Original Paper

Ann Nutr Metab 2016;69:15–23 Received: March 11, 2016

Accepted after revision: June 6, 2016
DOI: 10.1159/000447498
Published online: July 9, 2016

Beneficial Effects of Enteral Docosahexaenoic

Acid on the Markers of Inflammation and
Clinical Outcomes of Neonates Undergoing
Cardiovascular Surgery: An Intervention Study
Mariela Bernabe-Garcia a Mardia Lopez-Alarcon a Raul Villegas-Silva b, e
Javier Mancilla-Ramirez c Maricela Rodriguez-Cruz a
Jorge Maldonado-Hernandez a Karina A. Chavez-Rueda d
Francisco Blanco-Favela d Lilia Espinoza-Garcia b Sandra Lagunes-Salazar b
a
Medical Research Unit in Nutrition, b Neonatal Intensive Care Unit, and c Escuela Superior de Medicina, IPN,
Hospital de la Mujer, d Medical Research Unit in Immunology, Hospital de Pediatria, Centro Medico Nacional Siglo
XXI, Instituto Mexicano del Seguro Social, and e Neonatal Intensive Care Unit, Hospital Infantil de Mexico Federico
Gomez, Paseo de la Salud, Col. Doctores, SSA, Mexico City, Mexico

Key Words vascular surgery. Inflammation was evaluated by percentage

Docosahexaenoic acid · Clinical outcomes · Neonate ·
Inflammatory response · Sepsis · Organ dysfunction ·
Neonatal intensive care unit stay · Cytokines
Abstract
Background: Neonates undergoing surgery are at risk for
uncontrolled inflammatory response and adverse clinical
outcomes. Docosahexaenoic acid (DHA) ameliorates inflam-
mation, improving clinical outcomes. However, its effect has
not been evaluated in neonates undergoing surgery. We
evaluated the effect of DHA on markers of inflammation and
clinical outcomes in neonates undergoing surgery. Meth-
ods: A double-blind clinical trial evaluated the effect of en-
teral DHA (DHA group) versus sunflower oil (SO group) peri-
operatively administered in neonates scheduled for cardio-
of cells+ for cytokines and CD69 in mononuclear cells at
baseline, 24 h and 7 days post surgery. Clinical outcomes
measured were sepsis, organ dysfunctions (ODs), length of
stay in intensive care and bleeding. Repeated measures anal-
ysis of variance and logistic regression were applied. Results:
Sixteen neonates received DHA and 18 received SO. Cells+
from neonates in the DHA group showed an early increase
in receptor antagonist of interleukin (IL)-1+ (IL-1ra+) and IL-
10+ and a late decrease in IL-6+. IL-1β+ and IL-10+ changes
were different between groups. After adjusting for con-
founders, less cells from DHA group were IL-1β+, IL-6+, IL-
1ra+ and IL-10+. DHA group presented less sepsis, ODs and
shorter stay, but no difference in CD69+CD4+ cells or bleed-
ing between groups. Conclusions: Administration of enteral
DHA ameliorates markers of inflammation and improves
clinical outcomes in surgical neonates. © 2016 S. Karger AG, Basel
172.16.7.111 - 7/15/2016 2:23:04 PM
Verlag S. KARGER AG, BASEL

© 2016 S. Karger AG, Basel Mariela Bernabe-Garcia, PhD

0250–6807/16/0691–0015$39.50/0 Medical Research Unit in Nutrition
Av. Cuahutemoc 330, Col. Doctores, Del. Cuauhtemoc
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E-Mail karger@karger.com
DF, Mexico City 06725 (Mexico)
www.karger.com/anm
E-Mail marielabernabe1 @ gmail.com
 Introduction Table 1. Fatty acid composition of interventions

