Polyunsaturated fatty acids levels and initial presentation of somatic symptoms induced by interferon-alpha therapy in patients with

chronic hepatitis C viral infection

Jane Pei-Chen Chang1,2,5, Hsueh-Chou Lai2,6, Hui-Ting Yang3, Wen-Pang Su2,6, Cheng-

Yuan Peng2,6, Piotr Gałecki4, Anna Walczewska4, Carmine M. Pariante7, Kuan-Pin

Su,1,2,5,7*

1Graduate Institute of Neural and Cognitive Sciences, China Medical University, Taichung, TAIWAN

2School of Medicine, China Medical University, Taichung, TAIWAN

3Department of Nutrition, China Medical University, Taichung, TAIWAN

4Medical University of Łódź, Łódź, POLAND

5Department of Psychiatry & Mind-Body Interface Laboratory (MBI-Lab), China Medical University Hospital, Taichung, TAIWAN

6Department of Hepatogastroenterology, China Medical University Hospital, Taichung, TAIWAN

7Institute of Psychiatry, King’s College London, United Kingdom

*Address correspondence to:

Prof. Kuan-Pin Su

Department of Psychiatry, China Medical University Hospital,

No. 2, Yuh-Der Road, Taichung 404, Taiwan

Telephone number: 886-4-22062121 ext. 4126

Fax number: 886-4-22361230

E-mail: cobolsu@gmail.com

Conflicts of Interest: None

1

Acknowledgement

The work was supported by the following grants: MOST103-2320-B-039-MY3,

MOST103-2320-B-038-012-MY3, NSC 103-2923-B-039-002-MY3, 102-2911-I-039-

501, 101-2628-B-039-001-MY3 and 101-2320-B-038-020-MY2 from the Ministry of

Science and Technology and CMU103-S-03, DMR-103-078, 102-068 and 101-081

from the China Medical University in Taiwan.

2

Abstract

Objectives: Somatic symptoms are common in depressive disorder and are similar to

sickness behaviors due to inflammatory activation after cytokine administration.

Omega-3 polyunsaturated fatty acids (PUFAs) are natural anti-inflammatory agents

and may reduce inflammation-induced behavioral changes. The aim of this study was

to investigate the role of PUFAs on the development of somatic symptoms and

depression in patients of hepatitis C virus infection (HCV) receiving interferon-alpha

therapy (IFN-α) in a prospective manner.

Methods: In this 24-week, prospective cohort study, 43 patients with chronic HCV

ongoing IFN-αtherapy were assessed with the Mini-International Neuropsychiatric

Interview for major depressive episodes and Neurotoxicity Rating Scale (NRS) for

somatic symptoms.

Results: One-third later developed IFN-α-induced depression (DEP group). As

compared subjects without depression, DEP group had higher NRS scores (p<0.001),

lower eicosapentaenoic acid (EPA) levels (p=0.038) at week 2. Somatic symptoms,

regardless of painful/nonpainful characteristics, had positive association with

arachidonic acid (AA) (p<0.05), and negative association with EPA (p<0.05).

3

Conclusion: This study implies that early intervention with omega-3 PUFAs might be

a promising strategy to prevent depression and somatic symptoms in patients

receiving cytokine therapy.

Keywords: depression, painful symptoms, n-3, PUFAs, omega-3

4

Introduction

Depressive disorders with predominant somatic presentation, including general

weakness, malaise, fatigue, muscle and joint aches, loss of interest, poor appetite and

poor concentration, are the most common forms of depression.1-2 On the other hand,

somatic symptoms had been closely associated with immune/inflammatory system.3

Somatic symptoms has been described as the manifestation of activation of brain

cytokines system sensitization responding to immune reactions,4-6 which are similar to

inflammatory activation associated sickness behaviors of animals or humans after

receiving cytokine/prostaglandins E2 (PGE2) administration.5-6 The most supportive

evidence for the ‘inflammation theory’ of depression is that a therapeutic

administration of the interferon-alpha (IFN-α) induced depression, where in about a

third of patients with chronic hepatitis C viral infection (HCV) or cancers receiving

IFN-αdevelop depression.6-8

Previous genetic association and cross-sectional studies have demonstrated that

polyunsaturated fatty acids (PUFAs) may mediate in somatic symptoms in

depression.7 There are two main types of PUFAs: the omega-6 (n-6) series (cis-

linoleic acid [LA,18:2], γ-linolenic acid [GLA, 18:3, n-6], dihomo-GLA [20:3, n-6],

arachidonic acid [AA,20:4, n-6]); and the omega-3 (n-3) series (α-linolenic acid

[ALA, 18:3], eicosapentaenoic acid [EPA, 20:5, n-3], docosahexaenoic acid [DHA]).

