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European Child & Adolescent Psychiatry

, Volume 26, Issue 12, pp 1433–1441 | Cite as

Changes in serum levels of kynurenine metabolites in paediatric patients affected by ADHD

  • Melania Evangelisti
  • Pietro De Rossi
  • Jole Rabasco
  • Renato Donfrancesco
  • Luana Lionetto
  • Matilde Capi
  • Gabriele Sani
  • Maurizio Simmaco
  • Ferdinando Nicoletti
  • Maria Pia Villa
Original Contribution

Abstract

This study aims at determining serum levels of tryptophan and other metabolites of the kynurenine pathway in children with attention deficit hyperactivity disorder (ADHD) compared to healthy controls. Such metabolites interact with glutamate receptors in the central nervous system, potentially modulating mechanisms that are pivotal in ADHD and thus potentially representing peripheral biomarkers of the disorder. We measured serum levels of tryptophan and some metabolites of the kynurenine pathway in 102 children with ADHD and 62 healthy controls by liquid chromatography–tandem mass spectrometry (LC–MS/MS). As compared to healthy controls, children with ADHD showed a reduction in serum levels of anthranilic acid (−60%), kynurenic acid (−11.2%), and xanthurenic acid (−12.5%). In contrast, serum levels of tryptophan (+11.0%) and kynurenine (+48.6%) were significantly enhanced, and levels of quinolinic acid were unchanged in children with ADHD. In a logistic regression model, the presence of ADHD was predicted by low anthranilic acid and high tryptophan levels. These findings support the involvement of the kynurenine pathway in the pathophysiology of ADHD and suggest that anthranilic acid and tryptophan levels should be investigated as potential peripheral biomarker for ADHD.

Keywords

ADHD Biomarker Kynurenines Children Neurobiology 

Introduction

Attention Deficit Hyperactivity Disorder (ADHD) is a neurobehavioral childhood disorder characterized by inattention, hyperactivity, impulsivity, or a combination of these symptoms. The disorder is typically diagnosed in childhood, but affected persons frequently remain symptomatic in adulthood [1].

The world-wide prevalence in developmental age is 5.29% and is higher in boys than girls [2].

The diagnosis in children focuses on behavioural symptoms and, according to DSM-5 criteria, is based on the presence of at least six out of nine symptoms in the two domains of inattention and hyperactivity–impulsivity [3].

The aetiology of ADHD can be explained by a combination of inherited and environmental factors that critically influence the developmental trajectory of the prefrontal cortex [4]. In fact, Shaw and colleagues have found that persistence of ADHD is associated with divergence away from typical developmental trajectories [5].

Several studies from genomics to metabolomics as well as measurements of plasma levels of monoamines, hormones (e.g., corticol and oxytocin), and neurotrophic factors have been performed in an attempt to elucidate the biological mechanisms involved in ADHD and to identify potentially predictive peripheral and genetic markers [6, 7, 8, 9, 10]. These studies, however, are not conclusive and further investigation is needed.

The kynurenine pathway of tryptophan metabolism generates neuro-active compounds that are able to interact with neurotransmitters receptors in the central nervous system (CNS). The pathway is activated by either indolamine-2,3-dioxygenase or tryptophan-2,3-dioxygenase, which catalyzes the conversion of l-tryptophan into N-formylkynurenine. l-Kynurenine generated from N-formylkynurenine can be (1) transaminated into kynurenic acid by types 1 and 2 kynurenine aminotransferases (KATs); (2) hydroxylated into 3-hydroxykynurenine by kynurenine mono-oxygenase (KMO); or (3) converted into anthranilic acid by kynureninase. 3-Hydroxykynurenine is sequentially metabolized into 3-hydroxyantranylic acid and quinolinic acid, which is the direct precursor of nicotinamide. Xanturenic acid and cinnabarinic acid are by-products of the kynurenine pathway, generated from 3-hydroxykynurenine and 3-hydroxyanthranilic acid, respectively [11]. Kynurenic acid acts as a competitive antagonist at the glycine site on the GluN1 NMDA receptor subunit, thereby inhibiting NMDA receptor activation [12]. In contrast, quinolinic acid acts as an orthosteric agonist at the GluN2 NMDA receptor subunits [13]. Recent evidence suggests that xanthurenic acid activates mGlu2 and mGlu3 metabotropic glutamate receptors [14], although it has been argued that xanthurenic acid acts primarily by inhibiting the vesicular glutamate transporters, thereby enhancing non-vesicular release of glutamate from nerve endings [15]. Cinnabarinic acid acts as a weak orthosteric agonist of mGlu4 receptors [16]. Given the involvement of glutamatergic system (especially with respect to NMDA receptor-related genetic variants) in the development of ADHD [17] and the at least partial inconsistency of studies that have tried to shed light on biological mechanisms underlying ADHD, a study on kynurenine metabolites levels in this disorder is warranted.

