Altered Kynurenine Pathway Metabolism in Drug-naive Children and Adolescents with Generalized Anxiety Disorder

Abstract

Objective

Generalized anxiety disorder (GAD) is among the most common psychiatric conditions in children and adolescents. Although its aetiology remains unclear, emerging evidence highlights the role of immune-metabolic pathways in its development. This study aimed to investigate alterations in the kynurenine pathway (KP) metabolites in drug-naive paediatric patients with GAD compared to healthy controls.

Methods

Twenty-six drug-naive children with GAD and thirty-two age- and sex-matched healthy controls were enrolled. Serum levels of tryptophan (TRP), kynurenine (KYN), kynurenic acid (KYNA), and quinolinic acid (QUIN) were measured using ELISA. Enzymatic activity indices, including KYN/TRP (IDO/TDO), KYNA/KYN (KAT), and QUIN/KYN (KMO), were calculated. Group differences and correlations with clinical anxiety scores were analysed. Additionally, ROC analysis was performed to evaluate the diagnostic performance of TRP levels in predicting GAD.

Results

TRP levels were significantly elevated in the GAD group (median 62.8 μmol/L) compared to controls (42.9 μmol/L, p < 0.001), while KYN (54.5 vs. 66.8 nmol/L, p = 0.042) and QUIN (9.9 vs. 11.9 μmol/L, p = 0.004) levels were reduced. The KYN/TRP ratio was positively correlated with RCADS-CV total anxiety scores (r = 0.430, p = 0.028). ROC analysis indicated that TRP had good diagnostic performance for GAD, with an AUC of 0.824, sensitivity of 73%, and specificity of 78%.

Conclusion

These findings suggest that disrupted tryptophan metabolism, characterized by reduced KP activation and altered neurotoxic metabolite levels, may contribute to the pathophysiology of paediatric GAD. The KP may represent a promising target for future biomarker and therapeutic research in anxiety disorders.

INTRODUCTION

Generalized anxiety disorder (GAD) is a prevalent and disabling psychiatric condition affecting children and adolescents, often manifesting through excessive and uncontrollable worry about various aspects of daily life. It is frequently underdiagnosed in youth due to its overlapping somatic symptoms and comorbid presentations [1,2]. GAD presents with both physical and psychological symptoms. Patients commonly report physical complaints such as palpitations, sweating, trembling, dry mouth, shortness of breath, chest pain, and difficulty swallowing. Psychological symptoms include restlessness, fatigue, difficulty concentrating, irritability, and sleep disturbances. Interestingly, patients often seek medical attention for physical symptoms rather than psychological ones [3]. GAD symptoms usually begin in early childhood, fluctuate throughout adolescence, and may intensify in mid/late adolescence [4]. GAD often co-occurs with other psychiatric disorders, such as depression, attention deficit hyperactivity disorder (ADHD), and obsessive-compulsive disorder (OCD) [5].

Although the exact aetiology of GAD remains unclear, it is thought to be a multifactorial interaction of environmental and genetic factors [1]. In this context, chronic stress has been identified as an important modulator of neuroimmune interactions. Prolonged stress can affect the hypothalamic-pituitary-adrenal (HPA) axis and the immune system, leading to hypercortisolemia and increased levels of pro-inflammatory cytokines [6]. These cytokines can trigger neuroinflammation, exacerbating the clinical symptoms of psychiatric diseases [7-9]. The kynurenine pathway (KP), the main metabolic pathway of tryptophan (TRP), plays a key role in neuroinflammation and central nervous system (CNS) changes [10,11]. Imbalances in TRP metabolism, especially changes in the ratios of its neuroactive metabolites, have been associated with many neurological and psychiatric disorders [12]. Disruptions in KP may worsen CNS-related conditions.

