Linkage of kynurenine pathway metabolites to neuroinflammation in drug-naive children with attention-deficit/hyperactivity disorder: A cross-sectional, case–control study

Abstract

BACKGROUND

Attention-deficit/hyperactivity disorder (ADHD) is a neurobehavioral disorder that causes psychological, social, academic, and occupational impairments. Despite numerous studies, its etiopathogenesis remains incompletely understood.

AIM

To compare serum kynurenine pathway (KP) metabolite levels and metabolite ratios between drug-naive children with ADHD and healthy controls.

METHODS

The study included 51 drug-naive children with ADHD and 36 age- and gender-matched healthy controls. Serum tryptophan, kynurenine (KYN), kynurenic acid (KYNA), and quinolinic acid (QUIN) levels were measured. Ratios reflecting the activities of KP enzymes, namely KYN/tryptophan, KYNA/KYN, and QUIN/KYN, were measured, and the neurotoxic (QUIN/KYNA) and neuroprotective (KYNA/QUIN) indices were calculated.

RESULTS

Compared with controls, serum KYNA levels and the KYNA/QUIN ratio (neuroprotective index) were significantly lower in the ADHD group, while the QUIN/KYNA ratio (neurotoxic index) was significantly higher (P = 0.023, 0.025, and 0.029, respectively). There were no significant differences between groups in other KP metabolites or their ratios (P > 0.05).

CONCLUSION

These findings support the hypothesis that neuroinflammatory imbalance in the KP contributes to the pathogenesis of ADHD and highlight the importance of new biomarkers for the development of targeted therapies.

Key Words: Attention deficit hyperactivity disorder; Kynurenine; Kynurenine pathway; Neuroinflammation; Tryptophan metabolism alterations

Core Tip: This study examined the kynurenine pathway (KP) in drug-naive children with attention-deficit/hyperactivity disorder (ADHD), focusing on the balance between neuroprotective and neurotoxic metabolites. Serum kynurenic acid (KYNA) levels and the KYNA/quinolinic acid ratio were significantly lower in ADHD patients, while the neurotoxic quinolinic acid/KYNA ratio was higher compared to controls. These findings suggest an imbalance between excitotoxic and neuroprotective mechanisms and suggest that dysregulation of the KP may play a role in the pathophysiology of ADHD. Although diagnostic accuracy is low, the results highlight the potential of KP-derived indices as biomarkers of ADHD. Larger longitudinal studies will be necessary to determine causal relationships and assess the therapeutic modulation of KP activity.

INTRODUCTION

Attention-deficit/hyperactivity disorder (ADHD) is a prevalent neurodevelopmental disorder characterized by persistent symptoms of inattention, hyperactivity, and impulsivity[1]. These symptoms generally emerge in early childhood and often persist into adolescence and adulthood[2,3]. Individuals with ADHD frequently face challenges in social relationships and academic performance. In addition, patients suffer from deteriorated peer interactions, behavior problems, and occupational failure. ADHD is linked to various comorbidities, including depression, anxiety, drug addiction, criminal behavior, and health problems primarily related to accidental injury[4,5]. ADHD is one of the most prevalent psychiatric disorders in children, with numerous studies investigating its prevalence in the population[6]. Current evidence estimates that 5%-7% of children and adolescents worldwide are affected by ADHD[7]. Although ADHD is generally accepted as a childhood disease, symptoms continue in 30%-60% of patients throughout adolescence and adulthood[4].

The etiology of ADHD involves both genetic and environmental factors. Among these, prenatal environmental factors are considered to make significant contributions to the development of ADHD in children, such as maternal overweight, maternal smoking exposure, preeclampsia, and hypertension during pregnancy[8,9]. Despite great effort, the etiology and pathogenesis of ADHD have not been fully defined[10,11]. Over time, the role of neuro-inflammation in the pathophysiology of major psychiatric disorders has become increasingly clear[12]. This understanding has prompted extensive research into inflammatory processes across conditions such as autism spectrum disorder, depression, schizophrenia, and bipolar disorder[13].

The kynurenine pathway (KP) plays a critical and complex role in the central nervous system, particularly in regulating inflammation[14,15]. Approximately 90% of tryptophan (TRP) metabolism occurs through the KP[16]. In this pathway, TRP is converted into kynurenine (KYN) by the enzyme indoleamine 2,3-dioxygenase (IDO). This critical step initiates the downstream production of various metabolites involved in immune, neurological, and metabolic functions, which subsequently diverge into two distinct functional branches[17]. In the first branch, KYN is converted to kynurenic acid (KYNA) by the kynurenine aminotransferases. As an endogenous antagonist of glutamatergic receptors, including N-methyl-D-aspartic acid (NMDA) receptors KYNA regulates excitatory neurotransmission and provides neuroprotection[18]. Conversely, in the second branch, KYN is converted to 3-hydroxykynurenine (3-HK) by KYN monooxygenase (KMO) in microglia. Subsequently, 3-HK is metabolized to 3-hydroxyanthranilic acid and then to quinolinic acid (QUIN) through the action of kynureninase and 3-hydroxyanthranilate oxidase. QUIN, which functions as an NMDA receptor agonist, exhibits excitotoxic properties that contribute to neurotoxicity[19].

