Accepted Manuscript

Kynurenine Pathway Changes in Late-life Depression

YJ Wu , XM Zhong , NK Mai , YG Wen , DW Shang , LJ Hu ,

B Chen , M Zhang , YP Ning

PII: S0165-0327(17)32311-X
DOI: 10.1016/j.jad.2018.04.007
Reference: JAD 9661

To appear in: Journal of Affective Disorders

Received date: 7 November 2017

Revised date: 5 March 2018
Accepted date: 2 April 2018

Please cite this article as: YJ Wu , XM Zhong , NK Mai , YG Wen , DW Shang , LJ Hu , B Chen ,
M Zhang , YP Ning , Kynurenine Pathway Changes in Late-life Depression, Journal of Affective Dis-
orders (2018), doi: 10.1016/j.jad.2018.04.007

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 ACCEPTED MANUSCRIPT

Highlights
 TRP metabolism was found in LOD; low level of TRP and shift of KYN metabolism
were found in EOD.

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Kynurenine Pathway Changes in Late-life Depression

Wu YJ #, Zhong XM #, Mai NK, Wen YG, Shang DW, Hu LJ, Chen B, Zhang M, Ning YP *.

The Affiliated Brain Hospital of Guangzhou Medical University (Guangzhou Huiai Hospital),
Guangzhou, Guangdong, China

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#
These authors contributed equally to this work.

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Conflict of interest: All authors declare no potential conflict of interests.
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*
Corresponding author: Ning YP,
Address: No.36, Mingxin Road, Liwan District, Guangzhou, China
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Tel: (86-20) 8189 1425,

Fax: (86-20) 8189 1391
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Email: ningjeny@126.com
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Abstract
Background: Kynurenine pathway (KP) activation is associated with several neuropsychiatric
diseases, including major depression disorder (MDD). Although several investigations have been
conducted on MDD, these have seldom shed light on KP changes in late-life depression (LLD).
Objective: We aimed to investigate whether tryptophan (TRP) metabolism and kynurenine (KYN)
metabolism are imbalanced in LLD patients and to explore the differences in KP characteristics
between early onset depression (EOD) and late onset depression (LOD) patients.
Methods: We investigated 170 LLD patients (EOD 90, LOD 80) and 135 normal controls. Serum

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concentrations of TRP, KYN and kynurenic acid (KYNA) were detected by the liquid

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chromatography-tandem mass spectrometry method. Depressive symptoms were assessed by the

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17-item Hamilton Depression Scale (HAMD-17).
Results: LLD patients exhibited lower levels of TRP, KYN, KYNA and KYNA/KYN ratio and a higher

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level of KYN/TRY ratio than normal controls. The decrease in TRP and the increase in KYN/TRP ratio
were found in LOD patients. A low TRP level without increased KYN/TRP ratio was found in EOD
patients. The “Depression” factor, which was extracted from HAMD-17 by the principal component
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factor analysis, was correlated with the TRP level and KYNA/KYN ratio in the EOD group, but no
such correlation was found in the LOD group.
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Conclusions: KP changes were observed in LLD patients; LOD patients showed profound shifts in
TRP metabolism, while EOD patients showed low TRP level and a shift in KYN metabolism.
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Keywords: Late-life depression; Tryptophan; Kynurenine; Kynurenic acid

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Introduction
Depression in old age has become an increasingly severe global health problem because of high
morbidity and comorbidity associated with increasing age. Approximately 3.6% to 4.6% of elderly
people have been found to experience late-life depression (LLD) (Weyerer et al., 2013). LLD is
associated with multiple symptoms, including apathy, cognitive deficits, and poor treatment
responses (Marin, Butters, Mulsant, Pollock, & Reynolds, 2003; Sheline et al., 2006; Tedeschini et
al., 2011). Depression can increase medical expenses by 50–100% (Zivin, Wharton, & Rostant,
2013), and depressed patients have increased risk of Alzheimer's disease (Heser et al., 2016) as

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compared to non-depressed patients. Because of the enormous burden on the economy and the

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healthcare system, LLD is an urgent problem that requires further study (Caraci, Copani, Nicoletti,

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& Drago, 2010).
According to the established evidence, the kynurenine pathway (KP) plays an important role in the