Sepsis and organ dysfunctions (ODs) remain among %/total weight Interventions
the leading causes of death in critically ill patients, espe- DHA SO
cially in developing countries where infections cause ∼1.6
C 10:0 capric 1.0 0.0
million deaths annually in neonates alone [1]. C 12:0 lauric 4.3 0.0
Neonates are highly susceptible to the harmful effects C 14:0 myristic 14.0 0.1
of pro-inflammatory cytokines such as tumor necrosis C 16:0 palmitic 12.2 5.2
factor (TNF)-α, interleukin (IL)-1β and IL-6, due likely C 16:1 palmitoleic 1.9 0.1
to a diminished production of anti-inflammatory cyto- C 18:0 stearic 0.7 3.8
kines IL-10 and receptor antagonist of IL-1 (IL-1ra), C 18:1 n-9 oleic 18.9 58.9
C 18:2 n-6 linoleic 1.3 29.2
among others [2, 3]. The excessive inflammatory re-
C 18:3 n-6 γ-linolenic 0.4 0.3
sponse known as systemic inflammatory response syn- C 18:3 n-3 α-linolenic <0.1 1.2
drome (SIRS), together with cell-mediated paralysis that C 20:1 eicosenoic <0.1 0.3
follows major surgery, appears to be responsible for the C 20:4 n-6 arachidonic <1.0 0.0
increased susceptibility for the development of sepsis C 20:5 n-3 eicosapentaenoic <0.1 0.0
and ODs [4, 5]. Interestingly, SIRS involves excessive C 22:0 behenic 0.2 0.7
production of both pro- and anti-inflammatory cyto- C 22:6 n-3 docosahexaenoic 44.3 0.0
C 24:1 nervonic 0.1 0.0
kines, but their interaction between SIRS and a compen-
satory anti-inflammatory response syndrome may re-
sult either in restoration of homeostasis or ODs [6], re-
sulting in an elevated mortality risk between 40 and 80%
Materials and Methods
[7].
The altered inflammatory response may also be detect- Study Design and Patients
ed by expression of CD69, a marker of early lymphocyte A randomized double-blind parallel group trial was conducted
activation that exhibits an early rise after surgical stress. at the NICU of a pediatric hospital affiliated with the Mexican In-
Such increased expression has been associated with rejec- stitute of Social Security (IMSS) in Mexico City from March 2007
to June 2011. This study was approved by the Ethics Committee of
tion of cardiac allograft in children [8], but its relation- the IMSS (No. 2006-785-069). Trial registration: www.clinicaltrials.
ship with sepsis and ODs after surgery in neonates has not gov with ID number NCT01049529. Written informed consent
been studied. was obtained from both parents before study inclusion. Eligibility
Administration of n-3 long-chain polyunsaturated criteria were newborns younger than a month after birth with car-
fatty acids (LC-PUFAs), eicosapentaenoic acid (EPA) diovascular malformations, scheduled for Blalock–Taussig shunt
or aortoplasty as surgical treatment, >34 weeks of corrected gesta-
and docosahexaenoic acid (DHA) to adults who under- tional age, adequate weight for gestational age, and tolerating at
go surgical trauma has resulted in decreased inflamma- least minimum enteral feeding. Those patients scheduled for car-
tory cytokines [7], reduced risk of developing infections diopulmonary bypass or with data of SIRS were excluded [10].
and shorter stays in intensive care unit (ICU) [9]. Such
possible beneficial effects have been scarcely investigat- Intervention
Neonates were randomly assigned to receive 75 mg/kg of base-
ed in neonates who present a high susceptibility and risk line weight/day of DHA (DHA group) in sunflower oil (SO) as
of death; therefore, it is relevant to evaluate new strate- excipient (Neuromins for Kids, Martek Life Enriched Inc., Parsip-
gies to improve their clinical outcomes. In addition, pany, N.J., USA) or SO group, with similar volume, appearance
there are concerns about the risk of bleeding events at- and consistency between interventions. A table of random num-
tributable to the use of n-3 LC-PUFAs in critically ill bers was used to allocate the treatments: ‘A’ letter for DHA or ‘B’
for SO, stored in opaque envelopes sequentially numbered, and
neonates. opened once written informed consent was obtained. The daily
In this study, we evaluated the effect of enteral DHA dose was administered in 2 fractions through an enteral feeding
administration to neonates scheduled for cardiovascular tube with a sterile syringe before human milk or formula feeding.
surgery on the percentage of positive cells to intracellular DHA or SO administration was initiated 2 days before surgery,
pro- and anti-inflammatory cytokines, to CD69 in CD4- suspended 4 h pre-surgery, restarted and continued for 6 days after
surgery. Abdominal and clinical conditions were previously evalu-
lymphocytes, development of sepsis, ODs and length of ated by a neonatologist. All doses were prepared using aseptic tech-
stay in the neonatal ICU (NICU), as well as bleeding nique by a nurse who did not participate in patient care. Fatty acid
events. composition of the interventions is presented in table 1.
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16 Ann Nutr Metab 2016;69:15–23 Bernabe-Garcia et al.