Both n-3 and n-6 PUFAs are converted to eicosanoids for their biological functions

and are essential for survival of humans and other mammals.9 N-6 PUFAs becomes

the precursor of the pro-inflammatory arachidonic acid (AA), precursor of

prostaglandin (PGs) 2 series, thromboxanes (TXs) and Leukotrienes (LTs) 4 series.

5

Meanwhile, n-3 PUFAs forms the anti-inflammatory PGs 3 series and LTs 5 series. N-

3 PUFAs are critical in balancing immune function by reducing membrane n-6 PUFAs

and PGE2 synthesis and are associated with somatic manifestations in depression.10

Genetic variations on the key enzyme in PUFAs metabolism, the phospholipase A2

(PLA2), also have significant effect on somatic symptoms in both patients with IFN-

α-induced depression and the patients with major depression,7,11 possibly by affecting

the levels of n-3 PUFAs. In addition, a negative correlation between PUFAs levels and

somatization symptoms has also been reported in a cross-sectional study.12

The interpretation of previous cross-sectional case-controlled studies is limited by

possible confounders and weak cause-and-effect relationship. Therefore, the

prospective cohort of chronic HCV patients receiving IFN-αtherapy would provide an

excellent model to study the development of somatic symptoms in a prospective

human model. In this study, we aimed to investigate the roles of PUFAs on initial

somatic symptoms presentation induced by IFN-αtherapy in patients with chronic

HCV infection. We hypothesize that (1) the patients with HCV who later developed

depression during IFN-α treatment have higher levels of somatic symptoms at week 2,

(2) patients with HCV who later developed depression have higher levels of AA, and

(3) lower levels of EPA and DHA.

Methods

This was a 24-week, prospective cohort study. Eligible patients were adult patients

with chronic HCV, who were assessed by three hepatologists to be eligible for IFN-

αtherapy. Patients received combination therapy as their treatment for chronic HCV

for 24 weeks (1.5μg of peg-IFN-α-2beta per kilogram of body weight subcutaneously

6

once weekly, and 600-800 mg of ribavirin daily). Exclusion criteria included age over

70 years; any cause for liver disease other than HCV; any current psychiatric disorder

and current use of psychotropic agents. Patients were recruited from the Liver Center

of the China Medical University Hospital, Taichung, Taiwan by hepatologists and

referred to researchers to provide structured information about IFN-α-induced

neuropsychiatric adverse effects and depression, and the procedure of this study.

Patients had to fully understand and sign the informed consent before enrolment. The

study was approved by the China Medical University Hospital Institutional Review

Board.

Forty-three patients with chronic HCV were enrolled for weekly evaluation for

initial 2 weeks of IFN-αtherapy. At each assessment, patients were assessed with the

structured Mini-International Neuropsychiatric Interview (MINI) for confirmation of

diagnosis of major depressive episode and with self-reported Neurotoxicity Rating

Scale (NRS)13 for somatic and neuropsychiatric symptoms. Blood samples of 43

patients were available at baseline; however, only thirty-six participants were

available at week 2 of IFN-αtherapy for assesment of blood PUFAs level .