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development of depression. The kynurenine to tryptophan (KYN/TRP) ratio is correlated with
suicidality (Brundin et al., 2016) and is elevated in major depressive disorder (MDD) (Gabbay et al.,
2010). Kynurenic acid (KYNA) (Wichers et al., 2005), which is degraded from KYN, and TRP (Gabbay
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et al., 2010) are decreased in MDD. Furthermore, the severity of depression correlated with the
imbalance in KP (Reus et al., 2015), indicating that imbalanced KP might be one of the causative
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factors of depression.
TRP is an essential amino acid that can cross the blood-brain barrier. Approximately 95% of TRP is
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metabolised through KP. KYN is synthesised from TRP. The activity of indoleamine 2,3-dioxygenase
(IDO), the major rate-limiting enzyme, is enhanced by pro-inflammatory cytokines, which increase
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KYN production (Oxenkrug, 2010) as well as the KYN/TRP in an inflammatory condition. KYN as a
precursor was catabolised into KYNA or quinolinic acid (QUIN). The enzyme
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kynurenine-3-monooxygenase (KMO) is also activated by pro-inflammatory cytokines to induce

increased production of 3-hydroxykynurenine (OHK), and QUIN is synthesised from OHK. Therefore,
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in the presence of inflammation, KYN metabolism shifts to the QUIN arm and leads to a reduction
in KYNA/KYN (Myint, 2012).
A previous study showed that TRP catabolism is related to depressive symptoms in old age
(Capuron et al., 2011), which was simplified with the findings from MDD, indicating that the
relationship between TRP catabolism and depression still exists in old age. Although MDD and LLD
share the same diagnostic criteria, there are several differences between these two diseases. LLD is
characterised by cognitive impairment and poor prognosis relative to MDD (Mitchell &
Subramaniam, 2005). In addition, a meta-analysis found that LLD patients exhibited more agitation,
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hypochondriasis and somatic preoccupation (Hegeman, Kok, van der Mast, & Giltay, 2012). Based
on the clinical features mentioned above, some pathologic processes could explain both LLD and
MDD, but the majority of features remain different. The "vascular hypothesis" offers some
explanations about these differences (Alexopoulos et al., 1997). However, the mechanism
underlying LLD's clinical manifestation remains poorly understood. Further, LLD patients have a
higher risk of dementia, and thus, early recognition and intervention are extremely important.
According to a recent study, imbalance of the hypothalamic–pituitary–adrenal axis, excess
glucocorticoids and autoimmune reaction were associated with the changes in KP, which is

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identified as the probable cause of LLD. Thus, understanding the KP change in LLD can help us to

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understand the disease progression and might provide cues for early recognition.

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Based on the age at the first depressive episode, LLD could also be divided into early onset
depression (EOD) and late onset depression (LOD). According to a meta-analysis, EOD is
characterised by higher frequency of a family history of mood disorders than LOD; this suggested

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that the genetic effect was more crucial in the development of EOD than in LOD (Grayson &
Thomas, 2013). Similar to the production of KP, serotonin is an important target in the treatment
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of depression, and serotonin transporter polymorphisms play an essential role in depression
development(Alexopoulos et al., 2009). Thus, KP change might different between EOD and LOD,
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while the KP change between EOD and LOD still has not been clarified nowadays.
In this study, we aimed to: 1) detect the serum concentrations of TPR, KYN and KYNA of all
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participants; 2) compare the differences of KP changes between the EOD and LOD patients; and 3)
explore the relationship between KP changes and depressive symptoms of EOD and LOD patients.
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Materials and methods

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Participants and control subjects

We obtained written informed consent from each participant after a full explanation of the study.
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This study was approved by the ethics committee of the Affiliated Brain Hospital of Guangzhou
Medical University (Guangzhou Huiai Hospital). We investigated the LLD database in The Affiliated
Brain Hospital of Guangzhou Medical University, till 2017 Feb. All patients were recruited at the
outpatient and inpatient Department of Affiliated Brain Hospital of Guangzhou Medical University,
Guangzhou, Guangdong, China, and normal controls (NC) were recruited from the community.
All participants were interviewed through a clinical interview that was structured to meet the
inclusion and exclusion criteria. Depression was diagnosed according to DSM-IV criteria. The
presence of depressive symptoms was evaluated by the 17-item Hamilton Rating Scale for
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Depression (HAMD-17) by a well-trained psychiatrist. Exclusion criteria were as follows: 1) serious

suicidal behaviour; 2) serious medical conditions or concomitant medications likely to influence the
central nervous system or immunological function, including cardiovascular, respiratory, endocrine
and neurological diseases; and 3) a history of drug or alcohol abuse within 6 months or a history of
drug or alcohol dependence within 1 year. NC were required to have no first-degree relative with a
psychiatric disorder (Savitz, Dantzer, et al., 2015). We further divided LLD patients into EOD and

LOD groups by their first onset ages: ＜60 years old for EOD patients; ≥60 for LOD patients

(Hashem, M, Gomaa, & Khalaf, 2017).

