1. Introduction
Postmenopausal, obese women are vulnerable to chronic inflammation and associated diseases [
1]. Reducing chronic inflammation is associated with better health outcomes [
2]. The very long-chain n-3 polyunsaturated fatty acids (n-3 PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), found in high amounts in fatty fish [
3], reduce circulating inflammatory markers in some [
4,
5,
6] but not all studies [
7,
8]. Mechanisms underlying effects of EPA+DHA may include the low inflammatory potential of their metabolites [
9]; downregulation of pro-inflammatory cytokines, including interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα), [
10,
11]; and production of metabolites with strong inflammation resolving properties [
12].
PUFAs of the n-6 family, primarily linoleic acid (LA) and arachidonic acid (AA), also have been studied in relation to inflammation [
13,
14]. AA-derived eicosanoids are elevated in individuals with highly inflammatory conditions but seem to have both potent pro-inflammatory and anti-inflammatory effects [
15].
PUFAs have also been found to impact quality of life (QOL). Among older women with depression, supplementation with n-3 PUFAs was associated with significant improvements in physical and mental QOL [
16]. Postmenopausal women supplemented with n-3 PUFAs reported improvements in menopausal-specific QOL, but not significantly different from placebo [
17]. However, not all randomized controlled trials have reported benefits of n-3 PUFAs on QOL [
18].
Prior analyses of data from this intervention found that a diet low in added sugars, high in fiber, and high in n-3 PUFAs from fatty fish was associated with reduced inflammation in 14 post-menopausal women with obesity who completed a 12-week pilot study [
19]. In this analysis, we examined change in erythrocyte PUFAs and association with markers of inflammation and QOL in women who completed the pilot dietary intervention. We hypothesized that participants’ erythrocyte EPA and DHA would increase and be associated with lower concentrations of inflammatory markers and better QOL at end of intervention and follow-up compared to baseline.
2. Materials and Methods
The Ohio State University Institutional Review Board approved this study and all participants provided written informed consent. An experimental pre-post test, single group design was used. The pilot study, evaluating a 12-week dietary intervention to reduce inflammation, is described in detail elsewhere [
19]. Briefly, participants received weekly tailored nutrition counseling for 12 weeks. Specific study goals included increasing fatty fish intake to ≥3 servings/week, increasing fiber intake to ≥20 g/day, and reducing added sugar intake to ≤10% of total kcals/day. A 12-week maintenance period without contact from researchers followed the end of the intervention, and participants returned for one follow-up visit 24-weeks from baseline.
A sample size of 15 was chosen to provide >80% power to detect changes in nutritional biomarkers (e.g., plasma and red blood cell n-3 PUFAs), based on previously reported effect sizes [
20]. Inclusion criteria included females age ≥40 years with obesity (BMI > 30 kg/m
2) who were post-menopausal (defined as self-reported amenorrhea ≥12 months) and willing to make dietary changes. Exclusion criteria included self-reported current inflammatory disease, type 1 or type 2 diabetes mellitus, symptomatic heart disease, and daily use of NSAIDs, aspirin, hypoglycemic medications, or high dose n-3 PUFA supplement (>360 mg EPA + DHA/day). Volunteers were recruited from central Ohio and enrolled in 2015. An in-person visit confirmed eligibility via height and weight measurement to calculate BMI.
Outcome measurements were collected at baseline, end of intervention (week 12) and follow-up (week 24). Body weight and height were measured using a research quality scale and stadiometer (Seca Co., Hamburg, Germany). Usual dietary intake over the previous twelve weeks was assessed using Vioscreen, a validated web-based tool including a graphical self-administered electronic food frequency questionnaire (VioCare, Inc., Princeton, NJ, USA) [
21]. Self-reported QOL was assessed using the 12-Item Short-Form Health Survey (SF-12), a valid and reliable tool for measuring physical and mental QOL [
22].
Fasting whole blood was collected and stored at −80 °C. Serum inflammatory markers, IL-6, TNFα receptor-2 (TNFα R-2), and high sensitivity C-reactive protein (CRP), were analyzed by electrochemilluminescence and read using the Meso Quick Plex SQ 120 (Meso Scale, Discovery, Rockville, MD, USA) at the Ohio State University Clinical Research Center as previously reported [
19].
Lipids were extracted from erythrocytes, and fatty acids (FAs) were methylated using boron trifluoride as previously described [
23,
24,
25]. FA methyl esters were analyzed using a gas chromatograph (Shimadzu Scientific Instruments, Columbia, MD, USA) equipped with a 30-m Omegawax TM 320 fused silica capillary column (Supelco, Bellefonte, PA, USA) according to established methods [
26]. Retention times of samples were compared to standards (Matreya, LLC, Pleasant Gap, PA, USA; Supelco, Bellefonte, PA, USA; and Nu-Check Prep Inc., Elysian, MN, USA). FAs are reported as a percent of total FAs [
27]. Representative coefficients of variation from eight duplicate samples were: EPA, 6.6%; DHA, 3.8%; LA, 1.2%; AA, 0.9%.