The NRS is a checklist questionnaire used for evaluation of cytokine therapy

related neuropsychiatric symptoms and is categorized into general symptoms, non-

painful somatic symptoms and painful somatic symptoms.13 With each item rated

from 0 to 10 on a visual analogue scale, the score ranges from 0-390. The general

symptoms include anxiety, health worries, sadness/depression, restlessness, no

interest in people, no interest in activities, difficulty making decisions, strange

thoughts, all-over sick feeling, distractibility, episodes of confusion, word-finding

7

problems, memory problems, irritability, decreased motivation, hallucinations, lack of

emotions, mood swings, tension, slowed movements, loss of interest in sex,

nightmares/dreams, and other unspecified symptoms; The non-painful somatic

symptoms include nausea, vomiting , tiredness/fatigue, tremors/shakiness, walking

problems, vision problems, bowel-bladder problems, fever, headache, difficulty getting

to sleep, difficulty staying asleep, sleeping too much, loss of appetite; The painful

symptoms include body aches, joint pain, other pain.

Fatty acid composition of erythrocyte membranes was analyzed and the level of

individual fatty acid was measured with gas chromatography of methyl esters (Lipid

Standards, FAME, Sigma Co., St. Louis, MO, USA). Fatty acid profiles were

identified by comparing the retention times with those of appropriate standard fatty

acid methyl esters. The phospholipids extracted from samples were dissolved in 1 ml

of 14% boron trifluoride methanol (BF3-methanol, SIGMA®), and 100ml of

heptadecanoic acid (Margaric acid, C17:0, SIGMA®) was added as internal standard,

then methylated reaction was performed by water bath heating at 100℃ for 30 min.

Finally, 2ml pentane and 1ml H2O were added and centrifuged for 10 min, took the

supernatant and redissolved in 100 l n-hexane then injected into gas chromatography

for fatty acid profile analysis.

The capillary gas chromatography (Trace GC, Thermo Finnigan) was equipped

with a 30m, 0.32mmid capillary column (cross-linked polyethylene glycol-TPA phase,

8

Sulpelco®) and frame ionized detector. The injector and detector temperatures were

230℃ and 270℃, respectively, and the split ratio was 100:1. Oven temperature was

set at 160℃ for 4 minutes at the initial stage, and was increased by 2.5℃/min to

225℃ then and held for 20 minutes. Peaks were recorded and interpreted by a

programmable integrator (Chromcard for Trace). Fatty acid profiles were identified

according to the retention time of appropriate standard fatty acid methyl esters.

Researchers who participated in the laboratory were blind to the information of coded

samples.

All statistical analysis was carried out with the Statistical Package for the Social

Science (SPSS), version 11.0 for windows. Demographic and clinical characteristics

of patients between depression (DEP) and non-depression (Non-DEP) groups were

compared by t-test or Chi-Square test where appropriate. Pearson correlation was used

to evaluate association between NRS scores and PUFAs levels, p value <0.05

indicates statistical significance.

Results

Fifteen out of 43 participants (34%) developed IFN-α-induced depression (DEP

group), and they had a significant higher second week NRS total scores (p < 0.001)

and greater increase in NRS total scores at the end of second week (p < 0.001) (Table

1). However, there were no differences between the DEP and Non-DEP groups in

terms of age and sex distribution, baseline NRS scores, blood PUFAs (AA, EPA,

9

DHA) levels at baseline and at week 2, and within 2-week changes of blood AA and

EPA levels. Moreover, after initiating IFN-α therapy, Non-DEP group had greater

drop in the changes of blood DHA level between week 2 to baseline when compared

to the DEP group (p = 0.031) (Table 1).

-----------------------------------------------

Insert Table 1 about Here

-----------------------------------------------

Correlation analysis further showed negative correlation between week 2 EPA

and NRS total, painful, and nonpainful symptom scores at weeks (p=0.032, 0.037, and

0.029, respectively; Table 2). We also found positive correlation between week 2

blood AA levels and NRS total, painful, and nonpainful symptom score changes

within week 1 (p=0.014, <0.001, =0.004, respectively; Table 3); and with painful

symptom scores at week 1 (p<0.005, Table 3). Furthermore, positive correlations was

found between within-2 week changes of AA blood levels and within-1 week painful

symptoms scores changes (p=0.036). However, no correlation was made between

NRS total, painful, non-painful symptoms scores and blood DHA levels (Data not

shown in the tables).

