EXPERIMENTAL & CLINICAL CARDIOLOGY Title: "Oral Administration of N-3 Long-chain Fatty Acids Reduce...

EXPERIMENTAL & CLINICAL CARDIOLOGY

Volume 20, Issue 6, 2014

Title: "Oral Administration of N-3 Long-chain Fatty Acids Reduce Inflammatory Response andImprove Clinical Outcomes in Patients with Cardiovascular Surgery"

Authors: Mariela Bernabe-Garcia, Mardia Lopez-Alarcon , Armando Mansilla-Olivares , Jorge Maldonado-Hernandez , Francisco Blanco-Favela , Luis Chavez-Sanchez, Karina Chavez-Rueda ,Javier Mancilla-Ramirez , Lourdes Arriaga-Pizano and Carlos Riera-Kinkel How to reference: Oral Administration of N-3 Long-chain Fatty Acids Reduce InflammatoryResponse and Improve Clinical Outcomes in Patients with Cardiovascular Surgery/MarielaBernabe-Garcia, Mardia Lopez-Alarcon , Armando Mansilla-Olivares , Jorge Maldonado-Hernandez , Francisco Blanco-Favela , Luis Chavez-Sanchez, Karina Chavez-Rueda ,Javier Mancilla-Ramirez , Lourdes Arriaga-Pizano and Carlos Riera-Kinkel /Exp Clin Cardiol Vol20 Issue6 pages 145-160 / 2014

Experimental and Clinical Cardiology

Oral administration of n-3 long-chain fatty acids reduce

inflammatory response and improve clinical outcomes in

patients with cardiovascular surgery

Original Research

______________

Mariela Bernabe-Garcia PhD1*, Mardia Lopez-Alarcon PhD1, Armando Mansilla-

Olivares PhD2, Jorge Maldonado-Hernandez MSc1, Francisco Blanco-Favela PhD3, Luis

Chavez-Sanchez PhD3, Karina Chavez-Rueda PhD3, Javier Mancilla-Ramirez PhD4,

Lourdes Arriaga-Pizano PhD5, Carlos Riera-Kinkel MD6

1 Unidad de Investigación Médica en Nutrición, Hospital de Pediatría, Centro Médico Nacional Siglo XXI, Instituto Mexicano del

Seguro Social (IMSS) México City, México. *Corresponding author: Address Apartado Postal C-029 C.S.P.I. “Coahuila”, Coahuila

No. 5, Col. Roma, C.P. 06703 México, D.F., México. Tel. & Fax: +52 55 5627 6944. E-mail: [email protected]

2 Unidad de Investigación Biomolecular, Hospital de Cardiología, Centro Médico Nacional Siglo XXI, IMSS, México City, México

3 Unidad de Investigación Médica en Inmunología, Hospital de Pediatría, Centro Médico Nacional Siglo XXI, IMSS, México City,

México

4 Departamento de Infectología e Inmunología, Instituto Nacional de Perinatología, México City, México

5 Unidad de Investigación Médica en Inmunoquímica, Hospital de Especialidades, Centro Médico Nacional Siglo XXI, IMSS, México

City, México

6 Servicio de Cirugía Cardiotorácica, Hospital de Cardiología, Centro Médico Nacional Siglo XXI, IMSS, México City, México

_____________________

2013 et al.; licensee Cardiology Academic Press. This is an open access article distributed under the terms of the Creative

Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and

reproduction in any medium, provided the original work is properly cited.

Oral Administration of N-3 Long-chain Fatty Acids Reduce Inflammatory Response and Improve Clin...

Exp Clin Cardiol, Volume 20, Issue 6, 2014 - Page 145

ABSTRACT

Background: Exacerbated post-surgery inflammation likely leads to adverse clinical outcomes (ACO). Given the anti-

inflammatory properties of n-3LC-PUFA potential beneficial effects are expected in patients with myocardial

revascularization.

Objective: To evaluate the effect of oral omega-3 LC-PUFA administration on the inflammatory response and ACO in

subjects undergoing cardiovascular surgery.

Methods: A double-blind randomized clinical trial was carried in 23 patients scheduled to myocardial

revascularization. Twelve patients received orally 1.6g/day EPA + 0.8g DHA (O3-G) throughout 7 days starting 1-day

previous surgery; 11 patients who received placebo were controls (CO-G). Intracellular expressions of IL-1beta, TNF-

alpha, IL-6, IL-1ra, IL-10 and CD69 co-stimulatory molecule were measured in whole blood by flow-cytometry at 1-d

before surgery, and at 2h, 24h, 48h and 7-day post-surgery. The development of infection, and cardiovascular and

respiratory dysfunctions was evaluated daily; hospital stay was also assessed.