DOI: 10.1159/000447498
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 Plasma Collection and Fatty Acid Profile
At baseline, venous blood was collected into EDTA tubes for
determination of DHA, EPA, α-linolenic acid, linoleic acid, arachi-
donic acid (AA) and oleic acid in erythrocyte membranes. Blood
was centrifuged at 2,000 g for 15 min at 4 ° C to separate erythro-
cytes. The pellet was washed at least 3 times, suspended with 0.9%
of saline solution, and frozen at –70 ° C until analysis by gas chro-
matography [11]. Results of fatty acids were expressed as weight
percentages (%/total weight). Whenever possible, a blood sample
was obtained when ordered by a neonatologist for determination
of another blood parameter to avoid multiple punctures.
Markers of Inflammation
To determine markers of inflammation, the positive cells for
intracellular cytokines and CD69 were measured in blood collect-
ed into heparin tubes by the method followed in other studies and
in our group of research [3, 12–14]. Briefly, one milliliter of fresh
whole blood was diluted 1:1 in RPMI-1640 (Invitrogen, Carlsbad,
Calif., USA) and distributed in 24-well plates (Nunc, Denmark).
Brefeldin A, a protein transport disrupter (BD-Biosciences, San
Jose, Calif., USA), was added to block cytokine secretion. Part of
the sample was stimulated with phorbol 12-myristate 13-acetate
plus ionomycin (Sigma-Aldrich, St. Louis, Mo., USA). Plates were
incubated at 37 ° C with 5% CO2 for 5 h, then, fixation and per-
meabilization solutions (DAKOCytomation, Glostrup, Denmark)
were added to cells alternating incubations and washings with PBS.
The intracellular cytokine staining was performed with anti-hu-
man monoclonal antibodies TNF-α-FITC, IL-1β-FITC, IL-6-PE,
IL-1ra-PE and IL-10-APC (BD Pharmingen, San Jose, Calif.,
USA). Cells were incubated for 15 min in the dark at 25 ° C after
staining. Autofluorescence was adjusted with unstimulated and
unstained samples, while nonspecific fluorescence was adjusted by
isotype-matched antibodies (BD Biosciences), both at every time
of the study.
Samples were run using a FACS Aria flow cytometer (BD Bio-
sciences) and analyzed using an electronic gate for monocytes plus
lymphocytes with FlowJo software (Tree Star, Ashland, Oreg.,
USA). This gate represents the mononuclear cell producers of in-
tracellular cytokines, which were measured as percentage of posi-
tive cells (cells+), determined before surgery (baseline), at 24 h and
at 7 days post surgery. Results are presented as changes be-
tween these 3 study times between groups. CD69 was PE-labeled
in CD4-APC-labeled into non-incubated, unstimulated blood (BD
Pharmingen, San Jose, Calif., USA) following the same study
times.
Clinical Outcomes
Clinical data included the development of sepsis and ODs eval-
uated by an expert neonatologist according to an International Pe-
diatric Consensus [10], blinded to the intervention. Our primary
outcomes were severe sepsis, respiratory and cardiovascular ODs.
In addition, as secondary outcomes, we also evaluated hemato-
logic, hepatic and renal ODs, as well as the length of stay in NICU.
Briefly, sepsis was defined as data of SIRS related to suspected or
proven infection, whereas severe sepsis was sepsis plus cardiovas-
cular or respiratory dysfunction or ≥2 other ODs. Length of stay
in NICU and follow-up of the study were considered from the time
of surgery until NICU discharge.
Severity of illness was evaluated with the Score for Neonatal
Acute Physiology at baseline and 24 h post surgery [15]. Duration
DHA on Inflammation and Clinical
Outcomes in Neonates
of cardiovascular surgery, aortic clamp, concurrent medications,
total parenteral nutrition (TPN), human milk and formula intake
were prospectively registered during follow-up.
Adverse Events
Bleeding volume during surgery, bleeding and vomiting events
or death throughout NICU stay were registered.
Statistical Analyses
Group comparisons were performed using Fisher’s exact test,
Student t test or Mann–Whitney U test when data were not nor-
mally distributed after log transformation. To adjust for con-
founding variables, repeated measures analysis of covariance
(ANCOVA) with Dunnett’s post test, and logistic regression were
performed as appropriate. Events of sepsis, severe sepsis, patients
who developed OD, vomit and mortality events were analyzed be-
tween groups by intention to treat (ITT). Minitab software (Minit-
ab 14.2, State College, Pa., USA) was used for statistical analysis;
p ≤ 0.05 was considered statistically significant.
The sample size was computed with circulating IL-6 in surgical
adults who received fish oil by TPN [16], because there were no
data in neonates when this study was planned. The estimation con-
sidered an effect size of 30% in IL-6 reduction for the DHA group,
with 80% of sample power and 5% of significance level, resulting
in 18 patients per group. However, a preliminary analysis after ad-
