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Original Research: Asthma |

The Effect of Omega-3 Fatty Acids on Bronchial Hyperresponsiveness, Sputum Eosinophilia, and Mast Cell Mediators in AsthmaOmega-3 Fatty Acid Supplements in Asthma FREE TO VIEW

John D. Brannan, PhD; Johan Bood, PhD; Ahmad Alkhabaz, MD; David Balgoma, PhD; Joceline Otis, BSc; Ingrid Delin, BSc; Barbro Dahlén, MD, PhD; Craig E. Wheelock, PhD; Parameswaran Nair, MD, PhD; Sven-Erik Dahlén, MD, PhD; Paul M. O’Byrne, MB, FCCP
Author and Funding Information

From the Firestone Institute for Respiratory Health (Drs Brannan, Alkhabaz, Nair, and O’Byrne and Ms Otis), St. Joseph’s Healthcare and McMaster University, Hamilton, ON, Canada; and Department of Medical Biochemistry and Biophysics (Drs Balgoma and Wheelock), National Institute of Environmental Medicine (Drs Bood, Balgoma, and S.-E. Dahlén and Ms Delin), Department of Medicine, Karolinska University Hospital Huddinge (Drs Bood and B. Dahlén), and Centre for Allergy Research (Drs Bood, B. Dahlén, Wheelock, and S.-E. Dahlén and Ms Delin), Karolinska Institutet, Stockholm, Sweden.

CORRESPONDENCE TO: John D. Brannan, PhD, Department of Respiratory and Sleep Medicine, Westmead Hospital, Darcy Rd, Westmead, NSW 2145, Australia; e-mail: john.brannan@sydney.edu.au


Part of this article has been presented in abstract form (Brannan JD, Alkhabaz A, Otis J, et al. Effect of omega-3 fatty acid [O3FA] from fish oils on mannitol-induced airway hyperresponsiveness [AHR] and sputum eosinophils in patients with asthma. Eur Respir J. 2009;34[suppl 33]:333s).

FUNDING/SUPPORT: Dr Brannan was supported by an Asthma Foundation of New South Wales (Australia) Martin Hardie Traveling Fellowship. Dr Nair holds a Canada Research Chair in Airway Inflammometry. Dr Balgoma was supported by the Innovative Medicines Initiative project U-BIOPRED (Unbiased Biomarkers in Prediction of Respiratory Distress Outcomes). The clinical study was funded by the Firestone Institute for Respiratory Health, and the work at Karolinska Institutet was supported by funding to Drs B. Dahlén, Wheelock, and S.-E. Dahlén from several Swedish sources (Heart and Lung Foundation, Medical Research Council, Stockholm County Council, VINNOVA, the Åke Wibergs Stiftelse, Jeanssons Stiftelse, Fredrik and Ingrid Thurings Stiftelse, and Centre for Allergy Research).

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details.


Chest. 2015;147(2):397-405. doi:10.1378/chest.14-1214
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BACKGROUND:  Omega-3 fatty acid supplements have been reported to inhibit exercise-induced bronchoconstriction (EIB). It has not been determined whether omega-3 supplements inhibit airway sensitivity to inhaled mannitol, a test for bronchial hyperresponsiveness (BHR) and model for EIB in people with mild to moderate asthma.

METHODS:  In a double-blind, crossover trial, subjects with asthma who had BHR to inhaled mannitol (n = 23; 14 men; mean age, 28 years; one-half taking regular inhaled corticosteroids) were randomized to omega-3 supplements (4.0 g/d eicosapentaenoic acid and 2.0 g/d docosahexaenoic acid) or matching placebo for 3 weeks separated by a 3-week washout. The primary outcome was the provoking dose of mannitol (mg) to cause a 15% fall in FEV1 (PD15). Secondary outcomes were sputum eosinophil count, spirometry, Asthma Control Questionnaire (ACQ) score, serum triacylglyceride level, and lipid mediator profile in urine and serum.

RESULTS:  PD15 (geometric mean, 95% CI) to mannitol following supplementation with omega-3s (78 mg, 51-119 mg) was not different from placebo (88 mg, 56-139 mg, P = .5). There were no changes in sputum eosinophils (mean ± SD) in a subgroup of 11 subjects (omega-3, 8.4% ± 8.2%; placebo, 7.8% ± 11.8%; P = .9). At the end of each treatment period, there were no differences in FEV1 % predicted (omega-3, 85% ± 13%; placebo, 84% ± 11%; P = .9) or ACQ score (omega-3, 1.1% ± 0.5%; placebo, 1.1% ± 0.5%; P = .9) (n = 23). Omega-3s caused significant lowering of blood triglyceride levels and expected shifts in serum fatty acids and eicosanoid metabolites, confirming adherence to the supplements; however, no changes were observed in urinary mast cell mediators.

CONCLUSIONS:  Three weeks of omega-3 supplements does not improve BHR to mannitol, decrease sputum eosinophil counts, or inhibit urinary excretion of mast cell mediators in people with mild to moderate asthma, indicating that dietary omega-3 supplementation is not useful in the short-term treatment of asthma.

TRIAL REGISTRY:  ClinicalTrials.gov; No.: NCT00526357; URL: www.clinicaltrials.gov.

