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Clinical Benefits of n-3 PUFA and ɤ-Linolenic Acid in Patients with

Rheumatoid Arthritis.

Article  in  Nutrients · January 2017

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Mirjana Veselinovic Dragan Vasiljevic

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 nutrients
Article
Article
Clinical Benefits of n-3 PUFA and ɤ-Linolenic
Clinical Benefits -Linolenic Acid in
Article
Patients
Patients
Clinicalwith Rheumatoid
withBenefits
Rheumatoid Arthritis
Arthritis
of n-3 PUFA and ɤ-Linolenic Acid in
Mirjana Patients with Rheumatoid Arthritis
MirjanaVeselinovic
Veselinovic ,1 ,Dragan
1
DraganVasiljevic
Vasiljevic,2Vesna
2
, VesnaVucic
Vucic, 3Aleksandra
3
, Aleksandra Arsic
Arsic, Snjezana
3 3 Petrovic
, Snjezana
3, 3
Petrovic ,
Aleksandra
Aleksandra Tomic-Lucic
Tomic-Lucic 1,, Maja
1
Maja Savic
Savic
44, Sandra Zivanovic 55
, Sandra Zivanovic , ,Vladislava
VladislavaStojic
Stojic
6 6and Vladimir
and
Mirjana Veselinovic 1, Dragan Vasiljevic 2, Vesna Vucic 3, Aleksandra Arsic 3, Snjezana Petrovic 3,
Jakovljevic 7,*
Vladimir Jakovljevic 7, *
Aleksandra Tomic-Lucic 1, Maja Savic 4, Sandra Zivanovic 5, Vladislava Stojic 6 and Vladimir
Department
11 ofof
Jakovljevic
Department Internal
7,*
Internal medicine,
medicine, Faculty
Facultyofof Medical
MedicalSciences,
Sciences,University
UniversityofofKragujevac,
Kragujevac,Kragujevac
Kragujevac34000,
34000,
Serbia;
Serbia;veselinovic.m@sbb.rs
1 veselinovic.m@sbb.rs
(M.V.);
(M.V.); sanlusa@ptt.rs
sanlusa@ptt.rs (A.T.-L.)
(A.T.-L.)
Department of Internal medicine, Faculty of Medical Sciences, University of Kragujevac, Kragujevac 34000,
22 Department of Hygiene, Institute for Public Health, Faculty of Medical Sciences, University of Kragujevac,
Department ofveselinovic.m@sbb.rs
Serbia; Hygiene, Institute for Public
(M.V.); Health, Faculty
sanlusa@ptt.rs of Medical Sciences, University of Kragujevac,
(A.T.-L.)
Kragujevac
Kragujevac 34000,
34000,
2 Department Serbia;
Serbia;dvg_gana@yahoo.com
dvg_gana@yahoo.com
of Hygiene, Institute for Public Health, Faculty of Medical Sciences, University of Kragujevac,
33
ArticleCentre
Centre ofofResearch
Research
Kragujevac Excellence
Excellence
34000, inin
Serbia; Nutrition
Nutrition and
and
dvg_gana@yahoo.com Metabolism,
Metabolism, Institute
InstituteforforMedical
MedicalResearch,
Research,University
Universityofof