justment for confounders showed anticipated statistical signifi-
cance, and the study was finalized.
Results
Demographic and Baseline Characteristics
Fifty-five neonates were recruited. The demographic
and baseline characteristics of dropouts were comparable
to the 34 patients who completed the follow-up
(CONSORT flow diagram; fig. 1). Of these, 16 neonates
received DHA and 18 received SO. Neonates in DHA
group and SO group presented unique hypoplastic ven-
tricle (0 vs. 1), pulmonary atresia or stenosis without
within-ventricle communication (6 vs. 11) and aortic co-
arctation (10 vs. 6; p > 0.05), respectively. No differences
were observed in characteristics at baseline or fatty acid
content in erythrocyte membranes between groups
(table 2).
Effect of Enteral DHA Supplementation on Markers of
Inflammation
Baseline percentage of positive cells to intracellular cy-
tokines was similar between groups (data not shown).
Changes in positive cells between baseline and 24 h post
surgery showed a significant increase of IL-1β+ cells fol-
lowed by a decrease between 24 h and day 7 post surgery
in SO group, but no significant changes in positive cells
of DHA group, leading to a borderline difference between
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Ann Nutr Metab 2016;69:15–23 17
DOI: 10.1159/000447498
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Enrollment Assessed for eligibility at NICU
Allocated to control intervention (n = 28)
– Received allocated intervention (n = 28)
– Did not receive allocated intervention (n = 0)
Lost to follow-up (n = 10) due to:
– Discontinued intervention by attendant (n = 0)
– Did not underwent a surgery (n = 2)
– Surgery required cardiopulmonary bypass
(n = 4)
– Deceased before surgery (n = 1) or during
follow-up (n = 3)
Analyzed PP (n = 18)
Excluded from analysis (n = 10)
ITT analyzed (n = 28)
Fig. 1. Flow diagram of CONSORT for the study. Control group received SO. DHA group received enteral DHA.
PP = Per protocol; ITT = intention to treat.
groups. Percentage of TNF-α+ cells significantly increased
in SO group between 24 h and day 7 post surgery. Mean-
while, the percentage of positive cells from DHA group
did not change significantly. IL-6+ cells decreased in DHA
group from 24 h to day 7 post surgery with no significant
changes in positive cells from SO group. There were no
significant differences in changes of TNF-α+ cells and IL-
6+ cells between groups (fig. 2).
Regarding anti-inflammatory cytokines, IL-10+ and
IL-1ra+ cells tended to increase earlier and greater in DHA
group than in SO group, between baseline to 24 h post
surgery, but only IL-1ra+ cells reached statistical signifi-
cance; IL-10+ cells decreased in DHA group between 24 h
and day 7 post surgery in opposition to SO group (fig. 3).
After adjusting by sepsis, anti-inflammatory medication,
n-3 content in erythrocytes and human milk intake, the
mean percentage of IL-1β+, IL-6+, IL-1ra+ and IL-10+ cells
was lower in DHA group compared with SO group during
follow-up (repeated measures ANCOVA p = 0.0005, p =
18 Ann Nutr Metab 2016;69:15–23
DOI: 10.1159/000447498
Color version available online
(n = 83)
Excluded (n = 28)
– Not meeting inclusion criteria (n = 26)
– Declined to participate (n = 2)
Randomized (n = 55)
Allocation
Allocated to DHA intervention (n = 27)
– Received allocated intervention (n = 27)
– Did not receive allocated intervention (n = 0)
Follow-up
Lost to follow-up (n = 11) due to:
– Discontinued intervention by hemodynamic
instability (n = 2)
– Did not underwent a surgery (n = 3)
– Surgery required cardiopulmonary bypass
(n = 4)
– Deceased during follow-up (n = 1)
– Aortic coarctation after aortoplasty (n = 1)
Analysis
Analyzed PP (n = 16)
Excluded from analysis (n = 11)
ITT analyzed (n = 27)
0.0016, p = 0.013 and p = 0.025), respectively, except for
TNF-α (p = 0.78). There was no interaction between treat-
ment and time in ANCOVA models (p > 0.10). Finally,
CD69+CD4+ cells were not different between DHA and
SO group at baseline, 24 h and 7 days post surgery (1.50 ±
2.25 vs. 1.88 ± 2.86%; 2.19 ± 2.08 vs. 3.0 ± 2.45%, and
1.74 ± 3.66 vs. 1.97 ± 3.53%, respectively; all p > 0.05).
Effect of Enteral DHA Supplementation on Clinical
Outcomes
The DHA group tended to present fewer events of sep-
sis and severe sepsis than SO group (table 3). The raw rel-
ative risk (RR) to develop sepsis in DHA group did not
reach statistical significance (RR 0.62, 95% CI 0.366–1.04;
p = 0.065). However, frequency of ODs and patients who
develop ≥1 ODs were lower in DHA group (table 3). Raw
and adjusted RR for patients who develop ≥1 ODs in DHA
group were also lower than SO group after adjusting by
human milk intake, anti-inflammatory medication and
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Bernabe-Garcia et al.
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 Color version available online
1.0 0.4 0.5 SO
a
0.8 0.3 0.4 DHA
0.6 0.3