Results: After adjusting by confounders, O3-G patients exhibited lower cytokine and CD69 expressions than CO-G.

The frequency of ACO (33% vs. 82%, P=0.03), and the hospital stay (P<0.05) were also lower in O3-G than in CO-G.

Conclusion: The oral administration of n-3 LC-PUFA reduces inflammatory response and protects surgical patients

against adverse clinical outcomes.

Key Words: Fish oil, postoperative patients, clinical outcome, inflammatory response, cytokines

_____________________

1. INTRODUCTION

Ischemic heart disease is the first leading cause of death worldwide [1], and myocardial revascularization is

increasing as treatment in adults. Major surgery induces an inflammatory response manifested by increased pro-

inflammatory cytokine production, such as interleukin IL-1 beta (β), TNF-alpha (α) and IL-6, which are

physiologically down-regulated by the anti-inflammatory cytokines IL-1ra and IL-10. The net result is that, their

interaction modulates the intensity of the inflammatory response along with the lipid-derived mediators eicosanoids

and lipoxins [2]. However, excessive pro-inflammatory cytokine production elicits a systemic inflammatory response



syndrome (SIRS),which in turn leads to adverse clinical outcomes from infection and sepsis to respiratory and

cardiovascular dysfunction, as the most frequent postoperative complications [3]. In addition, it is accepted that

sepsis increases 20 to 50% the mortality risk, and have the potential to trigger organ dysfunctions, which carries a

mortality risk of 40-80% in critically ill patients [4, 5].

Blocking cytokines alone provides no beneficial effects to the host, probably because the inflammatory response is

already active or because the cytokine production is not modulated, favoring an imbalance in cellular and cytokine

homeostasis [6]. Based on these, it appears that the goal of the intervention must be directed to modulate both pro-

inflammatory and anti-inflammatory mediators, rather than to inhibit selectively, in order to restore the homeostasis.

In addition, CD4-lympocytes direct the cellular immune response toward either Th1 or Th2, leading the pro-

inflammatory or anti-inflammatory production accordingly. On this regard, CD69 co-stimulatory molecule is the

earliest marker of activation and proliferation of T cells, including CD4 [7], but such molecule has been scarcely

studied in surgical patients.

On the other hand, long chain polyunsaturated fatty acids from the omega 3 family (n-3 LC-PUFA), eicosapentaenoic

(EPA) and docosahexaenoic acids (DHA) regulate several inflammatory mediators. The mechanisms used by n-3 LC-

PUFA to attenuate inflammation include: competition with arachidonic acid (from omega 6 family) as substrate for

producing biologically less potent eicosanoids/docosatrienes, first derived from EPA and second from DHA;

alteration of gene expression through modifying intracellular signal transduction; participation as ligands of the

transcription factor activation like peroxisome proliferator-activated receptor (PPAR) α and , and inhibiting the

nuclear factor κB (NFkB) to attenuate cytokine production [8] . In addition, resolvins and protectins, a family of lipid

mediators involved in the process of inflammation resolution are synthesized from EPA and DHA with higher

efficacy than lipoxins [9]. Most recently identified is the lipid raft hypothesis which suggests that incorporation of n-3

LC-PUFA modifies microdomains of the phospholipid bilayer in cell membranes modifying the interactions between

cells, their activation, and intracellular signaling [10]. Overall effect of these molecular actions gives the reduction of

the inflammatory response.

Studies in humans who undergo an abdominal surgery have shown that the n-3 LC-PUFA beneficial clinical

outcomes are dose- response related [11], but their administration is mostly through parenteral lipid emulsions,

which carries additional risks and high cost [12]. Cardiovascular surgery patients have intact gastrointestinal tract,

hence, they can receive this treatment by oral administration from admission through hospitalization. Besides, it

appears that there are no studies evaluating the effects of n -3 LC-PUFA on cytokines and CD69 expression in patients

who are exposed to cardiovascular surgery. Therefore the aim of the present study was to evaluate if n-3 LC-PUFA

administered orally, provide beneficial effect on the cytokines and CD69 expression, and in the frequency of adverse

clinical outcomes such as infections, organic dysfunctions and hospital stay in subjects who undergo myocardial

revascularization.