The balance between KYNA, a neuroprotective compound, and QUIN, a neurotoxic compound, is crucial for maintaining nerve homeostasis. Persistent imbalance in the KP metabolites are closely associated with various neurological and psychiatric disorders, suggesting the pathway’s critical role in neuropsychiatric pathogenesis[19,20]. Consequently, the QUIN/KYNA ratios and QUIN levels are significant indicators of pathological changes in neuro-central diseases[19]. Furthermore, the KYNA/KYN ratio, which reflects kynurenine aminotransferase activity, also indicates neuroprotective status in KP[21].

Despite growing interest in the kynurenine pathway (KP), neurotoxic–neuroprotective balance in drug-naive children with attention-deficit/hyperactivity disorder (ADHD) remains poorly characterized. This study compared serum KP metabolites (TRP, KYN, KYNA, QUIN) and their ratios between drug-naive children with ADHD and healthy controls, with particular focus on QUIN/KYNA and KYNA/QUIN as indicators of inflammatory imbalance. Findings may contribute to understanding KP dysregulation and its biomarker potential in pediatric ADHD.

MATERIALS AND METHODS

Study participants

The current study adhered to the ethical principles outlined in the Declaration of Helsinki, including its subsequent amendments, as well as other comparable ethical standards. Approval was obtained from the University’s Clinical Research Ethics Board. Participants were informed about the study details, and an informed consent form was obtained from each participant.

The study was conducted at the Gazi University Hospital. Participants were recruited from the Child and Adolescent Psychiatry Outpatient Clinic, and biological samples were analyzed at the Department of Medical Biochemistry of the same institution. An expert psychiatrist conducted a semi-structured psychiatric interview to ensure that participants met the inclusion and exclusion criteria.

The inclusion criterion for the patient group consisted of children and adolescents aged 8 years to 18 years diagnosed with ADHD based on the Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime Version (K-SADS-PL). The healthy control group consisted of individuals aged 8-18 years, recruited from a university-affiliated outpatient psychiatry service. These participants were evaluated using the K-SADS-PL and the Diagnostic and Statistical Manual of Mental Disorders, 5^th Edition. Individuals with any psychiatric diagnosis were excluded from the control group.

Exclusion criteria for all participants were: (1) A diagnosis of neurological disorders (e.g., cerebral palsy, epilepsy), metabolic disorders (e.g., phenylketonuria), diabetes mellitus, cancer, allergic, rheumatologic, inflammatory, or autoimmune diseases; (2) History of an infectious disease within the last 4 weeks; (3) Use of anti-inflammatory medications within the previous 4 weeks; (4) Immunosuppressive treatment in the past 6 months; (5) History of substance use in the last 3 months; (6) A clinical suspicion of pediatric autoimmune neuropsychiatric disorders associated with streptococci; (7) Children meeting obesity criteria (body mass index ≥ 95^th percentile); and (8) Presence of any psychiatric disorder other than ADHD (e.g., depression, anxiety disorders, autism spectrum disorder, bipolar disorder, or psychotic disorders), as evaluated with K-SADS-PL.

Socio-demographic data form

The researchers designed a socio-demographic questionnaire to identify family dynamics (e.g., parents’ marital status and number of siblings) and participants’ socioeconomic indicators (e.g., household income and parents' educational level).

Schedule for Affective Disorders and Schizophrenia for School-age Children - Present and Lifetime version (K-SADS-PL)

The structured diagnostic interview, K-SADS-PL, was developed by Kaufman et al[22] for the diagnosis of psychiatric disorders and is used with children and adolescents. It assesses five main diagnostic categories: Mood disorders, anxiety disorders, psychotic spectrum disorders, disruptive behavior disorders, and substance use disorders. These categories cover a wide range of mental health problems, allowing clinicians to identify both the primary diagnosis and comorbid conditions. This range of diagnoses makes the instrument applicable to a variety of clinical presentations, enabling a comprehensive, structured assessment of mental health in young individuals. The Turkish adaptation of the K-SADS-PL was validated to ensure its cultural and linguistic applicability, and its reliability was demonstrated in a 2018 study by Ünal et al[23].

