Paixão et al.

Nutrition Journal (2017) 16:71

DOI 10.1186/s12937-017-0295-9

RESEARCH Open Access

The effects of EPA and DHA enriched fish

oil on nutritional and immunological
markers of treatment naïve breast cancer
patients: a randomized double-blind
controlled trial
Elemárcia Martins da Silva Paixão1*, Ana Carolina de M. Oliveira1, Nathalia Pizato1,
Maria Imaculada Muniz-Junqueira2, Kelly G. Magalhães3, Eduardo Yoshio Nakano4 and Marina K. Ito1

Abstract
Background: We evaluated the effects of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids enriched fish
oil (FO) on nutritional and immunological parameters of treatment naïve breast cancer patients.
Methods: In a randomized double blind controlled trial, the FO group (FG) patients were supplemented with 2 g/
day of FO concentrate containing 1.8 g of n-3 fatty acids during 30 days. The placebo group (PG) received 2 g/ day
of mineral oil. At baseline and after the intervention, plasma levels of n-3 fatty acids, dietary intake, weight, body
composition, biochemical and immunological markers were assessed.
Results: At the end of the intervention period, no between group differences were observed regarding anthropometric
parameters. There was a significant increase in the plasma phospholipid EPA (p = 0.004), DHA (p = 0.007) of the FG
patients. In FG patients the percentages of peripheral blood CD4+ T lymphocytes and serum high sensitivity C-reactive
protein (hsCRP) levels were maintained while in PG patients there was a significant increase in hsCRP (p = 0.024). We also
observed a significant reduction in the percentage of CD4+ T lymphocytes in the peripheral blood (p = 0.042) of PG
patients. No changes in serum proinflammatory cytokine and prostaglandin E2 levels were observed.
Conclusions: Supplementation of newly diagnosed breast cancer patients with EPA and DHA led to a significant change
in the composition of plasma fatty acids, maintained the level of CD4+ T cells and serum levels of hsCRP, suggestive of a
beneficial effect on the immune system and less active inflammatory response.
Trial registration: Brazilian Clinical Trials Registry (REBEC): RBR-2b2hqh. Registered 29 April 2013, retrospectively
registered.
Keywords: Breast cancer, N-3 fatty acids, Fish oil, Immunonutrition, Cytokines, Eicosapentaenoic acid (EPA)

* Correspondence: [email protected]
1
Post Graduate Program in Human Nutrition, University of Brasília, Brasilia,
Federal District 70910-900, Brazil
Full list of author information is available at the end of the article

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Paixão et al. Nutrition Journal (2017) 16:71 Page 2 of 11