Figures in this Article

Dietary supplementation using omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) plays an uncertain role in the management of asthma, which has led to calls for more prospective studies to investigate whether there are any benefits from an omega-3-enriched diet in asthma.1 Results have not been consistent when assessing high doses of dietary omega-3s on bronchial hyperresponsiveness (BHR), a key feature of asthma. Early studies administering omega-3s daily over 10 weeks demonstrated no attenuation of exercise-induced bronchoconstriction (EIB)2 while causing a small decrease of the late airway response to inhaled allergen.3 More recently, 3 weeks of omega-3s were reported to inhibit mild airway responses to exercise and eucapnic voluntary hyperventilation (EVH) in both elite athletes and symptomatic subjects with asthma.46 This was observed in association with reductions in markers of airway inflammation, such as reduced cytokine levels, reduced excretion of the urinary eicosanoids 11β-prostaglandin F2α (11β-PGF) and leukotriene E4 (LTE4), and decreased sputum eosinophil counts, suggesting that the benefits of an omega-3-enriched diet on EIB was through antiinflammatory mechanisms.

Inhaled mannitol as a test for BHR has been demonstrated as a useful model for EIB,7 and all pharmacotherapies effective at inhibiting EIB inhibit BHR to mannitol.811 Mannitol causes bronchoconstriction through an increase in the osmolarity of the airway surface fluid, leading to mediator release from mast cells similar to the proposed mechanism for EIB.12 Evidence for mediator release to mannitol is an increase in urinary eicosanoids 11β-PGF and LTE4 following a mannitol challenge11 similar to what has been observed with exercise and EVH.13,14 BHR to mannitol identifies EIB in elite athletes15 and predicts the severity of EIB in people with asthma.7

Considering these similarities, we evaluated whether dietary supplementation with omega-3 fatty acids could inhibit BHR to inhaled mannitol to assess the efficacy of omega-3s in people with mild to moderate BHR. We assessed the effect of 3 weeks of dietary omega-3s on BHR to mannitol, sputum eosinophil counts, spirometry, and asthma symptoms in subjects with clinically defined steroid-naive asthma as well as in those using regular inhaled corticosteroids (ICSs). Urine and serum were collected during the study to determine the biochemical effects of the omega-3 supplementation on mast cell mediators derived from fatty acids.

Study Design

The study used a randomized, double-blind, placebo-controlled, and crossover trial design. Subjects attended the laboratory in the morning on four occasions occurring before and after two 3-week treatment periods. People taking ICSs were required to attend an extra initial visit 2 weeks prior to the commencement of the trial in order to perform a mannitol challenge to document that their airway sensitivity in the presence of ICS was reproducible over the run-in period and that their asthma was clinically stable.

Each visit comprised self-administered questionnaires (Asthma Control Questionnaire and Asthma Quality of Life Questionnaire); taking blood to assess fasting blood triglyceride, omega-3, and omega-6 fatty acid levels; obtaining a urine sample to assess urinary eicosanoid counts; assessing baseline lung function using spirometry; and performing a mannitol challenge followed by sputum induction. More details are provided in e-Appendix 1. Upon meeting entry criteria, subjects were randomized to a 3-week supply of a daily dose of 10 capsules of either a matched placebo containing a blend of omega-6 and omega-9 or a ratio of omega-3 fatty acids comprising 400 mg EPA and 200 mg DHA (40/20EE capsules; Ocean Nutrition Canada). This equated to a daily dose of 4.0 g EPA and 2.0 g DHA. Each treatment period was separated by a 3-week washout. More details are provided in e-Figure 1.

The protocol was approved by the ethics review board of St. Joseph’s Healthcare, Hamilton, Ontario, Canada (R.P.#06-2750), and Health Canada (Approval No. 120532). All subjects gave written informed consent.

Subjects

Nonsmoking subjects with clinically diagnosed asthma and current asthma symptoms with an FEV1 > 70% predicted were entered into the trial (Fig 1, Table 1). Short-acting β2-agonists were withheld for 8 h and long-acting β2-agonists for 48 h, and no ICSs were taken on the morning of the study. Antihistamines and leukotriene antagonists were not permitted throughout the whole study period. No caffeine-containing food or drink and no vigorous exercise were permitted on the study day prior to the study visit. Subjects were asked to abstain from fish meals or other significant sources of omega-3s for at least 2 weeks prior to the study and throughout the study.

Figure Jump LinkFigure 1 –  Participant flow diagram. BHR = bronchial hyperresponsiveness; ICS = inhaled corticosteroid; PD15 = provoking dose of mannitol (mg) to cause a 15% fall in FEV1; p.r.n = as needed; Rx = supplement.Grahic Jump Location
Table Graphic Jump Location
TABLE 1 ]  Anthropometric Data, Medication, FEV1, Response to Mannitol, and % Eosinophils in Sputum at Baseline

/ = in combination; BEC = beclomethasone; BUD = budesonide; Ef = eformoterol; F = female; FL = fluticasone; ICS = inhaled corticosteroid; M = male; PD15 = provoking dose of mannitol (mg) to cause a 15% fall in FEV1; prn = as needed; S = salbutamol; Sm = salmeterol.

a 

Geometric mean.

b 

95% CI.

Biochemical Measurements

Serum triacylglyceride levels and the fatty acid composition of the serum phospholipids were analyzed for triglycerides as well as omega-3 and omega-6 fatty acids. The effects of the dietary intervention on eicosanoid levels of 11β-PGF and LTE4 in urine were assessed by enzyme immunoassay,11,14 and an ultraperformance liquid chromatography-tandem mass spectrometry platform16 that measures > 90 different lipid mediators. More details are provided in e-Appendix 1.

