Effect of Dietary Supplemental Fish Oil in Alleviating Health Hazards Associated With Air Pollution

The investigators conduct a randomized controlled trial among 70 healthy college students in Shanghai, China. These students fulfilling the recruitment criteria will be randomly divided into two parallel groups to receive either fish oil \[marine-derived n-3 polyunsaturated fatty acids (PUFA)\] or sunflower seed oil. Participants in fish oil group receive 2.5 g/day (two 1.25-g capsules daily) in divided doses. The sunflower seed oil capsules are identical. All interventions started at 7:30 a.m.to avoid issues related to diurnal variation. Health endpoints, including blood pressure, skin color, fractional exhaled nitric oxide and pulmonary function were evaluated and biological samples such as morning urine, fast blood, saliva sample,buccal cells and skin tape stripping will be collected at week 1 before supplemenatation, and week 8, week 10, week 12 as well as week 14 during the supplementation phase.The investigators measure personal real-time exposure to PM2.5 and personal temperature and relative humidity.

• Fish oil supplementation (Dietary supplement): Participants of this randomized double-blind placebo-controlled trial include 70 healthy Chinese adults. The investigators randomly assign participants to two parallel groups at baseline using a random-number table. Compliance is determined by directly observed supplement intake. Participants in fish oil group are assigned to receive 2.5 g/day in divided doses. Each capsule contains 60% n-3 PUFA \[24% docosahexanoic acid (C22:6 n-3 DHA) and 36% eicosapentaenoic acid (C20:5 n-3 EPA)\].
• Sunflower seed oil supplementation (Dietary supplement): Participants of this randomized double-blind placebo-controlled trial include 70 healthy Chinese adults. The investigators randomly assign participants to two parallel groups at baseline using a random-number table. Compliance is determined by directly observed supplement intake. Participants in sunflower seed oil group receive 2.5 g/day in divided doses, and the capsules are identical as the fish oil capsules.