Through the enzymatic actions of indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO), TRP is shunted away from serotonin synthesis toward the KP, producing neuroactive metabolites such as kynurenine (KYN), kynurenic acid (KYNA), and quinolinic acid (QUIN) (Fig. 1) [13-15]. Cytokines like interferon-gamma and tumor necrosis factor-alpha activate IDO, while inflammatory and stress conditions indirectly stimulate its activity [16]. These metabolites exert diverse effects on glutamatergic neurotransmission, oxidative stress, and neuroinflammation, potentially contributing to psychiatric symptoms. Increased IDO activity redirects TRP from serotonin production to the KP. The first stable product, KYN, is a pivotal point in the KP and is further metabolized into neuroprotective KYNA in astrocytes or neurotoxic QUIN in microglia and macrophages [17]. The KYNA/KYN ratio reflects the neuroprotective status, while QUIN/KYNA ratios are used as markers of neurotoxic activity [18].

Fig. 1.

The kynurenine pathway of tryptophan metabolism.

IDO, indoleamine 2,3-dioxygenase; TDO, tryptophan 2,3-dioxygenase; KAT, kynurenine aminotransferases; KMO, kynurenine 3-monooxygenase.

Rising evidence suggests that the KP, an important pathway of TRP metabolism, is relevant to the neuroinflammatory mechanisms of a broad range of psychiatric illnesses. Alterations in KP function have been reported in major depressive disorder (MDD) [11], ADHD [19], and social anxiety disorder [20], implying that immune-metabolic dysregulation may be a trans-diagnostic process of various types of psychopathology. These findings emphasize the neuroimmune crosstalk role in psychiatric disorders and suggest disturbances in KP metabolites, such as KYN, KYNA, and QUIN, may influence the clinical presentation and severity of anxiety. GAD may share common neurobiological mechanisms with these disorders, particularly in the context of chronic stress and HPA axis dysregulation. Chronic stress activates the HPA axis, leading to sustained hypercortisolemia and increased production of pro-inflammatory cytokines, which influence KP activity and neuroinflammation [6].

These converging mechanisms suggest that alterations in the KP may play a role in the pathogenesis of GAD, yet this area remains largely unexplored in paediatric populations. To our knowledge, this is the first study to examine the relationship between the TRP–KP and GAD in children and adolescents. Specifically, we assessed the serum concentrations of TRP, KYN, KYNA, and QUIN, along with enzymatic activity indices including KYN/TRP (IDO/TDO activity), KYNA/KYN (KAT activity), and QUIN/KYN (kynurenine 3-monooxygenase [KMO] activity) ratios. Furthermore, we investigated the associations between these biochemical markers and clinical features of GAD to better elucidate the underlying biological mechanisms and contribute to a more comprehensive understanding of its aetiopathogenesis.

METHODS

Study Design and Ethics

This cross-sectional study was conducted at the Gazi University Faculty of Medicine (GUFM), Ankara, Türkiye. The study protocol adhered to the ethical principles outlined in the Declaration of Helsinki. Ethical approval was obtained from the Gazi University Clinical Research Ethics Board (Decision No.: 189, dated 07/03/2022). Written informed consent was obtained from all participants and their legal guardians prior to study enrolment.

Participants and Recruitment

This study was conducted at the GUFM. Participants were selected from the GUFM hospital’s Child and Adolescent Psychiatry Outpatient Clinic, and samples were analysed at the Department of Medical Biochemistry of the Gazi University Hospital. The researcher conducted a semi-structured psychiatric interview for the patient group to ensure compliance with the inclusion and exclusion criteria.

Inclusion criteria for the patient group were as follows: children and adolescents aged between 8 and 18 who were diagnosed with GAD based on the Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime Version (K-SADS-PL-DSM-5) and Revised Child Anxiety and Depression Scales (RCADS). Exclusion criteria were carefully defined to ensure sample homogeneity and minimize confounding factors. The exclusion criteria applied to all participants are summarized in Table 1.

Table 1.

