Association between plasma polyunsaturated fatty acids and depressive among US adults

Background Depression is associated with greater functional impairment and high societal costs than many other mental disorders. Research on the association between plasma polyunsaturated fatty acids (PUFAs) levels and depression have yielded inconsistent results. Objective To evaluate whether plasma n-3 and n-6 PUFAs levels are associated with depression in American adults. Methods A cross-sectional study included 2053 adults (aged ≥20 y) in the National Health and Nutrition Examination Survey (NHANES), 2011–2012. The level of plasma n-3 and n-6 PUFAs were obtained for analysis. Self-reported Patient Health Questionnaire-9 (PHQ-9) was used to identify the depression status. Binary logistic regression analysis was performed to evaluate the association between quartiles of plasma n-3 and n-6 PUFAs and depression after adjustments for confounders. Results The study of 2053 respondents over 20 years of age with a weighted depression prevalence of 7.29% comprised 1,043 men (weighted proportion, 49.13%) and 1,010 women (weighted, 50.87%), with a weighted mean (SE) age of 47.58 (0.67) years. Significantly increased risks of depression over non-depression were observed in the third quartiles (OR = 1.65, 95% CI = 1.05–2.62) for arachidonic acid (AA; 20:4n-6); the third quartiles (OR = 2.20, 95% CI = 1.20–4.05) for docosatetraenoic acid (DTA; 22:4n-6); the third (OR = 2.33, 95% CI = 1.34–4.07), and highest quartiles (OR = 1.83, 95% CI = 1.03–3.26) for docosapentaenoic acid (DPAn-6; 22:5n-6); and the third (OR = 2.18, 95% CI = 1.18–4.03) and highest quartiles (OR = 2.47, 95% CI = 1.31–4.68) for docosapentaenoic acid (DPAn-3; 22:5n-3); the second (OR = 2.13, 95% CI = 1.24–3.66), third (OR = 2.40, 95% CI = 1.28–4.50), and highest quartiles (OR = 2.24, 95% CI = 1.08–4.69) for AA/docosahexaenoic acid (DHA; 22:6n-3) ratio compared with the lowest quartile after adjusting for confounding factors. Conclusion Higher plasma levels of AA, DTA, DPAn-6, DPAn-3 PUFAs, and AA/DHA ratio may be potential risk factors for depression in US adults.


Introduction
Depression is a common and serious mental disorder and affects more than 280 million people globally (1).In fact, the World Health Organization ranked severe depression as the third cause of burden of disease worldwide as early as 2008 and projected that the disease will be the leading cause of disease burden worldwide by the year 2030 (2).In recent years, many studies have reported understanding the role of different influence factors, such as neurotransmitter, inflammatory markers and nutritional factors, to elucidate the underlying pathophysiology of depression in adults (3)(4)(5).Polyunsaturated fatty acids (PUFAs), as important nutrients, exhibit significant effects on the composition of the intestinal microflora as well as the function of the brain (6), and participate in numerous biological processes such as oxidation, neurotransmission, and inflammation (7,8).Notably, PUFAs may play an important role in depression and its symptoms.Increasing evidence suggests that PUFAs could be associated with the pathophysiology of depression, as well as with the mechanisms underlying the therapeutic actions of antidepressants (9)(10)(11).
PUFAs are a class of fatty acids with two or more carbon-carbon double bonds (10).In human health, some PUFAs are considered essential nutrients, mainly including n-3 (primarily from fish, walnuts, wheat germ, and flaxseed) and n-6 PUFAs (primarily from refined vegetable oils such as corn, sunflower, and soybean), which cannot be synthesized in the body and must be obtained from dietary sources (8,9).Various mental disorders such as Alzheimer disease (AD), dementia, attention-deficit/hyperactivity disorder (ADHD), autism spectrum disorder (ASD), schizophrenia, Bipolar disorders (BD) and depression have been suggested to be associated with altered levels and functions of PUFAs (6,(12)(13)(14).However, inconsistent conclusions remain, especially in the studies on depression.
The association between n-3 PUFAs and depression has been extensively investigated.Many studies showed that higher levels of n-3 PUFAs, mainly eicosapentaenoic acid (EPA; 20:5n-3) and DHA, were associated with a lower risk of depression (15-22).However, a few studies observed no apparent association with n-3 PUFAs (23)(24)(25)(26)(27), and, in particular, a recent longitudinal study did not support a protective effect of n-3 PUFAs on depression risk (28).In contrast to the cumulative evidence indicating the association of n-3 PUFAs with depression, the relationship between n-6 PUFAs and depression have received much less attention.Some studies found higher levels of n-6 PUFAs related to higher severity of depression, although the results of the relevant studies have been inconsistent (27,(29)(30)(31)(32)).Okubo's study performed among Japanese breast cancer survivors indicated that a higher blood levels of n-6 PUFAs may increase the risk of depression, while the Avon Longitudinal Study (31) and Thesing's study (33) reported no association in British and Dutch populations.Moreover, studies on the association of n-6/n-3 ratio and depression are also controversial.Some cross-sectional or longitudinal studies suggested a positive association (18,(34)(35)(36), whereas several other studies reported a negative association (37) or no association (25,38).
Therefore, we comprehensively estimated the association of plasma n-3 and n-6 PUFAs and depression in a nationally representative sample of US adults aged 20 years and older.In order to provide a reference for elucidating the role of PUFAs on depression and a safer and more effective strategy to prevent or mitigate depressive symptoms in US adults.