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We investigated the serum levels of TRP, KYN and KYNA in 170 patients with the diagnosis of LLD,

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and 135 healthy elderly subjects as NCs. For further study, LLD patients were divided into two

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subgroups: an EOD group (90 patients), which included subjects whose first depressive episode
occurred before the age of 60 years; and an LOD group (80 patients), consisting of the patients

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with depressive episodes appearing after the age of 60 years.
Laboratory analyses
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Peripheral blood samples were collected between 8:00 and 9:00 AM after an overnight fast. Blood
samples were collected in vacutainers without further additives. After 0.5 h of coagulation, the
samples were centrifuged at 3,000 r/min for 10 min, and the supernatant was aliquoted into
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Eppendorf tubes (Eppendorf, Hamburg, Germany) and immediately frozen at -80℃ until assay.
We used a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS)
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method to detect TRP, KYN and KYNA serum concentrations. L-TRP, L-KYN, KYNA and activated
charcoal were purchased from Sigma-Aldrich, and Kyna-d5 was supplied by Toronto Research
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Chemicals, Inc. (Toronto, Canada). Methanol was obtained from Merck KGaA (Darmstadt,
Germany), and ammonium formate was purchased from Sigma-Aldrich Corporation (Bangalore,
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India). Purified water was produced by a Milli-Q water purification system (Millipore Corporation,
Billerica, MA, USA). Calibrators and controls were established by serial dilution of stock solution
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with methanol. The final concentrations were used for 7-point calibration as follows: TRP: 1, 2, 5,
10, 20, 40 and 50 μg/mL; KYN: 0.1, 0.2, 0.5, 1, 2, 4 and 5 μg/mL; KYNA: 1, 2, 5, 10, 30, 50 and 60
ng/mL. Final concentrations of QC were as follows: TRP: 2, 8 and 40 μg/mL; KYN: 0.2, 0.8 and 4
μg/mL; KYNA: 2, 8 and 50 ng/mL (Hu et al., 2016).
Statistics analyses
Statistical analysis was performed with Statistical Package for Social Sciences software version 22.0
(SPSS IBM, Chicago, Illinois, USA). Demographic and clinical variables were analysed using a
chi-square (χ2) test for the categorical variables, and a t-test and one-way analysis of variance
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(ANOVA) were used for the continuous variables. Further, to control significance between group
effects for difference in age and gender, we used a generalised linear model with diagnosis as the
independent factor and age and gender as covariates. A principal component factor analysis with
varimax rotation was performed on the HAMD-17. Correlations among depressive symptoms and
changes in the KYN pathway were examined by Pearson correlation coefficients, and stepwise
multiple regression analysis was used to examine the relationships between depressive symptoms
and changes in KP in patients and NC.
The level of significance was set as 2-tailed P values of 0.05.

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Results

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Demographic and clinical characteristics
Demographic and clinical variables of the LLD and NC groups are shown in Table 1. LLD patients
had fewer years of education than NC (P<0.001). LLD patients also exhibited higher HAMD scores

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than NC (P<0.001). No difference in gender and age was found between these two groups (all
P>0.05).
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In the EOD group, 26 patients were medication-free. Twenty patients received serotonin
noradrenaline reuptake inhibitors (SNRIs), 20 patients received selective serotonin reuptake
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inhibitors (SSRI), 4 patients received tricyclic antidepressants (TCAs), 20 patients received

noradrenergic and specific serotonergic antidepressants (NaSSA) and 54 patients received
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benzodiazepines (BZ) as a combination treatment within the last 3 months. In the LOD group, 21
patients were medication-free. Thirteen patients received SNRI, 30 received SSRI, 1 patient
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received TCAs, 15 received NaSSA and 43 patients received BZ as a monotherapy or combination

treatment within the last 3 months. No difference was found between the EOD and LOD groups in
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terms of antidepressant treatments (χ2=5.96; P=0.21) and BZ (χ2=0.67; P=0.41).