Statistics

The primary outcome was the airway sensitivity to a provoking dose of mannitol (mg) to cause a 15% fall in FEV1 (PD15). Secondary outcomes were sputum eosinophil counts, baseline FEV1, and asthma symptoms scores. Spearman rank correlation was performed on nonnormally distributed data. For comparisons between groups, the Student t test was used for paired measurements, with P < .05 considered significant. A sample size for each treatment group for PD15 was based on a prior study demonstrating a clinically significant improvement in association with decreased airway sensitivity to inhaled mannitol using ICS.17 When raw data for PD15 values were log2 converted, this study demonstrated a 1.9 doubling dose shift in the presence of ICS and a within-subject SD of 0.7, yielding a sample size of four subjects at 80% power and a significance of .05. When calculating a minimal important difference of 1 doubling dose, assuming the same within-subject SD, this yielded a sample size of seven at 80% power and a significance of .05. The rate of spontaneous recovery of lung function following the challenge was calculated as an area under the % fall in FEV1 vs time curve.

Omega-3 fatty acid supplements were not effective at attenuating BHR to mannitol. The geometric mean PD15 was 88 mg (95% CI, 56-139 mg) after placebo treatment and 78 mg (95% CI, 51-119 mg) after omega-3 fatty acid supplements (P = .5). This represented a doubling dose shift in favor of placebo of −0.25 (SD, 1.1) and less than the minimal important difference of 1 doubling dose. Although the airway sensitivity to mannitol was in the moderate range for most subjects (PD15 < 155 mg), those taking β2-agonists as needed were more sensitive to mannitol than those taking ICSs (P = .03) (Table 2). However, no differences were observed based on subjects’ daily treatment (Fig 2, Table 2, e-Fig 2). In addition, there were no differences in the spontaneous recovery from mannitol between the omega-3 supplement and placebo groups. However, a subgroup analysis found those taking ICS had a slower recovery while on omega-3 supplements compared with placebo, and recovery time for the whole group was longer in the presence of omega-3 supplements compared with baseline (Table 2, e-Fig 3).

Table Graphic Jump Location
TABLE 2 ]  Total Group or Individual Treatment Groups of Symptoms, FEV1 % Predicted, PD15, and Spontaneous Recovery in Lung Function and Baseline Urinary Metabolites Either at Baseline or Following Omega-3 Supplementation or Placebo

Data are presented as mean ± SD or geometric mean (95% CI) unless otherwise indicated. 11β-PGF = 11β-prostaglandin F2α; ACQ = Asthma Control Questionnaire; AQLQ = Asthma Quality of Life Questionnaire; AUC = area under the curve; %fall.min = % fall in FEV1 vs time; LTE4 = leukotriene E4. See Table 1 legend for expansion of other abbreviations.

a 

ICS with or without long-acting β2-agonist.

b 

P < .05 with baseline.

c 

P < .05 with placebo.

d 

For β2-agonist group, urine data only in 11 subjects.

Figure Jump LinkFigure 2 –  The individual data and geometric mean (95% CI) for PD15 in subjects with asthma following 3 wk of daily omega-3 fatty acid supplementation compared with placebo, demonstrating no change in PD15. (n = 23, P = .5). = inhaled corticosteroid group; ○ = β2-agonist group; + = two subjects who did not have a PD15 value. See Figure 1 legend for expansion of abbreviation.Grahic Jump Location

The majority of subjects who could produce an adequate sputum sample had evidence of eosinophils (> 2%) (Table 1), and there was a significant relationship between the percentage of eosinophils and mannitol PD15 (rs = 0.53, P = .047, n = 14). However, in subjects who provided suitable sputum samples at the end of each treatment period (n = 11), there were no differences in eosinophil counts following omega-3 supplementation (8.4% ± 8.2%) compared with placebo (7.8% ± 11.8%) (Fig 3).

Figure Jump LinkFigure 3 –  The individual data and mean (SE) for the percentage of eosinophils in sputum in a subgroup of subjects with asthma following 3 wk of daily omega-3 fatty acid supplementation compared with placebo, demonstrating no change in percent eosinophils (n = 11, P = .9). = inhaled corticosteroid group; ○ = β2-agonist group.Grahic Jump Location

There were no significant improvements in either lung function (baseline FEV1) or asthma symptoms with the omega-3 supplements, with no changes in asthma control or asthma quality of life scores (Table 2). However, there was a 27% reduction in fasting blood triglyceride levels in subjects taking the omega-3 supplements compared with those taking placebo (P < .001) as well as evidence of raised EPA and DHA serum levels in the presence of the omega-3 supplements compared with placebo (P < .001) (Figs 4, 5). This finding indicated biologic activity of the omega-3 supplements and that subjects were adherent to the dietary supplementation.

Figure Jump LinkFigure 4 –  The individual data and mean (SE) for serum triglyceride levels in subjects with asthma following 3 wk of daily omega-3 fatty acid supplementation compared with placebo, demonstrating a significant decrease in serum triglyceride concentration following omega-3 fatty acid supplementation (n = 23, P = .0011). *P < .0011. = inhaled corticosteroid group; ○ = β2-agonist group.Grahic Jump Location
Figure Jump LinkFigure 5 –  The individual data and mean (SE) for combined omega-3 fatty acids in serum (weight [wt] as a percentage of total phospholipid) in subjects with asthma following 3 weeks of daily omega-3 fatty acid supplementation compared with placebo, demonstrating a significant increase in omega-3 fatty acid levels in serum following omega-3 fatty acid supplementation (n = 23, P < .001). *P < .001. = inhaled corticosteroid group; ○ = β2-agonist group.Grahic Jump Location

As further evidence of the effectiveness of the intervention, serum levels (mean ± SEM) of the omega-3 fatty acid EPA-derived metabolites 12-hydroxyeicosapentaenoic acid (HEPE) and 15-HEPE increased significantly following omega-3 supplementation (0.6 ± 0.1 pM vs 6.9 ± 2.6 pM, P = .022) compared with placebo (0.1 ± 0.02 pM vs 0.6 ± 0.1 pM, P = .000024). Measurement of the analogous omega-6 fatty acid arachidonic acid-derived metabolites 12- and 15-hydroxyeicosatetraenoic acid showed no changes (11 ± 2 pM vs 17 ± 5 pM and 1.3 ± 0.1 pM vs 1.3 ± 0.1 pM, respectively).