Introduction Materials & methods

Cell-mediated immune response (IR) plays an important Study population
role in cancer immunoediting. CD4+ and CD8+ T cells Breast cancer patients attending the University Hospital
are the main lymphocytes involved in cell-mediated im- of Brasilia and the Base Hospital of the Federal District
munity. It is established that an effective anti-tumor IR were invited to participate. Inclusion criteria were
requires the participation of both types of T lympho- treatment-naïve patients between 18 and 70 years of age,
cytes cells, since the CD4+ T cells are critical for the with mammographic image classification 4C or higher
generation of tumor-specific cytotoxic T cells as well as according to Breast Imaging-Reporting and Data System
memory T cells expansion [1]. Fatty acids are modula- (BI-RADS), and with surgery as primary treatment op-
tors of lymphocyte functions. Both the type of fatty acids tion. BI-RADS 4C denotes “finding of moderate concern
present in the diet and their serum levels may influence of being cancer” and patients in this category are advised
lymphocyte proliferation, cytokine production and T- to perform biopsy exams. Exclusion criteria were pa-
lymphocyte migration [2, 3]. tients with metastatic or recurrent disease, comorbidity
Breast cancer patients have altered cell-mediated IR or other disease that prevented the use of fish oil or
compared to healthy controls. In newly diagnosed pa- affected the blood parameters being studied, pacemaker
tients, low peripheral blood CD4+ cell counts have been users and those unable to be weighted or with edema.
observed [4, 5]. Moreover, results contrary to the above All patients signed an informed consent before entering
[6] or those who showed no difference between patients the study.
and controls [7] have also been published. Furthermore,
even in the early stages, breast cancer patients have in- Study design
creased serum levels of prostaglandin E2 (PGE2) [8] and A randomized, controlled, double-blind study was con-
proinflammatory cytokines such as tumor necrosis factor ducted between the period of February 2012 and March
alpha (TNF-α), interleukin (IL)-1β and IL-6 [9]. Elevated 2013. The study was carried out in compliance with
serum levels of C-reactive protein (CRP) at the time of Good Clinical Practice and the Consolidated Standards
diagnosis were observed and associated with shorter for Reporting of Trials (CONSORT) statement. The
disease-free survival and overall survival of breast cancer study protocol was approved by the Human Research
patients [10]. Ethics Committees of the University of Brasilia and of
Diagnosis of cancer can motivate patients to alter their the Federal District Health Secretariat. The Brazilian
dietary habits on its own. Nutritional supplement intake Registry of Clinical Trials (ReBEC) is one of the primary
such as fish oil, which is the principal source of n-3 fatty registry site of the WHO International Clinical Trials
acids eicosapentaenoic acid (EPA) and docosahexaenoic and the study was registered as RBR-2B2hqh. The
acid (DHA), is highly prevalent among breast cancer pa- randomization was done beforehand and performed by
tients [11]. The benefit of n-3 fatty acid intake on breast manual raffling the blocks of ten sequential numbers
cancer incidence has been reported in a recent system- with five chances of being raffled to one of the two
atic review of prospective cohort studies, that suggested groups. A laboratory technician not involved in the re-
a dose response relationship of 5% lower risk for each search performed the randomization, assigned fish oil
0.1 g/day increment of marine n-3 fatty acid intake [12]. group (FG) or placebo group (PG) to the sequential
For those already with the disease, specific human inter- numbers and kept the randomized sequence secret to
vention studies are limited and the results have been the project team members and patients until the last pa-
variable [13–15]. In metastatic breast cancer patients, tient’s data collection were finished. Patients were ran-
oral supplementation with DHA during chemotherapy domized only after positive biopsy confirmation for
potentially improved patient survival [13] and in patients malignancy. The same technician provided the blinded
under chemo or radiotherapy for other types of cancer, supplement (which was identified only with the sequen-
the supplementation with EPA and DHA increased body tial numbers) to the research team. The supplements
weight [16] and reduced serum CRP [17], proinflamma- were supplied in white plastic bottles containing 30 cap-
tory cytokines and PGE2 levels [14, 16, 17]. However, sules (sufficient for 15 days). Patients entering the study
there is a gap of knowledge on the potential benefit of were assigned to the sequential identification number.
n-3 fatty acid intake for breast cancer patients at early The intervention lasted 30 days, immediately following
stages of treatment. the diagnosis and before the surgical procedure. Thirty
Thus, the aim of this study was to investigate whether days was the mean time needed for patients to go
supplementation with EPA and DHA, immediately fol- through pre-surgery exams. Patients were scheduled to
lowing the diagnosis of breast cancer but prior to treat- return in the middle of the intervention period, when
ment, would have a positive impact on patient’s the second supplement bottle was given. At the final
nutritional and selected immune parameters. visit, patients were asked to return any unused capsules.
Paixão et al. Nutrition Journal (2017) 16:71 Page 3 of 11