Table 1. Demographic and clinical characteristics of LLD patients and NC
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LLD NC Statistics

Mean ± SD Mean ± SD t/z df P

N 170 135

Men N (%) 25.29 31.11 1.26 1 0.26

Age (years) 66.52±7.01 66.99±6.77 0.35 304 0.55

Education (years) 8.11±3.91*** 9.80±3.39 3.98 304 <0.001

HAMD-17 11.48±8.88*** 1.75±2.83 150.83 304 <0.001

Abbreviations: LLD, late-life depression; NC, normal control; HAMD, Hamilton Depression Scale.
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Compared with NC, ***P＜0.001

The serum levels of TRP, KYN and KYNA in LLD patients and NC are provided in Table 2. The LLD
group had lower TRP, KYN and KYNA serum levels than the NC group (all P<0.05). Then, we
calculated the KYN/TRP ratio to present the shift in TRP to the KYN arm, and the kynurenic acid to
kynurenine (KYNA/KYN) ratio to reflect the imbalance between KYNA and QUIN. The LLD group
showed a higher KYN/TRP ratio (P=0.011) and a lower KYNA/KYN ratio than the NC group (P=0.032).
However, significant differences were found between the LLD and NC groups, including a higher
KYN/TRP ratio and still lower KYN serum concentration; this finding contradicted the results of the

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previous study (Strasser, Becker, Fuchs, & Gostner, 2017), and we think that further exploration of

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this issue is needed.

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Table 2. TRP, KYN and KYNA levels of LLD patients and NC

LLD NC Statistics

Fa

TRP (ng/ml)
Mean ± SD

10388.79±2607.70*** US
Mean ± SD

11849.04±1965.64 28.11
df

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P

<0.001
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KYN (ng/ml) 308.82±84.94** 333.93±66.92 7.15 1 0.008

KYNA (ng/ml) 6.676±2.83*** 7.85±3.06 12.79 1 <0.001

KYN/TRP 0.031±0.011* 0.028±0.0054 6.58 1 0.011

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KYNA/KYN 0.021±0.0081* 0.023±0.0082 4.65 1 0.032

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Abbreviations: LLD, late-life depression; NC, normal control; TRP, tryptophan; KYN, kynurenine; KYNA, kynurenic
acid; KYN/TRP, kynurenine to tryptophan ratio; KYNA/KYN, kynurenic acid to kynurenine ratio
Fa: a generalised linear model with diagnosis as the independent factor and age and gender as covariates
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Compared with NC, *P＜0.05, **P＜0.01, ***P＜0.001.

Demographics and clinical characteristics of the EOD, LOD and NC groups are presented in Table 3.
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EOD and LOD patients had fewer years of education than NC (P=0.003; P=0.004). Further, EOD
patients were younger than NC subjects (P=0.002), while LOD patients were older than NC subjects
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(P=0.027). EOD patients exhibited the first depressive episode in an earlier stage of life than LOD
patients (P<0.001). Both EOD and LOD patients showed more severe depressive symptoms than
the NC group according to HAMD scores (P<0.001), and no difference in HAMD scores was found
between the LOD and EOD groups (P=1.000). There was no difference in gender among these three
groups (all P>0.05).
Table 3. Demographic and clinical characteristics of EOD patients, LOD patients and NC
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LLD NC Statistics
EOD LOD
Mean ± SD Mean ± SD Mean ± SD F /z df P
N 90 80 135
Men N (%) 24.44 26.25 31.11 1.33 2 0.513
Age (years) 63.92±5.78*a 69.44±7.15*a 66.99±6.77 14.95 2, 302 <0.001
Education (years) 8.11±3.77**a 8.11±4.08**a 9.80±3.39 7.91 2, 302 <0.001
b
First onset age (years) 51.39±9.79*** 66.66±6.57 1.57 2, 302 <0.001
a a
HAMD-17 11.39±9.02*** 11.59±8.77*** 1.75±2.83 75.19 2, 302 <0.001

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Abbreviations: EOD, early-onset depression; LOD, late-onset depression; NC, normal control; HAMD, Hamilton

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Depression Scale.

