Flaxseed modulates inflammatory and oxidative stress biomarkers in cystic fibrosis: a pilot study

Abstract

Background: Cystic fibrosis (CF) leads to advanced lung disease despite aggressive care. Persistent inflammation and oxidative stress contribute to exacerbations and disease progression. Flaxseed (FS), a dietary botanical supplement with high fiber, lignan phenolics, and omega-3 fatty acids has anti-inflammatory and antioxidant properties in murine models of acute and chronic lung injury. This pilot study was designed to determine whether CF patients could tolerate FS, evaluate circulating FS metabolites, and study biomarkers of lung damage, as a prelude to studying clinical outcomes.

Methods: 10 CF patients and 5 healthy volunteers consumed 40 g of FS daily for 4 weeks with safety and tolerability being assessed. Urine was evaluated for systemic oxidative stress and plasma for FS metabolites (enterolignans) and cytokine levels. Buccal swabs were analyzed for gene expression of Nrf2-regulated antioxidant enzymes including Heme Oxygenase-1 (HO-1) and NAD(P)H Quinone Oxidoreductase 1 (NQO1).

Results: All subjects completed the study without serious adverse events. Plasma levels of enterolignans were detectable in both healthy controls and CF volunteers. CF patients were stratified based on plasma enterolignan levels after 2 weeks of FS administration into high- (174 to 535 nM ED and 232 to 1841 nM EL) and low- (0 to 32 nM ED and 0 to 40 nM EL) plasma lignan cohorts. The low enterolignan level cohort experienced a statistically significant drop in urinary inflammatory IsoP and plasma TNFα levels, while demonstrating higher average NQO1 mRNA levels in buccal epithelium compared to high-lignan patients.

Conclusions: This pilot study demonstrated that FS is tolerated by CF patients. FS metabolites could be detected in the plasma. Future studies will assess appropriate dosing and target populations for FS, while exploring clinical outcomes.

Trial registration: ClinicalTrials.gov identifier: NCT02014181 .

Figures

Figure 1

Study Enrollment Flow Diagram. Study enrollment opened July 2012. 67 CF cases were reviewed to determine eligibility. All cases were reviewed by 3 board-certified pulmonary and critical care physicians actively caring for adults with CF. 50 patients immediately met pre-determined exclusion criteria: actively infected, actively on intravenous antibiotics, recently had surgery, had gut issues or were known to be unreliable (>3 office no shows). 17 CF patients met eligibility criteria so were interviewed during the 3-month open enrollment period. Another 5 patients refused to participate due to distance from Philadelphia, concerns about meeting the requirements of the study, or were just not interested. Among the 12 patients that consented and began the trial, another 2 withdrew for personal reasons. Between July – November 2012, 10 CF patients completed the study.

Figure 2

Study Schematic. Total duration of the study was 8 weeks. Patients (10 adult cystic fibrosis patients and 5 healthy volunteers) were enrolled at T = 0 at which time blood, urine and buccal swabs were collected to establish baseline levels of lignans, cytokines, isoprostanes, and 8-oxo-dGuo. Urine and blood samples were drawn during each visit at 1, 2, 4, and 8 weeks. Buccal swabs were performed at weeks 0, 4, and 8 (4 weeks post-FS).

Figure 3

Safety and Tolerability of FS in Adult Cystic Fibrosis Patients. A five question survey was conducted on each clinic visit to assess: 1) adherence to the sample diet; 2) methodology of ingestion, i.e. as smoothie, condiment, etc.; 3) presence or absence of adverse events or other side effects; 4) changes in bowel habits (a known side effect of large volume flaxseed ingestion; and 5) subjective evaluation of mood and perception of wellness, How do you feel today compared to before starting flaxseed…”

Figure 4

Detection of Enterodiol in Plasma. Levels of plasma enterodiol were quantitated at weeks 0, 1, 2, 4, and 8 of the trial using LC/MS/MS. No enterodiol was detected at week 0, prior to start of trial, and at week 8, which corresponds to 4 weeks after FS was discontinued. Data is presented as plasma concentration (nM). Panel A displays comparison between healthy controls (n = 5) and all cystic fibrosis patients (n = 10). Panel B displays comparison between healthy controls (n = 5), cystic fibrosis patients with low lignans (n = 6), and cystic fibrosis patients with high lignans (n = 4). * signifies p < 0.05 for comparison between any time point in each respective cohort versus baseline (pre-FS).