Clinical Benefits of n-3 PUFA and ɤ-Linolenic Acid in

Belgrade, Beograd
3 Centre
Belgrade, Beograd 11000,
of Research Serbia; vesna.vucic.imr@gmail.com
11000, Excellence (V.V.);
in Nutrition and Metabolism,(V.V.);
Serbia; vesna.vucic.imr@gmail.com vesna.vucic.imr@gmail.com
Institute (A.A.);
for Medical Research, University
vesna.vucic.imr@gmail.com of
(A.A.);
snjezana570.imr12@gmail.com
Belgrade, Beograd
snjezana570.imr12@gmail.com (S.P.) (S.P.)
11000, Serbia; vesna.vucic.imr@gmail.com (V.V.); vesna.vucic.imr@gmail.com (A.A.);
Patients with Rheumatoid Arthritis
44 Department
Departmentsnjezana570.imr12@gmail.com
ofofPharmacy,
Pharmacy,Faculty (S.P.)
FacultyofofMedical
MedicalSciences,
Sciences,University
UniversityofofKragujevac,
Kragujevac,Kragujevac
Kragujevac34000,
4 Department of Pharmacy, Faculty of Medical Sciences, University of Kragujevac, Kragujevac 34000, Serbia;
34000,Serbia;
Serbia;
maja.jovanovic.2008.38@gmail.com
maja.jovanovic.2008.38@gmail.com
55 Faculty ofmaja.jovanovic.2008.38@gmail.com
Hotel Management and Tourism, Department ofofNatural Sciences and
Mirjana Faculty
Veselinovicof Hotel
5 Faculty
Management
1, Dragan Vasiljevicand 2, Tourism,
Vesna Vucic Department
3, Aleksandra Natural
Arsic Sciences andmedicine,
3, Snjezana 3, University
medicine,
Petrovic Universityofof
of Hotel Management and Tourism, Department of Natural Sciences and medicine, University of
Kragujevac,
Kragujevac,
Aleksandra Kragujevac
Tomic-LucicKragujevac
1, Maja34000,
34000,
SavicSerbia;
Serbia; zivanovicsandra@hotmail.com
4, Sandra zivanovicsandra@hotmail.com
Zivanovic 5, Vladislava Stojic 6 and Vladimir
Kragujevac, Kragujevac 34000, Serbia; zivanovicsandra@hotmail.com
66 Faculty of Medical Sciences, University of Kragujevac, Kragujevac 34000, Serbia;
Faculty
Jakovljevic 7,*6 of Medical Sciences, University of Kragujevac, Kragujevac 34000, Serbia;
Faculty of Medical Sciences, University of Kragujevac, Kragujevac 34000, Serbia;
vladisavastojic@medf.kg.ac.rs
vladisavastojic@medf.kg.ac.rs
vladisavastojic@medf.kg.ac.rs
1 Department of Internal medicine, Faculty of Medical Sciences, University of Kragujevac, Kragujevac 34000,
77 Faculty 7of Medical
Faculty Faculty of Sciences,
of Medical
Serbia; veselinovic.m@sbb.rs
Sciences,
Medical University
University
Sciences,
(M.V.);
ofof
University
sanlusa@ptt.rs
Kragujevac,
Kragujevac, Physiology,
of Kragujevac,
(A.T.-L.)
Physiology,
Physiology,Kragujevac
Kragujevac
Kragujevac 34000,
34000,Serbia
34000, Serbia
Serbia
** Correspondence:
Correspondence:
2 Department
drvladakgbg@yahoo.com
* ofCorrespondence: drvladakgbg@yahoo.com
drvladakgbg@yahoo.com
Hygiene, Institute for Public Health, Faculty of Medical Sciences, University of Kragujevac,
Kragujevac
Received: 34000,
Received:Received:
28 Serbia;
28November
November dvg_gana@yahoo.com
28 November
2016; 2016; Accepted:
2016;Accepted:
Accepted: 88March 8 March
March 2017;2017;
2017; Published:
Published:
Published: 25 25
25MarchMarch
March 20172017
2017
3 Centre of Research Excellence in Nutrition and Metabolism, Institute for Medical Research, University of
Belgrade, Beograd
Abstract:Abstract:
Abstract: (1)
11000,
(1)Serbia;
(1) Background:
Background: vesna.vucic.imr@gmail.com
Background:Marine
Marine n-3polyunsaturated
Marine
n-3
(V.V.); vesna.vucic.imr@gmail.com
polyunsaturated
n-3 polyunsaturated fatty
fattyfatty acids
acids
acids (PUFA)
(PUFA)and
(PUFA)
(A.A.);
and ɤ-linolenic
and ɤ-linolenic
-linolenicacid acid
acid
snjezana570.imr12@gmail.com (S.P.)
(GLA) are (GLA) are
areofwell-known
well-knownwell-known anti-inflammatory
anti-inflammatory agents agents
that that may
mayhelp help
helpin in
inthe the treatment
the treatment
treatment of inflammatory
of inflammatory
inflammatory
(GLA)
4 Department Pharmacy, Facultyanti-inflammatory
of Medical Sciences, agents
Universitythatofmay
Kragujevac, Kragujevac of
34000, Serbia;
disorders. disorders.
Their Their effects
effects were were examined
examined in in patients
patients with with rheumatoidarthritis;
rheumatoid arthritis; (2)
(2) Methods:
Methods: Sixty Sixty
maja.jovanovic.2008.38@gmail.com
disorders. Their effects were examined in patients with rheumatoid arthritis; (2) Methods: Sixty
patients with activeandrheumatoid arthritis ofwere involved in and
a prospective, randomized trial of a 12
patients with
5 Faculty of Hotel
patients with active
Management
active rheumatoid
rheumatoid Tourism,arthritis
Department
arthritis were involved
Natural
were involved in
Sciences a prospective, randomized
medicine, University of trial aof12a
Kragujevac, week supplementation
Kragujevac 34000, Serbia; with fish oil (group I), fish oil in
zivanovicsandra@hotmail.com
a prospective,
with primrose evening randomized
oil (grouptrial ofwith
II), or
12 week
week supplementation
supplementation withwith
fishfish oil (group I), fish oil withprimrose
primroseevening
evening oil(group(group II),or orwith
with
6 Faculty no supplementation
of Medical (group
Sciences, University ofoil (group
III). I),
Clinical
Kragujevac, fish
and oil
Kragujevac with
laboratory
34000, evaluations were oil
Serbia; done at theII), beginning
no supplementation
no supplementation (group
and at the end(group
vladisavastojic@medf.kg.ac.rs
III). Clinical
III). Clinical
of the study;
and laboratory
and The
(3) Results: laboratory evaluations
evaluations
Disease Activity
were
Score were
done
28 (DAS doneat the beginning
at the number
28 score), beginning and
of
at the
7 Faculty end
and atofthe of
Medical
tender the study;
end Sciences,
of theand
joints (3)
study; Results:
University
visual The
(3)analogue
Results:
of Disease Activity
ThePhysiology,
Kragujevac,
scale Disease
(VAS) score Score
Activity
Kragujevac 28
Score
decreased (DAS
34000, 28 28
(DAS
Serbia
notably score), number
28supplementation
after of tender
score), numberinof
joints and
* Correspondence:
tender jointsvisual
groups and analogue scaleIn(VAS)
drvladakgbg@yahoo.com
I and II (p <analogue
visual 0.001). scalescore
plasma decreased
phospholipids
(VAS) notably
the
score decreased after
fatty supplementation
n-6/n-3notably acids declined in
ratiosupplementation
after fromgroups
15.47inI
and II28(p
groups
Received: ±<5.51
0.001).
IIto
INovember
and <In
(p10.62plasma
± 5.07
0.001).
2016; Accepted:phospholipids
In(pplasma
=8 March
0.005),
phospholipids
2017; n-6/n-3
the18.15
and Published:
from the
25 fatty
±March
5.04 to acids
13.50
n-6/n-3
2017 ± ratio
fatty 4.81 (pdeclined
acids = 0.005)
ratio from
in 15.47
groups
declined ±
I and
from 5.51
II
15.47
±to5.51
10.62 ± 5.07
to respectively.
10.62 (p =The
± 5.07 0.005),
(p andand
= combination
0.005), fromof 18.15 ± 5.04
n-3 18.15
from PUFA ±and5.04to
GLA 13.50
to ±±4.81
(group
13.50 II)
4.81 (p
increased ɤ-linolenic
(p== 0.005)
0.005) in groups
in groups acidII(0.00
and±IIII
and
respectively.
Abstract: (1)0.00 The
Background:combination
to 0.13 Marine
± 0.11, of
p <n-3 n-3 PUFA
whichandwasGLA
polyunsaturated
0.001), fatty(group
acids II)
undetectable inincreased
(PUFA) and ɤ-linolenic
all groups -linolenic
before theacidacid (0.00 ± (4)
treatments; 0.00
respectively. The combination of n-3 PUFA and GLA (group II) increased ɤ-linolenic acid (0.00 ±
(GLA)
to are ±
0.13 well-known
Conclusion:
0.11, p < anti-inflammatory
Daily which
0.001), was agents
supplementation thatn-3
with
undetectable may in help
fattyall in the
acids
groups treatment
alone or in the
before oftreatments;
inflammatory
combination with(4)
GLA exerted
Conclusion:
0.00 to 0.13 ± 0.11, p < 0.001), which was undetectable in all groups before the treatments; (4)
disorders. Their effectsclinical
significant
Daily supplementation were with
examined
benefits in patients
n-3 and
fattycertain
acids with rheumatoid
changes in in
disease arthritis; (2)
activity. Methods: Sixty significant
Conclusion:
patients with activeDaily supplementation
rheumatoid with
arthritis were n-3 alone
involved
or
fattyinacids combination
alone
a prospective, or randomizedwith
in combination GLA exerted
with
trial of a 12GLA exerted
clinical benefits
significant clinicaland certainand
benefits changes
certaininchanges
disease activity.
in disease activity.
week supplementation
Keywords:with fish oilɤ-linolenic
n-3 PUFA; (group I), acid;
fish oil with primrose
rheumatoid evening oil (group II), or with
arthritis
no supplementation (group III). Clinical and laboratory evaluations were done at the beginning
Keywords: n-3 PUFA; -linolenic acid; rheumatoid arthritis
andKeywords:
at the end n-3of the study;ɤ-linolenic
PUFA; (3) Results:acid; rheumatoid
The Disease Activity arthritis
Score 28 (DAS 28 score), number of
tender joints and visual analogue scale (VAS) score decreased notably after supplementation in
groups I and 1. II
Introduction
(p < 0.001). In plasma phospholipids the n-6/n-3 fatty acids ratio declined from 15.47
± 5.51 to 10.62 ± 5.07 (p = 0.005), and from 18.15 ± 5.04 to 13.50 ± 4.81 (p = 0.005) in groups I and II
1. Introduction Rheumatoid arthritis (RA) is one of the most common autoimmune disorders. The main
respectively.
1. Introduction The combination
characteristic of RAofisn-3thePUFA
onsetand GLA (group changes
of pathological II) increased
in the ɤ-linolenic
lining ofacid the (0.00 ± Due to the
joints.
0.00 to Rheumatoid
0.13infiltration
± 0.11, p arthritis
<of0.001), which
(RA) is
macrophages, was
Boneundetectable
ofand
cells, theCD4+
most inhelper
all groups
common T cells before
autoimmune
into thethe synovial
treatments;
disorders. (4) synovium
stroma, The main
Rheumatoid
Conclusion: Dailyof arthritis
is the(RA)
supplementation isofn-3
with one of the
fatty acidsmost common autoimmune disorders. The to main
characteristic
proliferates RA causing onset
swelling pathological
and pain ofalone or in[1,2].
changes
joints combination
in the lining
Furthermore,withofGLAthe exerted
the joints. Due
overproduction the
of
characteristic
significant clinicalof RA
benefits
inflammatory is the
and onset
certain
molecules, of pathological
changes
prostaglandinin disease
E2 changes
activity.
(PGE2), tumour in the lining
necrosis
infiltration of macrophages, B cells, and CD4+ helper T cells into the synovial stroma, synovium of
factor the
(TNF), joints. Due
interleukin to the
(IL)-1,
infiltration
proliferatesandof macrophages,
cytokines
causing inducesand
swelling Bchronic
cells, and CD4+
paininflammation
of joints helper
[1,2].[3,4]. T cells into
Especially
Furthermore, thethe
IL-1 andsynovial
TNF playstroma,
overproduction important synovium
roles in
of inflammatory
Keywords:
proliferates n-3 PUFA;
causing ɤ-linolenic
swelling acid;
andrheumatoid
pain ofarthritis
joints [1,2]. Furthermore, the overproduction of
molecules, prostaglandin
Nutrients E2 (PGE2), tumour necrosis factor (TNF), interleukin
2017, 9, 325; doi:10.3390/nu9040325 (IL)-1, and cytokines
www.mdpi.com/journal/nutrients
inflammatory molecules, prostaglandin E2 (PGE2), tumour necrosis factor
induces chronic inflammation [3,4]. Especially IL-1 and TNF play important roles (TNF), interleukin (IL)-1,
in inflammation
and cytokines induces chronic inflammation [3,4]. Especially IL-1 and TNF play important roles in
1. Introduction
Nutrients
Nutrients2017,
2017,9,9,325;
325;doi:10.3390/nu9040325
doi:10.3390/nu9040325 www.mdpi.com/journal/nutrients
www.mdpi.com/journal/nutrients
Rheumatoid arthritis (RA) is one of the most common autoimmune disorders. The main
characteristic of RA is the onset of pathological changes in the lining of the joints. Due to the
infiltration of macrophages, B cells, and CD4+ helper T cells into the synovial stroma, synovium
 nd ɤ-Linolenic Acid in
ritis, Vesna Vucic , Aleksandra Arsic , Snjezana Petrovic ,
ljevic 2 3 3 3