Exclusion criteria applied to study participants

Kriter No.	Exclusion criteria
(i)	Current or past diagnosis of neurological (e.g., cerebral palsy, epilepsy), metabolic (e.g., phenylketonuria), endocrinological (e.g., diabetes mellitus), oncological, allergic, rheumatological, inflammatory, or autoimmune disorders.
(ii)	History of any infectious disease within the past 4 weeks.
(iii)	Use of anti-inflammatory medication in the past 4 weeks.
(iv)	Receipt of immunosuppressive therapy within the last 6 months.
(v)	Substance use history within the past 3 months.
(vi)	Clinical suspicion of paediatric autoimmune neuropsychiatric disorders associated with streptococci.
(vii)	Meeting the criteria for obesity (BMI ≥ 95th percentile).
(viii)	Diagnosis of any psychiatric disorder other than specific phobia, including major depressive disorder, obsessive-compulsive disorder, attention-deficit/hyperactivity disorder, post-traumatic stress disorder, psychotic disorders, or intellectual disability.

Clinical Evaluations

Clinical assessments were carried out by an experienced child and adolescent psychiatrist to prevent any influence on study outcomes. The assessment included a semi-structured diagnostic interview, standardized rating scales, and a sociodemographic questionnaire.

The Sociodemographic Data Form, developed by the researchers, was used to collect information on the age, gender, number of siblings, family structure, level of parental education, and socioeconomic status of the participants’ household.

The K-SADS-PL-DSM-5 was used to assess psychiatric diagnosis and determine group eligibility. This semi-structured interview assesses current and lifetime psychiatric disorders in children and adolescents between the ages of 6 and 18 years according to DSM-5 criteria [21]. The validity and reliability of the Turkish version of the instrument were demonstrated by Ünal et al. (2018) [22].

The Revised Children’s Anxiety and Depression Scales—Child and Parent Versions (RCADS-CV & RCADS-PV) is used in the assessment of anxiety and depressive symptoms. The 47-item scale assesses general anxiety severity and includes subscales for separation anxiety disorder, GAD, panic disorder, social phobia, OCD, and MDD [23]. Gormez et al. (2017) [24] performed the adaptation and psychometric testing of the scale in Turkish.

Biochemical Procedures

Venous blood samples from drug-naive GAD patients and the control group were collected in the morning (between 08:00 and 10:00) after an overnight fasting period of at least 8 hours, and then centrifuged for 15 minutes at 4,000 rpm to obtain serum for biochemical analysis. Haemolytic or icteric samples were excluded to maintain data integrity. Serum levels of TRP, KYN, KYNA, and QUIN were determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits (BT LAB; Cat. Nos: TRP – E4244Hu, KYN – EA0098Hu, KYNA – E3982Hu, QUIN – E3983Hu), following the manufacturer’s protocols. Assays were performed in duplicate. The detection limits of the ELISA kits were 0.23 μg/ml for TRP, 1.24 pmol/ml for KYN, 0.21 nmol/L for KYNA, and 0.103 nmol/ml for QUIN. TRP concentrations were converted from μg/ml to μmol/L using a molecular weight-based conversion factor of 4.89. KYN and QUIN levels were expressed in nmol/L and μmol/L, respectively. All other biochemical parameters were reported in their original units. Haemolytic or icteric samples were excluded.

Calculation of enzyme activity ratio

Various ratios reflecting the enzymatic activities of the measured biomarkers were calculated. The KYN/TRP ratio served as an indicator of IDO and TDO activity, the KYNA/KYN ratio reflected kynurenine aminotransferase (KAT) activity, and the QUIN/KYN ratio indicated KMO activity. Additionally, the KYNA/QUIN and QUIN/KYNA ratios were evaluated as neuroprotective and neurotoxic indices, respectively. Clinical data also included the duration since symptom onset for each participant.

Statistical Analyses

Statistical calculations were made using the SPSS Statistics version 23.0 (IBM Corp.). Descriptive statistics were presented as frequencies (n, %) for categorical data and as means ± standard deviation or medians with interquartile ranges (IQR) for numerical data. The chi-square test was used to compare categorical variables. The normality of numerical variables was assessed with the Kolmogorov-Smirnov test. For correlation analyses, clinical scores used included the RCADS-CV Generalized Anxiety Disorder subscale (RCADS-CV GAD) and total anxiety score (RCADS-CV Total Anxiety), as well as the RCADS-PV GAD subscale and total anxiety score. Depending on data distribution, appropriate correlation coefficients (Pearson or Spearman) were calculated. Correlation coefficients (r) were interpreted as follows: 0.0−0.39 (no relationship), 0.40−0.69 (moderate), 0.70−0.89 (strong), and 0.90−0.99 (very strong). A pvalue of < 0.05 was considered statistically significant, with results evaluated at a 95% confidence interval. To assess the diagnostic utility of TRP in diagnosing GAD, the area under the curve (AUC) and threshold values were determined using receiver operating characteristic (ROC) curve analysis.