To the best of our knowledge, only two research groups have examined serum levels of kynurenine metabolites in patients affected by ADHD with contrasting results [18, 19]. On one hand, Aarsland and colleagues found that ADHD in adults is associated with lower serum concentrations of tryptophan, kynurenic acid, xanthurenic acid, and 3-hydroxyanthranilic acid [18].

On the other hand, Oades and colleagues describe lower levels of 3-hydroxykynurenine in children with ADHD and interpret this finding as consistent with the delayed brain maturation peculiar to the disorder [19]. However, the study by Oades and colleagues is based on a rather small sample, while Aarsland and colleagues focused on adults, a population where age-related changes might have occurred.

The aim of our study was to investigate the serum levels of tryptophan and several metabolites of the kynurenine pathway in children with ADHD and healthy controls.

Methods

One hundred and two drug-naïve children, referred to the Clinic for Developmental Neurology and Psychiatry of the S. Pertini Hospital in Rome and to the Paediatric Department of Sant’Andrea Hospital in Rome, and diagnosed with ADHD according to DSM-5 criteria, were enrolled. Children, as well as their parents, underwent a semi-structured psychiatric interview, i.e., the Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime Version (K-SADS-PL) [20]. In addition, parents filled out the ADHD Rating Scale, adapted for the Italian population [21].

A detailed medical history was obtained for all subjects. All participants to the study underwent a neurological and physical examination to detect co-morbid medical and neurological conditions. Children with genetic disorders, cerebral palsy, autism, neuromuscular disease, and associated chromosomal syndromes were excluded.

Serum samples from fasting patients and controls were obtained between 10 am and 12 am. Blood was sampled in anticoagulant-free tubes and kept at room temperature for 1 h before the serum was separated (centrifugation at 2000g for 10 min). Aliquots of serum were stored at −80 °C until analysis.

A control group of 62 healthy Caucasian children, age and sex matched, were randomly recruited from a community-based survey in the same area of children with ADHD, in Rome. A diagnosis of ADHD or other disorders was subsequently ruled out through the ADHD Rating Scale and the K-SADS-PL as previously described for children diagnosed with ADHD.

All children underwent a cognitive assessment by the Italian validated version of the Wechsler Intelligence Scale for Children—Third Edition Revised [22], and a full-scale intelligence quotient was obtained. Any child with an Intelligence Quotient (IQ) <70 was excluded.

None of the subjects showed any signs of topic eczema or other allergic or rheumatic diseases. Parents of all children gave written informed consent to the study. The study was approved by the local ethical committee of S. Andrea Hospital in Rome, on the basis of the Helsinki criteria.

Clinical assessment

Parents filled out the ADHD Rating Scale, consisting of 18 items divided into two subgroups of nine questions that investigate inattention and hyperactive-impulsive symptoms. Parents were requested to record the frequency of symptoms (0 = no symptoms; up to 3 = most of the time). The presence of at least six out of nine symptoms in either or both of the two subgroups (inattention and hyperactivity–impulsivity) was considered positive. The clinical diagnosis was subsequently confirmed by the K-SADS-PL administered by an experienced clinician (R. D.).

Cognitive assessment

IQ was obtained using the Wechsler Intelligence Scale for Children—Third Edition Revised [22]. This is a validated intelligence test for children between 6 and 16 years of age, which is usually administered in 75–80 min.

The test comprises ten core subtests and two supplemental tests. These subtests generate a full scale score, Total-IQ (T-IQ), and two composite scores known as indexes: the Verbal-IQ (V-IQ) (including Vocabulary, Similarities, Comprehension, Information, Arithmetic, and Digit Span as supplemental test) and the Performance-IQ (P-IQ) (including Block Design, Picture Stories, Picture Completion, Puzzle, Coding, and Mazes as supplemental test).