-----------------------------------------------

Insert Tables 2 & 3 about Here

-----------------------------------------------

10

Discussion

HCV patients receiving IFN-αtherapy who later developed depression (DEP

group) had significantly higher neurotoxicity symptom scores as early as the first two

weeks after initiating IFN-αtherapy in this prospective study. This is compatible with

our first hypothesis. Although IFN-α therapy may induce acute sickness behavior

manifestations similar to the presentation of depressive disorder,6 patients who later

developed IFN-α-induced depression tend to report a greater severity of the somatic

symptoms. Depressive disorders with predominantly somatic presentation are the

most common forms of depression.6 Moreover, previous reviews showed two of the

three most common symptoms (low mood: 76%, fatigue: 73%, sleep disturbances:

63%) reported during a current depressive episode were somatic.14 Hence, it is

important to note that the depressive symptoms of patients receiving IFN-α therapy

may be masked by the acute sickness behavioral symptoms at the beginning of the

treatment. Moreover, the manifestation of the somatic symptoms may be due to (1)

the inflammatory reactions induced by IFN-α via the induction of the nitric oxide

synthase (iNOS) and nitric oxide (NO) release and down-regulation of heme

oxygenase-1 (HO-1) expression,15 and (2) inflammatory and oxidative and nitrosative

reactions reported in patients with depression.16

Our study results echoes part of our second hypothesis in that DEP group had

lower EPA levels at week 2, but not in DHA or AA levels. Previous clinical trials and

meta-analyses have suggested that EPA, rather than DHA, might be the most active

component of omega-3 PUFAs’ antidepressant effects.17-21 Moreover, a 2-week,

double-blind, placebo-controlled trial comparing EPA, DHA, and placebo for the

11

prevention of IFN-α-induced depression showed that the incident rates of IFN-α-

induced depression were significantly lower in EPA-treated but not in DHA treated

patients (10% and 28%, respectively, versus 30% for placebo, p= 0.037).8 Genetic

variations of PLA2 and cyclooxygenase 2 (COX2) genes, the two key enzymes in the

metabolism of omega-3 PUFAs have also been associated with the development of

major depressive disorder and IFN-α-induced depression.7,22 PLA2 BanI GG or

COX2 rs4648308 AG genotypes have a higher risk of IFN-α-induced depression.7 In

addition, the at-risk PLA2 polymorphism is associated with lower EPA levels and the

at-risk COX2 polymorphism is associated with lower levels of both DHA and EPA

during IFN-α therapy.7

In this study, Non-DEP group also had a greater drop in blood DHA levels after

two weeks of IFN-αtherapy than DEP group. The initial drop in blood DHA level of

Non-DEP group may implicate that protective effect of releasing more free form

blood DHA to be available in non-depressed patients to counteract against depression.

IFN-α may activate PLA2 and decrease membrane DHA and increases free DHA.

Moreover, lower endogenous DHA had been identified as a risk factor for IFN-α-

induced depression in previous study;7 which further reflects less endogenous anti-

inflammatory capability in those who later develop depression.7 In addition, both

clinical 18 and cellular studies also supported the protective effect of DHA against

depression and associated somatic symptoms via modulation of the inflammatory

reactions, such as reducing expressions of tumor necrosis factor-α, IL-6, NOS, and

COX2, and inducing upregulation of HO-1.15

Omega-3 fatty acids modulation on inflammation had also been proposed as the

12

shared link between depression,23-24 cardiovascular diseases25 and associated somatic

symptoms.26 This study further supported role of PUFAs in depression associated

somatic symptoms by showing significant positive association between painful

symptom scores with blood AA levels and negative association with blood EPA levels

in HCV patients with IFN-α. Previous study showed inverse correlation between EPA

level and somatic symptoms,12 which further implicated that perception of somatic

complaints may be associated to EPA depletion changes. Moreover, EPA acts as the

precursor for eicosanoids and a modulator of cytokines.27 It has been proposed that

depression is accompanied by increased secretion of eicosanoids, such as

prostaglandins, and by an excessive secretion of proinflammatory cytokines.28 EPA

can act as the inhibitor of PLA2 to reduce the secretion of eicosanoids and

proinflammatory cytokines,29 which might have been associated with the

improvement of somatic symptoms in patients with depression.6 On the other hand,

the positive correlation between blood AA levels and painful somatic symptoms

maybe associated the actions of AA-derived-PGs. PGE2 and other pro-inflammatory

cytokines including interleukin IL-1 Beta and IL-6 tend to promote neuropathic pain

