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Neuroendocrinology Letters Volume 43 No. 3 2022
ISSN: 0172-780X; ISSN-L: 0172-780X; Electronic/Online ISSN: 2354-4716
Web of Knowledge / Web of Science: Neuroendocrinol Lett
Pub Med / Medline: Neuro Endocrinol Lett

Elevated ratio of serum anandamide to arachidonic
acid intake in community-dwelling women withhigh depressive symptoms

1 Institute of Medical, Pharmaceutical & Health Sciences, Kanazawa University, Kanazawa 920-1192,Japan
2 Department of Health and Psychosocial Medicine, Aichi Medical University School of Medicine,

Nagakute 480-1195, Japan
3 Department Hygiene and of Public Health, Graduate School of Medicine, Kanazawa University,

Kanazawa 920-8640, Japan
4 Department of Bioinformatics and Genomics, Graduate School of Advanced Preventive Medical

Sciences, Kanazawa University, Kanazawa 920-8640, Japan
Correspondence to:

Hirohito Tsuboi, MD, PhD
Institute of medical, pharmaceutical and health sciences, Kanazawa University,
Kakuma-machi, Kanazawa 920-1192, Japan
tel: + 81 76 234 4403, e-mail: tohtori17@gmail.com

Submitted: 2022-01-19
Key words:

Accepted: 2022-07-08

NEL430322A03 © 2022 Neuroendocrinology Letters • www.nel.edu

OBJECTIVES: The purpose of the present study was to investigate the serum
levels of endocannabinoids (eCBs; anandamide: AEA and 2-arachidonoylglycerol: 2-AG) and daily intake of polyunsaturated fatty acids (PUFAs; arachidonic
acid: ARA, docosahexaenoic acid: DHA, and eicosapentaenoic acid: EPA) among
subjects with high and low depressive symptoms.
METHODS: The participants comprised female community-dwellers aged 40 years
or older in Japan. Among 208 females, fourteen participants with high depressive
symptoms and ten participants with low depressive symptoms were selected forthis study. The depressive symptoms were measured by the Japanese version of theCentre for Epidemiologic Studies Depression Scale (CES-D). The daily intake
of PUFAs were assessed utilising the brief-type self-administered diet history
questionnaire. The blood samples were analysed for AEA, 2-AG, and the CB
receptor 1 gene (CNR1) single nucleotide polymorphism (SNP) rs806377.
RESULTS: The ratio of AEA serum level to ARA intake (AEA/ARA) in highdepressive participants was significantly higher compared with those in low
depressive participants even after controlling for confounders, whereas there
were no significant differences in the serum concentrations of eCBs, daily intake
of PUFAs, as well as the CNR1 SNP (rs806377) between the high and low CES-D
scored groups.
CONCLUSION: The elevated level of AEA/ARA among high depressive participants suggests that the conversion rate of ARA to AEA may be accelerated indepressive individuals.
To cite this article: Neuroendocrinol Lett 2022; 43(3):145–153

A R T I C L E

Anandamide; Arachidonic acid; 2-arachidonoylglycerol; Depression;
Docosahexaenoic acid (DHA); Eicosapentaenoic acid (EPA); Endocannabinoids;
Female population; Polyunsaturated fatty acids

Neuroendocrinol Lett 2022; 43(3):145–153 PMID: 36179725

Abstract

Published online: 2022-07-10

O R I G I N A L

Hirohito Tsuboi1, Masahiro Matsunaga2, Hiromasa Tsujiguchi3, Takayuki Kannon4,
Kazuyoshi Hosomichi4, Takehiro Sato4, Atsushi Tajima4, Naoko Yoshida1,
Akinori Hara2, Hiroyuki Nakamura2

Tsuboi et al: Anandamide, arachidonic acid, and depression
Abbreviations:eCBs- endocannabinoids
CNS- central nervous systems
2-AG- 2-arachidonoylglycerol
AEA- arachidonoylethanolamide (anandamide)
ARA- arachidonic acid
BDHQ.
- brief-type self-administered diet history
questionnaire
BDI- Beck Depression Inventory
BMI- body mass index
CB1R- endogenous cannabinoid receptors-1
CB2R- endogenous cannabinoid receptors-2
CNR1- CB1R gene
COVID-19- coronavirus disease 2019
DHA
- docosahexaenoic acid
DHEA
- ethanolamide derivatives docosahexaenoyl
ethanolamine
DHQ- diet history questionnaire
EPA- eicosapentaenoic acid
EPEA
- eicosapentaenoyl ethanol-amide
FAAH- fatty acid amide hydrolase
MAGL- mono-acylglycerol lipase
MDD- major depressive disorder
PUFAs- polyunsaturated long-chain fatty acids
QC- quality control
S.D.
- standard deviation
S.E.
- standard error
SNP- single nucleotide polymorphism