Compared with NC, *aP＜0.05, **aP＜0.01, ***aP＜0.001

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Compared with LOD patients, *** bP＜0.001

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The serum levels of TRP, KYN, KYNA, KYN/TRP ratio and KYNA/KYN ratio in EOD, LOD and NC groups
are shown in Table 4. In pairwise comparisons of TRP levels, both EOD (P<0.001) and LOD (P<0.001)
groups had significantly lower TRP levels than the NC group, and no difference was found between
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the EOD and LOD groups (P=0.520). The EOD group exhibited a lower KYN level than the NC group
(P=0.003), and there was no difference in the KYN level between the LOD and NC groups (P=0.181),
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and between the EOD and LOD groups (P=0.160). With regard to the KYNA level, both EOD
(P<0.001) and LOD (P=0.05) groups were significantly lower than the NC group, and no difference
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was found between the EOD and LOD groups (P=0.112). The LOD group showed a significantly
higher KYN/TRP ratio than the NC group (P=0.011), and there was no difference in the KYN/TRP
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ratio between the EOD and NC groups (P=0.098), and between the EOD and LOD groups (P=0.396).
The only difference in the KYNA/KYN ratio was observed between the EOD and NC groups
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(P=0.011), and there was no difference in the KYNA/KYN ratio between the LOD and NC groups
(P=0.351), and between the EOD and LOD (P=0.166) groups.
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Table 4. TRP, KYN and KYNA levels of the EOD, LOD, and NC groups
LLD NC Statistics
EOD LOD
Mean ± SD Mean ± SD Mean± SD Fa df P
TRP (ng/ml) 10235.31±2486.95*** 10561.46±2742.66*** 11849.04±1965.64 14.23 2 <0.001
KYN (ng/ml) 295.49±80.81* 323.82±87.44 333.93±66.92 4.57 2 0.011
KYNA (ng/ml) 6.27±2.81*** 7.13±2.81* 7.85±3.06 7.70 2 0.001
KYN/TRP 0.030±0.011 0.032±0.011* 0.028±0.0054 3.65 2 0.027
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KYNA/KYN 0.021±0.0072* 0.022±0.0089 0.023±0.0082 3.30 2 0.038

Abbreviations: EOD, early-onset depression; LOD, late-onset depression; NC, normal control; TRP, tryptophan;
KYN, kynurenine; KYNA, kynurenic acid; KYN/TRP, kynurenine to tryptophan ratio; KYNA/KYN, kynurenic acid to
kynurenine ratio
Fa: a generalised linear model with diagnosis as the independent factor and age and gender as covariates

Compared with NC, * p ＜0.05, ** p ＜0.01, *** p ＜0.001

To further explore the potential influencing factors of KYN pathway changes, a factor analysis of
HAMD-17 was conducted in all 305 subjects and described in Table 5; from this analysis, we

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determined 4 principal factors. The first factor accounted for 21.968% of total variance, and we

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named it as "Anxiety", which comprised 6 items: anxiety: psychic, anxiety: somatic, somatic
symptoms: gastrointestinal, somatic symptoms: general, hypochondriasis and loss of weight. The

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second factor occupied 19.797% of total variance and was named "Depression"; it also consisted of
6 items: depressed mood, feelings of guilt, suicide, work and activities, agitation and insight. The

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third factor explained 13.723% of total variance, and we termed it as"Sleep" because it was
characterised by insomnia early, insomnia middle and insomnia late. The fourth factor accounted
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for 8.766% of total variance, and we named it "Somatisation" as it was formed by retardation:
psychomotor and genital symptoms. The four principal factors explained 64.254% of total variance.
Table 5. Factor analysis on the 17-item HAMD
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Factor 1 Factor 2 Factor 3 Factor 4

Anxiety Depression Sleep Somatisation
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12. Somatic symptoms: gastrointestinal 0.76

11. Anxiety: somatic 0.75
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15. Hypochondriasis 0.74

13. Somatic symptoms: general 0.64
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10. Anxiety: psychic 0.64

16. Loss of weight 0.59
17. Insight 0.77
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1. Depressed mood 0.71

7. Work and activities 0.66
2. Feelings of guilt 0.65
9. Agitation 0.57
3. Suicide 0.51
5. Insomnia middle 0.83
4. Insomnia early 0.82
6. Insomnia late 0.74
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14. Genital symptoms 0.85

8. Retardation: psychomotor 0.58
total 21.96 19.79 13.72 8.76
Four factors explained 64.254% of total variance
Anxiety, Depression, Sleep, and Somatisation factor scores of the EOD, LOD and NC groups are
presented in Table 6. The EOD and NC groups showed significant differences in Anxiety (P<0.001),
Depression (P<0.001) and Sleep (P=0.014). The LOD and NC groups showed significant differences
in Anxiety (P<0.001), Depression (P<0.001), Sleep (P=0.007) and Somatisation (P=0.002). However,