The measurements of urinary levels (ng/mmol creatinine) of LTE4 and the prostaglandin D2 (PGD2) metabolite 11β-PGF revealed no differences between the two treatments (Table 2); thus, further characterization of the effect of omega-3 supplements on lipid mediator profiles using ultra-performance lipid chromatography-tandem mass spectrometry was performed in a subset of subjects (n = 5). No changes were found in the urinary excretion of any of the key metabolites of the primary prostaglandins or LTE4. However, there was a decrease in urinary prostaglandin E2 (PGE2) by omega-3 supplements compared with placebo (29 ± 19 units vs 141 ± 52 units, respectively; P = .048). A decrease was also observed for 8,12-iso-iPF-VI after omega-3 supplementation compared with placebo (581 ± 103 units vs 730 ± 129 units, respectively; P = .0117). The levels of all lipid mediators measured in the urine are provided in e-Table 1.

A post hoc power calculation of sample size using the data for PD15 obtained in this study showed that the total sample size of 23 was of sufficient power for assessing a minimal important difference of one doubling dose. The within-subject SD for a comparison of PD15 values at the beginning of each treatment period before any treatment was administered and following washout (each separated by 6 weeks), was 1.2. The SD for the PD15 values in the presence of the placebo and omega-3 fatty acid treatments (each also separated by 6 weeks) was 1.1. This yielded a sample size of 13 and 12 subjects, respectively, at a power of 80% and a significance of 0.05. Individual data for PD15 can be found in e-Table 2. The doubling dose difference comparing the PD15 values at the commencement of each treatment period was 0.25.

This study demonstrates that a high daily dose of an omega-3 fatty acid supplement has no effect on either BHR to mannitol or sputum eosinophil percentage in subjects with mild to moderate asthma in association with no changes in asthma symptom control. The lack of effect was seen irrespective of whether the subjects had baseline treatment with as-needed β2-agonists or were maintained on ICS. Neither did the omega-3 fatty acid supplementation change the resting levels of key arachidonic acid-derived mediators of mast cell responses, such as the cysteinyl leukotrienes and prostaglandin D2. However, we did see an increase in the serum levels of the omega-3 fatty acid-derived products 12- and 15-HEPE after the active supplementation in association with the expected lowering of serum triglyceride levels, which together provide firm evidence that the subjects were adherent to the treatment.

The findings are consistent with the initial observation that omega-3 fatty acids had no effect on EIB2 but contradictory to other studies where similar doses of omega-3 supplements over the same treatment duration of 3 weeks were found to inhibit EIB4,5 and EVH.6,18 The most obvious difference is that we used inhaled mannitol, a bronchial provocation challenge that has been derived from the understanding of the effects of dry air hyperpnea-induced BHR, which is a well-established alternative challenge stimulus to study EIB. There is strong evidence that the airway response to mannitol closely mimics central components of EIB, such as the activation of mast cells and eosinophils, resulting in the release of bronchoconstrictive mediators.7,913,19,20 However, the differences may lie in the intensity of the stimulus to the airways. The studies showing benefit performed the exercise challenge until volitional exhaustion, which is a test that has a known low sensitivity to identifying EIB and which may induce milder airway responses in those with EIB.4,5 In both exercise and EVH studies by Mickleborough and colleagues,4,5 no data were provided in relation to the intensity or reproducibility of the exercise and ventilatory stimulus administered or whether this achieved the minimum requirements for each protocol to induce EIB.

Moreover, the current findings are consistent with earlier studies demonstrating that a 10-week treatment period with omega-3 supplements does not attenuate EIB using the standardized 8-min exercise protocol.2 The authors assessed airway resistance (specific airway conductance) and not FEV1 as a measure of airflow limitation. The same investigators, however, did demonstrate a small effect of omega-3s on the late airway response to allergen.3 Interestingly, in a treatment study, the same group also found that omega-3 supplements changed the profile of blood fatty acids and lipid mediator biosynthesis in circulating leukocytes but did not improve the clinical course of rhinitis or asthma.21

In contrast to Mickleborough and colleagues,4,5 we did not find any significant reductions in resting levels of inflammatory markers, such as sputum eosinophils or the urinary metabolites of PGD2 and LTE4 with omega-3 fatty acids. The present findings are consistent with studies in mice showing no effect of a 6-week treatment with EPA and DHA (using a similar equivalent dose) on suppressing ovalbumin-augmented eosinophils in an asthma mouse model, with evidence that DHA alone augments eosinophilia.22 However, it was also shown in mice that both EPA and DHA can suppress PGE2 in BAL. We also observed significant reductions in PGE2 excretion (its source likely from the kidney), which suggests that omega-3 fatty acids can modify prostaglandin metabolism. PGE2 in the airway is believed to have a beneficial effect as an endogenous bronchodilator, providing bronchoprotection after exercise.23 We did not notice any detrimental effect of the omega-3 supplements other than a small, but statistically significant prolonged recovery from bronchoconstriction to mannitol in the presence of the omega-3 treatment; however, the clinical relevance of this is unclear. Slower recovery from mannitol challenge does suggest a direct role of leukotrienes.10,19 One study in humans has demonstrated worsening of daily peak flow and increased β2-agonist use with longer treatment of a daily dose of 3 g omega-3 fatty acids over 6 weeks in subjects with asthma and aspirin sensitivity.24