INTRODUCTION
The endocannabinoids (eCBs) are endogenous ligands
for cannabinoid receptors and the eCB system is implicated in various psychosomatic processes such as
depression, anxiety, chronic pain, and obesity (Chanda
et al. 2019). The eCB system is a neuromodulator
comprised of two endogenous cannabinoid receptors-1(CB1R) and -2 (CB2R), as well as their two primaryendogenous ligands, arachidonoylethanolamide (AEA;
a.k.a. anandamide) and 2-arachidonoylglycerol (2-AG)(Hillard, 2015). Although cannabinoid binding sites
exist throughout the central nervous systems (CNS) andthe peripheral nervous systems, circulating eCB concentrations are hypothesized to be biomarkers of CNS eCB
signalling, and are also implicated in psychologicaldepression (Hillard, 2018). Women with major depressive disorder (MDD) had lower serum 2-AG concentrations compared to non-depressed controls (Hill et al.
2009), and symptoms of depressed mood were inversely
correlated with the 2-AG concentration (Stensson
et al. 2018). Meanwhile, another study found significantly higher circulating concentrations of both AEA
and 2-AG in MDD patients (Romero-Sanchiz et al.
2019). A study in a sizable group of participants withand without a history of psychiatric disorders showeda relationship between the circulating levels of eCBs
and affect regulation (Coccaro et al. 2018). The strongest relationship was observed between the affect intensity and the circulating AEA levels, even in the absenceof relationships categorised by the Diagnostic andStatistical Manual of Mental Disorders, Fifth Edition(DSM)-5 or state dimensional measures of depression
or anxiety (Coccaro et al. 2018). Furthermore, the eCBs

146

system can play a role in the remission of depression.
Administration of an eCB agonist to healthy volunteers
induced a network-wide shift of the brain from a bias
for negative emotional content towards a bias for positive emotional content (Bossong et al. 2013). The activation of the CB1R, which is encoded by the CB1R gene
(CNR1), may be effective as a treatment for depression.
By analysing the single nucleotide polymorphism (SNP)
variability in the CNR1 gene (rs806377) (Matsunaga
et al. 2014), we showed that the subjective happinesslevel was higher in the C carriers, which have a highbinding capability to the eCB receptor compared to the
TT genotype. However, other researchers indicated no
influence of the rs806377 on the overall susceptibility
to MDD (Chakrabarti and Baron-Cohen, 2011).
AEA and 2-AG are the most well-studied eCBs, which
are derived mainly from arachidonic acid (ARA), oneof ω-6 polyunsaturated long-chain fatty acids (PUFAs)(Dyall, 2017, Hansen and Artmann, 2008). Moreover,
AEA and 2-AG are degraded into ARA primarily by theserine hydrolase enzymes fatty acid amide hydrolase(FAAH) and mono-acylglycerol lipase (MAGL), respectively (Blankman & Cravatt, 2013). It was reported that
depressive symptoms of white-collar workers were
negatively correlated with the percentage of ARA in theserum (Tsuboi et al. 2013). Among the PUFAs, not only
ARA but also ω-3 PUFAs are also involved in the function of the eCB system. Docosahexaenoic acid (DHA)
and eicosapentaenoic acid (EPA) are major ω-3 PUFAs,
which can convert to their ethanolamide derivatives
docosahexaenoyl ethanolamine (DHEA) and eicosapentaenoyl ethanol-amide (EPEA), respectively. DHEA
and EPEA exhibit binding affinity and agonist activity,
though low, on eCB receptors (McDougle et al. 2017).
Namely, DHEA and EPEA function as CB1R and CB2R
agonists (Brown et al. 2010). Furthermore, cytochrome
P450 epoxygenase (CYP) converts DHEA and EPEA
into epoxydocosapentaenoic acid-ethanolamide (EDPEA) and epoxides epoxyeicosatetraenoic acid-ethanolamide (EEQ-EA), respectively, both of which are
CB1R and CB2R agonists (McDougle et al. 2017). In
epidemiological studies, it is well-known that the intake
of DHA or EPA has an inverse association with depression, especially in women (Colangelo et al. 2009, Tsuboi
et al. 2019). Thus, DHA and EPA intake can be thought
to be highly relevant with AEA and 2-AG functions.
We have been investigating factors that maintain
psychological wellbeing in everyday life. Although
MDD and depressive symptoms in the non-clinicalpopulation should be distinguished, assessing depressive symptoms and the prevention of depression is
crucial, because depression is associated with anincreased risk of mortality in general community populations, as well as in patient populations with chronic
illnesses (Cuijpers et al. 2014). Additionally, in the last
few years in Japan, coronavirus disease 2019 (COVID19) pandemic increased the rates of probable depression by 2- to 9-fold during the second wave compared