Changes of TNF-į, log

Changes of IL-1DŽ, log

0.2

Changes of IL-6, log

a p = 0.05
0.4 0.2
0.2 0.1 0.1
0 0 0
–0.2 –0.1 –0.1
–0.4 –0.2
–0.2
–0.6 –0.3
–0.8 –0.3 –0.4
a
–1.0 –0.4 –0.5
24 h – baseline 7 days – 24 h 24 h – baseline 7 days – 24 h 24 h – baseline 7 days – 24 h

Fig. 2. Changes of positive cells for intracellular pro-inflammatory cytokines during follow-up in neonates un-
dergoing cardiovascular surgery. Bars represent the mean ± SEM of the positive cells (‘y’ axe in logarithm 10)
between baseline and 24 h, and between 24 h and 7 days after surgery. a Within-group difference, p < 0.05.

Table 2. Demographic and baseline characteristics of neonates un- baseline severity of illness (RR 0.41, 95% CI 0.229–0.75;
dergoing cardiovascular surgery p = 0.006 and RR 0.050, 95% CI 0.003–0.776; p < 0.001),
respectively. In addition, complications (sepsis plus ODs)
Group p value
were positively correlated with CD69+CD4+ cell expres-
DHA SO sion (rho = 0.303, p = 0.05). Length of stay in NICU was
(n = 16) (n = 18)
shorter in DHA group compared with SO group (table 3).
At birth After an ITT approach, the main clinical outcomes re-
Gestational age, weeks 38.6±1.1 38.1±2.0 0.398 vealed similar results compared with per protocol (PP)
Gender, male:female, n 10/6 8/10 0.327 analysis. The events of the development of sepsis did not
Apgar score, n reach the statistical significance between DHA and SO
Minute 5, <8:≥8 2:16 4:16 0.660
group (14/27 vs. 19/28, p = 0.175), as well as the raw RR
At baseline for DHA group (RR 0.67, 95% CI 0.343–1.299; p = 0.228).
Postnatal age, days 18.8±13.7 21.3±10.1 0.567
Weight, g 2,862±600 2,987±509 0.499
The events of severe sepsis were also not different be-
Recumbent length, cm 50.0±2.6 49.9±2.0 0.929 tween DHA and SO group (12/27 vs. 14/28, p = 0.444,
Head circumference, cm 33.8±2.0 34.0±1.4 0.824 respectively). The number of patients who developed ≥1
Surgery, n (%) ODs remained significantly lower in the DHA group
Blalock–Taussig shunt 7 (47) 12 (67) compared with SO group (12/27 vs. 21/28, p = 0.020, re-
Aortoplasty 9 (53) 6 (33) 0.300
Severity of illness (SNAP-II) 5.0 (0–18) 5.0 (0–35) 0.237 spectively). Likewise, the raw RR for the development of
Erythrocyte fatty acids, %/total weight ODs remained significantly lower in DHA group com-
Oleic acid 20.7±0.9 20.9±1.5 0.880 pared with SO group (RR 0.45, 95% CI 0.218–0.837; p =
LA 8.9±1.2 8.8±1.2 0.954 0.023).
LNA 0.67±1.8 0.51±1.9 0.274
EPA 0.56±2.0 0.38±1.7 0.117 Regarding confounders, there were no differences be-
DHA 3.3±1.9 3.4±1.9 0.870 tween groups in illness severity at baseline (table 2) or at
Total n-3 24 h post surgery (p = 0.482), in duration of surgery and
(LNA + EPA + DHA) 5.0±1.5 4.7±1.5 0.649 aortic clamp, use and duration of anti-inflammatory
AA 10.2±1.7 12.0±1.7 0.264
Total n-6 (LA + AA) 20.0±1.3 21.5±1.3 0.492
medication or TPN duration (p > 0.05). A trend to higher
cumulative dose of aspirin in the SO than DHA group was
Data are mean ± SD or median (minimum–maximum) unless observed (105.1 ± 28.0 vs. 34.5 ± 13.0 mg/kg, p = 0.065).
otherwise indicated. All neonates received antibiotics. The proportion of neo-
LA = Linoleic acid; LNA = α-linolenic acid; EPA = eicosapen- nates who received any human milk during follow-up in
taenoic acid; DHA = docosahexaenoic acid; AA = arachidonic
acid. DHA group versus SO group was 15/16 versus 17/18, but
only 4/16 neonates versus 1/18 neonates (p = 0.161), re-
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DHA on Inflammation and Clinical Ann Nutr Metab 2016;69:15–23 19