Conner Parent Rating Scale-48 (CPRS-48)

The CPRS-48 is widely used to assess children’s ADHD and other behavioral symptoms through parental report[24]. The Turkish adaptation of the CPRS-48 was undertaken with due attention to cultural and linguistic appropriateness and has demonstrated strong psychometric properties in reliability and validity studies[25]. This comprehensive instrument consists of subscales assessing attention problems, hyperactivity, conduct problems, psychosomatic problems, and anxiety. Items are scored using a 4-point Likert scale: 0 = not at all, 1 = a little, 2 = a fair amount, 3 = a great deal. Total and subscale scores can provide valuable insights into symptom patterns and their potential impact on cognitive, emotional, and social functioning. Because it is a standardized measure, it also assists clinicians and researchers in diagnosing and monitoring therapeutic effects.

Biochemical analyses

Venous blood samples were collected from drug-naive ADHD patients and controls and then centrifuged at 4000 rpm for 15 minutes to separate serum for biochemical analysis. Samples showing signs of hemolysis or jaundice were carefully excluded from analysis to maintain data integrity. The obtained serum was stored at -80 °C until the day of analysis. Serum levels of TRP (category No. E4244Hu), KYN (category No. EA0098Hu), KYNA (category No. E3982Hu), and QUIN (category No. E3983Hu) were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits (BT LAB, China). Detection limits of serum TRP, KYN, KYNA, and QUIN are 0.23 μg/mL, 1.24 pmol/mL, 0.21 nmol/L, and 0.103 nmol/mL, respectively. Ratios reflecting KP enzyme activities (KYN/TRP, KYNA/KYN, QUIN/KYN) and neurotoxic index (QUIN/KYNA) were calculated.

Statistical analyses

Statistical analysis were made using the SPSS version 23.0 package program (Chicago, IL, United States). Categorical variables were summarized as n (%) and continuous variables were reported as means with standard deviations or medians with interquartile ranges (IQR) based on distribution. The χ² test was utilized to examine differences in categorical data. The Kolmogorov-Smirnov test was employed to evaluate the normality of numerical variables, and non-normally distributed data were analyzed using the Mann-Whitney U test. Spearman’s rank correlation analysis was conducted to examine relationships among non-normally distributed variables. Statistical significance was set at a P value less than 0.05, with findings interpreted within a 95% confidence interval. Additionally, receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic performance of the QUIN/KYNA neurotoxic index for detecting ADHD, by calculating the area under the curve (AUC) and determining the optimal threshold values.

RESULTS

Socio-demographic and clinical characteristics

The study included 51 drug-naive participants with ADHD (34 boys/17 girls; 67%/33%) and 36 healthy controls (17 boys/19 girls; 47%/53%). The median age was 151 months (115-169) in the ADHD group and 154 months (123-180) in the control group. No statistically significant differences were observed between the groups in terms of gender (P = 0.070) and age (P = 0.438). Although the proportion of males was higher in the ADHD group, this difference was not statistically significant and was not thought to influence the results. No significant differences were observed between the patient and control groups in terms of body mass index percentiles or smoking status. However, a family history of psychiatric disorders was significantly more common in the ADHD group compared to the control group (P = 0.043). The median (IQR) months of the time since symptoms onset among ADHD patients, defined as the time elapsed since the formal clinical diagnosis, was 36 (24-60).

When comparing CPRS-48 subscale scores, children in the ADHD group exhibited significantly higher scores across all subscales and the total CPRS-48 score than those in the control group (P < 0.001). Moreover, in the control group, no significant correlations were observed between CPRS-48 subscale scores and socio-demographic variables, suggesting that elevated scores were specific to the ADHD group. Socio-demographic and clinical characteristics, as well as CPRS-48 scores of all participants, are presented in Table 1.

Table 1 Socio-demographic, clinical characteristics, and Conner Parent Rating Scale-48 subscale scores of children with attention-deficit/hyperactivity disorder and control, n (%)/median (interquartile range).

	ADHD group (n = 51)	Control group (n = 36)	P value
Males/females	34/17 (67/33)	17/19 (47/53)	0.070
Smoking (yes)	5 (9.8)	-	0.074
Psychiatric illness in the family	22 (43.1)	8 (22.2)	0.043
Age (months)	151 (115-169)	154 (123-180)	0.438
BMI percentile	52 (24.8-74.5)	66.4 (27.9-75.4)	0.957
Time since symptoms onset (months)	36 (24-60)	-	-
CPRS-48 subscale scores
Conduct problems	10 (5-16)	2 (0-3)	< 0.001
Learning problems	7 (6-10)	1 (0-3)	< 0.001
Psychosomatic	6 (2-9)	0.5 (0-3.5)	< 0.001
Impulsive-hyperactive	7 (5-9)	2.5 (1-5)	< 0.001
Anxiety	8 (5-12)	4 (2-6)	< 0.001
Total score	49 (32-69)	42 (7-24.5)	< 0.001

BMI: Body mass index; CPRS-48: Conner Parent Rating Scale-48.