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there were no differences between the EOD and LOD groups with regard to these factor scores (all

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P>0.05).
Table 6. Anxiety, Depression, Sleep, and Somatisation factor scores of the EOD, LOD and NC groups

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LLD NC Statistics
EOD LOD
Fa
Anxiety
Depression
Mean ± SD
0.27±1.15***
0.41±1.15***
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Mean ± SD
0.30±1.26***
0.33±1.18***
Mean± SD
-0.36±0.44
-0.46±0.39
17.52
31.98
df
2,303
2,303
P
<0.001
<0.001
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Sleep 0.14±1.14* 0.18±1.11* -0.19±0.77 4.93 2,303 0.008
Somatisation 0.49±0.69 0.25±0.76* -0.18±0.21 5.04 2,303 0.007
Abbreviations: EOD, early-onset depression; LOD, late-onset depression; NC, normal control; TRP, tryptophan;
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Compared with NC, * p ＜0.05, ** p ＜0.01, *** p ＜0.001

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In correlation analysis, we determined whether Anxiety, Depression, Sleep, and Somatisation

factors had effects on TRP metabolism or KYN metabolism. The results showed that in all LLD
patients, KYNA was negatively associated with the "Depression" factor (r=-0.171, P=0.026). The
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KYNA/KYN ratio was also associated with the "Depression" factor (r=-0.220, P=0.004). In EOD
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patients, TRP was inversely associated with "Depression" factor (r=-0.221, P=0.037), while the
KYNA/KYN ratio was associated with "Depression" factor (r=-0.208, P=0.049). In LOD patients, the
KYNA/KYN ratio was negatively associated with the "Depression" factor (r=-0.230, P=0.040). No
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correlation was found in NC. In the stepwise multiple regression for association with KYNA in total
LLD patients, the "Depression" factor (β=-0.171, t=-2.251, P=0.026) remained significant. The
KYNA/KYN ratio was associated with the "Depression" factor (β=-0.198, t=-2.822, P=0.005). In the
stepwise multiple regression for association with TRP in EOD patients, the "Depression" factor
(β=-0.221, t=-2.121, P=0.037) remained significant. The KYNA/KYN ratio was associated with the
"Depression" factor (β=-0.380, t=-2.860, P=0.005). No correlation was found in the LOD group after
regression analysis.
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Discussion
To our best knowledge, the present study is the first to explore KP changes between LLD patients
and normal elderly people as well as to compare between EOD patients and LOD patients. The
main findings of our study were as follows: (1) LLD patients exhibited significantly different levels
of TRP, KYN, KYNA, KYNA/KYN ratio and KYN/TRP ratio compared to NC, indicating a significant KP
change in LLD. (2) The decrease in TRP level and the increase in the KYN/TRP ratio were found in
LOD patients; this reflected that the KP change of LOD was mostly disrupted in the TRP metabolism

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to KYN stage. (3) EOD patients showed low levels of TRP metabolites without a change in the

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KYN/TRP ratio; they also showed decreased KYNA/KYN ratio compared to NC, thus suggesting that

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the change from KYN to KYNA, the later part of KP, was disrupted in EOD. (4) The “Depression”
factor had an impact on KYN metabolism in LLD patients, and it was associated with the TRP level

regression analysis. US
and KYNA/KYN ratio in the EOD group, while no such correlation was found in the LOD group in