2. METHODS

2.1 Patients

A double blind, randomized controlled trial was carried out at the Cardiothoracic Surgery Service of the

Cardiology Hospital in the Mexican Institute of Social Security (IMSS) in Mexico City. Patients were selected from

October 2006 to September of 2008 if male, adults, not taking omega-3 supplements, and with ischemic heart disease

scheduled to myocardial revascularization. Those patients with systemic inflammatory response manifested by the

presence of at least two of the following data one of which must be abnormal central temperature (>38.5°C o <36°C):

abnormal leukocyte count ( >12,000 cells/μl or <4,000/µL), normal white blood cell count but with >10% immature

forms, tachycardia (>90 beats/min), tachipnea (>30 respirations/min), altered mental status, or PaCO2 < 32 mm Hg [13],

were excluded. Other exclusion criteria included the use of anti-thrombotic treatment, clotting alterations, previous

coronary bypass, allergy against fish protein, unstable diabetes mellitus, and renal or hepatic insufficiency. The

protocol was approved by the Ethics Committee of the Hospital of Cardiology (2006-3604-04). All patients provided

written informed consent.

2.2 Procedures

After signing the written consent form, patients were randomly assigned to receive 1600 mg EPA and 800 mg DHA

(Omega Rx, Zone labs inc., Massachusetts, USA) daily (O3-G) or corn starch (CO-G), distributed in two capsules with

breakfast plus two capsules with dinner, starting at one day before surgery, and during six days post-surgery.

Randomization was conducted using a table of random numbers; assigned treatment was previously placed into

sealed opaque envelopes by one researcher who did not participate in recruitment, clinical evaluation or

determination of cytokines. Before surgery, weight and height were measured with a Tanita scale (BWB-700, Tokio

Japan) and a stadiometer (SECA 206, CA, USA). Weight was registered in grams and height to the nearest 0.1 cm.

Body mass index (BMI) was calculated as weight (in kg) divided by the square of height (m2). The mean of three

measurements was used. Severity of illness before surgery was evaluated by APACHE II [14]. All medication and

surgery variables, such as bleeding volume, time of aortic clamp and cardiovascular bypass, were prospectively

recorded.

A blood specimen was centrifuged at 3000 rpm at baseline, and separated erythrocytes were used to determine fatty

acid profile [15]. After blood collection, erythrocytes were washed at least three times with 0.9% of saline solution,

suspended in a minimal volume of saline solution and frozen at -20 °C until analysis. Total fat from erythrocytes was

extracted with isopropanol and butylated hydroxytoluene as antioxidant. Fatty acids were methylated and methyl

esters were extracted and analyzed by gas chromatography (589 Series II, Hewlett Packard, Avondale, PA, USA)



using a 100-m x 0.2 mm inside diameter fused silica column coated with 0.2 µm CP Sil 88 (Chrompact, The

Netherlands). Afterwards, fatty acids were identified using standards for each fatty acid. In addition, heptadecanoic

acid was added to each sample as internal standard. Results are expressed as weight percentages (%/total weight) of

total fatty acids [16].

2.3 Mediators of inflammatory response: Intracellular cytokines and co-stimulatory molecule CD69 expression

Venous blood was collected in a tube with heparin after 12h overnight fast and before surgery (baseline). Blood was

also collected at 2h, 24h, 48h and day 7 post-surgery. Blood samples were diluted 1/1 in RPMI 1640 medium

(Invitrogen, Carlsbad, CA) to measure cytokines and CD69 expression.

2.3.1 Cytokines IL-1β, TNF-α, IL-6, IL-1ra and IL-10. Diluted whole blood was distributed in 24-wells plates (Nunc,

Roslike, Denmark) (500 µl/well), and 10 minutes before stimulation, golgistop (BD Biosciences, San Jose, CA) was

added to each well at a concentration of 10 µg/ml. After, diluted blood was stimulated with phorbol 12-myristate 13-

acetate (PMA), at a concentration of 40 ng/ml and ionomycin (0.5 µg/ml). As a negative control, diluted blood was

incubated only in culture medium in the absence of PMA and ionomycin, and both treatments were incubated for 5

hours. Further, 25 µl of diluted blood was stained with anti-human CD4-Cy5 for CD-4 lymphocytes. Then, 50 µL of

fixation and permeabilization solution (Intrastain kit, DAKO Cytomation, Denmark) was added to cells, and washed

with PBS (containing 1% BSA and 1% sodium azide buffer, Sigma-Aldrich) after fixation/permeabilization. Afterward,

cells were separately stained with monoclonal antibodies anti-human TNF-alpha-FITC, IL-1 beta-FITC, IL-6-PE,

antagonist of IL-1 (IL-1ra)-PE and IL-10-APC (BD Biosciences). Nonspecific fluorescence was adjusted by isotype-

matched antibodies (BD Biosciences). Cells were incubated 15 min in the dark at 25°C after adding each antibody,

fixation or permeabilization solution.