RESULTS

Socio-demographic and Clinical Profiles

The socio-demographic and clinical characteristics of treatment-naive GAD patients (n = 26) and controls (n = 32) are detailed in Table 1. The median [IQR] ages for the GAD and control groups were 170 [138−190] and 154 [124−175] months, respectively. Both groups were similar concerning age, gender, BMI percentile, smoking habits, COVID-19 history, and parental and family characteristics (p > 0.05), except for the prevalence of psychiatric illnesses within the family. The GAD group had a significantly higher proportion of individuals with a family history of psychiatric disorders compared to the control group (p = 0.011). The median [IQR] duration since symptom onset among GAD patients was 10 [6−24] months. Consistent with their diagnosis, the GAD group exhibited significantly higher scores on all subscales of the RCADS-CV compared to the control group (p < 0.001 for all subscales). The sociodemographic, clinical characteristics, and RCADS scores of all subjects are shown in Table 2.

Table 2.

Sociodemographic, clinical characteristics, and scores of children with GAD and control

Parameters	GAD group (n = 26)	Control group (n = 32)	p
Sex (female/male)	19/7 (73.1/26.9)	17/15 (53.1/46.9)	0.119
Family history of chronic illnesses	13 (52.0)	21 (65.6)	0.298
Family history of psychiatric disorders	15 (57.7)	8 (25.0)	0.011*
Age (yr)	170 [138−190]	154 [124−175]	0.205
BMI percentile	45.4 [21.5−79]	61 [22.8−79.6]	0.617
Duration since symptom onset (mo)	10 [6−24]	-	-
RCADS-CV scores
SAD	59.5 [53−66.8]	44.5 [41−52]	< 0.001*
GAD	58.5 [53.3−68]	42 [39−46]	< 0.001*
PD	68 [51.3−80]	43.5 [39−51]	< 0.001*
SP	53.5 [42−71.5]	39 [33−45]	< 0.001*
OCD	60.5 [52.5−69]	46 [38.5−52]	< 0.001*
MDD	60.5 [44−73]	42.5 [36−48]	< 0.001*
Total	65 [50.8−75]	41 [37−48]	< 0.001*

Values are presented as number (%) or median [interquartile range].

RCADS-CV, Revised Child Anxiety and Depression Scale-Child Version; SAD, separation anxiety disorder; GAD, generalized anxiety disorder; PD, panic disorder; SP, social phobia; MDD, major depressive disorder; OCD, obsessive-compulsive disorder; -, not available.

*p < 0.05.

Comparison of Kynurenine Pathway Measures between the GAD and Control Groups

In biochemical experiments with 26 GAD patients and 32 controls, serum levels of TRP, KYN, KYNA, and QUIN were analysed. Various ratios were calculated to assess specific enzyme activities: the KYN/TRP ratio was used to evaluate IDO/TDO activity, the KYNA/KYN ratio for KAT activity, and the QUIN/KYN ratio for KMO activity. The KYNA/QUIN and QUIN/KYNA ratios were evaluated as neuroprotective and neurotoxic indices, respectively. All parameters are presented as median [IQR] in Table 3.

Table 3.

