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ru
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org,
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olyu
ated
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uble
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significantly from natural triglycerides. The stereochemistry of fatty acid in acylglycerols did not
in Gre
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een fo
and se
ociatio
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ined
o oil
2. Methods
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com
Prostaglandins, Leukotrie
Essential Fatty Aci
workshop at the 29th yearly meeting of The European Society for Clinical
Prostaglandins, Leukotrienes and Essential Fatty Acids 83 (2010) 137–141and 36 men aged 23–55 years) volunteered for the study.E-mail address: jdcon@post4.tele.dk (J. Dyerberg).2.1. Subjects
Seventy-two healthy subjects (36 women aged 21–56 years
0952-3278/$ - see front matter & 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.plefa.2010.06.007
Investigation: n-3 Fatty acids: prevention and treatment in vascular disease.
S.D. Kristensen, E.B. Schmidt, R. de Caterina, S. Endres (Eds). Springer Verlag,
London, 1995
n Correspondence to: Lille Fredensvej 6A, DK 2920, Charlottenlund, Denmark.
Tel.: +45 39636688; fax: +45 39636689.$The main results have been previously published in the proceedings of aingested in order to reach an appropriate dose of n-3 PUFA have
prompted the development of more concentrated compounds [8].
plasma TG, cholesterol esters and phospholipids was exam
after intake of five different n-3 FA formulations or placeb
(corn oil, CO) for 2 weeks.potentially the use of n-3 PUFA supplements for prevention
coronary heart disease [3]. Supplementation with various n-3
PUFA formulations has served as the primary tool for obtaining an
exact dose of n-3 PUFA and to perform blinded, controlled studies.
Initially, deodorized fish oils, e.g. cod liver oil (CLO) and fish body
oils (FBO), were used. In these preparations, the n-3 PUFA are
esterified as triglycerides (TG). Problems of patient compliance
due to the relatively large amounts of such oils that have to be
reconverted to glycerides. Some conflicting results have arisen
from the rather few studies that have dealt with the bioavail-
ability of EPA and DHA from various concentrated n-3 PUFA
formulations [9–14]. The lack of a controlled study comparing the
five presently commonly used fish oil supplements (natural TG in
fish body oil and CLO, EE, FFA and rTG) led us to undertake a
blinded, placebo-controlled study in healthy volunteers, using
generally available products. The enrichment of EPA and DHA inFree fatty acids
Fish oil
Stereoisomery
1. Introduction
Since our original observations
association between dietary intake
unsaturated n-3 fatty acids (n-3 PUF
functions [1,2] much interest has b
health benefits of marine n-3 PUFA
Based on this, national heart ass
bodies have recommended an incr& 2010 Elsevier Ltd. All rights reserved.
enland Eskimos of an
arine long-chain poly-
biological and cellular
cused on the potential
afood in the diet [3–7].