Primary outcomes

• Biomarkers of Antioxidant Activity-Total Antioxidant Capacity (TAC) and Superoxide Dismutase (SOD) [Blood samples are obtained at the end of each round of 4 follow-ups with an interval of 2 weeks in the last 2 months of intervention.]: Peripheral blood samples (5 ml) were drawn, separated into serum, and stored at -80 ℃ within 30 minutes. We measure 2 biomarkers of antioxidant activity, including TAC and SOD. The values in the following Outcome Measure Data Table refer to the means of measurements at 4 follow-ups.
• Biomarkers of Inflammation-Interleukin-6 (IL-6) and Tumour Necrosis Factor-α (TNF-α) [Blood samples are obtained at the end of each round of 4 follow-ups with an interval of 2 weeks in the last 2 months of intervention.]: 2 inflammatory biomarkers in our study including IL-6 and TNF-α are measured. The values in the following Outcome Measure Data Table refer to the means of measurements at 4 follow-ups.
• Cogulation Biomarker-von Willebrand Factor (vWF) [Blood samples are obtained at the end of each round of 4 follow-ups with an interval of 2 weeks in the last 2 months of intervention.]: We measure vWF level in serum, one of the measured cogulation biomarkers in our study. The values in the following Outcome Measure Data Table refer to the means of measurements at 4 follow-ups.
• Biomarkers of Endothelial Function and Stress Hormone [Blood samples are obtained at the end of each round of 4 follow-ups with an interval of 2 weeks in the last 2 months of intervention.]: We measure endothelial function biomarkers, including E-selectin and endothelial nitric oxide synthase (eNOS), and 4 stress hormones, including corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH), cortisol and serotonin. The values in the following Outcome Measure Data Table refer to the means of measurements at 4 follow-ups.
• Endothelial Function Biomarker-Endothelin-1(ET-1) [Blood samples are obtained at the end of each round of 4 follow-ups with an interval of 2 weeks in the last 2 months of intervention.]: We measure ET-1 level in serum, an endothelial function biomarker. The values in the following Outcome Measure Data Table refer to the means of measurements at 4 follow-ups.
• Cogulation Biomarker-Fibrinogen [Blood samples are obtained at the end of each round of 4 follow-ups with an interval of 2 weeks in the last 2 months of intervention.]: We measure the serum level of fibrinogen, one of the two measured cogulation biomarkers in our study. The values in the following Outcome Measure Data Table refer to the means of measurements at 4 follow-ups.
• High-sensitivity C-reactive Protein (Hs-CRP)-an Inflammatory Biomarker [Blood samples are obtained at the end of each round of 4 follow-ups with an interval of 2 weeks in the last 2 months of intervention.]: The serum levels of hs-CRP are measured. The values in the following Outcome Measure Data Table refer to the means of measurements at 4 follow-ups.
• Oxidized Low Density Lipoprotein (Ox-LDL)-an Oxidative Stress Biomarker [Blood samples are obtained at the end of each round of 4 follow-ups with an interval of 2 weeks in the last 2 months of intervention.]: Peripheral blood samples (5 ml) were drawn, separated into serum, and stored at -80 ℃ within 30 minutes. We measure the serum level of ox-LDL. The values in the following Outcome Measure Data Table refer to the means of measurements at 4 follow-ups.
• Glutathione Peroxidase (GSH-Px)-a Biomarker of Antioxidant Activity [Blood samples are obtained at the end of each round of 4 follow-ups with an interval of 2 weeks in the last 2 months of intervention.]: Peripheral blood samples (5 ml) were drawn, separated into serum, and stored at -80 ℃ within 30 minutes. We also measure GSH-Px. The values in the following Outcome Measure Data Table refer to the means of measurements at 4 follow-ups.