The LLD group showed a decreased TRP level and an increased KYN/TRP ratio in peripheral blood
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as compared to the NC group. As shown in previous articles, a decrease in TRP level and an
increase in the KYN/TRP ratio were associated with chronic immune activation (Savitz, Drevets, et
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al., 2015; Strasser et al., 2017), and the inflammation caused by overactivation of the immune
system is one of the primary pathological characteristics of depression (Depino, 2017).
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Furthermore, stress, especially chronic stress, causes glucocorticoid resistance, resulting in high
serum levels of glucocorticoids (Miller et al., 2008); high serum glucocorticoids were also found in
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older people with depression (Bremmer et al., 2007). Increasing levels of glucocorticoids can
enhance the activity of tryptophan 2,3-dioxygenase (TDO), which plays a major role in TRP
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metabolism in the liver. Under inflammation conditions and high glucocorticoid levels, enzymes
IDO and TDO were activated and played a major role in accelerating TRP breakdown. The
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measurements of glucocorticoid levels and pro-inflammatory factors are needed in a future study
to determine which factor plays a more crucial role in TRP disruption in LLD. In the current study,
the KYN/TRP ratio was considered as a rough estimate of TRP metabolism. In LLD patients, the TRP
metabolising process was enhanced, and the formation of KYN became more efficient. Thus, TRP
showed a general low level, and the KYN/TRP ratio became higher than that in physiological
circumstances, which hinted at the shift of TRP conversion into the KYN arm in LLD.
LLD patients also showed decreased levels of KYN, KYNA and KYNA/KYN ratio. A previous study
suggested that QUIN, one of the KYN metabolites, is an N-methyl-D-aspartate receptor (NMDA-R)
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agonist, which caused excitotoxic neurodegeneration (Schwarz, Guillemin, Teipel, Buerger, &
Hampel, 2013). While KYNA has a neuroprotective function, KYNA is an NMDA-R antagonist, which
could help to antagonises the QUIN’s toxic effect. The imbalance of KYN metabolism, the increase
in QUIN, and the decrease in KYNA made the astrocyte-microglia-neuronal network vulnerable
(Myint, 2012). In LLD, the higher KYN/TRP ratio and the lower KYNA/TRP ratio demonstrated the
shift in KYN metabolism into the QUIN arm. The decrease in KYNA synthesis was also detected,
indicating that the neurotoxic effect of QUIN was increased in LLD, but the determination of
concentration of QUIN is needed to further confirm this hypothesis. Surprisingly, KYN levels in LLD

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patients were significantly lower than those in NCs, while the KYN/TRP ratio was increased.

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Because we assumed an efficient KYN production from TRP, the LLD group was separated into EOD

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and LOD groups for further exploration.
Compared with MDD, LLD shows more varying symptoms(Blazer, 1986); this indicates that different
pathways are involved in disease development during different age of onset. Thus, LLD can divided

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into EOD and LOD according to the age of depression onset. As reported in a previous study, the
cut-off was usually set at age 60 years(Hashem et al., 2017). The development of EOD is associated
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with family history of mental disease(B & P, 2012), anxiety as a personality trait, and stressful
event(Miyata et al., 2016). On the other hand, because of serious cognitive deficits, the
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development of LOD might be correlated with somatic disease and neurodegenerative disease(RS
et al., 2014). Thus, there might be different pathogenetic mechanisms for EOD and LOD
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development, which might cause different pattern of KP change.

Comparison of the EOD and LOD groups revealed an interesting finding that EOD patients had low
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TRP levels, but the KYN/TRP ratio of EOD was not different from that of the NC group; this meant
that KP was not changed in EOD patients. The decrease in KYN and KYNA might be caused by
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deficient TRP. This phenomenon might be due to the relatively long history of depressive disease
associated with the significantly deficient TRP concentration in the EOD group (Grayson & Thomas,
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2013). As we described above, KYNA plays an essential role in neuroprotection, and the ratio of
KYNA to KYN could represent the shift of KYN to the QUIN arm indirectly. In EOD patients, the
decrease in the KYNA/KYN ratio might be associated with enhanced KMO, the concentration of
QUIN would be measured in further study to confirm. The low level of KYN in EOD may be
responsible for the low KYN level in LLD patients. Thus, in EOD, the deficits of TRP production and
the lack of neuroprotection from KYNA were profound, but TRP-KYN transformation was relatively
reserved. This phenomenon may be caused by the enhanced KMO activity, while the activity of IDO
and/or TDO was compensatory reserved due to chronic inflammation caused by the relatively
 ACCEPTED MANUSCRIPT

longer disease course. In LOD patients, a decrease in the TRP level and an increase in the KYN/TRP
ratio were found, while serum concentrations of KYN remained at similar levels to those of the NC
group. The shift of TRP conversion into the KYN arm occurred in LOD patients, and the enhanced
TRP breakdown resulted in high availability of KYN. KYNA was at a lower level than NC, thus
reflecting an imbalance between the neurotoxic and neuroprotective arms of KYN metabolism, but
according to KYNA/KYN, the shift in QUIN was less serious than that in the EOD group. Our
hypothesis was that the significant changes in TRP-KYN transformation may be caused by enhanced
activity of IDO and/or TDO resulting from a depressive situation, while in EOD, the relatively

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reserved KYN/TRP may be caused by compensatory reserved IDO and/or TDO activity resulting