2.3.2 Co-stimultory molecule CD69. A volume of 25 µL of diluted blood with unstimulated cells was stained with

anti-human monoclonal CD4-APC plus CD69-PE (BD Biosciences) for quantification of CD-69 co-stimulatory

molecules in CD4-lymphocytes using the same fixation protocol.

Cells were measured using a FACS Aria flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree

Star, San Carlos, CA, USA). Results are reported as percentage of the cytokine and CD69 expression.

2.4 Adverse clinical outcomes

Infection, sepsis, and respiratory and cardiovascular dysfunctions, were prospectively registered daily throughout

hospitalization and considered as adverse clinical events according to international definitions [13, 17]. Briefly,

infection was defined as a pathological process induced by a micro-organism; sepsis as an infection documented or



suspected plus the inclusion of parameters of systemic inflammatory response described above [13]; cardiovascular

dysfunction as hypotension with a mean arterial pressure lower than 70 mm Hg and/or the need of inotropic support

for at least 1 hour such as dopamine, dobutamine, epinephrine or norepinephrine; and respiratory dysfunction as the

need of ventilatory support with a PO2/FIO2 ratio less than 400 [17]. After surgery, medical attention continued at the

intermediate surgery ward, and once critical condition improved the patient was transferred to routine medical

wards. The sum of days in both was considered as total hospital stay.

2.5 Statistical analysis

Data are presented as mean ± standard error (SEM), non-normally distributed variables were log transformed.

Median and intervals are reported in those variables which did not fit a normal distribution after log transformation.

Significance value was set at P<0.05 for comparisons. The Minitab Software was used for statistical computations

(Minitab 14.2, State College, PA, USA). For comparisons of cytokines between groups, Student t test or U-Mann-

Whitney test were used as appropriate. Repeated measures ANOVA using the General Linear Model approach was

used to analyze the effect n3 LC-PUFA on cytokines during follow-up, adjusting for confounders. Adverse clinical

outcomes and other qualitative variables are reported as frequency and percentage. The risk of adverse clinical

outcomes was evaluated with logistic regression analysis. The Hosmer-Lemeshow statistic was used for goodness of

fit defined with P value > 0.05.

3. RESULTS

Twenty-four adults accepted to participate; one patient in the CO-G was voluntary excluded, but withdraw was not

associated with side effect of intervention. Twelve patients were assigned to the O3-G and 11 to the CO-G. The

expression of cytokines and CD69 were measured only in 9 subjects from the O3-G and 10 from the CO-g because 4

samples were coagulated (Figure 1). Diagnosis, severity of illness, erythrocyte fatty acids and surgical variables were

comparable between groups. Although the mean BMI was in the range of overweight in both groups, BMI of the CO-

G was higher than that of O3-G (Table 1). Comparisons of confounders demonstrated that the use of statins was

longer in the CO-G than in the O3-G; all the other studied variables were comparable (Table 2).

3.1 Mediators of inflammatory response: Intracellular cytokines and co-stimulatory CD69 expression

Raw data showed that intracellular cytokines in circulating CD4-lymphocytes were comparable at baseline. In

contrast, the expression of the pro-inflammatory cytokines IL-1β, IL-6 and TNF- was lower in the O3-G compared

with the CO-G during follow-up. Statistical significance was reached for IL-1β at 2h and 24 h, and IL-6 at 24h; despite

TNF- was lower during the whole follow-up in the O3-G than in CO-G, such difference was only borderline (Figure

2 A-C). Regarding anti-inflammatory cytokines, the O3-G presented also significantly lower expression of IL-1ra at 2h



and a trend to express less IL-10 at 2h was observed; the co-stimulatory molecule CD69+ also tended to be different at

48h post-surgery (Figure 2 D-F, P=0.09).

Except for IL-6, after adjusting by age, body mass index, volume of bleeding during surgery, cardiopulmonary

bypass, and medication with acetylsalicylic acid and statins, the O3-G group exhibited a significantly lower pro-

inflammatory and anti-inflammatory cytokines and CD69 expression than CO-G (Figure 3).

3.2 Adverse clinical outcomes

The frequency of adverse clinical outcomes analyzed separately was not different between groups. Nevertheless, the

sum of events was significantly lower in O3-G than in CO-G. Likewise, the total stay in the hospital and the days of

stay in the medical ward were both lower in O3-G than in CO-G (Table 3). Logistic regression analysis including

confounders demonstrated that the risk to develop one or more adverse clinical outcomes was lower in O3-G than in

CO-G (OR = 0.031; 95% CI = 0.001, 0.669; P = 0.027).