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 Color version available online
0.5 SO 0.5
0.4 a DHA 0.4 p = 0.04
0.3 0.3

Changes of IL-1ra, log

Changes of IL-10, log

0.2 0.2
0.1 0.1
Fig. 3. Changes of positive cells for intracel- 0 0
lular anti-inflammatory cytokines during –0.1 –0.1
follow-up in neonates undergoing cardio- –0.2 –0.2
vascular surgery. Bars represent the mean ± –0.3 –0.3
SEM of the positive cells (‘y’ axe in loga- –0.4 –0.4
rithm 10) between baseline and 24 h, and –0.5 –0.5
between 24 h and 7 days after surgery. 24 h – baseline 7 days – 24 h 24 h – baseline 7 days – 24 h
a Within-group difference, p < 0.05.

Table 3. Clinical outcomes in neonates undergoing cardiovascular 1.000), respectively. The incidence of patients who died
surgery was also not different by PP (0 vs. 5.6%, p = 1.000) or by
Group p value ITT (3.7 vs. 17.9%, p = 0.193), respectively. No death was
related to DHA administration.
DHA (n = 16) SO (n = 18)

Sepsis, n 3a 9b 0.060
Severe sepsis, n 1 4 0.207 Discussion
Total ODs, n 2 17 <0.001
Respiratory, n 1 6 0.061
Duration, h 8.0 44.0 (7–93) 0.317 This study strongly suggests that perioperative enteral
Cardiovascular, n 1 4 0.207 DHA administration decreased production of intracellu-
Duration, h 11.0 43.5 (22–93) 0.157
Hematologic, n 0 3 0.136 lar inflammatory cytokines IL-1β, IL-6 and anti-inflam-
Duration, days – 5 (4–9) – matory cytokines IL-1ra and IL-10 from mononuclear
Hepatic, n 0 1 0.529 cells during follow-up and improved clinical outcomes in
Duration, days – 3.0 –
Renal, n 0 3 0.136 neonates who underwent a cardiovascular surgery.
Duration, days – 1 (1–4) – Perioperative administration of DHA and EPA with
Patients with at least one OD, n 1 11 0.001
Length of stay in the NICU, days 7.2±2.0 11.5±2.2 0.041
parenteral lipid emulsions has demonstrated beneficial ef-
fects on clinical outcomes in surgical adults [16, 17] and in
Data are frequency, mean ± SD or median (minimum–maximum). septic neonates who received DHA during an episode of
OD = Organ dysfunction; NICU = neonatal intensive care unit.
a
Microorganisms isolated in neonates from DHA group were Staphylo-
sepsis [11]. However, neonates did not receive TPN if they
coccus epidermidis, Pseudomonas aeruginosa and Staphylococcus hominis. had a functional gastrointestinal tract; thus, enteral ad-
b
Microorganisms isolated in neonates from SO group were Klebsiella ministration of DHA may be an affordable and physiolog-
pneumoniae, Enterobacter cloacae, Staphylococcus hominis, Pseudomonas
aeruginosa, Staphylococcus epidermidis and Acinetobacter baumannii. Two
ical intervention without TPN-related complications. To
cases had no isolated microorganisms. our knowledge, this is the first study of DHA without EPA
supplementation at a similar dose as the content in human
milk (∼1% of DHA/total fatty acids or 75 mg of DHA/kg/
day) [18] that evaluates cytokine response from immune
spectively, received a clinically significant intake of hu- cells related to clinical outcomes in surgical neonates.
man milk (>50 kcal/kg/day). All neonates received simi- Lower percentage of positive cells to pro-inflammato-
lar standard nutrition (data not shown). ry cytokines from DHA group indicated lower magnitude
of the inflammatory response after surgery, and the early
Adverse Events increase of percentage of the positive cells to anti-inflam-
There was also no difference between DHA and SO matory cytokines may have favored reaching the equilib-
group in bleeding during surgery (10 ml (0.1, 100 ml) vs. rium among pro- and anti-inflammatory cytokines, lead-
10 ml (0.1, 50 ml), p = 0.869), bleeding (0/16 vs. 3/18, p = ing to homeostasis. This could have been reflected in im-
0.230) or vomiting events at NICU (2/16 vs. 2/18, p = proved clinical outcomes.
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20 Ann Nutr Metab 2016;69:15–23 Bernabe-Garcia et al.