Copyright © 2022 Neuroendocrinology Letters ISSN 0172–780X • www.nel.edu

Tsuboi et al: Anandamide, arachidonic acid, and depression

Fig. 1. Distribution of the Centre for Epidemiologic Studies Depression Scale (CES-D) scores among 208 female
participants. Fourteen high depressive and ten low depressive women were selected for the present study.

with the pre-COVID-19 levels (Fukase et al. 2021). The
COVID-19 pandemic is also contributed to the psychological status of many individuals all over the world
as reviewed in previous articles. (Dong et al. 2021,
Rodríguez-Fernández et al. 2021)
Thus, we hypothesized (1) that the serum eCB
concentrations in low depressive people were higher
compared with those in high depressive people, and(2) that people with low depressive symptoms tend
to consume higher ARA, DHA, and EPA. In the present
study, we selected high and low depressive women from
community-dwelling people, and measured the serum
concentrations of AEA and 2-AG. We also assessed thenutritional intake of PUFAs in the study participants. In
addition, the rs806377 SNP of CNR1 was considered as
one of the covariates. The purpose of the present study
was (1) to compare the AEA and 2-AG levels in the
peripheral blood between the high and low depressive
subjects, and (2) to investigate the associations among
eCBs, nutrient intake, and de-pressive symptoms.

PARTICIPANTS AND METHODS
Study Population
The participants were selected from individuals in the“Shika study” project, which was conducted since 2011
in Noto Peninsula, Ishikawa, Japan. The details of thisstudy have been reported elsewhere (Center, 2016). The
study protocol was approved by the Ethical Committee
at Kanazawa University on 18 December 2013 (receipt
number 1491). Among 492 subjects aged 40 or older,409 individuals [200 men: mean age 59.1 (standard
deviation: S.D. 11.5), 208 women: mean age 61.1 (S.D.
12.9), 1 unknown] provided written informed consent

on research participation. All the respondents wereliterate, understood the Japanese language well, andwere requested not to use proxy respondents.
For the current study, we selected 14 high and
10 low depressive women that were age-matched as
possible, using the Japanese version of the Centre for
Epidemiologic Studies Depression Scale (CES-D)(Radloff, 1977, Shima et al. 1985). The reason that
we selected only women was because the association
between depression and ω-3 PUFAs were stronger
in women than in men (Colangelo et al. 2009), andbecause of sex matching.
Procedures and questionnaires
A self-administrated questionnaires were sent by post
to the participants beforehand and collected on theexamination day, when unclear parts of the questionnaires were addressed and corrected by the interviewers.
The questionnaire contained demographic measures(age, sex, height, weight, present health status, diseases,
medication, etc.), lifestyle characteristics (smoking
status, alcohol consumption, etc.), and socio-economic
status (living with family or alone, years of education,etc.). The entire process was conducted in a mannerthat protected the attendees’ privacy.
Nutrient intake was assessed using the brief-type
self-administered diet history questionnaire (BDHQ).
The BDHQ is a four-page structured questionnaire thatassesses the consumption frequency of 58 foods andbeverages commonly consumed by the general Japanese
population (Sasaki, 2004). It is a modified form of theself-administered diet history questionnaire (DHQ)(Sasaki et al. 1998), and was designed to be completed
within approximately 15 minutes. The BDHQ estimates

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147

Tsuboi et al: Anandamide, arachidonic acid, and depression
Tab. 1. Comparison of variables between high and low depressive participants
VariableHigh depressive (n=14)

Low depressive (n=10)

p value

Basic values
Age61.8 (13.56)

60.6 (12.8)