Twelve hours fasted blood samples were collected for Blood analysis
biochemical and immunological analyses at baseline and Blood samples were obtained for biochemical (serum) and
at the end of the intervention period. For the evaluation immunological analysis (plasma). Biochemical analysis in-
of nutritional status, body weight, height and body com- cluded serum glucose, total cholesterol, high- and low-
position analyses were performed. Dietary intake was density lipoprotein cholesterol and triglycerides (Labtest®),
evaluated by 24-h recall method, two at baseline and complete blood count (CELL-DYN 3500 system), albumin
two at the end of intervention. and high sensitivity C-reactive protein (hsCRP) by immu-
nonephelometry (Siemens®). Immunological parameters
evaluated were peripheral blood mononuclear CD4+ e
Fish oil supplements CD8+ lymphocyte cell counts, plasma cytokines and
Bulk fish oil concentrate (MaxOmega 46/38 EE®, Equa- PGE2. Plasma phospholipid fatty acid profile was also ana-
teq Ltd., United Kingdom) and mineral oil were pur- lyzed as a marker of compliance.
chased and encapsulated (Relthy Laboratories Ltd.,
Brazil) in 1 g gel capsules. FG patients were asked to in- Flow-cytometric analysis
gest 2 g of fish oil concentrate (2 capsules) daily for The peripheral blood mononuclear cells (PBMC) were
30 days, at lunch and dinner times. Each gram of fish oil obtained by density gradient centrifugation with Histo-
concentrate contained 470 mg of EPA, 390 mg of DHA paque ® - 1077 (Sigma-Aldrich). Lymphocytes were sus-
plus 18:3n3 acid, in the form of ethyl esters, with a total pended in phosphate buffered saline at a concentration
of 1.81 g of n-3 fatty acids per day, according to the of 5 × 105 cells/ well. CD4+ and CD8+ cells were
manufacturer’s information and confirmed in our lab. counted with surface marker PE mouse anti-human
The fish oil capsules also contained 0.32% (w/w) of vita- CD4 and CD8 (BD Biosciences, USA). The analysis was
min E (α-tocopherol) as antioxidant. Placebo group pa- performed in a FACSCalibur flow cytometer equipped
tients were given 2 g per day of mineral oil of the same with CellQuest software (BD Biosciences, USA). Twenty
color and smell of the fish oil supplement, divided in 2 thousand events were acquired from each sample and
capsules of 1 g each. In our study, rather than masking the results were analyzed using FlowJo software, version
the typical odor of fish oil, the plastic bottles for mineral 10.0 (Treestar, Inc. USA).
oil capsules were previously treated with fish oil cap-
sules. This procedure added subtle fish oil smell to the Proinflammatory cytokines and prostaglandin E2
bottles of mineral oil, thus, all patients thought they Plasma IL-6, IL-1β and TNF-α cytokines were quantified
were receiving fish oil capsules. by the ELISA method (Bioscience, San Diego, USA).
Compliance was promoted by regular telephone con- Prostaglandin E2 metabolites were quantified by compe-
tact with the patients and was monitored by counting tition ELISA method using the Prostaglandin E Metabol-
the returned capsules at 15th and 30th day visits. Plasma ite EIA kit (Cayman Chemical Company, USA)
phospholipid fatty acid profile before and at the end of according to the manufacturer’s instructions.
the intervention was also analyzed for compliance
evaluation. Phospholipid fatty acid profile
Plasma lipid was extracted according to Folch et al. [20]
and phospholipids were separated by thin layer chroma-
Nutritional status and dietary intake tography with solvent system hexane: diethyl ether:
Weight and height were measured in a Toledo digital acetic acid (80:20:2 v/v/v) [21]. Phospholipid fatty acids
scale and a metal stadiometer attached to the scale, were esterified by acid methylation [21] and analyzed by
using standard procedure. Body mass index (BMI) was gas chromatography (GC) (Shimadzu, 17A model), using
calculated and classified according to the World Health SP2560 column (Supelco, Bellefonte, PA, USA). Fatty
Organization cutoff values [18]. acids were identified using external standards (Sigma®)
The bioelectrical impedance analysis was performed and the results were expressed as percentage of fatty
with BIA Quantum II instrument (RJL Systems®) accord- acid in relation to the total area of the fatty acids.
ing to the standardized procedure. The phase angle (PA)
was obtained from the arc tangent relationship of react- Statistical analysis
ance/ resistance × 180 / π [19]. Primary end points of this study (nutritional status/ body
Dietary intake was assessed by 24-h recall using the weight) have not been reported in breast cancer patients
method of multiple passes and nutrient composition was receiving n-3 fatty acids prior to treatment. The sample
calculated with NutWin (1.5.2.51 version) software. Nut- size calculation was performed on the basis of Bougnoux
Win uses the USDA food composition database for nu- et al. study [13], which assessed the effect of DHA in
trient calculation. breast cancer patients during chemotherapy (that study
Paixão et al. Nutrition Journal (2017) 16:71 Page 4 of 11

found objective response rate to treatment in 44% of pa- was set at p < 0.05. Analyses were performed using R free
tients). Assuming a hypothesis that no more than 5% of software.
placebo group would present a positive response (in im-
munological or nutritional parameter), we estimated that Results
a minimum sample of 16 subjects in each group would Study population
allow the detection of differences due to the effect of n-3 One hundred and eight patients were invited to partici-
use, with a 80% power and 5% significance level. pate in the study (Fig. 1). Of these, 77 (71%) accepted
Descriptive statistics were presented as percentages, the invitation, but 32 patients were excluded due to: loss
means and standard deviations or median (upper and of contact for the baseline visit (n = 6), surgery sched-
lower quartiles). Baseline results were analyzed using the uled to date shorter than 30 days (n = 16), change in
chi-square test for categorical variables and Mann Whitney clinical treatment (n = 3) and negative biopsy (n = 7).
test for continuous variables. To check for intra group dif- Thus, 45 patients were randomized. Of the randomized
ferences, the Wilcoxon test was used. Differences between patients, eight of them discontinued the study due to
groups were verified by a two-way repeated measures supplement intolerance (n = 2) and to change to neoad-
ANOVA for ordinal data with group (fish oil and placebo) juvant chemotherapy as primary treatment (n = 6).
as between subject factor and time as within subject factor Thirty seven patients completed the study, of whom 18
[22]. All tests were two-tailed and the significance level were supplemented with fish oil and 19 with placebo.