vic 4, Sandra Zivanovic 5, Vladislava Stojic 6 and Vladimir

Aleksandra Arsic 3, Snjezana Petrovic 3,
Nutrients
ic 5, Vladislava Stojic 6 and2017, 9, 325
Vladimir 2 of 11
aculty of Medical Sciences, University of Kragujevac, Kragujevac 34000,
sanlusa@ptt.rs (A.T.-L.)
rs, Public Health,
University Faculty of Medical
during
of Kragujevac, RA. Sciences,
Numerous
Kragujevac 34000,University
randomized of Kragujevac,
controlled clinical trials have reported a strong anti-inflammatory
@yahoo.com potential of marine n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFAs) and improved clinical
utrition
of MedicalandSciences,
Metabolism, Institute
University ofoffor Medical Research, University of
Kragujevac,
parameters RA [5,6]. These effects are mostly attributed to eicosapentaenoic acid (EPA, C20:5 n-3) and
sna.vucic.imr@gmail.com (V.V.); vesna.vucic.imr@gmail.com (A.A.);
docosahexaenoic acid (DHA, C22:6 n-3) acting in two ways. Firstly, they interfere with the enzymatic
)Institute for Medical Research, University of
f (V.V.);
Medical conversion
Sciences, University of arachidonic
of Kragujevac, acid (AA, C20:4 n-6) to pro–inflammatory prostaglandins (PGs) and
vesna.vucic.imr@gmail.com (A.A.); Kragujevac 34000, Serbia;
leukotrienes (LTs). Secondly, EPA is a direct precursor in biosynthetic pathway of anti-inflammatory
Tourism, Department
ersity of Kragujevac, PGs (series-3)
of Natural
Kragujevac andSerbia;
Sciences
34000, LTs
and(series-5).
medicine, Dietary
University n-3ofLC-PUFAs replace AA in the phospholipid bilayer of the
cell and then alter the membrane composition and fluidity, as well as cell signalling, gene transcription
ia; zivanovicsandra@hotmail.com
sity of Kragujevac,
Natural Sciences and Kragujevac
andmedicine, 34000,
metabolism
University Serbia;
of proresolving
of mediators [7–10]. Specialized proresolving mediators (resolvins,
mail.com protectins and maresins) are bioactive lipid regulators implicated in the termination of inflammation
sity of34000,
ujevac Kragujevac,
Serbia;Physiology,
by acting through Kragujevac 34000,GSerbia
specific protein-coupled receptors. The resolution of inflammation is mediated by
oo.com
the termination of neutrophil recruitment, stimulation of apoptotic cells phagocytosis by macrophages,
ology,
d: Kragujevac
8 March 34000,
2017; Published: Serbia
and their subsequent
25 March 2017 leaving of inflammatory tissue [11–14]. These novel lipid mediators provide
rationale for further research on the beneficial effects of fish-oil enriched diets.
eed:n-3 polyunsaturated
25 March 2017 fatty acids
Unlikely other (PUFA)
n-6 PUFAs,and ɤ-linolenic acid(C18:3n-6, GLA), found mostly in borage, black current
-linolenic acid
mmatory agents that may help in the treatment of inflammatory
seed and evening primrose oil (EPO), exerts anti-inflammatory activity. Namely, it can be converted to
mined
fatty in patients
acids (PUFA) withand
dihomo- ɤ-linolenic
rheumatoid
-linolenicarthritis; (2) Methods:
acid (C20:3n-6;
acid DGLA)Sixty and further to PGs series-1, another group of eicosanoids
rthritis
ay helpwere involved
in the with in
treatment aofprospective,
potential inflammatory randomizedand
anti-inflammatory trialimmunoregulatory
of a 12 effects. Although these oils also contain
ilrheumatoid
(group I), fish oillinoleic
with(2)
arthritis; primrose
Methods:
acid (C18:2evening
Sixty oil (group
n-6, LA), II), or with
a precursor of AA, and some dietary GLA can eventually form AA,
Clinical and laboratory
in a prospective, randomized
studies evaluations trial of
have shown were
a 12done
that at the beginning
anti-inflammatory effects of EPO intake is prevailing [6]. Thus, GLA may also
esults: The Disease
h primrose evening alleviate inflammation and disease severity inofpatients with RA, as shown in clinical trials using high
Activity
oil (group ScoreII), 28
or (DAS
with 28 score), number
scale (VAS) were
evaluations scoredoses
decreased
done thenotably
ofat1400–2800 beginningmg after
GLA supplementation
per day [15–23]. in
ma phospholipids
tivity Score 28 (DAS the n-6/n-3
28Although
score), fatty acids ratio
number
many of declined
studies from 15.47 fish oil supplementation in RA patients, the results
have addressed
nd from notably
creased 18.15 ± 5.04
after to 13.50 ±
supplementation 4.81 (p = 0.005)
in
are inconsistent. Large doses of at least in groups I and
2.7 gII EPA + DHA are likely required to achieve desirable
-6/n-3
n-3 fattyand
PUFA acids
GLAratio
outcomesdeclined
(group in from
II)RA, but15.47
increased theseɤ-linolenic
doses canacidincrease
(0.00 the
± risk of bleeding [24]. In this study, RA patients were
13.50 ± 4.81 (p = 0.005) in
supplemented groups I
eitherand II
with
hich was undetectable in all groups before the treatments; (4) n-3 PUFA or n-3 PUFA and GLA to examine possible anti-inflammatory
nroup
withII)n-3 fatty acids
increased ɤ-linolenic
effectsaloneandorclinical benefits
in combination
acid (0.00 ± compared
with GLAtoexerted
the control group of patients, who had no supplementation.
tain changes
in all groups We also
in disease
before tested
activity.
the the possible
treatments; (4) synergistic effect of the combined n-3 PUFA + GLA supplementation. Our
alone or in combination primary with outcome
GLA exerted measures were the alterations of PUFA in plasma phospholipids, with emphasis
e activity.
acid; on EPA, DGLA and AA, that would indicate changes in the eicosanoids generation. The secondary
rheumatoid arthritis
outcome measure was the level of disease activity as measured with the DAS 28 disease activity score
tis and the visual analogue scale (VAS).