4. DISCUSSION

Results of this study demonstrate that the administration of oral n-3 LC-PUFA short before entering to

revascularization protects patients against the development of adverse events and decrease hospital stay. Such

positive effect is likely mediated by a better modulation of the inflammatory response by n3 fatty acids. Our study is

relevant because to our knowledge it is the first to demonstrate that this short-term intervention, even orally, provides

beneficial effects in surgical patients.

Our results are consistent with those of other studies that found positive effects of n-3 LC-PUFA administration,

particularly in the frequency of infections [18-20] and organ failure [21]. However, last study included gamma-

linolenic acid and antioxidants in the intervention, making difficult to ascribe the benefit to any particular nutrient.

Comparable reduction in hospital stay to those found in our study was recently reported in meta-analyses from

patients with abdominal surgery or sepsis [18-20].

Regarding cytokine expression, our data are consistent with other authors who detected positive effects on cytokine

reduction in plasma from surgical patients who received DHA and EPA through parenteral nutrition [22-27], and

even a dose-dependent reduction of intracellular expression in cell cultures [28]. Interestingly, in our study we

reached same beneficial effect through oral administration of 2.4 g/day of EPA plus DHA, which is relevant because

another study did not find cytokine reductions with an oral dose of 1.665 g/day of EPA plus DHA [29]. Accordingly,

present study allows us to propose the use of n-3 fatty acids to patients who will suffer a surgery even if they are not

candidates for parenteral nutrition.



We found that anti-inflammatory cytokine expression was also decreased by n-3 LC-PUFA supplementation, which

seems conflicting with the pathophysiology of the inflammatory response since they down-regulate pro-inflammatory

cytokines [3]. Basic studies with Th2-skewed cells revealed that T cells express 15-lipooxigenase (15-LO), and 15-LO

dependent production of protectin (PD1) DHA-derived. PD1 blocked induced interferon-gamma and TNF-α

production from T-cells suggesting it is substantially involved in the Th-1 counteracting properties of Th2 cells; PD1

also induces T cell apoptosis. This findings showed that PD1 is a major DHA conversion product that promotes anti-

inflammation [30]. This might be the mechanisms through our intervention counteracted inflammatory response in

the O3-G without increasing anti-inflammatory cytokines, suggesting that not only expression but other more

mechanisms modulate the immune and inflammatory response.

A previous report found that CD69-deficient mice as a model of lung injury and acute inflammation exhibited lower

cytokine expression and less lung damage than wild type [31], which is consistent with our results. Likewise, lower

CD69 expression was found in ex vivo cell cultures of healthy humans after receiving DHA plus EPA during 4 weeks

[32]. However, another ex vivo study did not observe changes on CD69 expression after adding several concentrations

of EPA or DHA [28]. To our knowledge this is the first evaluation of CD69 expression after receiving n-3 LC-PUFA in

surgical patients, thus the clinical implications are unclear and require further research.

We recognize the small sample size is a limitation in our study, likely explaining the borderline statistical significance

in cytokines. Nevertheless, are consistent with others as the above discussed and in the same directions, suggesting

that our results are valid. On the other hand, our study also presents several strengths, such as the use of a feasible

and single intervention, and specific laboratory methods that make our results more reliable.

In conclusion, the oral administration of n-3 LC-PUFA reduces inflammatory response and protects surgical patients

against adverse clinical outcomes.

5. ACKNOWLEDGMENTS

We thank to all patients who agreed to participate in this study and to Dr. Gabriel Gonzalez, Nut. Karla Elide Zavala-,

Nut. Mariana Rodriguez, Carmen Rodriguez, Guadalupe Matute and Filiberto Jasso for their support in field work.

We also thank to medical surgeons and attendants Dr. German Ordonez, Dr. Moises Calderon, Dr. Rodolfo Garcia-

Lemus, Dr. Sergio Claire, Dr. Jose Angel Cigarroa, Dr. Antonio Barragan, Dr. Jose Luis Garcia, Dr. Ivan Galvan, Dr.

Serafin Ramirez, Dr. Cecilio Cruz, Dr. Cristo Kusulas, and Dr. Samuel Miyamoto.

This investigation was supported by a financial grant from Fondo para la Investigación en Salud (FIS), IMSS, México.

No. FIS/IMSS/PROT 095, 516 and MD09/727 (MBG).



Disclosures:

The authors had no financial or personal conflict of interest to declare.

6. REFERENCES

1. WHO. The top 10 causes of death: WHO 2014 [updated July 2013; cited 2014 April 15th]. Available from:

http://www.who.int/mediacentre/factsheets/fs310/en/.