Fig. 1 CONSORT flow chart

Paixão et al. Nutrition Journal (2017) 16:71 Page 5 of 11

Baseline characteristics According to the BMI classification, the majority of

The socio-demographic and clinic-pathological charac- the patients had excess weight, 43% being classified as
teristics of patients at baseline are shown in Table 1. overweight and 30% as obese. No between group differ-
Most of the patients had infiltrating ductal carcinoma ences existed in the anthropometric parameters and
(62%), clinical staging 0/ I/ II (56%), estrogen recep- intake variables. The daily consumption of EPA and
tor + (ER+) (72%), progesterone receptor + (PR+) (59%) DHA was low in both groups, with medians of 0.005 g/
and negative for human epidermal growth factor recep- day of EPA and 0.020 g/ day of DHA, among FG pa-
tor 2 (HER2) (56%). There was no significant difference tients and 0.005 g/day and 0.025 g/day, respectively, in
between the FG and PG groups with respect to these the PG.
variables. The percentage of baseline plasma phospholipid EPA
were 0.4% and 0.3% in FG and PG, respectively; while
DHA were 2.5% and 3.1%, with no group differences.
The FG had significantly lower percentage of oleic acid
Table 1 Socio-demographic and clinicopathological
characteristics of patients randomized according to the study (p = 0.027) and a higher ratio of 18.0/18.1 (p = 0.022)
group when compared with PG. The percentage of other fatty
Groups p a acids was similar between the groups.
There was no significant difference between the
Fish oil Placebo
(n = 18) (n = 19) groups regarding the baseline percentage and ratio of
Age (years) b 48.6 ± 9.0 53.4 ± 7.5 0.107 PBMN CD4+ and CD8+ lymphocytes, serum levels of
b proinflammatory cytokines (TNF-α, IL-6 and IL-1β),
Education level (years) 7.2 ± 3.9 9.1 ± 4.0 0.227
PGE metabolites and hsCRP. With the exception of
Menopause % (n)
monocytes, blood count and serum biochemical parame-
No 50.0 (9) 26.3 (5) 0.138 ters were similar between FG and PG.
Yes 50.0 (9) 73.7 (14)
Histological type % (n) Tolerability and compliance
Ductal carcinoma in situ (DCIS) 22.2 (4) 10.5 (2) 0.756 Among the patients who completed the study, 55% and
47% of the FG and PG patients, respectively, reported
Infiltrating ductal carcinoma (IDC) 72.2 (13) 78.9 (15)
side effects such as dizziness, nausea, frequent belching,
IDC + DCIS 5.6 (1) 5.3 (1)
increased bowel frequency, heartburn and gastric full-
No information – 5.3 (1) ness. However, no between group differences was ob-
TNM classification % (n) served for the presence of symptoms (p = 0.616).
Tumor in situ 16.7 (3) – – Despite the reported side effects, 92% and 93% of the
I 16.7 (3) 5.3 (1) 0.212 prescribed capsules were consumed in the FG and PG,
respectively, which was considered as good supplement
II 38.9 (7) 47.4 (9)
compliance.
III 27.8 (5) 42.1 (8)
No information – 5.3 (1) Intervention effects
Estrogen receptor (ER) % (n) The effects of the intervention on nutritional status and
ER+ 77.8 (14) 73.7 (14) 1.000 dietary intake are shown in Table 2. At the end of the
ER- 16.7 (3) 15.8 (3) intervention period, the FG patients presented signifi-
cant gain of fat mass (p = 0.029), but no difference was
No information 5.6 (1) 10.8 (2)
observed between the groups regarding this and other
Progesterone receptor (PR) % (n)
anthropometric parameters analyzed. There was no intra
PR+ 66.7 (12) 57.9 (11) 1.000 group difference in the macronutrient intake, both in
PR- 27.8 (5) 31.6 (6) the PG and FG patients. However, there was a between
No information 5.6 (1) 10.5 (2) group difference in energy (p = 0.038) and protein
Human epidermal growth factor receptor 2 (HER2) % (n) (p = 0.010) ingestion being higher in PG. The FG group
intake of monounsaturated, palmitic, stearic and oleic
HER2+ 33.3 (6) 26.3 (5) 1.000
fatty acids reduced significantly, however, with no be-
HER2- 61.1 (11) 63.2 (12)
tween group differences. The dietary EPA, DHA and
No information 5.6 (1) 10.5 (2) total n-3 fatty acids showed no intra or between group
TNM Tumor, node, metastasis differences at the end of intervention period (Table 2).
a
Mann-Whitney test for age and education level and chi-square for the
other variables
Significant increase in plasma total n-3 fatty acids
b
Mean ± standard deviation (p = 0.004) and decrease in n-6: n-3 ratio (p = 0.002)
Paixão et al. Nutrition Journal (2017) 16:71 Page 6 of 11