Differences in KYN pathway measures between the GAD and control groups

Markers	GAD group (n = 26)	Control group (n = 32)	p
TRP (mmol/L)	62.8 [19.7−82.3]	42.9 [11−52]	< 0.001
KYN (nmol/L)	54.5 [44.3−68.4]	66.8 [49−81]	0.042
KYNA (nmol/L)	27.4 [22.2−36.9]	30.3 [27−36]	0.270
QUIN (mmol/L)	9.9 [7.8−11.4]	11.9 [10−14]	0.004
KYN/TRP × 1,000 (IDO/TDO activity)	89.9 [62.1−115.7]	206.7 [109−491]	< 0.001
KYNA/KYN × 100 (KAT activity)	61.5 [38.4−79.8]	51.8 [35−70]	0.247
QUIN/KYN (KMO activity)	0.18 [0.1−0.3]	0.18 [0.1−0.3]	0.566
KYNA/QUIN (neuroprotective index)	2.8 [2.3−3.6]	2.5 [2.1−3.0]	0.235
QUIN/KYNA (neurotoxic index)	0.36 [0.3−0.4]	0.40 [0.3−0.5]	0.219

Values are expressed as median [interquartile range].

GAD, generalized anxiety disorder; TRP, tryptophan; KYN, kynurenine; KYNA, kynurenic acid; QUIN, quinolinic acid; IDO/TDO, indoleamine 2,3-dioxygenase/tryptophan 2,3-dioxygenase; KAT, kynurenine aminotransferases; KMO, kynurenine 3-monooxygenase.

Serum TRP levels of GAD patients were significantly higher than those of healthy controls (p < 0.001). Serum KYN and QUIN levels of GAD patients were significantly lower than those of healthy controls (p = 0.042 and p = 0.004, respectively). KYN/TRP ratio, which is considered to indicate IDO/TDO activity, was significantly lower in the GAD group compared to the control group (p < 0.001) (Fig. 2). The groups had no significant differences regarding other metabolites or ratios within the KP (p > 0.05).

Fig. 2.

Comparison of kynurenine pathway metabolites and indices between GAD patients and controls.

GAD, generalized anxiety disorder; TRP, tryptophan; KYN, kynurenine; QUIN, quinolinic acid.

Correlation Analysis

Spearman correlation analyses were performed to evaluate the associations between KP metabolites and clinical parameters, including anxiety symptom severity and duration since symptom onset. In the GAD group, disease duration positively correlated with RCADS-CV GAD subscale (r = 0.448, p = 0.022) and total anxiety scores (r = 0.547, p = 0.004), suggesting a progressive symptom burden over time. These relationships are detailed in Table 4 (GAD group) and Table 5 (healthy controls).

Table 4.

Correlations between serum KYN pathway metabolites and clinical variables in children and adolescents with GAD (n = 26)

	RCADS-CV GAD			RCADS-PV GAD			RCADS-CV Total Anxiety			RCADS-PV Total Anxiety
	r	p		r	p		r	p		r	p
TRP (mmol/L)	−0.170	0.405		−0.054	0.792		−0.269	0.184		−0.057	0.782
KYN (nmol/L)	0.340	0.089		0.241	0.236		0.350	0.080		0.252	0.215
KYNA (nmol/L)	−0.167	0.415		0.075	0.717		−0.314	0.118		−0.179	0.382
QUIN (mmol/L)	−0.207	0.311		−0.151	0.460		−0.328	0.102		−0.229	0.261
KYN/TRP (IDO/TDO activity)	0.330	0.100		0.180	0.378		0.430	0.028		0.201	0.325
KYNA/KYN (KAT activity)	−0.317	0.114		−0.088	0.669		−0.454	0.020		−0.291	0.149
QUIN/KYN (KMO activity)	−0.322	0.108		−0.199	0.331		−0.394	0.046		−0.239	0.240
KYNA/QUIN (neuroprotective index)	−0.067	0.746		0.101	0.623		−0.146	0.475		−0.114	0.580
QUIN/KYNA (neurotoxic index)	0.049	0.813		−0.112	0.585		0.141	0.491		0.104	0.614
Duration since symptom onset	0.448	0.022		0.237	0.244		0.547	0.004		0.256	0.207

Index values presented as: KYN/TRP × 1,000; KYNA/KYN × 100. Spearman correlational analysis.

GAD, generalized anxiety disorder; TRP, tryptophan; KYN, kynurenine; KYNA, kynurenic acid; QUIN, quinolinic acid; IDO/TDO, indoleamine 2,3-dioxygenase/tryptophan 2,3-dioxygenase; KAT, kynurenine aminotransferases; KMO, kynurenine 3-monooxygenase; r, correlation coefficient; RCADS-CV, Revised Children’s Anxiety and Depression Scale-Child Version; RCADS-PV, Revised Children’s Anxiety and Depression Scale-Parent Version.