2. Materials and Methods

one of the most common This prospective,

autoimmune double-blind
disorders. randomized-controlled
The main trial included 60 female patients (mean
age 63.1in±the
f pathological changes 9.6 lining
years) with RA,
of the recruited
joints. Dueattothe Rheumatology Department, Clinical Center Kragujevac,
the
, and CD4+
mmon Serbia, in
helper Tdisorders.
autoimmune 2014.
cells into The RA was
the synovial
main diagnosed
stroma, synoviumthe American College of Rheumatology 2010 revised
based on
ds pain
in theoflining criteria
jointsof [1,2]. [25]. The
Furthermore,
the joints. mean
Due to the duration (± standard
the overproduction of deviation) of the disease was 59 ± 60 months
ndin
cells E2 (PGE2),
into (12–180
tumour
the synovial months).
necrosis
stroma, All(TNF),
factor
synovium patients had the (IL)-1,
interleukin same anti-rheumatic therapy. Changes in the treatment
mmation
]. regime
[3,4]. Especially
Furthermore, (dose/type of drug, etc.) were
IL-1 and TNFofplay important roles
the overproduction predetermined
in exclusion criteria. Patients received low
doses of
ur necrosis factor (TNF), corticosteroids
interleukin (IL)-1, (<10 mg/day), oral methotrexate (mean dose 15 mg/week) and folic acid
10 mg/week, while www.mdpi.com/journal/nutrients
nonsteroidal anti-inflammatory drugs (NSAIDs) were given occasionally. Patients
ally IL-1 and TNF play important roles in
were eligible if the dosage of NSAIDs and/or corticosteroids had been constant for at least one month
www.mdpi.com/journal/nutrients
at the recruitment, and remained unchanged during the study, while the dose of disease-modifying
antirheumatic drugs (DMARDs) had to be stable for at least two months before and throughout
the trial.
Patients were randomly allocated to three groups of 20 patients. The first group was taking 5 g
of fish oil (5 Omega-3 Cardio® gel capsules, Natural Wealth, NBTY Inc., New York, NY, USA; each
containing 1 g of concentrated fish oil with 300 mg of DHA, 200 mg of EPA, and 100 mg of other n-3
PUFAs) daily after meals. The second group received 2 Omega-3 Cardio® gel capsules and two Evening
Nutrients 2017, 9, 325 3 of 11

Primrose Oil® gel capsules (Natural Wealth, NBTY Inc., New York, NY, USA; one gel capsule contains
1300 mg of evening primrose oil (Oenotherabiennis, seed) with 949 mg of LA and 117 mg of GLA)
daily after meals, and the third group received only the previously described rheumatologic therapy,
representing the control group.
For the clinical evaluation of RA, we used the disease activity score (DAS) 28, which includes the
28 different joints for calculation (proximal interphalangeal joints, metacarpophalangeal joints, wrists,
elbows, shoulders, and knees). The DAS 28 also takes into account the erythrocyte sedimentation
rate (ESR) and the visual analogue scale (VAS) score. The VAS used a 100-mm horizontal scale where
patients were asked to report the current pain intensity, by placing a line on a VAS scale between
“no pain” (left end, 0 mm) and “excruciating pain” (right end 100 mm). The DAS 28 score calculation
was performed by the automatic DAS 28 calculator V1.1-beta [26].
Laboratory tests and clinical evaluations were performed on two occasions: before the treatments
were administered and three months after the beginning of the study. No patients dropped out from
the study and compliance was checked at the end of each month by the researchers, who counted the
number of capsules.

2.1. Adverse Effects

Adverse effects related to the study treatment were mild and their rate was similar in the
two intervention groups. Mild gastrointestinal discomfort (mild diarrhoea, abdominal pain, dyspepsia
or nausea lasting less than 72 h) that did not require any additional intervention, was noted in
two patients in the first group.
The following exclusion criteria were used: serious chronic renal, liver or heart diseases, diabetes
mellitus, smoking habit (in the last five years), uncontrolled hypertension, hyperlipidaemia, a family
history of cardiovascular disease, premature menopause, a severe folic acid, vitamin B6 or vitamin B12
deficiency, and other forms of arthritis apart from RA. Clinical evaluations included anthropometric
measurements (height, weight). Standing height was measured without shoes on a wall-mounted
stadiometer (Perspective Enterprises, Kalamazoo, MI, USA). Body weight, body mass index (BMI),
and body fat percent were measured with a Tanita body composition analyser (TBF-300, Tanita
Corp., Tokyo, Japan). Written informed consent was obtained from all study participants prior to
the enrolment.
The study was approved by the Ethical Committee, Clinical Center, Kragujevac, Serbia,
No 01/543-21.01.2014. This study has been conducted in accordance with the principles of the
Declaration of Helsinki.