ns and governmental
intake of oily fish and
Thus, concentrates of marine oils containing up to 30–90% of
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)
have been developed. The n-3 PUFA are generally present in these
formulations as free fatty acids (FFA), ethyl esters (EE) or as re-
esterified TG (rTG). The term ’’re-esterified’’ is used for products
made from FBO, in which the app. 30% TG content is transferred to
ethyl esters and then molecularly distilled to remove the short
chain and the saturated fatty acids increasing the EPA and DHA
contents to around 60%. The ethyl esters are then enzymaticallyRe-esterified triglycerides
influence the bioavailability of EPA and DHA.Bioavailability of marine n-3 fatty acid
J. Dyerberg a,n, P. Madsen b, J.M. Møller c, I. Aardest
a Department of Human Nutrition, Faculty of Life Sciences, University of Copenhagen,
b Department of Clinical Biochemistry, Center for Cardiovascular Research Aalborg Hos
c Department of Gastroenterology, Aalborg Hospital, Aalborg, Denmark
d Department of Cardiology, Center for Cardiovascular Research Aalborg Hospital, Aalb
a r t i c l e i n f o
Article history:
Received 3 March 2010
Received in revised form
28 June 2010
Accepted 29 June 2010
Keywords:
Bioavailability
n-3 Fatty acids
a b s t r a c t
The use of marine n-3 p
development of concentr
compares three concentra
— with placebo oil in a do
arms. Seventy-two volun
docosahexaenoic acid (DH
serum triglycerides, chole
from re-esterified triglyc
bioavailability from ethylrmulations$
p b, E.B. Schmidt d
nhagen, Denmark
l, Aalborg, Denmark
Denmark
nsaturated fatty acids (n-3 PUFA) as supplements has prompted the
formulations to overcome compliance problems. The present study
preparations— ethyl esters, free fatty acids and re-esterified triglycerides
-blinded design, and with fish body oil and cod liver oil in single-blinded
s were given approximately 3.3 g of eicosapentaenoic acid (EPA) plus
aily for 2 weeks. Increases in absolute amounts of EPA and DHA in fasting
ol esters and phospholipids were examined. Bioavailability of EPA+DHA
es was superior (124%) compared with natural fish oil, whereas the
rs was inferior (73%). Free fatty acid bioavailability (91%) did not differ/locate/plefa
nes and
ds

Volunteers who had taken fish oil preparations within 2 months
prior to the study were excluded. The volunteer subjects were
instructed to avoid acetylsalicylic acid 2 weeks prior to and during
the study, and to abstain from alcoholic beverages for 1 day before
study visits. The study was approved by the local Ethics
Committee.
2.2. Experimental design
The subjects were randomized to six groups: four double-
blinded groups given concentrated fish oils or placebo, each
person taking five capsules twice daily at meal times for 2 weeks
and two single-blinded groups given CLO or fish body oil (FBO)
capsules twice daily for 2 weeks. The single-blinded design in the
methanolic sulphuric acid 1% [17]. The methylated FA were
extracted after addition of 500 mL NaCI 5% and 1500 mL
hexane. The hexane phase was washed with 2% NaHCO3, dried
under N2 and redissolved in 60 mL dichloromethane. Gas chro-
matography was performed isothermally at 220 1C on an HP 5700
gas chromatograph (Hewlett Packard, Avondale, PA, USA)
supplied with a 25 m0.53 mm FFAP-CB capillary column
(Chrompack, Middelburg, The Netherlands) with N2 as the carrier
gas (2 ml/minute).
When comparing the bioavailability of different n-3 FA
preparations, it should be noticed that despite the fact that the
total amounts of EPA plus DHA given to the volunteers were
almost equal (Table 1), the relative amounts of EPA and DHA
varied to some extent, for example, between FFA and CLO. The
and TG, but calculating the sums of EPA plus DHA in each class of
enoi
J. Dyerberg et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 83 (2010) 137–141138two natural fish oil groups was due to different number of
capsules taken by these groups. All supplements were from
Pronova Biocare, Sandefiord Norway: EPAX 5500 TG consisting of
rTG; EPAX 6000 FA consisting of FFA; EPAX 5500 EE consisting of
EE; EPAX 3000 TG consisting of a refined fish body oil (FBO); cod
liver oil (CLO); and corn oil (CO) as placebo.
The composition and amounts of the supplements are given in
Table 1. The daily intake of EPA plus DHA was 3.1–3.6 g. The
subjects were examined at baseline and after 2 weeks of
supplementation. Each examination was made in the morning
after an overnight fast. Blood was drawn from an antecubital vein
with minimal stasis. Serum was prepared by clotting whole blood
for 1 h at room temperature and centrifuged at 2000g for 15 min.
Serum was transferred to plastic tubes and stored at 70 1C until
analysis. All analytical works were performed before breaking the
randomization code.