Table 5.

Correlations between serum KYN pathway metabolites and RCADS-CV & RCADS-PV scores in healthy controls (n = 32)

	RCADS-CV GAD			RCADS-PV GAD			RCADS-CV Total Anxiety			RCADS-PV Total Anxiety
	r	p		r	p		r	p		r	p
TRP (mmol/L)	0.063	0.732		−0.034	0.853		0.065	0.724		0.004	0.982
KYN (nmol/L)	0.013	0.945		−0.072	0.695		−0.067	0.717		−0.125	0.496
KYNA (nmol/L)	−0.024	0.894		0.162	0.375		−0.028	0.878		0.176	0.335
QUIN (mmol/L)	−0.008	0.964		0.016	0.931		0.091	0.621		0.104	0.570
KYN/TRP (IDO/TDO activity)	−0.012	0.947		0.019	0.916		−0.066	0.721		−0.076	0.678
KYNA/KYN (KAT activity)	−0.042	0.818		0.207	0.255		0.068	0.710		0.221	0.223
QUIN/KYN (KMO activity)	−0.096	0.602		0.040	0.829		0.062	0.736		0.130	0.478
KYNA/QUIN (neuroprotective index)	0.082	0.656		0.100	0.585		−0.001	0.788		−0.036	0.844
QUIN/KYNA (neurotoxic index)	−0.083	0.652		−0.115	0.530		0.050	0.787		0.032	0.860

Index values presented as: KYN/TRP × 1,000; KYNA/KYN × 100. Spearman correlational analysis.

GAD, generalized anxiety disorder; TRP, tryptophan; KYN, kynurenine; KYNA, kynurenic acid; QUIN, quinolinic acid; IDO/TDO, indoleamine 2,3-dioxygenase/tryptophan 2,3-dioxygenase; KAT, kynurenine aminotransferases; KMO, kynurenine 3-monooxygenase; r, correlation coefficient; RCADS-CV, Revised Children’s Anxiety and Depression Scale-Child Version; RCADS-PV, Revised Children's Anxiety and Depression Scale-Parent Version.

Among KP indices, the KYN/TRP ratio (reflecting IDO/TDO activity) showed significant positive correlations with RCADS-CV Total Anxiety (r = 0.430, p = 0.028). Conversely, the KYNA/KYN ratio (KAT activity) was negatively correlated with total anxiety (r = −0.454, p = 0.020), and the QUIN/KYN ratio (KMO activity) (r = −0.394, p = 0.046) also demonstrated a negative association with anxiety severity. Correlations between RCADS-CV Total Anxiety and duration since symptom onset, KYN/TRP ratio, KYNA/KYN ratio, and QUIN/KYN ratio are presented in Figure 3.

Fig. 3.

Correlations between RCADS-CV total anxiety scores and KYN/TRP, KYNA/KYN, QUIN/KYN ratios, and duration since symptom onset in the GAD group.

GAD, generalized anxiety disorder; TRP, tryptophan; KYN, kynurenine; KYNA, kynurenic acid; QUIN, quinolinic acid; IDO/TDO, indoleamine 2,3-dioxygenase/tryptophan 2,3-dioxygenase; KAT, kynurenine aminotransferases; KMO, kynurenine 3-monooxygenase; RCADS-CV, Revised Children’s Anxiety and Depression Scale-Child Version.

ROC Analysis

TRP levels demonstrated good discriminative power in distinguishing GAD patients from healthy controls. The area under the ROC curve (AUC) was 0.824 (95% CI: 0.719−0.929, p < 0.001), indicating a strong ability to classify individuals based on serum TRP concentrations (Fig. 4). The optimal cut-off point determined for TRP was 52.78 μmol/L, yielding a sensitivity of 73% and a specificity of 78%.

Fig. 4.

The ROC to assess the diagnostic value of tryptophan for the prediction of GAD.

Diagonal segments are produced by ties.