The presumed mechanism for explaining the possible beneficial effects of omega-3 fatty acids on inflammatory pathways is through diversion of the production of bronchoconstricting prostaglandins and leukotrienes from the omega-6 substrate arachidonic acid to mediators biosynthesized from the alternative omega-3 substrates. This assumes that these alternative metabolites have weaker effects on the airway smooth muscle and inflammatory effector cells.4,25 However, it has been shown that the 5-series leukotrienes derived from omega-3 fatty acids have the same bronchoconstrictive potency as the 4-series leukotrienes derived from arachidonic acid.26 In addition, the generation of the 3-series prostaglandins with omega-3 supplements may not provide any benefits on airway inflammation and BHR in an animal model.22 Considering this, it may be that omega-3 supplements, even in high doses, fail to substantially alter the substrate flow in the tissue-residing cells, whereas in the blood, significant concentrations exist to divert metabolism. It is well established that omega-3 fatty acids can effectively decrease serum triglyceride levels, as we observed in this study. Thus, omega-3 supplements may be effective in altering plasma fatty acid composition; function of blood cells; and metabolism in other cells exposed to high levels of circulating omega-3 fatty acids, such as vascular endothelium and renal tubular cells. This is supported by the current finding that the intervention caused a decrease in urinary PGE2, which is known to be derived directly from the kidney.27 We suggest that it may be more difficult to change the metabolic profile of cells within tissues, such as that of the airway mast cell, where there is most likely still sufficient arachidonic acid available as the preferred substrate for the activation-induced biosynthesis of cysteinyl leukotrienes and PGD2. During stimulation, arachidonic acid is liberated mainly by group 4 phospholipase A2 (or cytosolic phospholipase A2). This enzyme has preference for phospholipids with arachidonic acid, which may explain why in acute processes, displacing arachidonic acid with omega-3s is not effective to shield the production of arachidonic acid-derived eicosanoids.28

The study was adequately powered for the primary outcome, and this was supported with the post hoc calculations. We powered the study expecting a clinically important difference based on the existing studies showing a 64% protection in the percent fall in FEV1 after exercise in subjects with asthma taking omega-3 fatty acids5 and considering this inhibition to be similar to what has been observed using ICS on both exercise and mannitol in subjects with asthma.17,29 Independent of ICS therapy, a patient with asthma who has a PD15 will demonstrate considerable room for improvement in PD15 in the presence of improved ICS treatment (higher dose or improvement in adherence)17,30 and acute therapy using cromolyn drugs and β2-agonists.11 A post hoc sample size analysis demonstrated that the individual treatment groups based on the presence of ICS therapy or β2-agonists as needed were either adequately powered or slightly underpowered, suggesting that a minimum of 12 to 13 subjects are required when using inhaled mannitol in a study assessing an intervention and looking for a minimal important difference of 1 doubling dose. Considering the 0.25 doubling dose difference in favor of placebo, this possible slight underpowering for each treatment group is likely of no significance.

In conclusion, a diet enriched with omega-3 fatty acids over a 3-week treatment period had no clinical benefits in either steroid-treated or steroid-naive subjects with asthma, as confirmed by no observed improvements on the two key features of asthma: BHR and airway inflammation. Furthermore, there was no evidence of significant changes in resting eicosanoid metabolism caused by the omega-3 supplements, even though expected changes in serum omega-3 fatty acid metabolism and decreases in serum triglyceride levels were seen. These data suggest that in people with asthma, high daily doses of omega-3 fatty acids from fish oils are not an alternative or additive treatment strategy for asthma. Further studies need to elucidate whether the degree of the stimulus to elicit EIB is a determining factor on the protective effect of omega-3 fatty acids and whether the same effect is observed following longer-term therapy. Considering this, the findings have general ramifications, indicating that distinct manipulation of tightly controlled biologic responses in tissues by dietary intervention of omega-3 fatty acids is difficult.

Author contributions: J. D. B. had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. J. D. B. and P. M. O. equally contributed to the study concept; J. D. B., P. N., and P. M. O. contributed to the trial design; J. D. B., J. B., A. A., J. O., and I. D. contributed to the gathering and entering of data for analysis; J. B., D. B., I. D., and C. E. W. contributed to the biochemical analysis; J. D. B., J. B., and S.-E. D. contributed to the data analysis; J. D. B. and J. B. contributed to writing the manuscript; and J. D. B., J. B., A. A., D. B., J. O., I. D., B. D., C. E. W., P. N., S.-E. D., and P. M. O. contributed to the data interpretation, revision of the manuscript, and final approval of the manuscript.

Financial/nonfinancial disclosures: The authors have reported to CHEST the following conflicts of interest: Dr Brannan receives a 10% portion of the royalties paid to his previous employer, the Sydney South Area Health Service, where the bronchial provocation test Aridol/Osmohale was developed. These royalties are paid by the manufacturer Pharmaxis Ltd. Drs Bood, Alkhabaz, Balgoma, B. Dahlén, Wheelock, Nair, S.-E. Dahlén, and O’Byrne and Mss Otis and Delin have reported that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Role of sponsors: The sponsors had no role or influence on study design, recruitment, analysis, or manuscript writing. The funding was provided to promote independent clinical research. The content and opinions expressed in this paper is solely those of the authors.

Other contributions: The authors thank Bruce Holub, PhD, University of Guelph, for technical assistance and advice and Ann Efthimiadis, BSc, and Karen Radford, BSc, for technical assistance. The authors also thank Mark Inman, MD, PhD, for assistance in the sample size analysis. The authors acknowledge Pharmaxis Ltd for the in-kind supply of the mannitol challenge test kit (Aridol). The use of the application for mannitol described in this study is covered by US patent No. 5,817,028 and internationally by PCT/AU95000086. The patent is owned by the Sydney South Western Area Health Service and is licensed to Pharmaxis Ltd, NSW, Australia.