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 Experimental models have reported that DHA can in-
hibit or ameliorate pro-inflammatory cytokines [19, 20].
DHA is a substrate to synthesize docosatrienes with low-
er biological potency to induce inflammation and also to
generate D resolvins, protectins and maresins. These are
anti-inflammatory pro-resolving mediators that inhibit
infiltration of neutrophils into sites of inflammation, di-
minishing production of IL-1β and TNF-α and reducing
inflammation in a shorter time. These biological activities
are mediated via specific G-protein-coupled receptors
such as GPR120, which binds DHA to induce signaling
that interferes with activation of NFκB, resulting in down-
regulation of proteins involved in the inflammatory re-
sponse such as pro-inflammatory cytokines [20, 21]. In
our study, we observed a trend to a higher cumulative
dose of aspirin in the SO group. This is important because
aspirin is a trigger of DHA-derived resolvins and protec-
tins [22]. We think that this outcome strengthens our re-
sults because the anti-inflammatory effect of aspirin in
the SO group likely attenuates the differences.
Monocytes and lymphocytes, also known as mononu-
clear cells, are a model of neonatal cytokine response [23];
thus, we analyzed these cells together. The relevance of
present study is that it demonstrates that enteral adminis-
tration of DHA modulates the pro- and anti-inflammatory
cytokines. These results are similar of a recent report which
demonstrated that mononuclear cells from neonatal cord
blood pretreated with DHA and then stimulated with LPS
endotoxin, produced a concentration-dependent inhibi-
tion of IL-1β, TNF-α and IL-6 secretion [23]. Similarly, the
circulating concentrations of IL-1β, TNF-α and IL-6 were
significantly lower in the infants who received intravenous
fish oil compared to control infants with cardiopulmonary
bypass [24]. Lower IL-1β production and plasma concen-
tration has been reported in the n-3 LC-PUFAs group
compared with a control group in surgical [13] and septic
adults [14]. Likewise, the lower postoperative IL-6 ob-
served in DHA group compared with SO group has been
previously reported in surgical adults [16, 17, 25]. Poor
clinical outcome was associated with high concentrations
of IL-6 [26]; thus, DHA effects appear to be beneficial for
neonates. A study in septic neonates who received enteral
DHA also found lower circulating IL-6 and IL-1β but no
difference in TNF-α after adjusting for confounders com-
pared with a control group, similar to our results [11].
Weiss et al. [16] also found no difference in postoperative
TNF-α between groups of surgical adults. This could be
due the large inter-individual variation of TNF-α [27].
Interestingly, DHA group showed a higher percentage
of cells for IL-1ra+ and IL-10+ between baseline and 24 h
DHA on Inflammation and Clinical
Outcomes in Neonates
post surgery compared with SO group, suggesting that
anti-inflammatory response was stronger during the ear-
ly phase of acute inflammation (fig. 3). This was consis-
tent with higher IL-10 production in stimulated mono-
nuclear cells from stable neonates who received DHA +
AA compared with a control group [28]. In addition, low-
er percentage of IL-1ra+ and IL-10+ cells in DHA group
after adjusting by confounders in the present study was
consistent with septic adults [14] and adults with myocar-
dial revascularization [13]. This early increase in IL-10
may be relevant for neonates because neonatally stimu-
lated T cells have an impaired IL-10 production capacity
that may compromise the appropriate counter regulation
of pro-inflammatory cytokines [2].
Regarding the CD69+CD4+ cells, the trend toward
lower expression in DHA group was consistent with stud-
ies in rodents where DHA suppresses activation of CD4+
cells in response to an activating stimulus, and tips the
balance from a pro-inflammatory Th1 to an anti-inflam-
matory Th2 phenotype. Specifically, it suppresses T cell
proliferation, lowering activation markers such as CD69
and subsequent secretion of cytokines [29]. These func-
tional effects have also been linked with disruption of the
formation of signaling platforms in the plasma mem-
brane (lipid raft) and modification of early transduction
events [20]. To our knowledge, this is the first time that