Table 2 Nutritional status and dietary intake at baseline and at the end of the study
Fish oil group (n = 18) Pa Placebo group (n = 19) Pa Pb
Initial Final Initial Final
Median IQR Median IQR Median IQR Median IQR
Nutritional status
Weight (kg) 67.3 62.0–74.1 67.5 62.8–77.6 0.078 66.6 57.7–73.2 67.5 57.5–71.7 0.776 0.079
BMI (kg/m2) 27.0 23.7–32.0 27.1 23.6–32.5 0.078 26.6 24.9–30.2 26.3 24.8–29.6 0.723 0.101
Lean body mass (kg) 41.5 39.6–45.7 41.0 45.0–43.2 0.170 40.5 34.8–44.0 40.4 34.8–43.8 0.660 0.406
Fat mass (kg) 26.3 19.5–33.0 26.8 21.9–34.6 0.029 26.5 21.6–30.2 24.3 22.1–29.8 0.977 0.101
% Body fat 37.7 28.1–44.9 38.1 33.2–46.0 0.149 38.9 37.0–43.8 39.4 35.9–42.0 0.820 0.298
SPA −0.6 −1.2 - -0.2 −0.7 −1.1 - 0.1 0.513 −1.2 −1.6 - -0.6 −1.1 −1.63 - -0.7 0.394 0.492
Dietary intake
Energy (kcal) 1451 1052–1755 1226 1011–1629 0.124 1162 991–1500 1289 1186–1480 0.520 0.038
Kcal/kg 21 16–27 17 14–24 0.173 20 14–22 20 16–23 0.877 0.259
Carbohydrates (g) 172 128–260 155 118–235 0.148 147 133–186 171 118–226 0.557 0.200
Protein (g) 64 48–80 47 42–60 0.124 51 44–68 62 47–81 0.184 0.010
Lipids (g) 45 34–66 42 37–51 0.124 42 33–53 38 27–53 0.546 0.686
Fat acids (g)
Saturated 11.1 7.6–15.7 9.4 8.1–13.5 0.163 8.9 7.5–13.4 9.5 6.7–13.5 0.936 0.536
Monounsaturated 11.6 9.1–19.3 10.9 7.5–13.7 0.039 10.5 8.6–14.7 9.4 6.9–15.0 0.673 0.747
Polyunsaturated 9.4 7.4–11.5 8.1 7.2–10.6 0.163 9.0 7.1–10.6 8.0 6.5–10.0 0.376 0.752
16:0 6.3 4.6–9.7 5.3 3.9–7.0 0.013 5.3 4.5–7.6 5.2 3.9–7.5 0.809 0.449
18;0 2.6 1.9–4.6 2.4 1.5–3.4 0.019 2.1 1.8–3.3 2.2 1.6–3.8 1.000 0.489
18:1n-9 10.7 8.4–17.8 10.1 7.0–12.7 0.049 9.5 7.7–13.6 8.5 6.3–14.6 0.629 0.838
18:2 n-6 7.8 6.9–9.9 7.0 6.3–9.7 0.177 7.9 6.1–9.3 7.0 5.6–8.5 0.243 0.842
18:3 n-3 0.9 0.7–1.1 0.8 0.7–1.1 0.981 0.8 0.6–0.8 0.8 0.5–0.9 0.794 0.776
20:4n-6 0.10 0.47–0.17 0.07 0.54–0.11 0.163 0.09 0.05–0.12 0.09 0.06–0.19 0.162 0.165
20:5n-3 (EPA) 0.005 0.000–0.007 0.005 0.000–0.010 0.633 0.005 0.000–0.010 0.005 0.000–0.015 0.395 0.334
22:6n-3 (DHA) 0.020 0.002–0.052 0.020 0.005–0.032 0.162 0.025 0.010–0.030 0.015 0.000–0.065 0.139 0.295
Total n-3 1.0 0.7–1.3 0.8 0.8–11 0.850 0.8 0.6–1.1 0.8 0.6–1.0 0.831 0.711
Total n-6 8.3 7.1–10.2 7.2 6.4–9.7 0.201 8.0 6.2–9.4 7.0 5.2–8.4 0.163 0.850
n-6/n-3 ratio 8.3 5.8–9.7 7.8 7.4–8.8 0.723 8.3 7.3–10.1 7.2 6.7–10.5 0.381 0.175
18:0/18:1 ratio 0.2 0.2–0.2 0.2 0.2–0.2 0.554 0.2 0.2–0.2 0.2 0.2–0.2 0.469 0.387
IQR Interquartile range, BMI Body mass index, SPA Standardized phase angle, EPA Eicosapentaenoic acid, DHA Docosahexaenoic acid
a
Intragroup differences according to Wilcoxon test
b
Interaction test of a two-way repeated measures ANOVA for ordinal data to verify the significance of differences between fish oil and mineral oil groups

was seen in FG patients, with a significant between group difference (FG Δ% = −5.9 [−35.4–74.12], PG
group differences (p = 0.005 and p = 0.012, respectively) Δ% = 17.2 [−0.16–91.99] p = 0.059) (Fig. 2). No signifi-
(Table 3). cant changes in serum TNF-α, IL-1β, IL-6 cytokines
Regarding the acute phase immunological response, were observed.
no significant change was observed in the FG (initial We observed a significant reduction in the percentage
median 0.1 [IQR 0.1–0.5], final median 0.3 [IQR 0.0– of CD4+ T lymphocytes in the peripheral blood of PG
0.7], p = 0.510) while in PG patients there was a signifi- patients (initial median 57.2 [IQR 47.7–71.8], final me-
cant increase in hsCRP (initial median 0.1 [IQR 0.0–0.2], dian 52.7 [IQR 42.3–57.9], p = 0.042) and no change in
final median 0.2 [IQR 0.1–0.3], p = 0.024). While hsCRP the percentage of CD8+ cells. In the FG, no change in
remained stable in patients supplemented with n-3 fatty the percentages of CD4+ and CD8+ T cells and CD4+/
acids, the PG patients had a more pronounced increase CD8+ ratio occurred. No between group effects of treat-
in serum hsCRP levels, with a non-significant between ment (Δ%) were observed for these parameters (Fig. 3).
Paixão et al. Nutrition Journal (2017) 16:71 Page 7 of 11