Serum KP metabolites levels and ratios in ADHD and control groups

The Median (IQR) serum KP metabolite levels and the ratios for both groups are shown in Table 2. Serum KYNA levels and KYNA/QUIN ratio (neuroprotective index) were significantly lower in the ADHD group than in the control group (P = 0.023, P = 0.025, respectively). While the QUIN/KYNA ratio (neurotoxic index) was significantly higher in the ADHD group compared to the control (P = 0.029) (Figure 1). No significant differences were found in other KP metabolites or ratios (P > 0.05). No significant differences were found among ADHD subtypes or between subtypes and controls for any metabolite or KP index Table 3, indicating that KP alterations are not subtype-dependent.

Figure 1 Comparison of kynurenine pathway metabolites and indices between attention-deficit/hyperactivity disorder patients and controls. ADHD: Attention-deficit/hyperactivity disorder; QUIN: Quinolinic acid; KYNA: Kynurenic acid.

Table 2 Serum kynurenine pathway metabolite levels and associated ratios in attention-deficit/hyperactivity disorder and control group, median (interquartile range).

	ADHD group (n = 51)	Control group (n = 36)	P value
TRP (μmol/L)	174 (94-276)	210 (59-259)	0.914
KYN (nmol/L)	65.6 (54-77)	61.6 (48-81)	0.543
KYNA (nmol/L)	25.8 (21-36)	31.3 (27-39)	0.023
QUIN (μmol/L)	12.7 (10-16)	12 (10-15)	0.721
KYN/TRP × 1000 (IDO/TDO activity)	40 (40-82)	34.7 (20-94)	0.816
KYNA/KYN × 100 (KAT activity)	39 (32-61)	56.5 (36-75)	0.083
KYNA/QUIN (neuroprotective index)	2.3 (2.0-2.7)	2.5 (2.3-3.1)	0.025
QUIN/KYNA (neurotoxic index)	0.44 (0.37-0.50)	0.40 (0.32-0.44)	0.029
QUIN/KYN (KMO activity)	0.19 (0.13-0.28)	0.20 (0.14-0.28)	0.756

ADHD: Attention-deficit/hyperactivity disorder; KYN: Kynurenine; TRP: Tryptophan; IDO/TDO: Indoleamine 2,3-dioxygenase/tryptophan 2,3-dioxygenase; KYNA: Kynurenic acid; KAT: Kynurenine aminotransferases; QUIN: Quinolinic acid; KMO: Kynurenine monooxygenase.

Table 3 Correlation between biochemical parameters and Conner Parent Rating Scale-48 subscale scores in the associated ratios in attention-deficit/hyperactivity disorder group.

n = 51	CPRS-48, CP		CPRS-48, LP		CPRS-48, P		CPRS-48, I-H		CPRS-48, A		CPRS-48, total score
	r	P value	r	P value	r	P value	r	P value	r	P value	r	P value
TRP (μmol/L)	-0.237	0.093	-0.061	0.672	0.127	0.376	-0.001	0.994	-0.070	0.628	-0.083	0.564
KYN (nmol/L)	-0.152	0.286	0.006	0.964	0.096	0.501	-0.100	0.485	-0.158	0.270	-0.151	0.289
KYNA (nmol/L)	0.016	0.911	0.046	0.747	0.184	0.196	0.146	0.307	0.001	0.993	0.062	0.668
QUIN acid (μmol/L)	0.007	0.963	-0.075	0.602	0.076	0.594	0.063	0.663	-0.024	0.869	-0.005	0.970
KYN/TRP × 1000 (IDO/TDO)	0.165	0.248	0.054	0.708	-0.090	0.529	-0.059	0.680	0.039	0.788	0.032	0.824
KYNA/KYN × 100 (KAT activity)	-0.165	0.248	0.099	0.490	0.165	0.247	0.210	0.138	0.096	0.501	0.183	0.199
KYNA/QUIN (neuroprotective)	0.055	0.703	0.237	0.094	0.265	0.060	0.173	0.224	0.126	0.379	0.166	0.243
QUIN/KYNA (neurotoxic)	-0.058	0.685	-0.237	0.095	-0.264	0.061	-0.176	0.216	-0.127	0.373	-0.167	0.240
QUIN/KYN (KMO activity)	0.090	0.530	-0.032	0.824	0.050	0.728	0.151	0.291	0.050	0.726	0.101	0.479

Spearman correlational analysis was performed. r: Correlation coefficient. CPRS-48: Conner Parent Rating Scale-48; CP: Conduct problems; LP: Learning problems; P: Psychosomatic; I-H: Impulsive-Hyperactive; A: Anxiety; KYN: Kynurenine; TRP: Tryptophan; IDO/TDO: Indoleamine 2,3-dioxygenase/tryptophan 2,3-dioxygenase; KYNA: Kynurenic acid; KAT: Kynurenine aminotransferases; QUIN: Quinolinic acid; KMO: Kynurenine monooxygenase.