2. Medzhitov R. Origin and physiological roles of inflammation. Nature 2008; 454: 428-35.

3. Oberholzer A, Oberholzer C, Moldawer LL. Cytokine signaling--regulation of the immune response in normal and

critically ill states. Crit Care Med 2000; 28: N3-12.

4. Calder PC. Immunonutrition in surgical and critically ill patients. Br J Nutr 2007; 98 Suppl 1: S133-9.

5. Claus RA, Otto GP, Deigner HP, Bauer M. Approaching clinical reality: markers for monitoring systemic

inflammation and sepsis. Curr Mol Med 2010; 10: 227-35.

6. Angele MK, Faist E. Clinical review: immunodepression in the surgical patient and increased susceptibility to

infection. Critical care 2002; 6: 298-305.

7. Shimaoka M, Hosotsubo K, Sugimoto M, Sakaue G, Taenaka N, Yoshiya I, Kiyono H. The influence of surgical

stress on T cells: enhancement of early phase lymphocyte activation. Anesth Analg 1998; 87: 1431-5.

8. Martin JM, Stapleton RD. Omega-3 fatty acids in critical illness. Nutrition reviews 2010; 68: 531-41.

9. Ji RR, Xu ZZ, Strichartz G, Serhan CN. Emerging roles of resolvins in the resolution of inflammation and pain.

Trends Neurosci 2011; 34: 599-609.

10. Yaqoob P. Mechanisms underlying the immunomodulatory effects of n-3 PUFA. Proc Nutr Soc 2010; 69: 311-5.

11. Bernabe-Garcia M, Lopez-Alarcon M, Blanco-Favela F, Mancilla-Ramirez J, Mansilla-Olivares A, Arredondo-

Garcia JL. Beneficial effects of the n-3 long-chain polyunsaturated fatty acids in surgical patients: updating the

evidence. Prostaglandins Leukot Essent Fatty Acids 2011; 85: 261-6.

12. Calder PC, Jensen GL, Koletzko BV, Singer P, Wanten GJ. Lipid emulsions in parenteral nutrition of intensive care

patients: current thinking and future directions. Intensive Care Med 2010; 36: 735-49.

13. Levy MM, Fink MP, Marshall JC, Abraham E, Angus D, Cook D, Cohen J, Opal SM, Vincent JL, Ramsay G. 2001

SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Intensive Care Med 2003; 29: 530-8.

14. Knaus WA, Draper EA, Wagner DP, Zimmerman JE. APACHE II: a severity of disease classification system. Crit

Care Med 1985; 13: 818-29.

15. Fekete K, Marosvolgyi T, Jakobik V, Decsi T. Methods of assessment of n-3 long-chain polyunsaturated fatty acid

status in humans: a systematic review. Am J Clin Nutr 2009; 89: 2070S-84S.

16. Evershed R. Gas chromatography of lipids, In: Hamilton RJ HS, editor. Lipid analysis A practical approach.

Oxford: Oxford University Press, 1992. 113-51.

17. Meisner M, Tschaikowsky K, Palmaers T, Schmidt J. Comparison of procalcitonin (PCT) and C-reactive protein

(CRP) plasma concentrations at different SOFA scores during the course of sepsis and MODS. Critical care 1999; 3: 45-

50.

18. Wei C, Hua J, Bin C, Klassen K. Impact of lipid emulsion containing fish oil on outcomes of surgical patients:

systematic review of randomized controlled trials from Europe and Asia. Nutrition 2010; 26: 474-81.

19. Chen B, Zhou Y, Yang P, Wan HW, Wu XT. Safety and efficacy of fish oil-enriched parenteral nutrition regimen on

postoperative patients undergoing major abdominal surgery: a meta-analysis of randomized controlled trials. JPEN

Journal of parenteral and enteral nutrition 2010; 34: 387-94.

20. Pradelli L, Mayer K, Muscaritoli M, Heller AR. n-3 fatty acid-enriched parenteral nutrition regimens in elective

surgical and ICU patients: a meta-analysis. Critical care 2012; 16: R184.

21. Pontes-Arruda A, Aragao AM, Albuquerque JD. Effects of enteral feeding with eicosapentaenoic acid, gamma-

linolenic acid, and antioxidants in mechanically ventilated patients with severe sepsis and septic shock. Crit Care Med

2006; 34: 2325-33.

22. Weiss G, Meyer F, Matthies B, Pross M, Koenig W, Lippert H. Immunomodulation by perioperative

administration of n-3 fatty acids. Br J Nutr 2002; 87 Suppl 1: S89-94.