Table 3 Blood fatty acids profile at baseline and at the end of the study in both groups
Fish oil group (n = 18) Pa Placebo group (n = 19) Pa Pb
Initial Final Initial Final
Median IQR Median IQR Median IQR Median IQR
Fatty acids
Saturated 58.7 50.4–63.5 58.4 50.6–64.4 0.863 52.2 49.1–60.7 55.2 48.9–59.1 0.601 0.857
Monounsaturated 10.1 9.0–11.3 9.8 8.5–10.9 0.130 9.8 9.2–12.2 10.6 8.5–11.9 0.809 0.326
Polyunsaturated 27.4 21.9–33.3 27.9 25.0–38.0 0.113 36.1 25.4–37.7 35.2 28.2–38.8 0.376 0.795
16:0 29.9 24.6–35.1 30.4 23.6–34.7 0.356 25.0 22.1–31.3 25.7 22.7–29.6 0.717 0.824
18:0 16.8 15.3–17.4 17.4 15.7–18.6 0.356 15.3 13.7–18.5 15.9 12.9–17.1 0.841 0.409
18:1n-9 4.3 3.5–5.2 4.4 3.7–5.8 0.943 5.2 4.5–6.5 5.1 4.4–6.5 0.629 0.525
20:4n-6 8.8 7.3–10.9 8.2 6.0–10.2 0.124 10.0 7.9–14.0 11.1 8.5–12.6 0.984 0.284
20:5n-3 (EPA) 0.4 0.1–0.8 1.5 0.9–2.1 0.004 0.3 0.0–0.8 0.5 0.0–1.2 0.293 0.034
22:6n-3 (DHA) 2.5 1.9–3.6 4.6 3.4–6.2 0.007 3.1 2.1–5.0 3.8 2.0–4.9 0.904 0.000
Total n-3 3.3 2.4–4.9 6.5 4.3–8.7 0.004 3.7 2.6–5.9 4.1 2.9–5.9 0.952 0.005
Total n-6 25.0 19.1–30.2 23.0 19.1–29.5 0.554 31.6 22.6–32.4 30.0 24.1–33.9 0.702 0.246
n-6:n-3 ratio 7.7 5.3–9.7 3.8 3.0–4.7 0.002 7.0 4.5–11.1 6.8 4.4–8.8 0.904 0.012
IQR Interquartile range, EPA Eicosapentaenoic acid, DHA Docosahexaenoic acid
a
Intragroup differences according to Wilcoxon test
b
Interaction test of a two-way repeated measures ANOVA for ordinal data to verify the significance of differences between fish oil and mineral oil groups

Serum PGE metabolite levels in both groups did not supplementation with n-3 fatty acids in newly diag-
change due to intervention. Serum glucose, total choles- nosed breast cancer patients, prior to treatment. In
terol and fractions, complete blood count and serum al- the study, the FG plasma EPA and DHA levels in-
bumin showed no within or between group differences creased significantly after 30 days of n-3 supplemen-
(Table 4). tation. In terms of immune parameters, whereas
hsCRP significantly increased and CD4+ reduced in
Discussion the placebo group, in the n-3 fatty acids suplemmen-
To our knowledge, this randomized controlled double ted patients serum hsCRP and CD4+ were kept at
blind trial is the first that investigated the effects of levels similar to baseline values.

Fig. 2 Changes in high sensitivity C-reactive protein (hsCRP) according to the study groups. a Fish oil group (FG), n = 15 (b) Placebo group (PG),
n = 16; p-value for the Wilcoxon test (c) Variation after treatment (Δ%) n = 15; p values for Mann Whitney test. Data are presented as medians,
upper and lower quartiles, maximum and minimum values. (•) Outlier values indicated in the chart were excluded from the statistical analyses
Paixão et al. Nutrition Journal (2017) 16:71 Page 8 of 11

Fig. 3 Variation after treatment (Δ%) of subpopulation of CD4+ T lymphocytes, CD8+ and CD4+/CD8+ ratio, according to the study groups. Fish oil
group (FG); Placebo group (PG); (a) CD4+ T lymphocytes, PG n = 12, FG n = 15; p-value for the Wilcoxon test (b) CD8+ T Lymphocytes, PG n = 14, FG
n = 13 (c) CD4+/CD8+ ratio, PG n = 12 and FG n = 15. Data are presented as medians, upper and lower quartiles, maximum and minimum values