When the ADHD subtypes and control group were compared in terms of biochemical measures, there was no statistically significant difference in serum TRP, KYN, KYNA, QUIN, KYN/TRP, KYNA/KYN, QUIN/KYN, KYNA/QUIN, and QUIN/KYNA between the presentation of combined, predominantly inattentive, and predominantly hyperactive/impulsive types of ADHD and the control group (P > 0.05) (Table 4).

Table 4 Correlation between biochemical parameters and time since symptoms onset, Conner Parent Rating Scale-48 subscale scores, Conner Parent Rating Scale-48 total score in the attention-deficit/hyperactivity disorder group.

n = 51	Time since symptoms onset		CPRS-48, CP1		CPRS-48, LP1		CPRS-48, P1		CPRS-48, I-H1		CPRS-48, A1		CPRS-48, total score1
	r	P value	r	P value	r	P value	r	P value	r	P value	r	P value	r	P value
TRP (μmol/L)	0.032	0.825	-0.237	0.093	-0.061	0.672	0.127	0.376	-0.001	0.994	-0.070	0.628	-0.083	0.564
KYN (nmol/L)	0.082	0.565	-0.152	0.286	0.006	0.964	0.096	0.501	-0.100	0.485	-0.158	0.270	-0.151	0.289
KYNA (nmol/L)	0.050	0.728	0.016	0.911	0.046	0.747	0.184	0.196	0.146	0.307	0.001	0.993	0.062	0.668
QUIN (μmol/L)	0.162	0.256	0.007	0.963	-0.075	0.602	0.076	0.594	0.063	0.663	-0.024	0.869	-0.005	0.970
KYN/TRP × 1000 (IDO/TDO)	-0.040	0.783	0.165	0.248	0.054	0.708	-0.090	0.529	-0.059	0.680	0.039	0.788	0.032	0.824
KYNA/KYN × 100 (KAT activity)	0.107	0.455	-0.165	0.248	0.099	0.490	0.165	0.247	0.210	0.138	0.096	0.501	0.183	0.199
KYNA/QUIN (neuroprotective)	-0.105	0.463	0.055	0.703	0.237	0.094	0.265	0.060	0.173	0.224	0.126	0.379	0.166	0.243
OUIN/KYNA (neurotoxic)	0.117	0.413	-0.058	0.685	-0.237	0.095	-0.264	0.061	-0.176	0.216	-0.127	0.373	-0.167	0.240
OUIN/KYN (KMO activity)	0.195	0.170	0.090	0.530	-0.032	0.824	0.050	0.728	0.151	0.291	0.050	0.726	0.101	0.479

¹Spearman correlational analysis was performed.

r: Correlation coefficient; CPRS-48: Conner Parent Rating Scale-48; CP: Conduct problems; LP: Learning problems; P: Psychosomatic; I-H: Impulsive-Hyperactive; A: Anxiety; KYN: Kynurenine; TRP: Tryptophan; IDO/TDO: Indoleamine 2,3-dioxygenase/tryptophan 2,3-dioxygenase; KYNA: Kynurenic acid; KAT: Kynurenine aminotransferases; QUIN: Quinolinic acid; KMO: Kynurenine monooxygenase.

ROC analysis

ROC curve analysis revealed an AUC of 0.64 (95% confidence interval: 0.52-0.75) for the QUIN/KYNA (neurotoxic index) (P < 0.001). Based on the determined threshold value, it had 65% sensitivity and 60% specificity for diagnosing ADHD. Although the AUC value for the QUIN/KYNA ratio was modest (0.64), this finding supports its potential role as part of a broader biomarker panel for identifying neurochemical alterations in ADHD.

Correlation analysis

In the patient group, correlation analyses were conducted to investigate potential associations between KP metabolites, their ratios, the time since symptoms onset, and CPRS-48 subscale and total scores. However, no statistically significant correlations were observed (P > 0.05) (Table 4).

DISCUSSION

In this study, we evaluated serum levels of KP metabolites (TRP, KYN, KYNA, QUIN) and derived ratios reflecting KP enzyme activities (KYN/TRP, KYNA/KYN, QUIN/KYN), including the QUIN/KYNA neurotoxic index, in drug-naive children with ADHD and healthy controls. Findings revealed that the ADHD group exhibited significantly lower serum KYNA levels and a reduced KYNA/QUIN ratio, indicating reduced neuroprotective capacity. Conversely, a significantly higher QUIN/KYNA ratio indicated increased neurotoxic activity compared to the control group. These findings highlight that dysregulation between neuroprotective and neurotoxic metabolites in ADHD potentially contributes to the excitotoxicity and synaptic dysfunction observed in its pathology, suggesting that altered KP metabolite dynamics may play a role in the symptomatology and cognitive impairments in these patients. To our knowledge, this is the first study in drug-naive children with ADHD to investigate both KYNA/QUIN and QUIN/KYNA ratios, providing an integrated downstream KP signature.