23. Wachtler P, Konig W, Senkal M, Kemen M, Koller M. Influence of a total parenteral nutrition enriched with

omega-3 fatty acids on leukotriene synthesis of peripheral leukocytes and systemic cytokine levels in patients with

major surgery. The Journal of trauma 1997; 42: 191-8.

24. Liang B, Wang S, Ye YJ, Yang XD, Wang YL, Qu J, Xie QW, Yin MJ. Impact of postoperative omega-3 fatty acid-

supplemented parenteral nutrition on clinical outcomes and immunomodulations in colorectal cancer patients. World

J Gastroenterol 2008; 14: 2434-9.

25. de Miranda Torrinhas RS, Santana R, Garcia T, Cury-Boaventura MF, Sales MM, Curi R, Waitzberg DL. Parenteral

fish oil as a pharmacological agent to modulate post-operative immune response: a randomized, double-blind, and

controlled clinical trial in patients with gastrointestinal cancer. Clinical nutrition 2013; 32: 503-10.

26. Wei Z, Wang W, Chen J, Yang D, Yan R, Cai Q. A prospective, randomized, controlled study of omega-3 fish oil fat

emulsion-based parenteral nutrition for patients following surgical resection of gastric tumors. Nutrition journal 2014;

13: 25.

27. Larsen BM, Goonewardene LA, Joffe AR, Van Aerde JE, Field CJ, Olstad DL, Clandinin MT. Pre-treatment with an

intravenous lipid emulsion containing fish oil (eicosapentaenoic and docosahexaenoic acid) decreases inflammatory

markers after open-heart surgery in infants: a randomized, controlled trial. Clinical nutrition 2012; 31: 322-9.

28. Jaudszus A, Gruen M, Watzl B, Ness C, Roth A, Lochner A, Barz D, Gabriel H, Rothe M, Jahreis G. Evaluation of

suppressive and pro-resolving effects of EPA and DHA in human primary monocytes and T-helper cells. J Lipid Res

2013; 54: 923-35.

29. Yusof HM, Cawood AL, Ding R, Williams JA, Napper FL, Shearman CP, Grimble RF, Payne SP, Calder PC.

Limited impact of 2 g/day omega-3 fatty acid ethyl esters (Omacor(R)) on plasma lipids and inflammatory markers in

patients awaiting carotid endarterectomy. Marine drugs 2013; 11: 3569-81.

30. Ariel A, Serhan CN. Resolvins and protectins in the termination program of acute inflammation. Trends Immunol

2007; 28: 176-83.

31. Yamauchi K, Kasuya Y, Kuroda F, Tanaka K, Tsuyusaki J, Ishizaki S, Matsunaga H, Iwamura C, Nakayama T,

Tatsumi K. Attenuation of lung inflammation and fibrosis in CD69-deficient mice after intratracheal bleomycin.

Respiratory research 2011; 12: 131.

32. Kew S, Mesa MD, Tricon S, Buckley R, Minihane AM, Yaqoob P. Effects of oils rich in eicosapentaenoic and

docosahexaenoic acids on immune cell composition and function in healthy humans. Am J Clin Nutr 2004; 79: 674-81.



TABLE 1

Characteristics of patients scheduled to myocardial revascularization

Group

Omega 3

N = 12

Control

n = 11

P

Baseline

Age, years 58.7 ± 2.7 66.0 ± 2.5 0.064

Weight, kg 73.9 ± 2.5 80.3 ± 3.5 0.152

Height, m 1.68 ± 0.0 1.66 ± 0.0 0.482

BMI, kg/m2 26.0 ± 0.6 28.9 ± 1.3 0.050

Diabetes 4 (33) 2 (18) 0.640

Hypertension 10 (83) 7 (64) 0.351

Previous infarction 8 (67) 6 (55) 0.680

Illness severity before surgery (Apache II) 5.2 ± 1.1 4.7 ± 1.2 0.681

Fatty acids in erythrocytes, % weight/weight

Linoleic (LA, 18:2 n-6) 11.5 ± 0.36 10.7 ± 0.37 0.146

- Linolenic (LNA, 18:3 n-3) 0.41 ± 1.1 0.44 ± 1.1 0.561

Arachidonic (AA, 20:4 n-6) 17.3 ± 1.0 17.3 ± 1.0 0.859

Eicosapentaenoic (EPA, 20:5 n-3) 0.67 ± 1.1 0.60 ± 1.1 0.511

Docosahexaenoic (DHA, 22:6 n-3) 6.04 ± 0.31 6.01 ± 0.53 0.205

Myocardial Revascularization

With cardiopulmonary bypass, n (%) 8 (67) 6 (55) 0.680

Time on cardiopulmonary bypass, min 71.5 ± 8.3 74.2 ± 6.9 0.819

Time with aortic clamp, min 48.7 ± 5.9 41.2 ± 4.9 0.366

Duration of surgery, min 224.3 ± 17.8 224.1 ± 36.2 0.881

Bleeding volume during surgery, L 1.0 ± 0.2 1.1 ± 0.2 0.930

Mean ± SEM

Frequency (%)