• Serum Level of Lipid Peroxidation (LPO)-a Biomarker of Oxdative Stress [Blood samples are obtained at the end of each round of 4 follow-ups with an interval of 2 weeks in the last 2 months of intervention.]: Peripheral blood samples (5 ml) were drawn, separated into serum, and stored at -80 ℃ within 30 minutes. We measure the serum level of LPO. The values in the following Outcome Measure Data Table refer to the means of measurements at 4 follow-ups.

Secondary outcomes

• Blood Pressure [Blood pressure are measured at the end of each round of 4 follow-ups with an interval of 2 weeks in the last 2 months of intervention.]: After sitting in a quiet room for at least 5 min, participants had their left upper arm blood pressure measured by trained technicians using an electronic sphygmomanometer at least three times with 3-min minimum intervals between measurements. The second and third sets of readings were averaged to obtain systolic BP (SBP) and diastolic BP (DBP). If the differences among the three measurements were bigger than 5 mmHg, a new round of measurements was arranged. The values in the following Outcome Measure Data Table refer to the means of measurements at 4 follow-ups.
• Insulin Resistance [Blood samples are obtained at the end of each round of 4 follow-ups with an interval of 2 weeks in the last 2 months of intervention.]: The biomarker of insulin resistance is measured, calculated by fasting glucose and fasting insulin using the formula of \[fasting insulin (mU/L) × fasting glucose (mmol/L)\]/22.5. The values in the following Outcome Measure Data Table refer to the means of measurements at 4 follow-ups.
• Skin Inflammation Biomarkers [Skin samplings are obtained at the end of each round of 4 follow-ups with an interval of 2 weeks in the last 2 months of intervention.]: 2 infammation biomarkers from skin samplings are measured, including interleukin-1 Alpha (IL-1α) and Interleukin-1 receptor antagonist (IL-1Rα). The values in the following Outcome Measure Data Table refer to the means of measurements at 4 follow-ups.
• Skin Oxidative Stress Biomarker-Carbonyl Protein [Skin samplings are obtained at the end of each round of 4 follow-ups with an interval of 2 weeks in the last 2 months.]: oxidative stress biomarker in skin samplings includes carbonyl protein. The values in the following Outcome Measure Data Table refer to the means of measurements at 4 follow-ups.
• Lung Function-Forced Vital Capacity (FVC) and Forced Expiratory Volume in 1 Second (FEV1) [Lung function is measured at the end of each round of 4 follow-ups with an interval of 2 weeks in the last 2 months of intervention.]: Indicators of lung function include forced vital capacity (FVC) and andforced expiratory volume in 1 second (FEV1). The values in the Outcome Measure Data Table refer to the means of measurements at 4 follow-ups.
• Lung Function-Peak Expiratory Flow (PEF) [Lung function is measured at the end of each round of 4 follow-ups with an interval of 2 weeks in the last 2 months of intervention.]: Additional indicator of lung function includes peak expiratory flow (PEF). The values in the Outcome Measure Data Table refer to the means of measurements at 4 follow-ups.
• Skin Antioxidant Biomarker-Total Antioxidant Capacity (TAC) [Skin samplings are obtained at the end of each round of 4 follow-ups with an interval of 2 weeks in the last 2 months.]: we measure TAC level, an antioxidant biomarker in skin samplings. The values in the following Outcome Measure Data Table refer to the means of measurements at 4 follow-ups.
• Skin Antioxidant Biomarker-Glutataione (GSH) [Skin samplings are obtained at the end of each round of 4 follow-ups with an interval of 2 weeks in the last 2 months.]: we measure another skin antioxidant biomarker-GSH, . The values in the following Outcome Measure Data Table refer to the means of measurements at 4 follow-ups.