0.83

BMI

22.4 (3.49)

23.0 (3.67)

0.71

24.4 (8.1)

0.8 (1.03)<0.0005

T/T

4

4

T/C

4

4

C/C

2

0

Non-smoker

12

9

Ex-smoker

01

Present smoker

20

No habit

11

8

Several days per week30

Depressive symptoms
CES-D scores
SNP (rs806377)
0.41

Smoking habit
0.24

Alcohol consumption

EverydayYears of education
Solitary life

0.93

0

2

11.5 (2.92)

10.4 (1.51)

0.31

2

1

0.37

Values are expressed as mean (S.D.), p values generated t test.

dietary intake in the last month using an ad hoccomputer algorithm. The validity of the BDHQ has
been demonstrated in previous studies (Kobayashi et al.
2011). In order to analyse nutrient data, the density
method was used to estimate intake per 1000 kcal.
Blood Collection
Fasting blood was sampled between 08.00 a.m. and12.00 p.m. from the forearm vein of each participant.
The samples were collected into heparinized andse-rum-separator vacutainer tubes, from which sera
and blood clots were obtained by incubating the tubes
until the sera and clots were separated. The samples
were then de-livered to Kanazawa University througha commercial laboratory (SRL Kanazawa Laboratory,
Kanazawa, Japan) where the sera were dispensed into
other tubes. The sera and the clots were frozen andstored at -30°C until the assay.
Measurement of AEA and 2-AG
The serum samples were analysed for AEA and 2-AG,
using an ELISA kit for AEA (Cloud-Clone Corp., TX,
USA) and an ELISA kit for 2-AG (Cloud-Clone Corp.,
TX, USA), respectively.
Genotyping of the CB1R Gene
Genomic DNA was extracted from blood lymphocytes using the QIAamp DNA Blood Maxi kit (Qiagen,148

Hilden, Germany) according to the manufacturer's
instructions or consigning the samples to a company
specializing in clinical laboratory testing (SRL, Inc.,Tokyo, Japan). Genome-wide SNP genotyping was
performed using the Japonica Array v2 (Kawai et al.
2015) (TOSHIBA Co., Ltd. Tokyo, Japan). The quality
control (QC) procedures for the obtained genome-wide
SNP genotype data have been described previously(Nomura et al. 2021). Briefly, QC filtering of SNPs andparticipants was based on gender identity between
karyotype and questionnaire, call rate, Hardy-Weinberg
equilibrium test, inbreeding coefficient, cryptic relatedness, and population structure. The genotypes of SNP
rs806377 (T/C), which is located in the CNR1 gene,
were extracted from the array data from 851 unrelated subjects (based on genome-wide π values), which
passed the QC. In the QC step, the call rate for the SNP
was 99.8%, and the deviation from the Hardy-Weinberg
equilibrium was not observed (p = .478). Subsequently,
18 participants data were used in the present study.
Statistical Analysis
The Japanese version of the IBM SPSS Statistics version
25 software (IBM Japan, Tokyo, Japan) was utilized
for data analysis. The high and low depressive individuals were compared using age, BMI, lifestyles,
nutritional intake, and serum indicators. These comparisons were analysed with unpaired t-tests, followed by

Copyright © 2022 Neuroendocrinology Letters ISSN 0172–780X • www.nel.edu

Tsuboi et al: Anandamide, arachidonic acid, and depression

a linear analysis to adjust for possible confounders.
Subsequently, hierarchical models were applied to identify the effects of confounding factors. Chi-square test
was applied for the comparison of the qualitative values.
The results were considered to be statistically significant when p < 0.05.

RESULTS
As indicated in the method section, 14 participants withhigh depressive symptoms and 10 with low depressive
symptoms were analysed (Figure 1). All the participants
lived independently, and none used nursing services.
There were no significant differences in variables
between the high and low depressive symptoms groups
with the exception of CES-D scores ( t-test: p < 0.0005)(Table 1); i.e., there were no significant differences inage, body mass index (BMI), CNR1 SNP (rs806377),
smoking habit, alcohol consumption, years of education, and whether the participant lives alone (Table 1:
t-test for quantitative variables and Chi-square test
for qualitative variables; p = 0.83, p = 0.71, p = 0.41,
p = 0.24, p = 0.93, p = 0.31, p = 0.35, respectively).
Meanwhile, t-test and linear models revealed that theratio of the serum levels of AEA to ARA intake volume(AEA/ARA) in the high depressive group was significantly higher compared with those in the low depressive group (Model 1, 3, 4, 5: p < 0.05, p < 0.05, p < 0.01,
p < 0.01, respectively) (Table 2). In contrast, there wereno significant differences in the ratios of the serum
2-AG levels to PUFA intake volume (2-AG/ARA) as
well as mere PUFA intake levels or serum eCBs levels