The QUIN/KYNA and KYNA/QUIN ratios represent reciprocal expressions of the same biological concept, namely the balance between neurotoxic and neuroprotective metabolites within the KP. In the present study, QUIN/KYNA was selected as the primary index to reflect neurotoxic dominance, whereas KYNA/QUIN was reported as its inverse ratio to illustrate reduced neuroprotective capacity. To avoid redundancy and enhance conceptual clarity, biological interpretations are primarily centered on the QUIN/KYNA ratio.

KYNA is a neuro-active compound that interacts with different types of glutamate receptors and acts as an antagonist of NMDA receptors by blocking the glycine binding site[26]. Persistent hyperactivation of NMDA receptors leads to excitotoxic degeneration of dendritic spines and, in some cases, the degeneration of the entire neuron that expresses NMDA receptors[27]. Due to this mechanism, KYNA exerts neuroprotective activities[28]. In contrast, QUIN is an endogenous agonist of NMDA receptors and may cause excitotoxic neuronal death[29].

Furthermore, the decreased serum KYNA levels in children with ADHD emphasize that dysregulation of the KP may underlie core symptoms of ADHD, such as inattention and hyperactivity. This reduction can enhance glutamatergic excitotoxicity, as KYNA plays a crucial role in modulating glutamate signaling; glutamatergic excitotoxicity has also been implicated in ADHD. A few prior studies have explored the involvement of KYNA in children with ADHD, but their results are conflicting.

No significant associations were found between KP metabolites or derived ratios and symptom dimensions, as measured by the CPRS-48, in this cohort (Table 4). These negative findings are compatible with the interpretation that peripheral KP changes reflect trait-like neurobiological alterations, rather than state-dependent biomarkers directly linked to concurrent symptom severity. Indeed, similar dissociations between peripheral KP markers and behavioral symptom scores have been observed in ADHD and other neuropsychiatric disorders, consistent with the concept that biochemical dysregulation may precede or occur independent of symptom expression.

Peripheral KP activation is strongly influenced by inflammatory cytokines such as interleukin-6, interferon-γ, and tumor necrosis factor-α, which upregulate rate-limiting enzymes including IDO and tryptophan 2,3-dioxygenase (TDO)[30]. Increased KMO activity shifts TRP metabolism toward 3-HK and QUIN production, reducing KYNA availability and promoting excitotoxicity[19]. It should be noted that TDO is predominantly expressed in the liver and is primarily regulated by glucocorticoids and stress-related mechanisms rather than by pro-inflammatory cytokines. In contrast, IDO is the enzyme mainly induced by inflammatory stimuli such as interferon-γ. Therefore, alterations in downstream KP metabolites observed in this study are more likely to reflect the combined effects of peripheral stress-related activation and immune-related modulation of the pathway, rather than a uniform inflammatory upregulation of all rate-limiting enzymes[31,32].

Although enzymatic activity and cytokines were not measured in this study, the decreased KYNA levels and elevated QUIN/KYNA ratio observed in the ADHD group are consistent with the cytokine-mediated KP imbalance observed in neuroinflammatory models of ADHD. However, it should be noted that these interpretations of KMO activity are based on derived metabolite ratios, and direct measurement of enzyme activity (e.g., KMO protein levels or functional assays) is required to confirm this hypothesis definitively. Furthermore, no significant differences were observed for any KP metabolites or enzymatic indices among ADHD subtypes or between subtypes and controls (Table 3). This finding suggests that KP dysregulation may represent a common biochemical feature across ADHD phenotypes rather than a subtype-specific alteration, supporting evidence that neuroimmune mechanisms underlying ADHD substantially overlap among clinical variants. Importantly, given that this study was conducted in children, further research is warranted to determine if these specific KP imbalances persist or change during adolescence and adulthood, as the KP is known to be subject to developmental regulation.

