infarction
patients, n=40
Oleic acid, EPA+DHA
Lipid values at baseline Normal Normal Normal
Reduction at the end Yes Yes No
% reduction vs. baseline 11 13 0
Carrero et al. [29]
Peripheral vascular
disease patients, n=60
Oleic acid, EPA+DHA
Lipid values at baseline B. High Normal Normal
Reduction at the end Yes Yes No
% reduction vs. baseline 6 0 0
Fonoll et al. [27] Healthy adults, n=297 Oleic acid, EPA+DHA
Lipid values at baseline B. High B. High Normal
Reduction at the end Yes Yes Yes
% reduction vs. baseline 4 6 10
Visioli et al. [22] Healthy adults, n=8 PUFA, EPA+DHA
Lipid values at baseline Normal Normal Normal
Reduction at the end No No Yes
% reduction vs. baseline 0 0 19
Estvez-Gonzlez [21] Healthy children, n=88 Oleic acid, PUFA
Lipid values at baseline Normal Normal Normal
Reduction at the end Yes Yes Yes
% reduction vs. baseline 7 10 13.2
Castro et al. [28] Healthy subject, n=18 PUFA, EPA+DHA
Lipid values at baseline Normal Normal Normal
Reduction at the end No No Yes
% reduction vs. baseline 0 0 +51
206 E. Lopez-Huertas / Pharmacological Research 61 (2010) 200207
8. Concluding remarks
There is wide scientic evidence showing that substitution of
SFAby oleic acidandPUFAproduces CVbenets by reducing blood-
lipid concentrations and beyond. Substitution of the milk fat by a
combination of oleic acid and sh oils is a good nutritional strat-
egy to reduce the intake of SFA in favour of healthier fatty acids.
The human studies published describe benecial CV effects and
include healthy adults (3), healthy children (1) and subjects with
mild hyperlipidemia (1). The studies carried out with metabolic
syndrome (1) myocardial infarction(1) andperipheral vascular dis-
ease patients (1) alsodescribe benets andindicate that these types
of milk could be useful to control risk factors as nutritional support.
By taking intoaccount the totality of the data it is reasonable tosug-
gest that the consumption of these type of enriched milks, in the
context of a balanced diet and healthy lifestyle, produces desirable
CV effects by improving the blood-lipid prole (mainly by LDL-
cholesterol reduction), especially in a situation of elevated values.
These studies also underline the fact that supplemented foods may
play an important role in CVD prevention without involving major
dietary changes inthe population. However, larger RCTs are needed
at the community and clinical levels with different target popu-
lations, dietary patterns and more control of confounding factors
before general recommendations can be made.
In recent years, differential effects on gene expression, cell func-
tion and physiology have been reported for EPA and DHA in their
puried forms (reviewed in [43]). Indeed, puried EPA and DHA
administered incapsules seemto have differential effects onserum
lipids andlipoproteins, LDL particle size, glucose andinsulinrelease
[44,45] but this is yet to be conrmed in with nutritional doses and
different populations with large volunteer trials. In addition, DHA
andEPApossess different afnityfor organs. DHAgenerallyexceeds
EPA5- to 30-foldinmost organs [15]. DHAfor example highly accu-
mulates in brain and retina, so it makes sense to use puried DHA
in foods to keep a good supply of this nutrient whilst studying their
functionality.
A clear application of DHA-enriched milks and one very active
line of research is the study of their effects in infants and adoles-
cents. After breastfeeding, the vast majority of milk products used
by infants are based on cows milk. However, the fatty acids com-
position of human breast milk and cows milk are rather different:
though very much inuenced by diet, breast milk possess half the
amount of SFA and is rich in oleic acid and PUFA [46], including
signicant amounts of DHA(usually in the 510mg/100mL range),
whilst cows milk is devoid of DHAand poor in PUFA. DHAin breast
milk has been shown to play an important role in brain and retina
development in infants [47]. As a consequence, most commercially
availableinfant formulas that replacebreast milkaresupplemented
also with DHA. Recent studies in young children and adolescents
also suggest benecial effects incognitive development andmental
performance produced by DHA supplementation [48,49].
Humans are the only animals who take milk as adults. Interest-
ingly, the nutritional requirements of humans change with age and
physiology: they are not the same after birth, in the childhood, dur-
ing adolescence, in the adult period, during pregnancy, lactation,
menopause, etc. For this reason, if we look for optimal nutrition, it
makes sense to adapt the compositionof standard cows milk to the
nutritional requirements and physiology of the different age peri-
ods. Besides, the composition of cows milk maybe ideal for calfs,
but not necessarily for humans. In fact, when breastfeeding is not
possible, the infant formulae available on the market try to imitate
the human breast milk, adapting their compositions to the nutri-
tional requirements at the different early ages: 06 months, 612
months, and 13 years. If these concepts are extended to the adult
age or the different physiological status, modifyingthe composition
of standard cows milk may have many advantages. The substitu-
tion of the saturated milk fat by oleic acid and omega 3 has shown
cardiovascular benets, commented in this review.
Milk is an everyday used drink which has been used as a carrier
to try to increase the intake of certainnutrients (vitamins, minerals,
protein, fats) and/or bioactives (phytosterols, probiotics, antioxi-
dants, isoavones, etc.). In the future we will see more types of
enriched or modied dairy drinks aimed to promote organ health
and function and adapted to the different ages and physiology, an
area of research that will certainly develop in the future.
Conict of interest
The author states no conict of interest.
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