The primary objectives of this analysis were to (1) determine the effects of a dietary intervention on erythrocyte PUFA composition; and (2) to examine the relationships between erythrocyte PUFAs and inflammatory markers. The secondary objectives were to determine correlations among self-reported intake of dietary PUFAs, QOL outcomes, and erythrocyte PUFAs. All analyses were conducted using JMP (version 11.2.1, SAS Institute Inc., Cary, NC, USA). Repeated measures analysis of variance and post-hoc t-tests evaluated changes over time in erythrocyte FAs, inflammatory markers, QOL outcomes, and dietary variables. Pearson correlations quantified cross-sectional associations between erythrocyte FAs, inflammatory markers, QOL outcomes, and dietary variables. All dietary data were energy adjusted prior to correlation analyses. Due to significant weight loss observed post-intervention and at follow-up [
19], partial correlations were used to adjust for percent weight change, a potential confounder of the relationships of erythrocyte FAs with inflammatory markers and QOL outcomes.
4. Discussion
The results of this analysis support the efficacy of this behaviorally-based dietary intervention to raise biomarkers of n-3 PUFA intake. However, despite significant increases in erythrocyte EPA and DHA, and reductions in TNFαR-2 over the course of the study [
19], erythrocyte n-3 PUFAs were not associated with any measured inflammatory marker. Higher erythrocyte AA was associated with significantly higher CRP. Higher erythrocyte LA was associated with better physical functioning on the SF-12 QOL assessment at the end of the intervention.
As women in this study increased fatty fish intake to reach the goal of three 3-oz. servings per week [
19], dietary EPA and DHA increased by 0.12 g/day (0.07% kcal) and 0.24 g/day (0.14% kcal) respectively, by end of the intervention. The mean value of the Omega-3 index increased by 1.37% from baseline, and self-reported intakes of EPA and DHA were significantly associated with their respective erythrocyte PUFAs at all time points. These results suggest that participants experienced a dose response increase in erythrocyte EPA and DHA with increasing dietary EPA and DHA, as reported by others [
25,
28,
29].
Potential explanations for the lack of association between erythrocyte EPA or DHA and TNFαR-2, IL-6 or CRP exist. Participants may not have consumed enough n-3 PUFAs to realize anti-inflammatory benefits despite meeting targeted fatty fish intake. Women in the current study achieved a mean intake of EPA+DHA of 0.52 g/day by the end of the intervention. Although within the range recommended for cardiovascular benefit [
29], this amount may not be sufficient to reduce chronic inflammation [
5], particularly as those with obesity may require a larger dose of n-3 PUFAs to achieve the same targeted erythrocyte level as lean counterparts [
4]. Additionally, women in this study had a low baseline Omega-3 index of 3.54%, even less than the typically low Omega-3 Index of ~4–4.5% reported in adults not taking fish oil supplements and consuming a Western-style diet [
29]. Participants reached a mean Omega-3 index of 4.91% by the end of the intervention but did not approach the level of 8% suggested by some researchers as optimal [
30], although this target is based on cardiovascular benefit. It is also possible that our sample size was too small to detect relationships between n-3 PUFAs and inflammation, as power analysis was based on change in PUFA biomarkers as described previously [
19].
Mean erythrocyte AA content significantly increased from baseline to follow-up in our participants; however, erythrocyte AA was not associated with dietary intake of AA. Increasing erythrocyte AA could be related to our participants’ weight loss. In some studies of obese adults with non-alcoholic fatty liver disease [
31,
32], AA in erythrocyte phospholipids was low compared to lean controls. Weight reduction following bariatric surgery resulted in normalization of erythrocyte AA in these patients [
32]. Similarly, diet-induced weight reduction of >5% increased erythrocyte AA, improved erythrocyte membrane fluidity, and improved insulin sensitivity in overweight and moderately obese women [
33]. The authors suggest that increasing membrane fluidity via increasing AA may contribute to some of the beneficial metabolic outcomes associated with weight loss [
33]. Despite this potential metabolic benefit, we observed a direct association between erythrocyte AA and inflammation (i.e., CRP) in women at follow-up, although overall CRP trended downward in our study. Similar to our results, AA levels in blood and tissue have been associated with increased inflammation in some studies [
4,
15]. Reductions in erythrocyte AA have been reported when supplemental EPA+DHA was provided to healthy lean adults, but not until doses reached 0.6 g/day [
14,
29], which is greater than our participants’ mean intake. Taken together, these studies support the crucial role of adequate, but not excessive, AA in cellular membranes.
At the end of the intervention, higher erythrocyte LA was significantly associated with a better physical function score on the SF-12 QOL measure after adjusting for percent weight change. Similarly, higher erythrocyte EPA approached significance (
p = 0.051) with better physical function score. Beneficial effects of EPA and DHA supplementation on physical and mental function scores from the SF-36, a more comprehensive QOL tool, have been reported in elderly, depressed women who were supplemented with 2.5 g of EPA+DHA versus paraffin placebo for two months in a randomized, double blind, controlled trial [
16]. In another randomized, controlled trial of elderly people with mild cognitive impairment, approximately 2 g of either EPA, DHA or LA were supplemented for 6 months; despite improvements in depressive symptoms with EPA and DHA versus LA, none of the supplements significantly affected QOL measured by SF-36 [
34]. Women in our study did not consume as high a dose of EPA and DHA as achieved in these supplementation studies, but still appeared to obtain some marginal benefit on self-reported QOL by increasing EPA and maintaining LA.
Limitations of this pilot study include the small sample size with minimal diversity and lack of a control group. Additionally, women lost weight during the intervention, which could have been a confounding factor; we addressed this by adjusting analyses for weight change. Strengths of this study include the use of a validated, objective biomarker of EPA and DHA intake and n-6 PUFA status, and measurement of multiple biomarkers of inflammation across time.