Study design and sample
This is a cross-sectional study, done using the 2011-2012 cycle of The National Health and Nutrition Examination Survey (NHANES) data (39).NHANES is a stratified multistage probability sampling design to represent the noninstitutionalized civilian US population.Participants completed the survey through a computerassisted personal interview and a medical examination at a mobile examination center (MEC).More detailed information regarding the survey design and data collection procedure are available elsewhere (40).The study protocol was approved by the National Center for Health Statistics research ethics review board.Written informed consent was obtained for all participants.The 2011-2012 cycle was utilized since all the main independent and dependent variables of interest were available only in this dataset (especially plasma PUFA).Since depression is more common in adult group (≥ 20 years), participants who were < 20 years were excluded for this study.Participants who did not fully respond to the depression screener questionnaire (PHQ-9) were excluded from the study (41).A total of 2053 adults from the United States were included in this study.

Determination and classification of depression status
Depression status were determined based on participant's responses to the PHQ-9 questionnaire in the mental health-depression screener of questionnaire data of NHANES 2011-2012 cycle.PHQ-9 is the 9-item self-report depression scale that asks questions about the frequency of symptoms of depression over the past 2 weeks.Each item can be scored from 0 (not at all) to 3 (nearly every day) (42).The PHQ-9 score ranges from 0 to 27 and thus, classified in two categories.The individuals with PHQ-score < 9 were classified as "no or mild depression" and those with PHQ-score of 10 or more, were classified as "moderate to severe depression" (43, 44).

Assessment of plasma n-3 and n-6 PUFAs
Thirty fatty acids analyzed by means of gas chromatographymass spectrometry and expressed in μmol/L were measured in serum with the goal of obtaining US reference ranges for most circulating fatty acids in a fasting subsample of participants.Briefly, total fatty acids were hexane-extracted from the matrix (100uL serum or plasma) along with an internal standard solution for fatty acid recovery.The extract was derivatized with pentafluorobenzyl bromide to form pentafluorobenzyl esters.The reaction mixture is injected onto a capillary gas chromatograph column (45).More details about plasma fatty acids profile analysis are available in the NHANES manual (40).

Assessment of life's essential 8
LE8 scoring algorithm consists of 4 health behaviors (diet, physical activity, nicotine exposure, and sleep health) and 4 health factors (body mass index [BMI], blood pressure, glucose, lipids) (46).This study selected 5 individual LE8 metrics (i.e., blood pressure, lipids, glucose, BMI and nicotine exposure) that were more relevant to the study variables (Table 1).LE8 has great potential to assess and promote cardiovascular health (CVH) across life course.CVH is associated with cardiovascular disease (CVD), as well as non-CVD outcomes such as cognitive impairment and depression (47).CVH-related factors routinely collected (i.e., BMI, smoking, hypertension, hypercholesterolemia, and diabetes) were more relevant to our study variables and could be used to accurately estimate individuals' overall CVH across time even when LE8 metrics are incomplete (48).

Other covariates
Based on existing literatures on PUFAs and depression, age, sex, race/ethnicity (Non-Hispanic White, Non-Hispanic Black, Mexican American, Other Hispanic and Other race), educational level and poverty income ratio (PIR) were included in this study as other potential confounders (28,31,49).Highest educational level was categorized into 3 levels: (1) Less than high school/general education development (GED), (2) High school graduate/GED or equivalent and (3) higher than high school graduate/GED.PIR represent the ratio of family or unrelated individual income to their appropriate poverty threshold (50).It was categorized into three groups: (1) low: ≤ 1.3, (2) medium: 1.3-3.5, (3) high: > 3.5 (51).