Sex: All, ages: 18 Years to 27 Years, accepts healthy volunteers

Inclusion Criteria:

* Equal to or older than 18 years old.
* No history of smoking and alcohol addiction.
* No chronic diseases or respiratory or allergic diseases,such as hypertension,diabetes,chronic obstructive pulmonary disease,other respiratory/cardiovascular diseases, asthma, rhinitis or other allergic diseases reported by volunteers.
* No allergies to n-3 PUFA or fish.

Exclusion Criteria:

* Current smokers or ever smokers
* Chronic drug use due on cardiovascular or respiratory diseases
* Participants are taking fish oil,anti-inflammatory drugs, or antioxidant supplements (such as vitamin C or vitamin E)

Participant flow

Outcome results

• Biomarkers of Antioxidant Activity-Total Antioxidant Capacity (TAC) and Superoxide Dismutase (SOD) (Primary), Mean, U/ml: Fish Oil Supplementation 6.09 +/- 2.15; Sunflower Seed Oil Supplementation 5.80 +/- 2.36
• Biomarkers of Inflammation-Interleukin-6 (IL-6) and Tumour Necrosis Factor-α (TNF-α) (Primary), Mean, pg/ml: Fish Oil Supplementation 0.72 +/- 0.63; Sunflower Seed Oil Supplementation 1.76 +/- 5.75
• Cogulation Biomarker-von Willebrand Factor (vWF) (Primary), Mean, mIU/ml: Fish Oil Supplementation 1669.70 +/- 1022.98; Sunflower Seed Oil Supplementation 2144.30 +/- 1674.32
• Biomarkers of Endothelial Function and Stress Hormone (Primary), Mean, ng/ml: Fish Oil Supplementation 12.23 +/- 5.38; Sunflower Seed Oil Supplementation 14.16 +/- 6.81
• Endothelial Function Biomarker-Endothelin-1(ET-1) (Primary), Mean, pg/ml: Fish Oil Supplementation 1.80 +/- 2.26; Sunflower Seed Oil Supplementation 2.82 +/- 4.92
• Cogulation Biomarker-Fibrinogen (Primary), Mean, µg/ml: Fish Oil Supplementation 2.25 +/- 1.06; Sunflower Seed Oil Supplementation 2.63 +/- 1.28
• High-sensitivity C-reactive Protein (Hs-CRP)-an Inflammatory Biomarker (Primary), Mean, µg/ml: Fish Oil Supplementation 12.81 +/- 19.91; Sunflower Seed Oil Supplementation 19.93 +/- 30.03
• Oxidized Low Density Lipoprotein (Ox-LDL)-an Oxidative Stress Biomarker (Primary), Mean, U/L: Fish Oil Supplementation 34.74 +/- 9.85; Sunflower Seed Oil Supplementation 40.11 +/- 22.83
• Glutathione Peroxidase (GSH-Px)-a Biomarker of Antioxidant Activity (Primary), Mean, mU/ml: Fish Oil Supplementation 625.95 +/- 157.42; Sunflower Seed Oil Supplementation 581.51 +/- 171.92
• Serum Level of Lipid Peroxidation (LPO)-a Biomarker of Oxdative Stress (Primary), Mean, µg/ml: Fish Oil Supplementation 0.54 +/- 0.15; Sunflower Seed Oil Supplementation 0.58 +/- 0.17
• Blood Pressure (Secondary), Mean, mmHg: Fish Oil Supplementation 110.01 +/- 11.39; Sunflower Seed Oil Supplementation 111.55 +/- 10.89
• Insulin Resistance (Secondary), Mean, mmol/L × mU/L: Fish Oil Supplementation 1.40 +/- 0.94; Sunflower Seed Oil Supplementation 1.42 +/- 0.91
• Skin Inflammation Biomarkers (Secondary), Mean, pg/ml: Fish Oil Supplementation 184.49 +/- 107.31; Sunflower Seed Oil Supplementation 230.90 +/- 163.97
• Skin Oxidative Stress Biomarker-Carbonyl Protein (Secondary), Mean, µg/ml: Fish Oil Supplementation 35.93 +/- 15.37; Sunflower Seed Oil Supplementation 44.89 +/- 24.01
• Lung Function-Forced Vital Capacity (FVC) and Forced Expiratory Volume in 1 Second (FEV1) (Secondary), Mean, L: Fish Oil Supplementation 3.18 +/- 0.48; Sunflower Seed Oil Supplementation 3.16 +/- 0.62
• Lung Function-Peak Expiratory Flow (PEF) (Secondary), Mean, L/s: Fish Oil Supplementation 6.55 +/- 1.44; Sunflower Seed Oil Supplementation 6.38 +/- 1.60
• Skin Antioxidant Biomarker-Total Antioxidant Capacity (TAC) (Secondary), Mean, nmol/mL: Fish Oil Supplementation 42.99 +/- 95.34; Sunflower Seed Oil Supplementation 35.73 +/- 17.55
• Skin Antioxidant Biomarker-Glutataione (GSH) (Secondary), Mean, ppb: Fish Oil Supplementation 2.21 +/- 1.70; Sunflower Seed Oil Supplementation 0.42 +/- 1.07
• Differences in Metabolite Levels Detected in Serum Metabolomics Between the Two Groups (Post hoc)

Adverse events

Time frame: At the end of week 8, week 10, week 12 and week 14 during the period of supllementation

• Results publication: Zhou L, Jiang Y, Lin Z, Chen R, Niu Y, Kan H. Mechanistic insights into the health benefits of fish-oil supplementation against fine particulate matter air pollution: a randomized controlled trial. Environ Health. 2022 Oct 29;21(1):104. doi: 10.1186/s12940-022-00908-1. PMID 36309727
• Results publication: Lin Z, Chen R, Jiang Y, Xia Y, Niu Y, Wang C, Liu C, Chen C, Ge Y, Wang W, Yin G, Cai J, Clement V, Xu X, Chen B, Chen H, Kan H. Cardiovascular Benefits of Fish-Oil Supplementation Against Fine Particulate Air Pollution in China. J Am Coll Cardiol. 2019 Apr 30;73(16):2076-2085. doi: 10.1016/j.jacc.2018.12.093. PMID 31023432

• Study Protocol and Statistical Analysis Plan (PDF, 2017-12-15)

• Haidong Kan, Study director, School of Public Health，Fudan University