Additional information: The e-Appendix, e-Figures, and e-Tables can be found in the Supplemental Materials section of the online article.

11β-PGF

11β-prostaglandin F2α

BHR

bronchial hyperresponsiveness

DHA

docosahexaenoic acid

EIB

exercise-induced bronchoconstriction

EPA

eicosapentaenoic acid

EVH

eucapnic voluntary hyperventilation

HEPE

hydroxyeicosapentaenoic acid

ICS

inhaled corticosteroid

LTE4

leukotriene E4

PD15

provoking dose of mannitol (mg) to cause a 15% fall in FEV1

PGD2

prostaglandin D2

PGE2

prostaglandin E2

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Brannan JD, Anderson SD, Gomes K, King GG, Chan HK, Seale JP. Fexofenadine decreases sensitivity to and montelukast improves recovery from inhaled mannitol. Am J Respir Crit Care Med. 2001;163(6):1420-1425. [CrossRef] [PubMed]
 
Brannan JD, Gulliksson M, Anderson SD, Chew N, Seale JP, Kumlin M. Inhibition of mast cell PGD2 release protects against mannitol-induced airway narrowing. Eur Respir J. 2006;27(5):944-950. [PubMed]
 
Anderson SD, Daviskas E. The mechanism of exercise-induced asthma is. J Allergy Clin Immunol. 2000;106(3):453-459. [CrossRef] [PubMed]
 
O’Sullivan S, Roquet A, Dahlén B, et al. Evidence for mast cell activation during exercise-induced bronchoconstriction. Eur Respir J. 1998;12(2):345-350. [CrossRef] [PubMed]
 
Kippelen P, Larsson J, Anderson SD, et al. Acute effects of beclomethasone on hyperpnea-induced bronchoconstriction. Med Sci Sports Exerc. 2010;42(2):273-280. [CrossRef] [PubMed]
 
Holzer K, Anderson SD, Chan HK, Douglass J. Mannitol as a challenge test to identify exercise-induced bronchoconstriction in elite athletes. Am J Respir Crit Care Med. 2003;167(4):534-537. [CrossRef] [PubMed]
 
Balgoma D, Larsson J, Rokach J, et al. Quantification of lipid mediator metabolites in human urine from asthma patients by electrospray ionization mass spectrometry: controlling matrix effects. Anal Chem. 2013;85(16):7866-7874. [CrossRef] [PubMed]
 
Brannan JD, Koskela H, Anderson SD, Chan HK. Budesonide reduces sensitivity and reactivity to inhaled mannitol in asthmatic subjects. Respirology. 2002;7(1):37-44. [CrossRef] [PubMed]
 
Mickleborough TD, Vaughn CL, Shei RJ, Davis EM, Wilhite DP. Marine lipid fraction PCSO-524 (lyprinol/omega XL) of the New Zealand green lipped mussel attenuates hyperpnea-induced bronchoconstriction in asthma. Respir Med. 2013;107(8):1152-1163. [CrossRef] [PubMed]
 
Brannan JD, Gulliksson M, Anderson SD, Chew N, Kumlin M. Evidence of mast cell activation and leukotriene release after mannitol inhalation. Eur Respir J. 2003;22(3):491-496. [CrossRef] [PubMed]
 
Kippelen P, Larsson J, Anderson SD, Brannan JD, Dahlén B, Dahlén SE. Effect of sodium cromoglycate on mast cell mediators during hyperpnea in athletes. Med Sci Sports Exerc. 2010;42(10):1853-1860. [CrossRef] [PubMed]
 
Thien FC, Mencia-Huerta JM, Lee TH. Dietary fish oil effects on seasonal hay fever and asthma in pollen-sensitive subjects. Am Rev Respir Dis. 1993;147(5):1138-1143. [CrossRef] [PubMed]
 
Schuster GU, Bratt JM, Jiang X, et al. Dietary long-chain omega-3 fatty acids do not diminish eosinophilic pulmonary inflammation in mice. Am J Respir Cell Mol Biol. 2014;50(3):626-636. [CrossRef] [PubMed]
 
Melillo E, Woolley KL, Manning PJ, Watson RM, O’Byrne PM. Effect of inhaled PGE2 on exercise-induced bronchoconstriction in asthmatic subjects. Am J Respir Crit Care Med. 1994;149(5):1138-1141. [CrossRef] [PubMed]
 
Picado C, Castillo JA, Schinca N, et al. Effects of a fish oil enriched diet on aspirin intolerant asthmatic patients: a pilot study. Thorax. 1988;43(2):93-97. [CrossRef] [PubMed]
 
Calder PC. Immunomodulation by omega-3 fatty acids. Prostaglandins Leukot Essent Fatty Acids. 2007;77(5-6):327-335. [CrossRef] [PubMed]
 
Dahlén SE, Hedqvist P, Hammarström S. Contractile activities of several cysteine-containing leukotrienes in the guinea-pig lung strip. Eur J Pharmacol. 1982;86(2):207-215. [CrossRef] [PubMed]
 
Samuelsson B, Granström E, Green K, Hamberg M, Hammarström S. Prostaglandins. Annu Rev Biochem. 1975;44:669-695. [CrossRef] [PubMed]
 
Leslie CC. Properties and regulation of cytosolic phospholipase A2. J Biol Chem. 1997;272(27):16709-16712. [CrossRef] [PubMed]
 
Subbarao P, Duong M, Adelroth E, et al. Effect of ciclesonide dose and duration of therapy on exercise-induced bronchoconstriction in patients with asthma. J Allergy Clin Immunol. 2006;117(5):1008-1013. [CrossRef] [PubMed]
 