Analysis of serum levels of tryptophan and selected metabolites of the kynurenine pathway

We assessed serum levels of tryptophan, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxyanthraniclic acid, xanthurenic acid, and quinolinic acid by a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method, as described previously [14]. In brief, serum samples were collected and stored at −80 °C until analysis. One hundred μl of serum samples were deproteinized using 100 μl of Internal Standard (IS) working solution (50 µM in TCA 4%). Samples were vortex-mixed centrifuged at 14,000 rpm for 5 min. Fifty μl of clean upper layer were injected into chromatographic system. The HPLC analysis was performed using an Agilent Liquid Chromatography System series 1100 (Agilent Technologies, USA) which included a binary pump, an auto-sampler, a solvent degasser, and a column oven. Chromatographic separation was performed on a pentafluorophenyl column (100 × 2.1 mm, Kinetex PFP, 2.6 μm, 100 Å pore size, Phenomenex, CA, USA) equipped with a security guard precolumn (Phenomenex, Torrance, CA, USA) containing the same packing material. The mobile phase consisted of a solution of 0.1% aqueous formic acid (eluent A) and 100% methanol (eluent B); elution was performed at flow rate of 300 μl/min, using an elution gradient. The mass spectrometry method was performed on a 3200 triple quadrupole system (Applied Biosystems, Foster City, CA, USA) equipped with a Turbo Ion Spray source. The detector was set in the positive ion mode. The ion spray voltage was set at 5000 V and the source temperature was 300 °C. The instrument was set in the Multiple Reaction Monitoring (MRM) mode. Data were acquired and processed by the Analyst 1.5.1 Software. The inter- and intra-assay coefficients were always lower than 20%.

The Kynurenine/tryptophan ratio was used as an index of tryptophan-2,3-dioxygenase/indoleamine 2,3-dioxygenase (TPO/IDO) activity.

Statistical analysis

The normal distribution of data was assessed by means of the Kolmogorov–Smirnov test. Continuous variables were expressed as arithmetic mean ± SD or median (IQR) depending on their distribution. In our study, the results analysed with a Student t test revealed that group 1 (children with ADHD, N  =  102) had a mean tryptophan and kynurenine metabolite serum levels of 1343.0 ng/ml (SD 279.5) and that group 2 (healthy children, N  =  62) had a mean of 1199.7 ng/ml (SD 341.3). The calculated p value was 0.004. The calculated effect size was 0.5, which is considered “medium” according to Cohen. To test our hypothesis and determine if this finding was real or due to chance (i.e., to find a significant difference), with an effect size of 0.5 and p of <0.05, we considered a sample size of approximately N  =  60 in each group with a power 0.80.

Pearson’s correlation was used to correlate the characteristics of ADHD children (age, sex, comorbidity presence, ADHD Rating Scale, and IQ scores) with tryptophan and kynurenine metabolites levels.

Comparisons between the socio-demographic characteristics of ADHD and healthy children were performed using Student’s t test and Chi-square test. Student’s t test was also used for comparisons of the levels of tryptophan and kynurenine metabolites between healthy children and children affected by ADHD. Multiple testing was corrected using the Benjamini–Hochberg method to control the false-discovery rate (FDR).

Multiple logistic regression analysis, stepwise method was used to assess the effect of potential confounders on the presence of ADHD between patients and healthy controls.

Receiver operating characteristic (ROC) curves were performed and the area under curve (AUC) was calculated to assess the diagnostic value of anthranilic acid (ng/ml) for the prediction of ADHD. Optimal cut-off values were obtained from the Youden’s index. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated at the optimal cut-off value for anthranilic acid.

An SPSS software (Version 19, SPSS Inc., Chicago Il, USA) was used for the analyses. “p” values of <0.05 were considered to be statistically significant.

Results

One hundred and two children with ADHD (mean age of ADHD diagnosis 9.3 ± 2.7 years, 75 males and 27 females, BMI: 18.0 ± 3.7 kg/m2) and 62 healthy controls (mean age 9.60 ± 1.74 years, 48 males and 14 females, BMI: 17.4 ± 2.4 kg/m2), all of Caucasian origin, were enrolled.