The results of Evangelisti et al[17] in 2017 are similar to those of the current study. Drug-naive children with ADHD showed a reduction in serum levels of KYNA and levels of QUIN that were unchanged when compared to healthy controls. In contrast, serum levels of TRP and KYN were significantly enhanced. It has been reported that low KYNA levels and metabolic imbalances in the KP in individuals with ADHD may be associated with neuroinflammation. Our study is consistent with the existing literature in demonstrating decreased KYNA levels. The combination of a decreased KYNA/QUIN ratio and an increased QUIN/KYNA ratio suggests an underlying inflammatory mechanism contributing to ADHD pathophysiology and a neurochemical shift toward excitotoxicity. Although the QUIN/KYNA neurotoxicity index demonstrates only moderate diagnostic accuracy (AUC = 0.64), its importance lies not only in its diagnostic value but also in reflecting a broader neurochemical imbalance. Single-pathway biomarkers in ADHD share modest AUC values, suggesting that KP-derived indices may be more informative when integrated into multimodal biomarker panels rather than used independently. The modest diagnostic utility (AUC = 0.64) may also reflect the intrinsic heterogeneity of the ADHD population and the influence of uncontrolled environmental and lifestyle factors (e.g., diet, gut microbiota status, and physical activity) known to modulate peripheral KP activity.

The study by Sağlam et al[33] in 2021 showed that the KYNA/KYN ratio, proposed as a neuroprotective index, was reduced in drug-naive children with ADHD. However, KYNA levels did not show a significant change in the ADHD combined presentation compared to the healthy control group. In the study by Dolina et al[34] in 2014, which examined KP metabolites in urine samples, KYNA, TRP, and KYN levels were higher in drug-naive patients with ADHD compared with healthy controls. Although KYNA/3-HK was considered a neuroprotective index in that study, it was lower in untreated patients than in the control group. The same study also evaluated patients with ADHD treated with Ritalin and reported that, while treatment did not alter the overall pattern of KP metabolism, it modified the balance among certain detected metabolites. In a study conducted by Oades et al[35] in 2010, the KP metabolites of children with ADHD were compared with those of the control group. In particular, KYNA levels were lower in drug-naive ADHD patients compared to the control group. However, this was not statistically significant, and treatment increased KYNA production in the ADHD group. Similarly, our study observed a significant decrease in KYNA levels and KYNA/QUIN ratio in the drug-naive ADHD patient group compared to healthy controls.

These results are supported by the fact that, in their study, Molina-Carballo et al[36] in 2021 noted increased serum KYNA levels following methylphenidate treatment in ADHD patients. The latter facts confirm our hypothesis that pharmacological interventions to modify KP metabolism can help restore a balance between neuroprotective/neurotoxic processes, which may reduce some neurochemical disturbances related to ADHD. Long-term treatment may restore the disrupted balance between neuroprotective and neurotoxic mechanisms, thus supporting the therapeutic potential of KP-targeting interventions.

There are also literature reviews addressing studies on the KP and its metabolites in children with ADHD, primarily based on serum and urine samples[35]. In addition, a growing body of research has investigated KP alterations in adult ADHD populations. Collectively, these studies highlight the relevance of TRP metabolism in ADHD across different age groups. This metabolic pathway has been implicated in the pathophysiology of ADHD, and emerging evidence suggests its possible role in modulating the risk of developing the disorder. Reviews presenting studies that address the KP and the underlying mechanisms of inflammation in ADHD can be found in the literature[37,38]. Most interestingly, our results on the neurotoxic and neuroprotective ratios, KYNA/QUIN and QUIN/KYNA, provide important insights into the KP’s influence on the nervous system. Low KYNA levels and decreased KYNA/QUIN ratio in ADHD patients indicate impaired neuro-protection mechanisms and increased susceptibility to neurotoxicity, which is confirmed by the high QUIN/KYNA ratio, reflecting the dominance of neurotoxic effects over neuroprotective effects. These findings support the view that glutamate dysregulation and synaptic dysfunction may contribute to ADHD pathophysiology[39].

Except for indices related to KYNA and QUIN, no significant changes were found in the TRP, KYN, and overall KP-derived ratios in children with ADHD. These negative findings suggest that ADHD may not be associated with a general alteration in the entire KP, but rather with a selective disruption of specific enzymatic branches, particularly those involved in KYNA production and QUIN accumulation. This notion is consistent with studies showing that inflammatory/stress-induced KP activation generally does not uniformly affect upstream metabolites but rather primarily affects the KYNA-QUIN balance due to differential interactions between the IDO/TDO and KMO pathways[14,31,40]. Such selective modulation supports the view that neuroprotective and neurotoxic branches are differentially affected, while upstream TRP availability and primary KYN conversion pathways are relatively preserved. Such findings may be common in clinical studies due to the heterogeneous influence of inflammatory and neuroimmune factors on different enzymatic branches. Here, downstream metabolites (KYNA, QUIN) show more significant disease-related shifts than upstream TRP-KYN changes. Differences in analytical platforms (high-pressure liquid chromatography or ELISA), biological matrices (urine or serum), medication status (drug-naive or medicated), and age-related metabolic variability provide plausible explanations for the different findings between Evangelisti et al[17], Sağlam et al[33], Dolina et al[34], and Oades et al[35]. These methodological and population-level differences are well-established sources of heterogeneity in KP studies and are now explicitly acknowledged to contextualize our results.