Turton JA, Glasgow NJ, Brannan JD. Feasibility and acceptability of using bronchial hyperresponsiveness to manage asthma in primary care: a pilot study. Prim Care Respir J. 2012;21(1):28-34. [CrossRef] [PubMed]
 

Figures

Figure Jump LinkFigure 1 –  Participant flow diagram. BHR = bronchial hyperresponsiveness; ICS = inhaled corticosteroid; PD15 = provoking dose of mannitol (mg) to cause a 15% fall in FEV1; p.r.n = as needed; Rx = supplement.Grahic Jump Location
Figure Jump LinkFigure 2 –  The individual data and geometric mean (95% CI) for PD15 in subjects with asthma following 3 wk of daily omega-3 fatty acid supplementation compared with placebo, demonstrating no change in PD15. (n = 23, P = .5). = inhaled corticosteroid group; ○ = β2-agonist group; + = two subjects who did not have a PD15 value. See Figure 1 legend for expansion of abbreviation.Grahic Jump Location
Figure Jump LinkFigure 3 –  The individual data and mean (SE) for the percentage of eosinophils in sputum in a subgroup of subjects with asthma following 3 wk of daily omega-3 fatty acid supplementation compared with placebo, demonstrating no change in percent eosinophils (n = 11, P = .9). = inhaled corticosteroid group; ○ = β2-agonist group.Grahic Jump Location
Figure Jump LinkFigure 4 –  The individual data and mean (SE) for serum triglyceride levels in subjects with asthma following 3 wk of daily omega-3 fatty acid supplementation compared with placebo, demonstrating a significant decrease in serum triglyceride concentration following omega-3 fatty acid supplementation (n = 23, P = .0011). *P < .0011. = inhaled corticosteroid group; ○ = β2-agonist group.Grahic Jump Location
Figure Jump LinkFigure 5 –  The individual data and mean (SE) for combined omega-3 fatty acids in serum (weight [wt] as a percentage of total phospholipid) in subjects with asthma following 3 weeks of daily omega-3 fatty acid supplementation compared with placebo, demonstrating a significant increase in omega-3 fatty acid levels in serum following omega-3 fatty acid supplementation (n = 23, P < .001). *P < .001. = inhaled corticosteroid group; ○ = β2-agonist group.Grahic Jump Location

Tables

Table Graphic Jump Location
TABLE 1 ]  Anthropometric Data, Medication, FEV1, Response to Mannitol, and % Eosinophils in Sputum at Baseline

/ = in combination; BEC = beclomethasone; BUD = budesonide; Ef = eformoterol; F = female; FL = fluticasone; ICS = inhaled corticosteroid; M = male; PD15 = provoking dose of mannitol (mg) to cause a 15% fall in FEV1; prn = as needed; S = salbutamol; Sm = salmeterol.

a 

Geometric mean.

b 

95% CI.

Table Graphic Jump Location
TABLE 2 ]  Total Group or Individual Treatment Groups of Symptoms, FEV1 % Predicted, PD15, and Spontaneous Recovery in Lung Function and Baseline Urinary Metabolites Either at Baseline or Following Omega-3 Supplementation or Placebo

Data are presented as mean ± SD or geometric mean (95% CI) unless otherwise indicated. 11β-PGF = 11β-prostaglandin F2α; ACQ = Asthma Control Questionnaire; AQLQ = Asthma Quality of Life Questionnaire; AUC = area under the curve; %fall.min = % fall in FEV1 vs time; LTE4 = leukotriene E4. See Table 1 legend for expansion of other abbreviations.

a 

ICS with or without long-acting β2-agonist.

b 

P < .05 with baseline.

c 

P < .05 with placebo.

d 

For β2-agonist group, urine data only in 11 subjects.

References

Reisman J, Schachter HM, Dales RE, et al. Treating asthma with omega-3 fatty acids: where is the evidence? A systematic review. BMC Complement Altern Med. 2006;6:26. [CrossRef] [PubMed]
 
Arm JP, Horton CE, Mencia-Huerta JM, et al. Effect of dietary supplementation with fish oil lipids on mild asthma. Thorax. 1988;43(2):84-92. [CrossRef] [PubMed]
 
Arm JP, Horton CE, Spur BW, Mencia-Huerta JM, Lee TH. The effects of dietary supplementation with fish oil lipids on the airways response to inhaled allergen in bronchial asthma. Am Rev Respir Dis. 1989;139(6):1395-1400. [CrossRef] [PubMed]
 
Mickleborough TD, Murray RL, Ionescu AA, Lindley MR. Fish oil supplementation reduces severity of exercise-induced bronchoconstriction in elite athletes. Am J Respir Crit Care Med. 2003;168(10):1181-1189. [CrossRef] [PubMed]
 
Mickleborough TD, Lindley MR, Ionescu AA, Fly AD. Protective effect of fish oil supplementation on exercise-induced bronchoconstriction in asthma. Chest. 2006;129(1):39-49. [CrossRef] [PubMed]
 
Tecklenburg-Lund S, Mickleborough TD, Turner LA, Fly AD, Stager JM, Montgomery GS. Randomized controlled trial of fish oil and montelukast and their combination on airway inflammation and hyperpnea-induced bronchoconstriction. PLoS One. 2010;5(10):e13487. [CrossRef] [PubMed]
 
Brannan JD, Koskela H, Anderson SD, Chew N. Responsiveness to mannitol in asthmatic subjects with exercise- and hyperventilation-induced asthma. Am J Respir Crit Care Med. 1998;158(4):1120-1126. [CrossRef] [PubMed]
 