2.2. Fatty Acid (FA) Extraction and Analysis

Venous blood samples were taken after 12 h of fasting at the beginning of the study and after
12 weeks of intervention. The method using a chloroform to methanol mixture (2:1 v/v) as a solvent
and 2,6-di-tert-butyl-4-methylphenol (BHT, 10 mg/100 mL) as an antioxidant, was applied to extract
total plasma lipids, as described previously [27]. In the next step, the phospholipid (PL) fraction
was separated from other lipid subclasses via silica thin-layer chromatography in a neutral solvent
system (petrol ether: diethyl ether: acetic acid, 87:12:1 v/v). The phospholipids fraction was placed in
a screw-capped glass test tubes for methylation. The FA methyl esters were prepared as described
previously [28] with slight modifications. In the test tube was added 1.5 mL hexane and 0.2 mL of
2 N NaOH in methanol and heated at 85◦ C for 1 h, and then 0.2 mL of 1M H2 SO4 in methanol and
heated at 85◦ C for 2 h. After cooling to room temperature and centrifugation on 1860× g, for 15 min,
the hexane layer was dried under a stream of nitrogen. Prepared methyl esters were dissolved in
10 µL of hexane. Fatty acid (FA) analysis was started with the injection of 1 µL of sample into the
gas-liquid chromatography device (Shimadzu GC 2014, flame ionization detector, Rtx 2330 column
60 m × 0.25 mm ID, film thickness 0.2 µm, Restek, Bellefonte, PA, USA). FA were identified according
to the retention times of samples in comparison with the standards (Sigma Chemical Co., St. Louis,
Nutrients 2017, 9, 325 4 of 11

Missouri, MO, USA) and (PUFA)-2 standard mixture (Restek Co., Bellefonte, Pennsylvania, PA, USA),
and expressed as relative percentages of total FA.

2.3. Statistical Analysis

Statistical data analysis was performed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA), considering
p ≤ 0.05 as significant. The results are presented as mean ± SD. The Kolmogorov-Smirnov test was
applied to confirm the distribution of the data. Differences among the three tested groups at baseline
and at the end of the study were assessed by one-way ANOVA followed by the Tukey post hoc test
or by Kruskal-Wallis and Mann-Whitney U test, depending on the normality. To assess the effects
of the supplementations, two-way (group and time) repeated measures ANOVA, was applied, with
Bonferroni post hoc tests, where appropriate.

3. Results
Patients were randomly divided into three groups, which were receiving the same anti-rheumatic
therapy. During 12 weeks, the first group took fish oil, the second one took fish oil + EPO and the third
one did not take supplements. Patients’ basic characteristics are presented in Table 1. Age, BMI and
the duration of RA were similar among the groups (Table 1).

Table 1. Baseline characteristics of the patients with rheumatoid arthritis.

Group I Group II Group III

Patients’ Characteristics p Value
n = 20 n = 20 n = 20
Age (X ± SD; years) 54 ± 8 57 ± 8 59 ± 7 NS
BMI (kg/m2 ) 26.48 ± 4.15 26.52 ± 4.37 24.65 ± 6.29 NS
Disease duration 6.6 ± 4.0 8.1 ± 2.7 7.2 ± 2.6 NS
Values are expressed as mean ± SD; BMI: body mass index; NS: not statistically significant, assessed by
one-way ANOVA.

A significant decrease in the DAS 28 score was found in the fish oil group (4.99 ± 0.88 to 3.91 ± 0.80,
p < 0.001, group I) and in the group II taking n-3 PUFA and EPO (4.76 ± 0.85 to 3.79 ± 0.72, group II).
The DAS 28 score in group III trended towards a significant decrease (4.66 ± 0.80 to 4.23 ± 0.66,
p = 0.053) (Table 2).

Table 2. Comparisons of clinical and laboratory results in patients at the start and the end of
the supplementation.

Patients’ Group I Group II Group III

Characteristics Timing
n = 20 n = 20 n = 20
Baseline 12.4 ± 8.2 16.0 ± 18.3 12.7 ± 7.2
CRP (mg/L)
End of study 7.3 ± 2.9 *** 7.1 ± 5.5 *** 6.9 ± 3.5 ***
Baseline 6.2 ± 2.0 5.4 ± 1.9 5.0 ± 2.0
Tender joint count
End of study 3.3 ± 1.5 *** 4.0 ± 1.4 *** 4.6 ± 1.6
Baseline 1.8 ± 1.0 1.5 ± 1.6 1.0 ± 1.3
Swollen joint count
End of study 0.8 ± 0.3 *** 0.3 ± 0.8 *** 0.4 ± 0.2 **
Baseline 55.7 ± 10.1 59.0 ± 9.1 61.5 ± 8.9
VAS (pain)
End of study 46.7 ± 7.1 *** 50.5 ± 7.0 *** 59.3 ± 6.9
Baseline 35.0 ± 24.1 36.7 ± 19.2 33.3 ± 17.1
ESR, mm/h
End of study 23.2 ± 16.6 *** 19.9 ± 10.8 *** 24.1 ± 13.9 **
Baseline 4.99 ± 0.88 4.76 ± 0.85 4.66 ± 0.80
DAS 28
End of study 3.91 ± 0.80 *** 3.79 ± 0.72 *** 4.23 ± 0.66
** Significantly different compared to the start value (p ≤ 0.01); *** (p ≤ 0.001). Values are expressed as mean ± SD;
CRP: C-reactive protein; VAS: visual analogue scale; ESR: erythrocyte sedimentation rate; DAS 28: disease activity
score 28.
 Nutrients 2017, 9, 325 5 of 11