and hyperalgesia of sickness response via effects on central/peripheral neurons, glia

and endothelial cells.30

The balance between EPA, DHA, and AA are critical in the manifestation of

painful and non-painful somatic symptoms. Since EPA and DHA tend to suppress pro-

inflammatory activation of AA and reduce PGE2 synthesis, inactivate IL-1 beta-

induced PGE2 activation,31 attenuate IL-1-induced changes of nucleus accumbens

neurotransmitter release,32 and antagonize peripheral and neural production of

13

eicosanoids and cytokines, and improve neural plasticity induced by chronic pain.32

Moreover, Protein kinase C (PKC) is also activated in peripheral-pain neurons

following injury/inflammation, and mitogen-activated protein kinase (MAPK) has

been suggested to regulate central sensitization in inflammatory and neuropathic

pain.33 Meanwhile, n-3 PUFAs are able to inhibit PKC and MAPK actions in rats’

brain and interrupt painful pathway signaling.34 Hence, n-3 PUFAs may mediate in

depression-associated somatic symptoms via its anti-inflammatory actions in cellular

and molecular levels.

There are several limitations to this study. This sample size is relatively small;

hence generalization of the study results may be limited. Although blood samples of

43 patients were available at baseline; only thirty-six participants were available at

week 2 of IFN-αtherapy for assessment of blood PUFAs level. However, this study is

one of the first prospective study on the somatic symptoms presentation in patients

with IFN-α-induced depression. Secondly, although there were no differences

between DEP and Non-DEP group in terms of blood AA and DHA levels at week two,

which may be due to small sample size; there is a trend of greater DHA level in Non-

DEP group. Moreover, our study would be more comprehensive if we were to include

data in regards to the diets of the participants.

Conclusion

In conclusion, HCV patients receiving IFN-αtherapy who perceived higher

depressive and somatic symptoms as early as week 2 of IFN-αtherapy are prone

14

develop depression later during IFN-αtherapy. Contrariwise, those HCV patients with

higher basal DHA levels, possibly more free form DHA levels, receiving IFN-

αtherapy may be protected from cytokine induced depression.

N-3 PUFAs (EPA and DHA) associate with pain reduction in both

observational/clinical studies and are reported to participate in immune regulation via

balancing neuronal membrane stability, neurotransmission, and signal transduction;

and has been related to brain dysfunctions associated somatic symptoms of depression

and sickness behaviors. This prospective longitudinal study further showed painful

symptoms correlate positively with blood AA levels and negatively with blood EPA

levels in HCV patients with IFN-α-induced somatic symptoms, which further supports

the assumption that AA increase PGE2 and inflammation and contribute to sickness

behavior, while EPA antagonize AA activity by competing with AA as a substrate for

cyclo- and lipooxygenase and reduce of inflammatory metabolites (PGE2) production.

This study further suggests PUFAs levels and associated somatic symptoms may act

as potential predictors for IFN-α-induced depression as early as two weeks of

treatment.

15

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Table 1. Demographic data between depression and non-depression groups.

Non-DEP(N= 28)

Depression(N=15) p-Value

Female (%) 29% 47% 0.246

Age, yrs (mean +SD) 45.04+ 9.97 49.40+9.85 0.177

NRS (mean+SD)

Baseline (Week 0) 15.92+21.73 16.42+17.30 0.945

Week 2 23.81+20.31 63.36+ 27.23 <0.001**

Changes in 2 weeks 6.53+23.72 47.83+30.00 <0.001**

AA (mean+ SD)

Baseline (Week 0) 5.47+0.25 5.61+0.28 0.100

Week 2 5.35+0.18 5.37+0.20 0.694

Changes in 2 weeks -0.12+0.27 -0.23+0.40 0.269

EPA (mean+ SD )

Baseline (Week 0) 0.81+0.040 0.801+0.05 0.336

Week 2 0.79+0.048 0.76+0.04 0.038*

Changes in 2 weeks -0.02+ 0.050 -0.04+0.05 0.264

DHA (mean+ SD )