BMI: Body Mass Index



TABLE 2

Medication received by patients with myocardial revascularization from admission until hospital discharge

Group

Omega 3

n = 12

Control

n = 11

P

Therapy

Steroid use 0 1 (9) 0.478

Duration of use, days 0 1 (9) NS

Non-steroidal anti-inflammatory 12 (100) 11 (100) NS

Duration of use, days 4.8 ± 1.2 6.5 ± 1.1 0.172

Antibiotics 12 (100) 10 (91) 0.478


Angiotensin-converting enzyme inhibitors 6 (50) 4 (36) 0.680


Statins 12 (100) 11 (100) NS


Acetylsalicylic acid 12 (100) 11 (100) NS

Duration of use, days 7.5 [3,11] 8 [7,16] 0.426

Buprenorphine 7 (58) 4 (36) 0.414

Duration of use, days 1 [1,6] 1.5 [1,2] 0.658

Frequency (%)

Mean SEM

Median [minimum, maximum]

NS: Not statistically significant



TABLE 3

Clinical outcomes of patients with myocardial revascularization from baseline until hospital discharge

Group

O3-G

n = 12

CO-G

n = 11

P

Infections or sepsis, n 1 (8) a 4 (36) a,b,c,d 0.131

Organ dysfunction, n

Cardiovascular 3 (25) 4 (36) 0.444

Respiratory 0 1 (9) 0.478

Adverse clinical outcomes: infections or sepsis or organ

dysfunctions, n

4 (33) 9 (82) 0.026

Intermediate surgery ward stay, days 3.2 ± 1.1 3.2 ± 1.1 0.440

Medical ward stay, days 4.5 ± 1.1 6.8 ± 3.5 0.037

Total hospital stay, days 8.3 ± 1.1 10.3 ± 1.1 0.038

Frequency (%)

Mean SEM

a Infection of the urinary tract but no microorganism isolation

b Sepsis by Enterobacter cloacae

c Neumonia by Staphyloccocus epidermidis

d Infection of surgical wound by Staphyloccocus aureis and Rautella ornitolítica



Figure 1. CONSORT diagram for study

Analysed for

Adverse clinical outcomes (n= 11)

Intracellular cytokines and CD69 in

lymphocytes (n= 10)

Lost to follow-up (n= 0)

Lost to follow-up (n= 1)

Assessed for eligibility: Patients scheduled for

myocardial revascularization (n= 70)

Excluded (n= 46)

Not meeting inclusion criteria (n= 44)

Declined to participate (n= 2)

Allocated to control group (n= 12)

Received corn starch capsules (n= 12) Allocated to omega 3 group (n= 12)

Received omega 3 capsules (n= 12)

Analysed for

Adverse clinical outcomes (n= 12)

Intracellular cytokines and CD69 in

lymphocytes (n= 9)

Allocation

Analysis

Follow-Up

Randomized (n= 24)

Enrollment


Exp Clin Cardiol, Volume 20, Issue 6, 2014 - 8

Figure 2. Intracellular cytokine expression of IL-β, TNF-α, IL-6, IL-1ra and IL-10, and CD69 co-stimulatory molecule in CD4-

lymphocytes from patients who underwent myocardial revascularization. Data are mean ± SEM of the percentage of expression.

Blue line represents omega 3 group (n= 9) and red line the control group (n=10). Study time with an asterisk indicates statistical

difference between groups (P<0.05); superscript indicates borderline statistical significance (P≤0.09)



Figure 3. Mean reduction of cytokine and CD69 expression in omega 3 group compared with control group during follow-up,

adjusting for confounders. Data indicate mean ± SEM in percentage of expression. Baseline of figure represents the percentage of

expression for control group.



EXPERIMENTAL & CLINICAL CARDIOLOGY Title: "Oral Administration of N-3 Long-chain Fatty Acids Reduce...

Documents

Transcript of EXPERIMENTAL & CLINICAL CARDIOLOGY Title: "Oral Administration of N-3 Long-chain Fatty Acids Reduce...