The number of painful joints and VAS score significantly decreased in both the supplemented
groups (p ≤ 0.001) after 12 weeks, but not in the control group (Table 2).
Because of the high fish oil intake, EPA, DHA, n-3 PUFA and total PUFA increased significantly
(p ≤ 0.05, Table 3) in plasma of the group I. Additionally, the n-6/n-3 PUFA ratio and MUFA, especially
vaccenic acid (C18:1n-7) markedly decreased (Table 3).
and ɤ-Linolenic Acid in
s of n-3 PUFAArticle
capsules daily. of n-3 PUFA and ɤ-Linolenic Acid in
Table 3. Fatty acid distribution in plasma phospholipids at baseline and after a 12-week intake of five
heumatoid Arthritis
Clinical
Omega-3 Cardio gelBenefits
Patients with Rheumatoid Arthritis
Vasiljevic 2, Vesna Vucic
Fatty3, Acid
Aleksandra Arsic 3, Snjezana Petrovic
Distribution 3,
Group I Statistical Significance
aja Savic 4, Sandra Zivanovic
Mirjana 5, Vladislava 1Stojic 6 and Vladimir
Veselinovic
Fatty acid , Dragan Vasiljevic
Baseline , Vesna Vucic
2 3, Aleksandra
After 12 wk Arsic 3, Snjezana Petrovic 3,
p value
Aleksandra 16:0 Tomic-Lucic 1, Maja
30.07 ± 5.22
Savic 29.47 ±
4, Sandra Zivanovic 5, Vladislava
2.31 Stojic 6 andNS
Vladimir
16:1 n-7*
Jakovljevic 7, 0.57 ± 0.15 0.53 ± 0.23 NS
ine, Faculty of Medical Sciences, University
18:0 of Kragujevac, Kragujevac
18.59 ± 2.82 34000, 16.77 ± 2.53 NS
1 Department of Internal medicine, Faculty of Medical Sciences, University of Kragujevac, Kragujevac 34000,
M.V.); sanlusa@ptt.rs (A.T.-L.) 18:1 n-9 9.32 ± 1.24 8.05 ± 1.05 NS
tute for Public Health, Faculty Serbia;
18:1veselinovic.m@sbb.rs
of Medical (M.V.);ofsanlusa@ptt.rs
n-7 Sciences, University Kragujevac, (A.T.-L.)
1.99 ± 0.33 1.58 ± 0.25 0.010
2 Department of Hygiene, Institute for Public Health, Faculty of Medical Sciences, University of Kragujevac,
_gana@yahoo.com 18:2 n-6LA 23.73 ± 2.43 26.03 ± 2.91 NS
Kragujevac 34000, Serbia; dvg_gana@yahoo.com
in Nutrition and Metabolism, 18:3 n-3ALA
Institute for Medical Research, ± 0.24
0.23 University of 0.21 ± 0.13 NS
3 Centre of Research Excellence in Nutrition and Metabolism, Institute for Medical Research, University of
bia; vesna.vucic.imr@gmail.com18:3 n-6GLA
(V.V.); 0.00 ± 0.00
vesna.vucic.imr@gmail.com (A.A.); 0.00 ± 0.00 NS
Belgrade, Beograd 11000, Serbia; vesna.vucic.imr@gmail.com (V.V.); vesna.vucic.imr@gmail.com (A.A.);
m (S.P.) 20:3 n-6DGLA 2.84 ± 0.80 2.46 ± 0.94 NS
snjezana570.imr12@gmail.com (S.P.)
20:4
ulty of Medical Sciences, University n-6AA
of Kragujevac, 11.05
Kragujevac ± 3.31
34000, Serbia; 10.22 ± 1.81 NS
4 Department of Pharmacy, Faculty of Medical Sciences, University of Kragujevac, Kragujevac 34000, Serbia;
20:5 n-3EPA 0.32 ± 0.22 1.01 ± 1.02 0.026
l.com maja.jovanovic.2008.38@gmail.com
22:4 n-6 0.45 ± 0.38 0.32 ± 0.13 NS
and Tourism, Department of 5 Natural
Faculty ofSciences and medicine,
Hotel Management University
and Tourism, of
Department of Natural Sciences and medicine, University of
22:5 n-3DPA 0.38 ± 0.17 0.58 ± 0.30 NS
Kragujevac, Kragujevac 34000, Serbia; zivanovicsandra@hotmail.com
, Serbia; zivanovicsandra@hotmail.com
22:6 n-3DHA 1.92 ± 0.91 2.74 ± 1.08
6 Faculty of Medical Sciences, University of Kragujevac, Kragujevac 34000, Serbia;
0.007
University of Kragujevac, Kragujevacn-3 34000, Serbia; 2.85 ± 1.14 4.55 ± 2.26 0.013
vladisavastojic@medf.kg.ac.rs
n-6 38.08 ± 3.80 39.05 ± 3.22
7 Faculty of Medical Sciences, University of Kragujevac, Physiology, Kragujevac 34000, Serbia
NS
n-6/n-3
University of Kragujevac, Physiology, 15.47 ± 5.51
Kragujevac 34000, Serbia 10.62 ± 5.07 0.005
* Correspondence: drvladakgbg@yahoo.com
g@yahoo.com SFA 47.17 ± 3.59 46.24 ± 3.97 NS
MUFA
Received: 11.89 ±81.33
28 November 2016; Accepted: 10.16 ±
March 2017; Published: 251.34
March 2017 0.012
ccepted: 8 March 2017; Published: 25 PUFAMarch 2017 40.61 ± 3.52 43.13 ± 3.17 0.013
Abstract:
Data (1) Background:
are expressed as the mean ±Marine
SD; NS: n-3 polyunsaturated
not statistically fattyLA:
significant; acids (PUFA)
linoleic and ɤ-linolenic
acid; GLA: acid
-linolenic acid;
Marine n-3 polyunsaturated ALA:(GLA)fatty
areacids
α-linolenic acid; (PUFA)
well-known and ɤ-linolenic
DGLA:anti-inflammatory
dihomo- acid
-linolenic acid;
agentsAA:that
arachidonic
may helpacid;in EPA: eicosapentaenoic
the treatment acid; DPA:
of inflammatory
nflammatory agents that docosapentaenoic
may helpTheir
disorders. inacid;
the DHA: docosahexaenoic
treatment
effects were acid;
of inflammatory
examined SFA: saturated
in patients fatty acids; MUFA:
with rheumatoid monounsaturated
arthritis; (2) Methods: fatty
Sixty
acids; PUFA: polyunsaturated fatty acids.
patients with active rheumatoid
e examined in patients with rheumatoid arthritis; (2) Methods: Sixty arthritis were involved in a prospective, randomized trial of a 12
oid arthritis were involved weekin supplementation with fish oil (group
a prospective, randomized trial ofI),a fish
12 oil with primrose evening oil (group II), or with
In the
no group that ingested
supplementation (group 2 gIII).
of Clinical
fish oil +and 2.6laboratory
g EPO/day (group II),
evaluations thedone
were concentrations of GLA
at the beginning
fish oil (group I), fish oil with primrose evening oil (group II), or with
and AAand (18:3 n-6) increased, as well as n-3
at the end of the study; (3) Results: The Disease Activity Score 28 (DAS 28 score), number oftotal
and 20:4 significantly EPA, DPA, DHA, n-3 PUFA, and
III). Clinical and laboratory evaluations were done at the beginning
tender4).
PUFA (Table joints and with
In line visualthis,
analogue
level scale
of SFA (VAS)
and score decreased
the n-6/n-3 PUFA notably after
ratio were supplementation in
significantly lower
(3) Results: The Disease Activity Score 28 (DAS 28 score), number of
after thegroups I and II (p < 0.001).
supplementation (TableIn4).plasma phospholipids the n-6/n-3 fatty acids ratio declined from 15.47
logue scale (VAS) score decreased
± 5.51 to 10.62notably after
± 5.07 (p supplementation
= 0.005), and from 18.15in± 5.04 to 13.50 ± 4.81 (p = 0.005) in groups I and II
plasma phospholipids therespectively.
n-6/n-3 fatty The
acids ratio
combination
declined from 15.47
of n-3 PUFA and GLAat(group ɤ-linolenic
Table 4. Fatty acid distribution in plasma phospholipids baselineII)and
increased
after a 12-week acidof(0.00
intake two ±
05), and from 18.15 ± 5.04 to
0.00 13.50
to ±
0.13 4.81
± (p
0.11, =
p 0.005)
< in
0.001), groups
which wasI and II
undetectable
Omega-3 Cardio gel capsules and two Evening Primrose Oil gel capsules daily. in all groups before the treatments; (4)
n of n-3 PUFA and GLAConclusion:
(group II) Daily
increased ɤ-linolenic with
supplementation acidn-3
(0.00 ± acids alone or in combination with GLA exerted
fatty
1), which was undetectable significant
in all
Fatty Acid clinical benefits and certain
groups before the treatments;
Distribution changes
Group in disease activity.
(4) II Statistical Significance
ntation with n-3 fatty acids alone or
Fatty acidin combination with GLA
Baseline exerted After 12 week p value
Keywords:
nd certain changes in disease activity.
16:0
n-3 PUFA; ɤ-linolenic acid; rheumatoid arthritis
30.08 ± 5.06 28.85 ± 2.62 NS
16:1 n-7 0.56 ± 0.13 0.58 ± 0.26 NS
18:0 16.93 ± 1.83 16.34 ± 1.41 NS
enic acid; rheumatoid arthritis
18:1 n-9 8.64 ± 1.03 8.44 ± 0.93 NS
1. Introduction
18:1 n-7 1.75 ± 0.24 1.63 ± 0.23 NS
18:2 n-6LA 26.20 ± 1.90 26.30 ± 2.41 NS
Rheumatoid arthritis (RA) is one of the most common autoimmune disorders. The main
18:3 n-3ALA 0.22 ± 0.18 0.18 ± 0.15 NS
characteristic
18:3 n-6GLA of RA is the onset
0.00of pathological changes
± 0.00 0.13 ±in0.11
the lining of the joints.
<0.001Due to the
infiltration of macrophages, B 2.59
20:3 n-6DGLA cells,
± and
0.63 CD4+ helper2.67
T cells into the synovial stroma,
± 0.63 NS synovium
A) is one of the most proliferates
common causing swelling
autoimmune
20:4 n-6AA and± pain
disorders.
10.52 The ofmain
1.81 joints 11.29
[1,2]. ±Furthermore,
2.14 the overproduction
p = 0.048 of
inflammatory
20:5 n-3EPAmolecules, prostaglandin
0.20 ± E2
0.10
nset of pathological changes in the lining of the joints. Due to the (PGE2), tumour
0.49 ±necrosis
0.23 factor (TNF), interleukin
p < 0.001 (IL)-1,
and cytokines induces chronic inflammation [3,4]. Especially IL-1 and TNF play important roles in
B cells, and CD4+ helper T cells into the synovial stroma, synovium
g and pain of joints Nutrients
[1,2]. Furthermore, the overproduction of
2017, 9, 325; doi:10.3390/nu9040325 www.mdpi.com/journal/nutrients
taglandin E2 (PGE2), tumour necrosis factor (TNF), interleukin (IL)-1,
c inflammation [3,4]. Especially IL-1 and TNF play important roles in
040325 www.mdpi.com/journal/nutrients
Nutrients 2017, 9, 325 6 of 11