Table 4 Biochemical parameters at baseline and at the end of the study in both groups
Fish oil group (n = 18) Pa Placebo group (n = 19) Pa Pb
Initial Final Initial Final
Median IQR Median IQR Median IQR Median IQR
Biochemical
RBC (×106/mm3) 4.7 4.5–5.1 4.7 4.5–4.9 0.254 4.7 4.4–5.2 4.7 4.4–4.9 0.493 0.941
Hemoglobin (g/dL) 14.0 12.8–15.2 13.7 13.0–14.70 0.069 14.0 13.1–14.9 13.8 13.0–14.4 0.623 0.426
Hematocrit (%) 41.7 38.3–45.7 41.1 39.1–43.6 0.139 42.2 38.6–44.0 41.6 39.0–43.6 0.877 0.785
Leucocytes (mm3) 6405 5342–7950 6910 5105–7750 0.795 5220 4150–6700 5780 5265–6590 0.653 0.521
Platelets (×103/mm3) 281.0 215.2–307.7 259.0 220.0–291.0 0.523 246.0 193.0–282.0 241.0 212.0–278.5 0.492 0.456
Albumin (g/dL) 4.4 4.1–4.5 4.2 4.1–4.4 0.153 4.3 4.2–4.5 4.3 4.0–4.4 0.319 0.818
Fasting Glucose (ml/dL) 95.0 87.0–103.2 91.0 83.2–101.5 0.351 90.0 84.0–98.0 92.5 84.7–98.2 0.410 0.126
Total cholesterol (mg/dL) 218.0 194.5–258.5 217.0 194.5–254.5 0.758 211.0 196.0–247.0 208.0 183.5–241.5 0.185 0.414
HDL (mg/dL) 45.0 39.5–50.2 44.0 38.0–48.5 0.476 46.0 41.0–50.0 47.0 40.5–54.7 0.905 0.689
LDL (mg/dL) 141.0 118.7–176.7 146.0 122.0–176.0 0.518 145.0 121.0–169.0 140.5 112.0–157.2 0.138 0.182
Triglycerides (mg/dL) 160.0 90.7–199.5 146.0 98.0 - 191.5 0.421 125.0 75.0–165.0 105.0 80.0–146.7 0.679 0.887
IQR Interquartile range, RBC Reed blood cells, HDL High-density lipoprotein, LDL Low-density lipoprotein
a
Intragroup differences according to Wilcoxon test
b
Interaction test of a two-way repeated measures ANOVA for ordinal data to verify the significance of differences between fish oil and mineral oil groups
Paixão et al. Nutrition Journal (2017) 16:71 Page 9 of 11

