2.1 Study design
The double-blind placebo-controlled trial took place from April 2015 until January 2016 and consisted of 3 visits in a time frame of 4 to 8 weeks. Participation in the study was voluntary. Prior to the commencement of the first visit (Screening visit, V1), written informed consent was required from each subject or from the parents of children under the age of 18 years.
At V1, the participants’ inclusion and exclusion criteria were confirmed, a lung function test administered using the MasterScreen spirometer, a skin prick test (SPT) performed, eNO measured and an asthma control questionnaire (ACQ) provided to participants.
On the second (V2, Randomization) and third visit (V3, End of study) a lung function test was conducted, eNO measured, an exercise challenge in a cold chamber (ECC) administered and blood collected. If the patient fulfilled the inclusion criteria `FEV1 decrease ≥ 15% in the ECC` at V2, they were randomised to one of the two study arms (interventional group with sc-LCPUFA supplementation or placebo group). V3 was performed after 4 weeks of supplementation. The study flow chart with assessments is shown in Fig.1. The study was approved by the ethics committee of Goethe University (reference number 360/14) Clinical trials registration number: NCT02410096.
Ninety-nine subjects aged 10 to 45 years with asthmatic symptoms while exercising were recruited from the outpatient clinic of the Department for Children and Adolescents, Division of Allergology, Pulmonology and Cystic fibrosis, Goethe University, Frankfurt, Germany by a public posting. Subjects with chronic asthma and regular use of inhaled corticosteroids (ICSs) or leukotriene receptor antagonists were excluded. Further exclusion criteria were: subjects with FVC < 75%, subjects > 18 years/ < 18 years with FEV1 < 60/ < 70%, oral corticosteroids, other known chronic disease or infection, pregnancy, documented alcohol, medication or drug abuse and inability to perform and understand all study procedures.
2.3 sc-LCPUFA supplementation and Placebo
The total amount of functional LCPUFA in the investigational product came to 1190 mg/day (710 mg EPA, 161 mg DHA, 175 mg GLA and 144 mg SDA). Therefore, the patients had to take 4 different capsules each morning combined with their breakfast:
1 capsule PlusEPA (containing EPA, Minami Nutrition, Aartselaar, Belgium); 1 capsule EPA/DHA/GLA (containing EPA/DHA/GLA, Peak Performance Products S.A., Grevenmacher, Luxemburg); 2 capsules Echiomega (containing SDA/GLA, Igennus Healthcare Nutrition, Cambridge, UK). The placebo compound (Allcura, naturheilmittel GmbH) was composed of 500 mg olive oil.
2.4 Pulmonary function test
Baseline pulmonary function tests were performed using the MasterScreen spirometer (CareFusion, Germany). The following parameters were recorded: forced vital capacity (FVC), FEV1 and FEV1/FVC.
2.5 Measurement of exhaled nitric oxide
Measurements of eNO were conducted using the NIOX1 gas analyzer (Aerocrine, Sweden). NIOX1 measures eNO in exhaled air according to ATS guidelines (25).
2.6 Exercise challenge in a cold chamber
The exercise challenge was performed according to the ATS guidelines for EIA (26) as recently described (7, 27). The subjects ran in a cold chamber (Ilkazell Isoliertechnik, Germany) cooled to 2–4 °C (microprocessor-based controller, Dixell, Emerson Climate Technologies, United Kingdom) on a treadmill (Schiller Intertrack 8100T Med, Germany) with an incline of 10% for 6 mins (≤ 12 years) or 8 mins (> 12 years). At 5, 10, 15 and 30 mins after running, spirometry were performed. A positive reaction was defined as a decline in FEV1 ≥ 15% from the baseline value at two points in time after exercise.
2.7 Asthma control questionnaire (ACQ)
The ACQ has strong evaluative and discriminative properties and can be used with confidence to measure asthma control (28).The ACQ assesses seven items, which include asking patients to recall their experiences in the previous week and to respond to questions about nighttime waking, symptoms on waking, activity limitations, shortness of breath, wheezing, required use of short-acting β2-agonists for rescue, and FEV1 percent predicted before bronchodilator on a 7-point scale (28).
The items were equally weighted and the ACQ score was the mean of the 7 items and therefore between 0 (totally controlled) and 6 (severely uncontrolled). Thus, patients with an ACQ < 0.75 have a good asthma control, patients with ACQ >1.5 have an uncontrolled asthma, in-between the asthma is partially controlled (29, 30). The ACQ is valid for use in children from the age of 6 (31).
2.8 Verification of PUFA incorporation by gas-chromatography
The fatty acid profile of blood plasma and blood cells were separately determined by fatty acid methyl esters (FAME) analysis as previously described (24).
The FAMEs of plasma and blood cells were analyzed by capillary gas chromatography (CGC) (Trace1300, Thermo Scientific, Dreieich, Germany) equipped with an autosampler AS1310 (Thermo Fisher Scientific, Dreieich, Germany). Fatty acid separation was achieved by using a capillary pre-column GuardGOLD, length: 2 m, i.d.: 0.25 mm (Thermo Fisher Scientific, Milan, Italy) and downstream a capillary column TRACE TR-FAME (70% cyanopropyl polysilphenylene siloxane), length: 60 m, i.d.: 0.25 mm, film thickness: 0.25 μm (Thermo Fisher Scientific, Dreieich, Germany).
The gas chromatographic conditions were the following: injector (SSL): 250°C, splitless and carrier gas: helium (purification 99%) at a flow of 20 mL x min-1. Compounds were monitored by flame-ionization detector (FID) at 250 °C. Fatty acids were identified by comparison of their retention times with those of external standard (Supelco 37-Component FAME Mix, Sigma-Aldrich, St. Louis, Missouri, United States). The column oven temperature was maintained at 60 °C for 0.5 min after injection and then programmed at 40 min to 180 °C (held for 2 min), then at 2 min-1 to 210°C (held for 3 min) and finally at 3 min-1 to 240°C (held for 10 mins). The total run-time was 44 min.
2.9 Statistical analyses
GraphPad Prism 5.01 (GraphPad Software Inc., La Jolla, CA, USA) and R 3.6 (R Foundation for Statistical Computing, Vienna, Austria) were used for statistical analyses. Data are presented as median and range or as mean and standard deviation (SD).
The primary outcome measure was the decrease in FEV1 after exercise challenge in cold air as measured by spirometry (FEV1, percent predicted). The secondary outcomes were the anti-inflammatory effects monitored by exhaled NO (eNO) before and after PUFA supplementation versus placebo, the incorporation of the different components of the sc-LCPUFA supplementation and the side effects of the sc-LCPUFA supplementation.
Intra-group and inter-group comparisons were calculated either by One-Way ANOVA, paired t-test or Wilcoxon test and unpaired t-test or Mann-Whitney test, respectively, according to the Kolmogorov-Smirnov test for normal distribution. P < 0.05 was considered as statistically significant. We also compared the results with a more complex multivariable linear mixed effect analysis, especially for potential interactions.
Power Calculation: For the sample size calculation the results of a previous study were used: “Predictors and reproducibility of exercise-induced bronchoconstriction in cold air” (27). Assuming an improvement of the maximal FEV1 decrease after ECC of 35% in the interventional group and a test power of 80% (corresponding to a probability of beta-error of β = 0.2) 30 patients in the interventional and placebo group for an effect with an alpha coefficient of 0.05 had to be investigated.