Table 4. Cont.

Fatty Acid Distribution Group II Statistical Significance

22:4 n-6 0.37 ± 0.18 0.34 ± 0.13 NS
22:5 n-3DPA 0.31 ± 0.08 0.45 ± 0.16 p < 0.001
22:6 n-3DHA 1.61 ± 0.55 2.22 ± 0.74 p = 0.006
n-3 2.34 ± 0.65 3.33 ± 1.00 p = 0.001
n-6 39.69 ± 3.08 40.75 ± 2.86 NS
n-6/n-3 18.15 ± 5.04 13.50 ± 4.81 p = 0.005
SFA 47.01 ± 3.62 45.19 ± 2.80 p = 0.039
MUFA 10.56 ± 1.46 10.67 ± 1.12 NS
PUFA 42.04 ± 3.38 44.07 ± 2.96 p = 0.020
Data are expressed as the mean ± SD; NS: not statistically significant; Abbreviations as in Table 3.

In the group III (patients without supplementation), no significant changes in FA proportions

were found between baseline and the end of the trial (Table 5).

Table 5. Fatty acid distribution in plasma phospholipids at baseline and after 12 weeks
without supplementation.

Fatty Acid Distribution Group III Statistical Significance

Fatty acid Baseline After 12 wk p value
16:0 32.21 ± 3.69 29.67 ± 3.88 NS
16:1 n-7 0.72 ± 0.61 0.57 ± 0.12 NS
18:0 16.19 ± 2.40 16.55 ± 1.82 NS
18:1 n-9 8.51 ± 1.45 8.35 ± 1.39 NS
18:1 n-7 1.69 ± 0.25 1.68 ± 0.30 NS
18:2 n-6LA 26.40 ± 3.47 27.49 ± 3.96 NS
18:3 n-3ALA 0.21 ± 0.14 0.19 ± 0.23 NS
18:3 n-6GLA 0.00 ± 0.00 0.00 ± 0.00 NS
20:3 n-6DGLA 2.44 ± 0.78 2.82 ± 0.97 NS
20:4 n-6AA 8.98 ± 1.84 9.60 ± 2.90 NS
20:5 n-3EPA 0.29 ± 0.18 0.34 ± 0.16 NS
22:4 n-6 0.32 ± 0.14 0.29 ± 0.13 NS
22:5 n-3DPA 0.32 ± 0.14 0.56 ± 0.69 NS
22:6 n-3DHA 1.66 ± 0.69 1.83 ± 0.72 NS
n-3 2.46 ± 0.90 2.66 ± 0.81 NS
n-6 38.14 ± 3.09 40.03 ± 2.71 NS
n-6/n-3 17.25 ± 5.51 16.24 ± 3.14 NS
SFA 48.41 ± 3.02 46.26 ± 3.67 NS
MUFA 9.72 ± 2.41 10.61 ± 1.74 NS
PUFA 40.49 ± 4.45 43.60 ± 4.22 NS
Data are expressed as the mean ± SD; Abbreviations as in Table 3.

After 12 weeks of supplementation, when all groups were compared, levels of EPA, DHA and n-3
PUFA were higher, and the n-6 to n-3 ratio was lower in both supplemented groups than in the control
patients. GLA and AA were higher in the group II (fish oil + EPO) than in the groups I and III (Table 6).
A repeated measures ANOVA revealed significant time × group interactions for the following
FAs: stearic acid (18:0, p = 0.034), vaccenic acid (18:1n-7, p = 0.010), and MUFA (p = 0.028).
Nutrients 2017, 9, 325 7 of 11

Table 6. Comparisons of fatty acid levels in plasma phospholipids after 12 weeks of intervention.

Fatty Acid Group I Group II Group III

16:0 29.47 ± 2.31 28.85 ± 2.62 29.67 ± 3.88
16:1 n-7 0.53 ± 0.23 0.58 ± 0.26 0.57 ± 0.12
18:0 16.77 ± 2.53 16.34 ± 1.41 16.55 ± 1.82
18:1 n-9 8.05 ± 1.05 8.44 ± 0.93 8.35 ± 1.39
18:1 n-7 1.58 ± 0.25 1.63 ± 0.23 1.68 ± 0.30
18:2 n-6LA 26.03 ± 2.91 26.30 ± 2.41 27.49 ± 3.96
18:3 n-3ALA 0.21 ± 0.13 0.18 ± 0.15 0.19 ± 0.23
18:3 n-6GLA 0.00 ± 0.00 0.13 ± 0.11 ***### 0.00 ± 0.00
20:3 n-6DGLA 2.46 ± 0.94 2.69 ± 0.63 2.67 ± 0.77
20:4 n-6AA 10.22 ± 1.81 11.29 ± 2.14 *# 9.60 ± 2.90
20:5 n-3EPA 1.01 ± 1.02 ** 0.49 ± 0.23 *# 0.34 ± 0.16
22:4 n-6 0.32 ± 0.13 0.34 ± 0.13 0.29 ± 0.13
22:5 n-3DPA 0.58 ± 0.30 0.45 ± 0.16 0.56 ± 0.69
22:6 n-3DHA 2.74 ± 1.08 * 2.22 ± 0.74 * 1.83 ± 0.72
n-3 4.55 ± 2.26 ** 3.33 ± 1.00 * 2.66 ± 0.81
n-6 39.05 ± 3.22 40.75 ± 2.86 40.03 ± 2.71
n-6/n-3 10.62 ± 5.07 ** 13.50 ± 4.81 * 16.24 ± 3.14
SFA 46.24 ± 3.97 45.19 ± 2.80 46.26 ± 3.67
MUFA 10.16 ± 1.34 10.67 ± 1.12 10.61 ± 1.74
PUFA 43.13 ± 3.17 44.07 ± 2.96 43.60 ± 4.22
* Significantly different from the group III (p ≤ 0.05); ** (p ≤ 0.01); *** (p ≤ 0.001). # Significantly different from the
group I # (p ≤ 0.05); ### (p ≤ 0.001). Assessed by one-way ANOVA followed by the Tukey post hoc test. Data are
expressed as the mean ± SD; Abbreviations as in Table 3.