between the high and low depressive groups (Table 2).
The AEA/ARA data remained significant even after
controlling for possible confounders including age,
BMI, current smoking status, alcohol consumption,
the CNR1 SNP (rs806377), and whether the participant
lives alone (Table 2). The eCBs/ARA distributions withthe average scores and standard error (S.E.) were represented in Figure 2.

DISCUSSION
The present study compared the serum eCB (AEA and2-AG) levels and daily intake volumes of PUFAs (ARA,
DHA, and EPA) among high and low depressive female
community-dwellers with possible confounders. We
found a significant elevated level of AEA/ARA amonghigh depressive participants, even when controlling forthe confounders. Contrarily, there were no significant
differences in other eCBs, PUFAs, or the ratios of eCBs/
PUFAs between the high and low depressive participants.
The reason that we evaluated the ratio of eCBs (AEA,
2-AG) to PUFAs (ARA, DHA, EPA) was that AEA and
2-AG derive from ARA, and that DHA and EPA (Dyall,
2017, Hansen & Artmann, 2008), including their metabolites DHEA, EPEA, EDP-EA and EEQ-EA, can work as
CB1R and CB2R agonists (Brown et al. 2010).
This study did not confirm our hypothesis that low
depressive individuals had higher serum levels of eCBs
and higher intake of ARA, DHA, and EPA. Nonetheless,we identified a new finding. Specifically, the association
between depressive degrees and AEA/ARA ratios incommunity-dwelling women.

Tab. 2. Comparison of variables between high and low depressive groupsHigh
depressive(n=14)

Low
depressive(n=10)

Model 1

AEA (ng/mL)

39.6 (29.25)

26.0 (23.73)

1.21

0.28

0.43

0.64

0.67

2-AG (ng/mL)

41.5 (33.91) a

34.3 (33.47)

0.50

0.19

0.32

0.56

0.53

ARA (g/1000kcal)

78.3 (19.12)

100.6 (41.51)

1.58

0.04

0.09

0.19

DHA (g/1000kcal)

262.6 (97.92)

378.4 (339.56)

1.05

0.14

0.04

0.05

EPA (g/1000kcal)

157.9 (66.22)

231.2 (226.89)

0.99

0.15

0.05

0.02

AEA/ARA

0.57 (0.480)

0.26 (0.183)

2.20*

0.43

0.59*

0.95**

0.96**

2-AG/ARA

0.53 (0.455) a

0.35 (0.301)

1.09

0.33

0.45

0.84

0.83

Variables

Model 2

|t| value

Model 3Model 4Model 5

Standardised |β| value

Serum eCB concentrations

Nutrients intake per day

Ratio of eCB to ARA*: p <0.05, **: p <0.01, data are represented as the mean (S.D.: standard deviation)
Model 1: Simple comparison by unpaired t-test.
Model 2: Comparison adjusted for age and BMI by analysis of covariance.
Model 3: Model 2 + adjusted for current smoking and alcohol consumption by analysis of covariance.
Model 4: Model 3 + adjusted for living alone.
Model 5: Model 4 + adjusted for CNR1.
a n=12 due to small volume of two serum samples.
eCB: endocannabinoid, AEA: anandamide, 2-AG: 2-arachidonoylglycerol, PUFA: polyunsaturated fatty acids, ARA: arachidonic acid, DHA:
docosahexaenoic acid, EPA: eicosapentaenoic acid
Neuroendocrinology Letters Vol. 43 No. 3 2022 • Article available online: www.nel.edu

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Tsuboi et al: Anandamide, arachidonic acid, and depression