Statistical analysis
Following the NHANES analytic guidelines, all analyses in this study accounted for sample weights, clustering, and stratification to generate nationally representative estimates (39).
Means and percentages of baseline characteristics were compared using t-tests for continuous variables and χ 2 test for categorical variables.PUFAs were grouped into quartiles and analyzed for the inter-quartile group trend.The lowest quartile (first quartile) was defined as the reference group in each model.Binary logistic regression analysis was used to assess the relation between quartiles of plasma PUFAs level and depression.These regression analyses employed 4 sets of covariates: model 1 was crude model; model 2 adjusted for age, sex and race/ethnicity; Model 3 adjusted the ratio of family income to poverty and personal highest education level on the basis of Model 2; and Model 4 additionally adjusted cardiovascular health on the basis of Model 3.
All statistical analyses were performed using SAS software, version 9.4 (SAS Institute, Cary, NC, United States).Statistical significance was set at 2-sided p < 0.05.This analysis was conducted using publicly available, deidentified data and was not subject to review by the Guangdong Pharmaceutical University's institutional review board.

Demographic characteristics of study population
A total of 2053 US adults aged 20-80 who participated in NHANES 2011-2012 enrolled into the study (Table 2).7.29% (weighted) subjects had a PHQ-9 score ≥ 10 and were categorized as depressed (moderate to severe depression), and 92.71% (weighted) subjects with PHQ-9 score < 10 were categorized as not depressed.The

Metric
Points and criteria

The association between PUFAs and depression: univariate analyses
The univariate analyses of PUFAs as continuous variables indicated that there was an association between part of plasma PUFAs levels and depression among US adults (p < 0.05).The weighted mean (SE) of those plasma PUFAs for GLA, DGLA, EDA, EPA, DTA, DHA, DPAn-6, DPAn-3 and AA/DHA ratio were 64.67 (1.46), 167.35 3); PUFAs as categorical variables indicated that there were a difference in quartiles of plasma PUFAs levels of DTA, DHA, DPAn-6, total n-6/n-3 ratio and AA/DHA ratio among US adults with depression and non-depression (p < 0.05; Table 4).

The association between PUFAs and depression: stratified analyses
Adjusted model was performed to observe the association between the PUFAs and depression by keeping all other study variables constant (Table 6).With increased plasma levels of AA, DPAn-3 and AA/DHA ratio as well as decreased plasma levels of DHA, women have an increased risk of depression.The risk of depression increases with increased plasma levels of AA and DPAn-6 among ≥60 years age group.Participants aged between 40 and 59 years old were more likely to develop depression due to elevated plasma DPAn-3 levels.Furthermore, elevated plasma AA/DHA levels in the 20-39 years age group may be a risk factor for depression.