4. Discussion
Fatty acids found in plasma phospholipids reflect food intake over the last several days and
weeks [29] as well as FA metabolism [30]. Altered FA composition was reported in many pathologic
and physiological states [30–33], including chronic inflammation [34]. However, FA profiles and
the course of diseases can be affected by PUFA supplementation [35,36]. In this study, the levels of
supplemented PUFA in plasma increased in the groups I and II depending on the dosages (Tables 3
and 4), demonstrating a good patient compliance and bioavailability of the supplements. The changes
in FA composition in response to the supplementation (groups I and II) confirmed that plasma
phospholipids are reliable biomarkers for fat intake (Tables 3 and 4). In particular, it can be seen in
Table 6, where the differences among the three groups after supplementation were most significant for
FA used for supplementation.
The results have shown improvements in several laboratory and clinical parameters in RA
patients with the active disease, who were taking anti-inflammatory oils along with the standard
therapy. The consumption of 5 g of fish oil by the patients in group I resulted in significantly
elevated EPA (Table 3). The AA content in phospholipids is a regulator of the biosynthesis of
the proinflammatory prostanoids and leukotriens, whereas EPA has the opposite effect. Therefore,
the AA/EPA ratio determines the degree of inflammation. Additionally, EPA and DHA support
the synthesis of proresolving mediators such as protectins, resolvins, and maresins. Accordingly,
a significant improvement in clinical variables, the severity of pain, the number of swollen/tender
joints, as well as disease activity parameters (DAS 28 and VAS scores), were also observed in the
supplemented group.
In a 24-week study, Berbert et al. supplemented RA patients with 3 g/day of n-3 FA (1.8 g
EPA and 1.2 g DHA) and found reduced pain and morning stiffness as well as better patient global
assessment [37]. The others reported that daily supplementation of approximately 3 g n-3 PUFA for
about three months was sufficient to achieve clinical improvements in these patients [38,39]. However,
some meta-analyses have revealed no effect of n-3 PUFAs intake on the inflammation of joints, other
Nutrients 2017, 9, 325 8 of 11

clinical variables or the global assessment of RA patients [40,41]. In this regard, our study gives
substantial contribution to recommendation for the use of n-3 PUFA in RA patients.
The combination of fish oil and EPO led to an increase in AA (Table 4). Namely, dietary GLA
can be elongated to DGLA and subsequently desaturated to AA by delta-5 desaturase [42]. Hence,
a slight increase of the AA content in phospholipids after GLA supplementation (group II; Table 4) was
expected. In addition, LA as the main precursor for AA synthesis, was also presented in EPO capsules.
Nevertheless, in our study supplementation with both fish oil and EPO resulted in significantly
improved clinical variables, the severity of pain and the number of swollen and/or tender joints.
These results indicate that the anti-inflammatory potential of GLA and DGLA, including the synthesis
of PG series 1, are more pronounced than the conversion into inflammatory AA.
The intake of n-6 LA was usually not controlled in studies on patients with RA. LA is ultimately
converted into AA and further to pro-inflammatory cytokines [40]. PUFA of n-3 series can diminish
this conversion by competitive inhibition [43]. It has also been proposed that restricting AA intake
is required to benefit from the n-3 intake in RA patients [44]. Additional studies addressing plasma
lipids and patient compliance measurements should confirm these findings.
Numerous investigations have shown the relationship between n-3 PUFA consumption and
reducing inflammatory parameters [40,41,45,46]. Here we found lower ESR in group I and II than
in the control patients, which was in line with other studies [37,47]. However, the link between n-3
PUFA and CRP is unclear. For instance, a 12-week supplementation with different doses of n-3 FA
(1.5–6 g/day) did not decrease CRP concentrations when compared to the placebo group, as in our
study [48]. It seems that inconsistent results come from different doses of n-3 PUFA needed for different
inflammatory markers. Besides, we have shown that the consumption of fish oil combined with EPO
also had positive effects in patients with RA, which were similar or even better (Table 2) than the fish
oil supplementation alone. Thus, this combination has a great potential as an adjuvant therapy in
patients with RA and other chronic inflammatory diseases.
Furthermore, chronic use of standard DMARDs, especially methotrexate, often leads to
cardiovascular problems, that could be alleviated by concomitant consumption of n-3 PUFA [49].
As well, intake of 3 g of n-3 PUFA markedly increases the AA/EPA ratio and EPA + DHA index, which
are highly reliable predictors of sudden cardiac death [50–52]. This is an added value, since patients
with RA have strongly elevated risk of cardiac death [53,54]. Nevertheless, the exact impact of the
control of chronic inflammation on the reduction of cardiovascular mortality should be elucidated [41].
The weakness of this study is the lack of a blinded placebo-controlled group, which would have
made the interpretation of the data stronger. However, placebo capsules typically contain soy oil, corn
oil or sunflower oil, which are all rich sources of n-6 PUFA. This could influence our results on FA
profiles in the control group and must be taken into account in the interpretation of the FA alterations.

5. Conclutions
In conclusion, the intake of fish oil alone or combined with EPO resulted in a higher incorporation
of PUFA precursors for the anti-inflammatory lipid mediators in plasma phospholipids. This further
induced a significant improvement in the clinical status of patients with rheumatoid arthritis. Further
research with a large number of patients over a longer duration is needed to confirm the long-term
efficacy of these supplementations.

Acknowledgments: This study was supported by Grants No. III 41030 and 175043 from the Ministry of Education,
Science and Technological Development of the Republic of Serbia.
Author Contributions: Vladimir Jakovljevic and Dragan Vasiljevic conceived and designed the experiments;
Dragan Vasiljevic, Aleksandra Tomic Lucic and Sandra Zivanovic performed the experiments; Mirjana Veselinovic
and Vladisava Stojic analyzed the data; Vesna Vucic, Aleksandra Arsic and Snjezana Petrovic performed fatty
acids analyses and critically revised the manuscript; Mirjana Veselinovic and Maja Savic drafted the paper.
Conflicts of Interest: The authors declare no conflict of interest.
Nutrients 2017, 9, 325 9 of 11

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