Baseline (Week 0) 3.40+0.40 3.25+0.1 0.147

Week 2 3.24+0.069 3.26+ 0.063 0.361

Changes in 2 weeks -0.160+ 0.38 0.02+0.11 0.031*

Note: AA, arachidonic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic

acid; N, number; Non-DEP, non-depression group; NRS, sum of total neurotoxicity

rating scale scores; SD, standard deviation; yrs, years. *indicates statistical

significance of p<0.05 with Chi-Square; ** indicates statistical significance of p<0.01

with Chi-Square.

21

Table 2. The correlations between painful and non-painful somatic symptoms of

NRS and EPA level in HCV patients after 2 weeks of Interferon-alpha treatment.

EPA level Baseline Week 2¶ Changes in week 2¶

Pearson Correlation

R

Pearson Correlation

R

Pearson Correlation

RTotal NRS

Baseline (Week 0) 0.084 0.024 -0.045

Week 1 -0.130 -0.249 -0.138

Week 2 -0.071 -0.363* -0.312

Changes in 1 week -0.242 -0.238 -0.032

Changes in 2 weeks -0.139 -0.342 -0.232

Somatic scores

Baseline (Week 0) 0.035 0.010 -0.019

Week 1 -0.209 -0.222 -0.045

Week 2 -0.182 -0.354* -0.208

Changes in 1 week -0.281 -0.207 0.032

Changes in 2 weeks -0.212 -0.312 -0.139

Painful scores

Baseline (Week 0) -0.061 0.075 0.126

Week 1 -0.067 0.147 0.204

Week 2 -0.298 -0.369* -0.124

Changes in 1 week -0.101 0.105 0.191

Changes in 2 weeks -0.204 -0.313 -0.147

Note: EPA, eicosapentaenoic acid; HCV, hepatitis C viral infection; NRS, neurotoxicity rating scale; total NRS, sum of total neurotoxicity rating scale scores; Painful scores, sum of painful symptoms scores of NRS; R, The Pearson correlation coefficient; Somatic scores, refer to sum of non-painful symptoms scores of NRS. ¶Blood samples of 36 patients were available for measurements of EPA at 2nd week and changes in the 2 weeks.*Pearson correlation test was employed to determine the relationship between clinical variables.* indicates statistical significance of p<0.05.

22

Table 3. The correlations between painful and non-painful somatic symptoms of

NRS and AA level in HCV patients after 2 weeks of Interferon-alpha treatment.

AA Baseline Week 2¶Changes in 2 weeks¶

Pearson Correlation

R

Pearson Correlation

R

Pearson Correlation

RTotal NRS

Baseline (Week 0) -0.021 -0.310 -0.164

Week 1 0.061 0.284 0.111

Week 2 0.090 0.011 -0.071

Changes in 1 week 0.020 0.436* 0.231

Changes in 2 weeks 0.184 0.187 -0.055

Somatic scores

Baseline (Week 0) -0.030 -0.328 -0.167

Week 1 0.019 0.327 0.171

Week 2 -0.007 -0.028 -0.011

Changes in 1 week -0.007 0.506** 0.294

Changes in 2 weeks 0.086 0.191 0.035

Painful symptoms scores

Baseline (Week 0) -0.194 -0.219 0.040

Week 1 -0.031 0.465** 0.291

Week 2 -0.047 0.114 0.107

Changes in 1 week -0.004 0.658** 0.379*

Changes in 2 weeks 0.057 0.290 0.120

Note: AA, arachidonic acid; HCV, hepatitis C viral infection; NRS, neurotoxicity rating scale; total NRS, sum of total neurotoxicity rating scale scores; Painful scores, sum of painful symptoms scores of NRS; R, The Pearson correlation coefficient; Somatic scores, refer to sum of non-painful symptoms scores of NRS. ¶Blood samples of 36 patients were available for measurements of AA at 2nd week and changes in the 2 weeks.*Pearson correlation test was employed to determine the relationship between clinical variables.* indicates statistical significance of p<0.05; ** indicates statistical significance of p<0.01.

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