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from the relatively longer disease history. The activity of KMO was still reserved at a relatively

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normal level against the neurotoxic effect, but further examination is needed to verify this. In
general, the results from the current study could help us to clearly distinguish the two pathological
processes of EOD and LOD patients, and further support the opinion that different pathologic

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processes exist in EOD and LOD, and the different activity of rate-limiting enzymes such as IDO,
TDO and KMO may be responsible for the different KP changes in EOD and LOD. However, both
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these factors can be enhanced by the pro-inflammatory factor. Thus, the reason for different KP
changes in LLD with different age of onset still requires further research.
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In the correlation analysis, we found that the “Depression” symptom was the major factor that
influenced the KYNA/KYN ratio. Previous studies have mentioned that neurodegenerative changes
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and loss of astrocytes were found in major depression, which might be partly due to the increase in
neurotoxic effects from QUIN (Myint, 2012; Strasser et al., 2017), and the KYNA/KYN ratio was
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used to present the situation of the KYNA production from KYN. We found that the KYNA/KYN ratio
was correlated to the “Depression” factor in LLD patients. The EOD group also showed the
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correlation of the KYNA/KYN ratio and the “Depression” factor, but such correlation was not found
in the LOD group after regression analysis. This result supported the opinion that the pathology of
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EOD and LOD was different in KP. In the EOD group, the TRP level and KYNA/KYN ratio changes
were relevant to the “Depression” factor, which partly explained why all KP metabolites (TRP, KYN
and KYNA) were at lower levels than those in the NC group; however, the same observation was
not noted in the LOD group in the current study. The current results partly verified our hypothesis
about EOD that these changes might be relative to the severity of depressive symptoms, and
relatively longer histories of depression may explain these phenomena. However, the lack of
accurate medical histories is one of the limitations of this study.
In addition to the single race of patients, the present study also has other limitations: (1) The study
 ACCEPTED MANUSCRIPT

lacked cognitive assessment, which might have affected the KP; but we plan to investigate the
relationship between cognitive function and KP change in LLD in the further study. (2) The relative
rate-limiting enzymes such as IDO, TDO and KMO, and measurements of their causative factors
such as pro-inflammatory factors and glucocorticoids were needed to determine the underlying
cause of different KP changes in the EOD and LOD groups. (3) The use of anti-depression
medication may have effects on the serum 5-HT level and cause slight KP changes, but these
changes are vague. No difference was found in medications used by the EOD and LOD groups.
Furthermore, the KP changes between the LLD and NC groups were significant; therefore, the

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effects of the medication may be ignored. However, the effect of medication on KP change could

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be a topic of future study. (4) In current study, the age of onset in EOD seemed later than our

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expectation. According to this issue, we considered that the recognition of depression in China was
paid less attention in the past, which made the relative delay diagnosis in depression, even though
patients might have appeared depressive symptoms rather early. But we are confident with the

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group separation in this study, because of the sample size and the strict inclusion and exclusion
criteria.
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In conclusion, KP changes were observed in LLD patients, including lower serum concentrations of
TRP, KYN and KYNA, and the shifts of TRP conversion into the KYN arm and KYN metabolism into
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the KYNA arm. Further, the patterns of KP changes in EOD and LOD patients were different. EOD
patients presented serum concentration changes in TRP, KYN and KYNA, and changes in KYN
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metabolism, while LOD patients showed the shift of TRP metabolism. KYN metabolism changes
were associated with the “Depression” factor in the EOD group; this hinted that serum level
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changes of TRP, KYN and KYNA in EOD patients were partly due to the severity of their depressive
symptoms, while profound KP shifts were found in LOD patients.
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Role of the funding source

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This work was supported by the National Natural Science Foundation of China [No. 81571333; and
No. 81701341]; Science and Technology Department of Guangdong Province major science and
technology [No.2016B010108003]; the National R & D program focused on precision medical
research of China [No. 2016YFC0906302]; the National Key Technology Research and Development
Program of the Ministry of Science and Technology of China [No. 2015BAI13B01]; the Science and
Technology Program of Guangzhou [No. 2014J4100135]; and Guangzhou municipal key discipline
in medicine (2017-2019). The funding agency had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
 ACCEPTED MANUSCRIPT

Conflict of interest

All authors declare no potential conflict of interests.

Acknowledgement
We thank Bin Sun for helpful discussion on statistical analysis.

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