2.3. Fatty acid analysis
Total lipids were extracted from serum according to Bligh and
Dyer [15]. Serum (400 mL) was mixed briefly with 500 mL
chloroform (CHCl3) containing internal standards (diheptadeca-
noyl phosphatidyIcholine, cholesteryl heptadecanoate, and trihe-
piadecanoin) and 1000 ml methanol containing butylated
hydroxytoluene as antioxidant. After the addition of 500 ml CHCl3
and 500 mL H2O and brief mixing, the tubes were centrifuged at
1000g for 2 mins for phase separation; 550 ml of the CHCI3 phase
was transferred to a SepPak NH2 column (Waters Corporation,
Milford, MA, USA), which had been prewashed with hexane. Lipid
classes were separated into phospholipids (PL), cholesterol esters
(CE) and monoglycerides, diglycerides and TGs as described by
Kaluzny et al. [16], except that the glycerides were collected as a
single class. The extracted lipids were dried under nitrogen (N2)
and redissolved in 100 mL toluene. Transmethylation was carried
out overnight at 45 1C under N2 after addition of 200 ml
Table 1
Dose and composition of capsules and relative and absolute amounts of eicosapenta
rTG FFA
Capsule weight (mg) 650 650
Capsules/day (morning+evening) 5+5 5+5
EPA (%) 28.5 33.5
DHA (%) 19.8 21.5
n-6 FA (%) 4.0 2.2
Monounsaturated FA (%) 13.3 12.1
Saturated FA (%) 1.0 2.8
Tocopherols (mg/g) 3.7 3.5
EPA (g/day) 1.85 2.18
DHA (g/day) 1.29 1.40
EPA+DHA (g/day) 3.1 3.6Abbreviations: rTG: re-esterified triglycerides. FFA: free fatty acids. EE: ethyl esters. FBplasma lipids, and using the grand total as a measure of
bioavailability, EE was the formulation giving the lowest
assimilation. The differences in the grand total of EPA plus DHA
(Fig. 1) were significant when comparing EE with rTG (p¼0.000I)
and EE with fish body oil (p¼0.024), but not when comparing EE
with CLO (p¼0.13) and EE with FFA (p¼0.29). Comparing the
assimilation of the other preparations (rTG, fish body oil, CLO and
FFA), again using the grand total sums of EPA plus DHA as an
index, the bioavailability of rTG was significantly better than that
of FFA (p¼0.006) and of CLO (p¼0.002), whereas it did not differ
c acid (EPA) and docosahexaenoic acid (DHA) administered in the six study groups.
EE FBO CLO CO
650 1000 500 650
5+5 6+7 17+7 5+5
28.8 15.7 8.1 0
21.4 11.4 11.0 0
3.9 2.5 2.2 56.7
15.7 25.9 51.8 29.0
6.0 27.5 16.0 13.4
3.9 1.1 1.0 2.8
1.87 2.04 1.38 0
1.39 1.48 1.87 0
3.3 3.5 3.2 0sum of the increase in EPA and DHA is consequently the best
marker in comparisons to the bioavailability of the products.
2.4. Statistics
The SPSS for Windows statistical software package, version 6.1,
was applied. Statistical comparisons between groups at baseline
and after 2 weeks of supplementation were made using the
Mann–Whitney U-test. A two-sided p value o0.05 was consid-
ered to be statistically significant.
3. Results
The increases in the amounts of EPA and DHA in plasma CE, PL
and TG, and in the sum of EPA and DHA in each lipid class are
given in Table 2. The increases in plasma total lipids (CE+PL+TG)
of EPA, DHA and in EPA plus DHA are illustrated in Fig. 1. Each n-3
PUFA preparation produced a significant increase in both EPA and
DHA in all lipid classes compared to placebo oil. The volunteers in
the CLO group received approximately 0.5 g less EPA and
approximately 0.5 g more DHA per day compared to the other
groups. Consequently, the sum of EPA and DHA gives a more
accurate picture of the differences in bioavailability (Fig. 1). By not
considering the increase in EPA and DHA individually in CE, PLO: fish body oil. CLO: cod liver oil. CO: corn oil. FA: fatty acids.