Fig. 2. Comparison of the ratio of the eCB serum level (ng/mL) to the PUFA intake volume (g/1000kcal) between high(n=14) and low (n=10) depressive participants. Dotted line represents average score and S.E. of each variable. The
AEA/ARA ratio was significantly higher in high depressive participants than that of low depressive participants(t-test: t = 2.22, p < 0.05). The significance remained even after controlling for possible confounders including age,
BMI, current smoking status, alcohol consumption, the CNR1 SNP (rs806377), and whether the participants live
alone (See Table 2). The depressive symptoms were assessed using CES-D.
*: p < 0.05, N.S.: not significant, eCB: endcannabinoid, PUFA: polyunsaturated long-chain fatty acid, CES-D: theJapanese version of the Centre for Epidemiologic Studies Depression Scale, S.E.: standard error

eCBs
The eCBs system influences a wide variety of neurobiological processes including affect regulation andemotionality as well as other neuropsychiatric functions(Coccaro et al. 2018). We have been focusing on theassociations between eCBs and psychological depression with relevance to PUFAs. In the current study, theratio of AEA/ARA in the high depressive group was
significantly higher than those in the low depressive
group. In addition, there was no significant differencein the ratio of 2-AG/ARA between the two groups. We
analysed the ratios, because ARA is a precursor of AEA
and 2-AG (Freitas et al. 2018, Hansen & Artmann,2008). The AEA/ARA ratio was higher in the highdepressive groups, which appears to contradict the
hypothesis that eCB deficiency contributes to depressive symptoms. Below, we attempt to explain thesecontradictory results.
Elevated ratios of AEA/ARA in high depressive
participants
Several studies have found that circulating concentrations of both AEA and 2-AG are significantly alteredin women diagnosed with depression. For example, theserum concentrations of AEA and 2-AG in women with
MDD were significantly lower than in matched controls(Hill et al. 2009). Another study reported a lower concentration of 2-AG in women newly diagnosed with MDD
compared to controls. This study also revealed a significant negative relationship between circulating 2-AG
and the duration of depressive symptoms (Hill et al.
2008). Contrarily, AEA concentrations were increased

150

in women diagnosed with minor depression, which theauthors argued could be a compensatory mechanism
to maintain an elevated mood (Hill et al. 2009). A recent
study of men and women indicated a significant inverse
correlation between serum 2-AG levels and the severityof depressive symptoms of MDD patients assessed by the
Beck Depression Inventory (BDI). Moreover, baseline
plasma levels of AEA and 2-AG did not differ between
MDD patients and healthy controls (Bersani et al. 2021).
It remains an unanswered question whether circulatingeCB concentrations are related to specific depression
symptoms or clusters of symptoms.
In the present study, there was no significant differences in serum AEA and 2-AG levels between thehigh and low depressive groups. One explanation forthis finding may be that the sample size was too small
to detect any differences. Additionally, the CES-D
scores of the high depressive group ranged from 17
to 33, with the exception of one MDD patient witha CES-D score 44. These CES-D scores may not be highenough to represent a high depressive group.
Functional Differences of AEA and 2-AG
Interestingly, only AEA/ARA, but not 2-AG/ARA,
showed significant differences between the high andlow depressive participants. These results may be dueto the functional differences between AEA and 2-AG
as well as different distributions of CB1R and CB2R
throughout the body. 2-AG activates both CB1R
and CB2R with similar potency, whereas AEA activates CB1R with a much higher potency than CB2R
(McDougle et al. 2017). Additionally, CB1R is foundCopyright © 2022 Neuroendocrinology Letters ISSN 0172–780X • www.nel.edu