Discussion
Our findings suggest that there was a significant association between plasma PUFAs with moderate or severe level of depression in American adults after controlling all potential confounders.There were higher odds of developing depression among people who have higher plasma levels of n-6 PUFAs (AA, DTA, and DPAn-6), n-3 PUFAs (DPAn-3) and AA/DHA ratio.Our analysis consolidated the risk role of higher plasma levels of AA and AA/DHA ratio in depression, and was the first to report a positive association between higher plasma DTA, DPAn-6, and DPAn-3 levels and depression in America adult.
The weighted prevalence of depression among subjects was 7.29% in our study.A report from NHANES, 2009-2012 data set has indicated that 7.6% of Americans aged 12 and over had depression (52).The incidence rate is approximately the same as our study.We assessed the participants' blood lipid levels by LE8, and the blood lipids score was 64.28 (1.14).In Lili Wang's research on the associations between LE8 and non-alcoholic fatty liver disease among US adults, the blood lipids score was 67.0 (2.7) which is also roughly in the same way as in our study (53).Overall, our findings suggest that the prevalence of depression and blood lipid levels in our study are roughly in line with the other relevant studies.
Our data provided evidence for existence of associations of depression with higher plasma levels of AA and AA/DHA ratio in American adult sample, which is consistent with some earlier research (54)(55)(56).This may reflect the opposing effects of AA and DHA on depression, although the association between plasma DHA and depression did not persist after controlling for all the potential confounding factors.AA, is an abundant n-6 PUFA in the membrane phospholipids, where it is stored in the sn-2 position of phospholipids  prostaglandins and thromboxanes.On the other hand, lipoxygenases generate both leukotrienes and anti-inflammatory eicosanoids like lipoxins.Consequently, an imbalance between these AA-derived eicosanoids has been suggested as a contributing factor to inflammatory effects.Inflammatory processes within the central nervous system (CNS) are essential for the development of brain pathologies, including depression (59).Increased levels of inflammatory cytokines have been reported in depressed patients (60), and found to promote abnormalities in neurotransmitter metabolism and neuroendocrine function, which are related to the pathophysiology of depression (61).Whereas DHA, an n-3 PUFA, has a certain anti-inflammatory effect through competition with AA.Furthermore, DHA also has a neuroprotective effect against decreased neurogenesis and increased neuronal apoptosis (22).Increased plasma levels of AA and/or decreased DHA may cause a disproportionate ratio of n-6 to n-3 and lead to depression through increased inflammatory processes.This study also found elevated plasma level of other n-6 PUFAs such as DTA and DPAn-6 in patients with moderate to severe depression.DTA, a 2-carbon elongation product of AA, can be desaturated to generate DPAn-6 (62).Previous studies have shown that DTA can impair neurobehavioral development by increasing reactive oxidative species production in Caenorhabditis elegans (63), and higher DPAn-6 status also showed an association with worse mental health in older adults with mild cognitive impairment (64) and children and adolescents with bipolar disorder (65).The Western diet, which is high in processed foods with fat and sugar, is increasing the risk of depression (66).In addition, reactive oxygen species and inflammatory cytokines that could be induced by unreasonable intake of PUFAs have the importance in the progression of depression (67).Previous studies have shown that some n-6 PUFAs are able to activate inflammatory responses and lead to the accumulation of reactive oxygen species and pro-inflammatory factors (68, 69).These alterations might affect the integrity of the cell membrane, leading to alteration of intestinal flora, systemic inflammation and abnormal neurotransmitter transport in the brain (6,70).In summary, abnormalities in plasma n-6 PUFAs metabolism may be involved in the progress of depression.The DPAn-3 is less studied as a new player in the n-3 PUFAs family, compared to its counterparts EPA and DHA.The literature on DPAn-3 is limited, however most of the available data suggests it has beneficial health effects which is contrary to our found that higher DPAn-3 was associated with depression.Our result agrees with a minority of reports concerning DPAn-3 (71).More research remains to be done to further investigate the biological effects of this DPAn-3.
There were also some studies that did not observe an association between plasma PUFAs levels and depression (31,33,49,72,73).But most of these studies have focused only on the total n-3 PUFAs and/ or the total n-6 PUFAs, or the canonical fatty acids among them.A longitudinal study in middle-aged Finnish men did not find evidence that serum polyunsaturated fatty acids would be associated with risk of depression (38).The composition ratio of sex may partially influence results.According to previous studies of dietary intervention in Western countries, the association appears to be observed more in women than in men (21, 74).We also observed a significant association for AA, DHA, DPAn-3, and AA/DHA ratio in women through a sub-analysis stratified by sex.However, this finding should be interpreted with caution in light of the source of PUFAs.
One of the strengths of this study is the use of data from a nationally representative survey with a large sample size from a multiracial/multiethnic population.Even a large number of analyses have been conducted to assess the role of PUFAs in depression from multiple perspectives, especially at the supplement and diet levels, but only a few studies with small sample sizes have examined the association between depression and plasma PUFAs.To our knowledge, our study is the first observational study to examine the association between plasma PUFAs and depression among American adult using part of the LE8 indicators as the primary covariate.
Another strength of this study is that we used plasma PUFAs, which better reflects tissue levels of PUFAs than the more subjective measures of the Food Frequency Questionnaires (FFQ) and food records.Several limitations also need to be acknowledged.A major limitation is that this study is only a cross-sectional exploratory investigation, so a causal relationship cannot be concluded.Additionally, the PHQ-9 items measure only a subset of the symptoms of depression.Apart from the nine symptoms of the PHQ, other depressive symptoms were not included in this study which may be counted as a limitation.

Conclusion
In conclusion, this population-based cross-sectional study examining the association between plasma PUFAs and depression risk in American adults revealed an association between higher plasma levels of AA, DTA, DPAn-6, DPAn-3 and AA/DHA ratio and increased risk of depression.The complexity of PUFAs metabolism, interactions and competition between n-3 PUFAs and n-6 PUFAs and the mechanism by which these may influence depression requires continued study.

TABLE 1
Scoring criteria of CVH-related factors metrics based on life's essential 8.

TABLE 2
Descriptive characteristics of all Participants for total sample and by depression level groups among US adults in NANES, 2011-2012.

TABLE 3
Univariate analyses results of the relationship between continuous plasma n-3 and n-6 polyunsaturated fatty acids and depression in American adults: NHANES, 2011-2012.

TABLE 4
Univariate analyses results of the relationship between quartiles of plasma n-3 and n-6 polyunsaturated fatty acids and depression in American adults: NHANES, 2011-2012.
(57).AA-derived eicosanoids from cyclooxygenase (COX) pathways or lipoxygenase (LOX) pathways are important lipid mediators involved in a number of physiological and pathophysiological processes ranging from inflammation, allergic responses, blood lipid regulation, to cell metabolism (58).Cyclooxygenases participate in the production of pro-inflammatory eicosanoids, such as

TABLE 5
Association of plasma n-3 and n-6 polyunsaturated fatty acids levels with depression in American adults: NHANES, 2011-2012 a .