ROC, receiver operating characteristic; GAD, generalized anxiety disorder.

DISCUSSION

In this study, serum levels of TRP, KYN, KYNA, and QUIN were compared between drug-naive children and adolescents (8−18 years) diagnosed with GAD and matched healthy controls. We aimed to identify biochemical alterations in the KP associated with GAD and evaluate their relevance to clinical symptomatology. To our knowledge, this is the first study investigating the KP in treatment-naive paediatric GAD patients, offering a unique contribution to the growing literature on immune-metabolic pathways in psychiatric disorders.

Our findings revealed significantly higher serum TRP levels, significantly lower KYN/TRP ratios in the GAD group, and reduced KYN and QUIN concentrations. This metabolic profile contrasts with previous reports in disorders such as MDD and schizophrenia (SCZ), where chronic inflammation increases IDO activity, shifting TRP metabolism toward the KP, resulting in decreased TRP and elevated KYN/TRP ratios [13,16,25-27]. These divergences suggest alternative or non-inflammatory mechanisms may govern TRP metabolism in paediatric GAD. Although elevated TRP is uncommon in psychiatric disorders, Evangelisti et al. (2017) [19] reported similar findings in children with ADHD, suggesting the existence of developmental or disorder-specific KP alterations. The presence of elevated TRP in both disorders, despite their divergent clinical and neurobiological profiles, suggests that developmental stage or age-specific immunometabolic regulation may influence TRP metabolism across distinct paediatric psychiatric conditions.

There are several mechanisms that may be behind the elevated levels of TRP in children with GAD. One of them is the relative deficiency or down-modulation of pro-inflammatory cytokine signalling that, under different conditions, induces IDO activity. In the absence of such immune activation, breakdown of TRP via the KP is reduced, and thus, circulating levels of TRP rise. Another possible contributing factor is the gut-brain axis. Gut microbiota are crucial in modulating the metabolism of TRPs, not just by direct enzymatic degradation but also by controlling host immune response. Dysbiosis or defective microbial catabolism may decrease peripheral use of TRP via the KP, which will contribute to its systemic augmentation [12]. Additionally, peripheral TRP levels are determined by nutritional status and competitive transport via the blood-brain barrier. TRP shares a transporter (LAT1) with other large neutral amino acids such as leucine and phenylalanine, and their relative accessibility can limit TRP entry into the CNS [12,25].

Therefore, elevated serum TRP is not necessarily a reflection of increased serotonergic activity in the brain, rendering interpretation of TRP as a direct measurement of neurochemical function tenuous. The possibility of enhanced serotonin synthesis as a compensatory mechanism also warrants consideration. TRP is the precursor for serotonin (5-HT), converted by tryptophan hydroxylase [4,13]. Although increased TRP could enhance serotonergic tone, the impact on anxiety is complex. Overactivation of specific serotonin receptors—particularly 5-HT2A—may exacerbate anxiety symptoms rather than ameliorate [28]. Therefore, elevated peripheral TRP does not straightforwardly translate into anxiolytic effects and may contribute to a dysregulated serotonergic system in GAD [29].

An equally striking finding was the significantly reduced QUIN levels in GAD patients. As a neurotoxic metabolite produced by activated microglia, QUIN has been implicated in excitotoxicity, oxidative stress, and neuroinflammation [17,18,30]. Elevated QUIN levels have been consistently linked to symptom severity in SCZ and bipolar disorder [31-33]. Notably, inflammation-related neuroprogression and glial dysfunction have been strongly implicated in the pathophysiology of bipolar disorder [34], reinforcing the link between neurotoxic KP metabolites and affective symptom severity. Thus, the reduction of QUIN in paediatric GAD may indicate a distinct, potentially protective, metabolic profile.