CRP is an acute-phase serum protein of the pentraxin of TCD8+ lymphocyte did not change, the possibility
family produced mainly by hepatocytes and is regulated that the lower number of TCD4+ lymphocytes might
at the transcriptional level by IL-6. Its plasma concentra- have impaired proliferative capacity of the TCD8+ cells
tion increases during inflammatory state [23]. In our cannot be ruled out, because helper function of TCD4+
study, the placebo group showed an increase in CRP lymphocytes is required to full activation of TCD8+ cells
levels, suggestive of an inflammatory response to the [28]. As the number of TCD4+ and TCD8+ lymphocytes
tumor, while, in n-3 fatty acids treated-breast cancer pa- and its ratio remained stable in the fish oil treated
tients, the CRP showed a more regulated response. We group, taken together, the results of our study could sug-
speculate that n-3 fatty acid suplementation might have gest a positive effect of fish oil supplement in the adap-
modulated the inflammatory response to tumor, which tive immunity. Surgery is the mainstay of treatment of
in turn could collaborate to a better evolution of the pa- these patients and this procedure induces substantial
tient during subsequent treatment period. The absence immunomodulation, with pro-inflammatory response
of similar increase in IL-6 in our study may relate to the and leukocytosis [29]. Thus, a balanced adaptive im-
differences in the kinetic of their production, in which mune response may help prevent postsurgery immuno-
IL-6 serum levels had already decreased while CRP was supression and risks such as tumor dissemination into
still increasing, when tested in the study [23]. the circulation [30].
These results are consistent with the idea of EPA and No significant changes were observed in serum proin-
DHA acting in the modulation of CRP dependent in- flammatory cytokines due to the intervention. Similar
flammatory responses. Similar results have been ob- results in patients with different types of cancer and an-
served in patients with advanced cancer [17, 24]. These tineoplastic treatments were reported [14, 31]. Faber
results are relevant, given that high levels of CRP have et al. [14] supplemented radiotherapy cancer patients
been previously associated with a worse prognosis in with 3.6 g of n-3 fatty acids for 7 days and changes in
breast cancer patients [10] and with the fact that the re- the serum proinflamatory cytokines were undetectable
sults could potentially be attributed to n-3 fatty acids to some and not significant to IL-6 and IL-8. Moreover,
supplementation. Our results are also consistent with unlike the results of the present study, they observed a
the potential preventive effect of n-3 fatty acids in breast reduction in serum PGE2 levels. Gomez-Candela et al.
cancer [12]. [31] did not observe reduction of proinflammatory cyto-
According to Calder [25], dietary n-3 fatty acids kines, but a tendency of increased serum IL-6 after sup-
should be incorporated into leukocyte membrane in plementation with EPA and DHA. Nevertheless, it
order to be an effective immunomodulator. In breast should be considered that cytokines are mainly produced
cancer patients, after oral supplementation with 3 g of at local levels, so that one can not exclude the possibility
polyunsaturated fatty acids n-3 (EPA and DHA) there that there were modifications in their local levels but
was a threefold increase in circulating total n-3 acids that they were not sufficient to modify the systemic
[26]. In the present study, plasma phospholipd fatty serum levels. We were unnable to find previous studies
acids were used as surrogate markers of compliance to reporting the effects of n-3 supplementation on circulat-
the n-3 intervention and after 30 days, the median in- ing cytokines of breast cancer patients.
crease was significant but inferior to those reported by Despite the plausibility of antineoplastic effect of n-3
Bagga et al. [26]. These differences in incorporation may fatty acids according to cell culture and animal studies,
relate to the amount of n-3 fatty acids supplemented in reports of clinical trials are scarce [32] and the results
our study (1.8 g/ day) that could have been insufficient are inconsistent, one of the reasons being the high vari-
for a higher incorporation. Of note, recent study has in- ability in the study design. To our knowledge, in the few
dicated that different lipid structures used for EPA and studies with breast cancer patients, fish oil was studied
DHA supplementation have similar rates of incorpor- only as adjuvant to chemotherapy [13, 15, 33]. In our
ation into the blood [27]. study, the lack of significant findings in relation to pro-
Low peripheral blood CD4+ counts [5, 6] have been inflammatory cytokines and PGE2 may be in part due to
observed even in the early stages of breast cancer pa- the amount of supplement used or the length of the
tients. Whereas the number of circulating T CD4+ lym- intervention, that could have been insufficient to be ef-
phocytes decreased in the placebo group, which is in fective. Our intervention have used n-3 dose similar to
line with the suppressor substances produced by tumor that used by Bougnoux et al. [13], who reported good
cells as its immune escape mechanisms, the mainten- tolerance and no side effects. However, according to
ance of the number of T CD4+ lymphocytes in the n-3 Mocelim et al. [34], when supplementation is carried out
fatty acid treated group may have been due to the prolif- during a short period, higher doses of n-3 fatty acids are
erative effect of fatty acids on lymphocyte functions [2]. required to have an antiinflammatory effect. Also, the
In patients of the placebo group, although the number use of α-tocopherol as antioxidants in fish oil capsules
Paixão et al. Nutrition Journal (2017) 16:71 Page 10 of 11

may have reduced the effect of n-3 fatty acids, as dem- Ethics approval and consent to participate
onstrated in experimental studies [35]. Other limitation The study protocol was approved by the Human Research Ethics
Committees of the University of Brasilia (Protocol n° 72/09) and the Federal
of the study pertains to the discrepancy between the District Health Secretariat (Protocol n° 383/2011). Informed consent was
number of invited patients (n = 108) and the patients ex- obtained from each participant before the commencement of the study.
amined (n = 37) which affected the study power. Carry-
Consent for publication
ing out the study with patients immediately after the Not applicable.
diagnosis of such severe disease was challenging for both
the research group and patients, and contributed to high Competing interests
The authors declare that they have no competing interests.
refusal and drop out rates. A positive feature of the
study was the good compliance to fish oil supplement
Publisher’s Note
(92%), similar to the study by Taylor et al. [24]. As well, Springer Nature remains neutral with regard to jurisdictional claims in
the use of mineral oil as placebo had the merit of avoid- published maps and institutional affiliations.
ing the confounding effect of n-6 fatty acids in the con-
Author details
trol group. As study participants were treatment naïve, 1
Post Graduate Program in Human Nutrition, University of Brasília, Brasilia,
the results may better reflect the patient’s metabolic re- Federal District 70910-900, Brazil. 2Laboratory of Cellular Immunology,
sponse to the effect of n-3 fatty acids. Department of Pathology, Faculty of Medicine, University of Brasilia, Brasilia,
Federal District 70910-900, Brazil. 3Laboratory of Immunology and
Inflammation, Department of Cell Biology, Institute of Biology, University of
Brasilia, Brasilia, Federal District 70910-900, Brazil. 4Department of Statistics,
Conclusions University of Brasilia, Brasilia, Federal District 70910-900, Brazil.
In conclusion, the supplementation of newly diagnosed
breast cancer patients with 1.8 g of EPA and DHA for Received: 2 June 2017 Accepted: 10 October 2017
30 days led to a significant change in the composition of
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