Tsuboi et al: Anandamide, arachidonic acid, and depression

predominantly in the CNS, and CB2R is found in both
the CNS and the peripheral immune cells (Ashton &Glass, 2007). Therefore, the function and distribution of AEA may elevate the AEA/ARA ratios in highdepressive participants in order to compensate formaintaining psychological health.
Unique function of ARA, DHA, and EPA
ARA, DHA and EPA are essential fatty acids thathumans cannot synthesize, so they need to be ingested
for maintaining good health (Freitas et al. 2018). ARA
is one of the main ω-6 PUFAs, which is converted into
AEA and 2-AG (Dyall, 2017, Hansen & Artmann,2008). On the other hand, DHA and EPA are major ω3
PUFAs, which are converted into DHEA and EPEA.
CYP converts DHEA and EPEA into the eCB epoxides
EDP-EA and EEQ-EA, respectively, both of which are
CB1R and CB2R agonists (Brown et al. 2010, McDougle
et al. 2017).
In the present study, there were no significant differences between high and low depressive groups in ARA,
DHA, EPA, 2-AG/ARA, AEA/DHA, 2-AG/DHA, AEA/
EPA, or 2-AG/EPA between the high and low depressive groups with the exception of AEA/ARA (Table 2).
Given that DHA and EPA functions as agonists of CB1R
and CB2R, the AEA/DHA, 2-AG/DHA, AEA/EPA, or
2-AG/EPA ratios in the high depressive group might beelevated to maintain the mental wellness. Since DHA
and EPA reduced depressive symptoms, the intake
volume of DHA and EPA in the high depressive group
might be lower. However, the sample size of this study
might be too small to establish significant results.
The presence or absence in the diet of certain PUFAs
is associated with changes in AEA or 2-AG levels inseveral brain regions as well as various tissues (Matias
et al. 2008). Although it is not possible to explain
the results of the present study, the following reports
may be pertinent to the results. For example, piglets
with diets rich in ARA and poor in DHA were shownto have significantly enhanced levels of AEA in thebrain (Berger et al. 2001). Moreover, in a rat study,
ω-3 PUFA deficiency resulted in increased 2-AG in
the whole brain and plasma of adults and developing
animals (Watanabe et al. 2003), while supplementation of ω-3 PUFA appeared to reduce the AEA levels(Wood et al. 2010). In addition, supplementation of ω-3
PUFAs reduced 2-AG levels, which was associated withan increase in DHA incorporation within brain phospholipid membranes (Piscitelli et al. 2011).
Other variables
Although lifestyle (smoking habit and alcohol consumption), socio-economic status (years of education) and
CNR1 SNP (rs806377) might be related to depressive
symptoms, there were no significant differences inthese variables between the high and low depressive
participants in the current study. However, these variables may be meaningful as confounding factors.

LimitationsFirstly, the serum levels of PUFAs were not measured.
Although we monitored ARA intake, the analysisof serum ARA concentrations to analysis would be
useful to elucidate the mechanism of ARA metabolism, because AEA and 2-AG are derived mainly from
ARA (Dyall, 2017, Hansen & Artmann, 2008), and are
degraded into ARA again (Blankman & Cravatt, 2013).
Secondly, since this was a cross-sectional study, causal
associations cannot be inferred. Thirdly, the numberof participants was small. We had selected 18 highand 18 low depressive women that were age-matchedas possible. This sample size was chosen because the
eCB ELISA kit for could accommodate 36 samples.
However, we mistakenly measured 12 serum samples
collected two years earlier (2013) with no CES-D
results, because some of the numbers written on thesample tubes were the same between 2015 and 2013.
Consequently, 14 serum samples from high depressive women and 10 serum samples from low depressive
women were used in the present analysis. Finally, also
other variables may be significantly different in womenwith high depressive disorder in larger sample.
Conclusions
In summary, among female community-dwellers
of Japan, we found an elevated ratio of serum AEA
to daily intake of ARA in high depressive participants
compared with those in low depressive participants.
This result suggested that the conversion rate of ARA
to AEA may be accelerated in depressive individuals.
ARA supplementation may be helpful to maintain
psychological wellbeing in daily life, although further
studies are needed to address this hypothesis.

ACKNOWLEDGEMENTS
The authors thank the clinical staff at the health checkup locations for their assistance with this project.
Moreover, we are grateful to all the study participants
who generously contributed to the study.

DISCLOSURE STATEMENT
Nothing to disclose.

FUNDING
This research was supported by a Grant-in-Aid for
Scientific Research (C) 16K01787 from the Ministryof Education, Culture, Sports, Science and Technology,
Japan.

CONFLICTS OF INTEREST
The authors declare that there are no conflictsof interest.

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Tsuboi et al: Anandamide, arachidonic acid, and depression

AUTHOR CONTRIBUTIONS
H.T. (Hirohito Tsuboi) performed the psychologicalassessment of the study, the data analyses, and drafted
the manuscript. M.M. contributed to the AEA and
2-AG detection. H.T. (Hiromasa Tsujiguchi) and A.H.
managed the participation of the subjects. T.K., A.T.,
K.H., and T.S. detected the SNPs. N.Y. curated data.
H.N. conducted the whole cohort study.

INSTITUTIONAL REVIEW BOARD
STATEMENTThe study was conducted according to the guidelinesof the Declaration of Helsinki, and approved by the
Ethical Committee at Kanazawa University (protocol
code 1491 on 18 December 2013).

INFORMED CONSENT STATEMENT
Written informed consent has also been obtained fromthe participants to publish this paper.
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