CD69 expression in surgical neonates is being reported.
Development of sepsis was borderline lower in DHA
group, probably related to the small sample size; however,
it followed the same direction of meta-analyses in adults
that reported a reduction in the infection rate [9, 30]. De-
spite the small sample size used in our study, as is often
the case in studies in critically ill newborns, we used strict
inclusion criteria. Therefore, it is probable that effects on
sepsis development may become significant in a larger
sample.
ODs have been scarcely studied after n-3 LC-PUFA
intervention. Our study found a significant clinical re-
duction in the frequency of ODs in DHA group, contrast-
ing with no difference in multiple ODs in surgical adults
[16]. However, in adults with sepsis-related SIRS, there
was a lower rate of ODs development, attributing the re-
duction of disease progression to an enteral intervention
containing EPA, γ-linolenic acid and antioxidants [31].
Extended hospital stay after a cardiovascular surgery
has been associated with increased risk of infection, poor
neurodevelopmental outcomes in neonates and increased
hospital costs [32]; thus, it is highly desirable to reduce
the length of the NICU stay. Shorter NICU stay in our
DHA group was consistent with shorter stay in ICUs for
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Ann Nutr Metab 2016;69:15–23 21
DOI: 10.1159/000447498
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the surgical adults who received n-3 LC-PUFAs reported
in meta-analyses [9, 31, 33].
Bleeding complications are major surgery-related
concerns. Interestingly, no differences were found in re-
gard to bleeding events between groups, consistent with
a review in adults [34]. Likewise, vomiting and mortality
were not different between groups, according to other re-
ports [14, 25, 35], including preterm infants who received
the same DHA source for an enteral intervention during
a mean of 34 days [36]. However, we recognize that the
present study was not statistically powered to explore dif-
ferences in adverse events.
Another limitation of this study was the oil used for
sham intervention in the control group, which was simi-
lar in physical characteristics but different in fatty acid
content due to the need to substitute the DHA; neverthe-
less, it was also the best available option in composition
and microbiological quality for neonates. DHA-contain-
ing oil had higher lauric, myristic and palmitic acid, but
lower oleic and linoleic acid. Lauric, myristic and palmit-
ic acids have the potential to activate NFκB and increase
inflammatory cytokines in macrophages [37, 38], where-
as linoleic and oleic acid had no effect [37]. Nonetheless,
DHA may prevent or ameliorate their actions on inflam-
matory signaling attenuating inflammatory cytokines
[19, 20, 39]; thus, we do not expect that the concentration
of these fatty acids would have biased the results.
In conclusion, results suggest that enteral administra-
tion of DHA ameliorates markers of inflammation and
improves clinical outcomes in neonates undergoing car-
diovascular surgery.
Acknowledgments
We thank the parents of the infants who agreed to participate.
We also thank Carmen Rodriguez for support in field work, to the
medical staff who contributed to the present work: Dr. Olivia Mad-
rigal-Muniz, Dr. Vanessa Campos-Lozada, Dr. Alfredo Ulloa-Ri-
cardez, Dr. Jose Estrada-Flores, Dr. Carina Feria-Kaiser, Dr. Joa-
quin Rodolfo Zepeda-Sanabria, Dr. Mario Franco-Gutierrez, Dr.
Jose Manuel Vera-Canelo, Dr. Guillermo Diego-Rodriguez and
Dr. Angelica Carrillo-Sierra, as well as the nursery staff from the
NICU of the Pediatric Hospital, CMN Siglo XXI, IMSS for their
support in field work. Sharon Morey, Scientific Communications,
Inc., assisted with the English editorial review.
M.B.G. received the Top New Investigator Award with this
work at the 9th Meeting of the International Society for the Study
of Fatty Acids and Lipids for her contribution in Lipids and Health
Category (Maastricht, Holland 2010).
This work was supported by grants from FIS/IMSS/PROT 094
and CONACYT Salud-2011-1-161643 to MBG; Mexico.
Disclosure Statement
The authors have no conflicts of interest to disclose.

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