Limitations

Several limitations of this study should also be acknowledged in interpreting these findings. The study sample size was relatively small. Alterations in the KP, particularly those involving upstream metabolites such as TRP and KYN and their derived ratios, show small effect sizes in neuropsychiatric populations and therefore generally require larger cohorts to achieve adequate statistical sensitivity. Many of the non-significant findings in our study, particularly those involving upstream metabolites (TRP, KYN) and their ratios, may reflect type II (false-negative) errors due to insufficient statistical power rather than the actual absence of metabolic differences. The samples were drawn from a single geographic location, which may not accurately reflect the broader population and its cultural diversity.

Additionally, the study focused primarily on specific KP metabolites, including KYNA and QUIN, as well as their ratios. However, other KP intermediates such as 3-HK and 3-hydroxyanthranilic acid, were not evaluated. This incomplete profiling may have limited the scope of biochemical investigations. Moreover, analysis based on the utilization of blood serum samples, which are quantified using ELISA, restricts the examination to peripheral KP activity that may not be indicative of central nervous system processes. Although liquid chromatography-tandem mass spectrometry/high-pressure liquid chromatography is considered the gold standard for quantifying KP metabolites, ELISA was used due to practical constraints; this methodological choice may influence absolute concentrations, although relative group differences remain interpretable. Variability in methods and measurement techniques across studies could be another reason for inconsistent findings in the literature. Lastly, because this was a cross-sectional design, causal inferences cannot be made, and longitudinal studies are needed to establish temporal relationships and treatment effects.

Strengths

Despite its limitations, this study offers several notable strengths and introduces genuinely novel contributions to the literature on the ADHD and the KP. First, it is among the very few investigations to concurrently evaluate both the neuroprotective (KYNA/QUIN) and neurotoxic (QUIN/KYNA) indices, offering a more integrated picture of excitatory-inhibitory imbalance within the KP than previous work. Importantly, this study is the first to examine the diagnostic performance of the QUIN/KYNA neurotoxic index using ROC analysis in a drug-naive pediatric ADHD population, revealing preliminary biomarker potential. The inclusion of drug-naive individuals and the application of strict exclusion criteria (including recent infection, inflammatory diseases, obesity, and suspected pediatric autoimmune neuropsychiatric disorders associated with streptococci) minimize confounding factors that commonly influence KP metabolism, providing a clearer biological signal. Furthermore, the assessment of metabolite profiles across ADHD subtypes provides valuable data suggesting that KP dysregulation may represent a core biological signature rather than a subtype-specific variation. The consistent pattern of low KYNA, low KYNA/QUIN ratio, and high QUIN/KYNA ratio strengthens the evidence for a shift toward excitotoxicity, highlighting a mechanistic pathway of increasing importance in neurodevelopmental disorders. These features collectively position the study as a meaningful and innovative contribution to biomarker-driven ADHD research.

Future directions

Future research should involve larger, more diverse, and multi-center cohorts to replicate the observed associations and increase the generalizability of the findings. Longitudinal studies are particularly needed to examine KP metabolite changes over time and their responses to pharmacological and behavioral interventions. Expansion of metabolic profiling to a broader range of KP intermediates, 3-HK, 3-hydroxyanthranilic acid, and xanthurenic acid, is anticipated to further enrich the characterization of overall pathway dynamics. The integration of neuroimaging, cognitive testing, and genetic data may further delineate how KP dysregulation interacts with neurodevelopmental processes in ADHD. Moreover, the exploration of KP activity in important biological matrices, such as cerebrospinal fluid, or the use of animal models, may offer insights into brain-specific mechanisms. Lastly, the examination of environmental and immunological modulators of the KP, such as stress, diet, and gut microbiota, can reveal modifiable risk factors and pave the way for mechanism-based therapeutic interventions.

CONCLUSION

In conclusion, while the complete etiopathogenesis of ADHD remains to be fully elucidated, our findings highlight a significant role of the kynurenine pathway (KP) in its pathophysiology. The observed reductions in KYNA levels and elevated neurotoxic QUIN/KYNA ratios suggest a pivotal involvement of neuroinflammation and excitotoxicity in ADHD pathophysiology. These findings suggest that KP-driven neuroinflammation and excitotoxicity are pivotal components of ADHD, offering a novel perspective on its underlying biochemical mechanisms and potential future biomarker applications.

ACKNOWLEDGEMENTS

We sincerely thank all children and their families for their participation and cooperation throughout the study. We also express our gratitude to the clinical and laboratory staff of the Department of Child and Adolescent Psychiatry and the Department of Medical Biochemistry for their valuable assistance during data collection and sample analysis. The authors gratefully acknowledge the Academic Writing Practice and Research Center of Gazi University for providing professional academic language editing services.