Anderson SD. Single-dose agents in the prevention of exercise-induced asthma: a descriptive review. Treat Respir Med. 2004;3(6):365-379. [CrossRef] [PubMed]
 
Brannan JD, Anderson SD, Freed R, Leuppi JD, Koskela H, Chan HK. Nedocromil sodium inhibits responsiveness to inhaled mannitol in asthmatic subjects. Am J Respir Crit Care Med. 2000;161(6):2096-2099. [CrossRef] [PubMed]
 
Brannan JD, Anderson SD, Gomes K, King GG, Chan HK, Seale JP. Fexofenadine decreases sensitivity to and montelukast improves recovery from inhaled mannitol. Am J Respir Crit Care Med. 2001;163(6):1420-1425. [CrossRef] [PubMed]
 
Brannan JD, Gulliksson M, Anderson SD, Chew N, Seale JP, Kumlin M. Inhibition of mast cell PGD2 release protects against mannitol-induced airway narrowing. Eur Respir J. 2006;27(5):944-950. [PubMed]
 
Anderson SD, Daviskas E. The mechanism of exercise-induced asthma is. J Allergy Clin Immunol. 2000;106(3):453-459. [CrossRef] [PubMed]
 
O’Sullivan S, Roquet A, Dahlén B, et al. Evidence for mast cell activation during exercise-induced bronchoconstriction. Eur Respir J. 1998;12(2):345-350. [CrossRef] [PubMed]
 
Kippelen P, Larsson J, Anderson SD, et al. Acute effects of beclomethasone on hyperpnea-induced bronchoconstriction. Med Sci Sports Exerc. 2010;42(2):273-280. [CrossRef] [PubMed]
 
Holzer K, Anderson SD, Chan HK, Douglass J. Mannitol as a challenge test to identify exercise-induced bronchoconstriction in elite athletes. Am J Respir Crit Care Med. 2003;167(4):534-537. [CrossRef] [PubMed]
 
Balgoma D, Larsson J, Rokach J, et al. Quantification of lipid mediator metabolites in human urine from asthma patients by electrospray ionization mass spectrometry: controlling matrix effects. Anal Chem. 2013;85(16):7866-7874. [CrossRef] [PubMed]
 
Brannan JD, Koskela H, Anderson SD, Chan HK. Budesonide reduces sensitivity and reactivity to inhaled mannitol in asthmatic subjects. Respirology. 2002;7(1):37-44. [CrossRef] [PubMed]
 
Mickleborough TD, Vaughn CL, Shei RJ, Davis EM, Wilhite DP. Marine lipid fraction PCSO-524 (lyprinol/omega XL) of the New Zealand green lipped mussel attenuates hyperpnea-induced bronchoconstriction in asthma. Respir Med. 2013;107(8):1152-1163. [CrossRef] [PubMed]
 
Brannan JD, Gulliksson M, Anderson SD, Chew N, Kumlin M. Evidence of mast cell activation and leukotriene release after mannitol inhalation. Eur Respir J. 2003;22(3):491-496. [CrossRef] [PubMed]
 
Kippelen P, Larsson J, Anderson SD, Brannan JD, Dahlén B, Dahlén SE. Effect of sodium cromoglycate on mast cell mediators during hyperpnea in athletes. Med Sci Sports Exerc. 2010;42(10):1853-1860. [CrossRef] [PubMed]
 
Thien FC, Mencia-Huerta JM, Lee TH. Dietary fish oil effects on seasonal hay fever and asthma in pollen-sensitive subjects. Am Rev Respir Dis. 1993;147(5):1138-1143. [CrossRef] [PubMed]
 
Schuster GU, Bratt JM, Jiang X, et al. Dietary long-chain omega-3 fatty acids do not diminish eosinophilic pulmonary inflammation in mice. Am J Respir Cell Mol Biol. 2014;50(3):626-636. [CrossRef] [PubMed]
 
Melillo E, Woolley KL, Manning PJ, Watson RM, O’Byrne PM. Effect of inhaled PGE2 on exercise-induced bronchoconstriction in asthmatic subjects. Am J Respir Crit Care Med. 1994;149(5):1138-1141. [CrossRef] [PubMed]
 
Picado C, Castillo JA, Schinca N, et al. Effects of a fish oil enriched diet on aspirin intolerant asthmatic patients: a pilot study. Thorax. 1988;43(2):93-97. [CrossRef] [PubMed]
 
Calder PC. Immunomodulation by omega-3 fatty acids. Prostaglandins Leukot Essent Fatty Acids. 2007;77(5-6):327-335. [CrossRef] [PubMed]
 
Dahlén SE, Hedqvist P, Hammarström S. Contractile activities of several cysteine-containing leukotrienes in the guinea-pig lung strip. Eur J Pharmacol. 1982;86(2):207-215. [CrossRef] [PubMed]
 
Samuelsson B, Granström E, Green K, Hamberg M, Hammarström S. Prostaglandins. Annu Rev Biochem. 1975;44:669-695. [CrossRef] [PubMed]
 
Leslie CC. Properties and regulation of cytosolic phospholipase A2. J Biol Chem. 1997;272(27):16709-16712. [CrossRef] [PubMed]
 
Subbarao P, Duong M, Adelroth E, et al. Effect of ciclesonide dose and duration of therapy on exercise-induced bronchoconstriction in patients with asthma. J Allergy Clin Immunol. 2006;117(5):1008-1013. [CrossRef] [PubMed]
 
Turton JA, Glasgow NJ, Brannan JD. Feasibility and acceptability of using bronchial hyperresponsiveness to manage asthma in primary care: a pilot study. Prim Care Respir J. 2012;21(1):28-34. [CrossRef] [PubMed]
 
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