Although KYNA levels and the KYNA/QUIN ratio did not differ significantly between groups, the overall profile—elevated TRP, decreased KYN and QUIN, and a lower KYN/TRP ratio—strongly suggests a downregulated or reconfigured KP in paediatric GAD. The observed associations between TRP metabolism and clinical severity support this hypothesis. We found that TRP levels and KYN/TRP ratios significantly correlated with RCADS anxiety scores, with specific correlations observed in our study: KYN/TRP1000 (an indicator of IDO/TDO activity) showed positive correlations with RCADS-CV Total Anxiety (r = 0.430, p = 0.028). Conversely, KYNA/KYN100 (reflecting KAT activity) correlated negatively with total anxiety (r = −0.454, p = 0.020). Additionally, QUIN/KYN (representing KMO activity) was negatively correlated with total anxiety (r = −0.394, p = 0.046) and total internalizing (r = −0.404, p = 0.041). This suggests that immune-metabolic shifts are not merely epiphenomenal but may play a mechanistic role in symptom expression [26,35]. In contrast, our GAD profile may indicate, though speculative, a lower inflammatory tone or a compensatory neuroprotective pattern.

Interestingly, prior research has demonstrated that certain low-inflammation subtypes of MDD exhibit reduced KYN/TRP ratios, in contrast to the elevated ratios typically seen in inflammation-driven depressive phenotypes [36]. This underscores the heterogeneity in immune-metabolic profiles within psychiatric disorders. In our study, the positive correlation observed between KYN/TRP ratio and anxiety severity within the GAD group may reflect an early or subthreshold stage of immune activation, wherein increasing symptomatology corresponds to modest elevations in IDO or TDO activity and progressive KYN production.

Additional support for the relevance of KP dysregulation in psychiatric conditions comes from studies like Mingoti et al. (2022) [37], who showed that systemic inflammation and oxidative stress contribute to depression via TRP depletion and neurotoxic metabolite production. While our findings diverge from some previous studies, they nevertheless highlight the crucial role of TRP metabolism as a dynamic marker in neuropsychiatric disorders, suggesting a need for more nuanced investigations.

Compared to the adolescent MDD literature — where TRP is often reduced and KYN/TRP elevated — our findings suggest a unique paediatric GAD phenotype characterized by a non-inflammatory or alternative KP regulation [11,25,26]. Even in ADHD, where TRP elevation was noted, the pattern of reduced KYN and QUIN seen here has not been previously documented together [19].

The absence of any significant correlations between biochemical measures and RCADS-CV scores in both the child and parent control groups further strengthens the significance of the findings in the patient groups. This suggests that the observed changes in these biochemical markers may be specific to the pathophysiology of anxiety and depressive disorders, indicating their potential role as biomarkers in distinguishing disease states from the normal population.

To our knowledge, this is the first study to investigate the KP in drug-naive paediatric patients with GAD. The study also benefits from its focus on treatment-naive participants, eliminating pharmacological confounders that often affect KP activity. Moreover, the use of standardised diagnostic tools and validated clinical scales strengthens the internal validity of the findings. However, several limitations must be acknowledged. First, the cross-sectional nature of the study precludes any inference of causality; whether KP abnormalities are a cause or consequence of GAD remains unclear. Second, while adequate for initial exploratory comparisons, the sample size limits statistical power and generalizability to broader populations. Third, peripheral serum metabolites may not fully reflect CNS metabolism due to the selective permeability of the blood–brain barrier. Forth, although all participants fasted overnight, individual differences in dietary TRP intake or nutritional status were not systematically assessed and may have influenced serum metabolite levels.

Future studies should incorporate longitudinal designs, include inflammatory and microbiota markers, and explore the central-peripheral correspondence of TRP metabolites using neuroimaging or cerebrospinal fluid analysis. Investigating the relationship between TRP metabolism and treatment response may also provide valuable insights for developing targeted interventions in paediatric anxiety disorders.

In conclusion, this study highlights the critical role of the KP in the pathophysiology of paediatric GAD. Our findings show that TRP levels are significantly higher in GAD patients, while KYN and QUIN levels are lower, and the KYN/TRP ratio is reduced. This unique TRP metabolism profile may point to the critical importance of immune-metabolic mechanisms in this disorder.

Our study provides a valuable contribution to the growing literature linking immune-metabolic pathways to psychiatric disorders by deepening our understanding of the biochemical basis of GAD. These findings provide a solid foundation for the development of personalized interventions targeting KP activity to alleviate anxiety symptoms in the future.