Figure 5

Detection of Enterolactone in Plasma. Levels of plasma enterolactone were quantitated at weeks 0, 1, 2, 4, and 8 of the trial using LC/MS/MS. No enterolactone was detected at week 0, prior to start of trial, and at week 8, which corresponds to 4 weeks after FS was discontinued. Data is presented as plasma concentration (nM). Panel A displays comparison between healthy controls (n = 5) and all cystic fibrosis patients (n = 10). Panel B displays comparison between healthy controls (n = 5), cystic fibrosis patients with low lignans (n = 6), and cystic fibrosis patients with high lignans (n = 4). * signifies p < 0.05 for comparison between any time point in each respective cohort versus baseline (pre-FS). # signifies p < 0.05 for comparison between any time point in each respective cohort versus baseline (pre-FS), in which mean is less than baseline.

Figure 6

Effect of FS on Urinary F2a-Isoprostane Levels as a Biomarker of Systemic Oxidative Stress. F2a-isoprostane levels, a key biomarker of oxidative stress, were determined from urine samples collected during and after FS supplementation at week 0, 1, 2, 4, and 8. Levels were determined by high-performance liquid chromatography-electrospray tandem mass spectrometry and normalized to urinary creatinine. Panel A displays comparison between healthy controls (n = 5) and all cystic fibrosis patients (n = 10). Panel B displays comparison between healthy controls (n = 5), cystic fibrosis patients with low lignans (n = 6), and cystic fibrosis patients with high lignans (n = 4). * signifies p < 0.05 for comparison between any time point in each respective cohort versus baseline (pre-FS).

Figure 7

Effect of FS on Levels of Urinary 8-oxo-dGuo as a Biomarker of Systemic Oxidative Stress. 8-oxo-dGuo, one of the major products of DNA oxidation resulting from oxidative stress, was determined from urine samples collected during and after FS supplementation at week 0, 1, 2, 4, and 8. Levels were determined by high-performance liquid chromatography-electrospray tandem mass spectrometry and normalized to urinary creatinine. Panel A displays comparison between healthy controls (n = 5) and all cystic fibrosis patients (n = 10). Panel B displays comparison between healthy controls (n = 5), cystic fibrosis patients with low lignans (n = 6), and cystic fibrosis patients with high lignans (n = 4).

Figure 8

Effect of FS on Levels of Plasma Pro-Inflammatory TNFα. Levels of pro-inflammatory cytokine TNFα were determined from plasma samples collected during and after FS supplementation at week 0, 1, 2, 4, and 8. Panel A displays comparison between healthy controls (n = 5) and all cystic fibrosis patients (n = 10). Panel B displays comparison between healthy controls (n = 5), cystic fibrosis patients with low lignans (n = 6), and cystic fibrosis patients with high lignans (n = 4). * signifies p < 0.05 for comparison between any time point in each respective cohort versus baseline (pre-FS).

Figure 9

Effect of FS on Levels of Plasma Pro-Inflammatory Interleukin-1β. Levels of pro-inflammatory cytokine IL-1β involved were determined from plasma samples collected during and after FS supplementation at week 0, 1, 2, 4, and 8. Values are reported as pg per ml of plasma. Panel A displays comparison between healthy controls (n = 5) and all cystic fibrosis patients (n = 10). Panel B displays comparison between healthy controls (n = 5), cystic fibrosis patients with low lignans (n = 6), and cystic fibrosis patients with high lignans (n = 4). * signifies p < 0.05 for comparison between any time point in each respective cohort versus baseline (pre-FS).

Figure 10

FS Modifies Heme Oxygenase 1 Gene Expression in Buccal Epithelium. Buccal swabs were performed at week 0, 4, and 8 during the study. Buccal epithelial cells were harvested for RNA isolation and subsequent qPCR analysis. Antioxidant gene expression levels were determined using Taqman specific primers and probes to HO-1. Values are reported as fold change from pre-FS. Panel A displays comparison between healthy controls (n = 5) and all cystic fibrosis patients (n = 10). Panel B displays comparison between healthy controls (n = 5), cystic fibrosis patients with low lignans (n = 6), and cystic fibrosis patients with high lignans (n = 4). * signifies p < 0.05 for comparison between any time point in each respective cohort versus baseline (pre-FS).

Figure 11

FS Modifies NAD(P)H Quinone Oxidoreductase Gene Expression in Buccal Epithelium. Buccal swabs were performed at weeks 0, 4, and 8 during the study. Buccal epithelial cells were harvested for RNA isolation and subsequent qPCR analysis. Antioxidant gene expression levels were determined using Taqman specific primers and probes to Nqo1. Values are reported as fold change from pre-FS. Panel A displays comparison between healthy controls (n = 5) and all cystic fibrosis patients (n = 10). Panel B displays comparison between healthy controls (n = 5), cystic fibrosis patients with low lignans (n = 6), and cystic fibrosis patients with high lignans (n = 4).