V-IQ, P-IQ, and T-IQ scores fell within the normal range in both groups (Table 1), with higher values in healthy children (p < 0.001).
Table 1

Differences in socio-demographic characteristics and IQ scores between ADHD and healthy children

 

ADHD (n = 102)

Controls (n = 62)

p

Age (years)

9.3 ± 2.7

9.60 ± 1.74

NS

Sex (males)

75 (73.5%)

48 (77.4%)

NS

Total IQ

101.14 ± 15.28

109.56 ± 12.99

0.001*

Performance IQ

100.43 ± 16.03

117.68 ± 10.24

0.001*

Verbal IQ

102.46 ± 15.22

114.91 ± 10.31

0.001*

Data are expressed as mean ± standard deviation

ADHD attention deficit hyperactivity disorder, IQ intelligence quotient, NS not significant

* Student’s t Test

The ADHD Rating Scale Total Score in children with ADHD was 37.13 ± 9.16. In 53/102 (52.0%), children with ADHD at least one comorbid condition was present (Table 2).
Table 2

Comorbities in children with attention deficit hyperactivity disorder (ADHD)

 

ADHD children (n = 102)

Comorbitity

53/102 (52%)

Oppositional defiant disorder (ODD)

20/102 (19.6%)

Anxiety disorders

12/102 (11.8%)

Language impairment

11/102 (10.8%)

Dyslexia

9/102 (8.8%)

Dysorthography

8/102 (7.8%)

Learning disability

8/102 (7.8%)

Bipolar disorder

7/102 (6.9%)

Mood disorders

6/10 (5.9%)

Deficit emotional self-regulation (DESR)

5/102 (4.9%)

Dyscalculia

5/102 (4.9%)

Tourette syndrome

3/102 (2.9%)

Serum levels of tryptophan and kynurenine metabolites and kynurenine/tryptophan ratio are shown in Table 3.
Table 3

Serum levels of l-tryptophan, selected metabolites of the kynurenine pathway and kynurenine/tryptophan ratio in children with ADHD and age-matched controls

 

ADHD (n = 102)

Controls (n = 62)

p

FDR

Tryptophan (ng/ml)

8914.9 ± 1776.3

8038.6 ± 2219.6

0.01*

0.025

Kynurenic acid (ng/ml)

3.2 ± 0.9

3.6 ± 1.4

0.03*

0.031

Xanthurenic acid (ng/ml)

1.4 ± 0.5

1.6 ± 0.6

0.04*

0.037

Anthranilic acid (ng/ml)

9.6 ± 7.3

24.0 ± 8.9

<0.001*

0.006

3-Hydroxyanthranilic acid (ng/ml)

4.57 ± 3.01

3.62 ± 2.02

0.15

0.050

Kynurenine (ng/ml)

440.3 ± 158.6

296.0 ± 148.7

<0.001*

0.012

Quinolinic acid (ng/ml)

33.8 ± 10.1

31.3 ± 8.6

0.10

0.044

Kynurenine/Tryptophan Ratio

0.05 ± 0.02

0.04 ± 0.02

<0.001*

0.019

Data are expressed as mean ± standard deviation

ADHD attention deficit hyperactivity disorder, FDR p value from Benjamini–Hochberg method control for false-discovery rate (FDR)

* Student’s t Test

Children with ADHD showed a substantial reduction in anthranilic acid levels (−60%; p < 0.001), slight reductions in kynurenic acid (−11%; p = 0.03) and xanturenic acid (−12.5%; p = 0.04) levels, and no changes in 3-hydroxyanthranilic acid levels. In contrast, kynurenine levels were largely increased in children with ADHD (+49%; p < 0.001). We also found a slight increase in l-tryptophan levels (+11%; p < 0.01) and values for the kynurenine/tryptophan ratio (+25%; p < 0.001) in children with ADHD with respect to healthy controls. We divided the ADHD children into two subgroups, which differed by the extent of changes in the kynurenine/tryptophan ratio with respect to healthy controls. ADHD children with values of the kynurenine/tryptophan ratio >1.5 SDS (Standard Deviation Score) with respect to the mean value of healthy controls were those in which—presumably—the kynurenine pathway was highly activated. In spite of this, these children (n = 17) showed lower levels of anthranilic acid with respect to the other ADHD children (5.5 ± 3.6 vs 10.4 ± 7.6 ng/ml, p < 0.02) and to healthy controls. This interesting finding suggests that the reduction in anthranilic acid is observed even in those cases in which large amounts of its metabolic precursor, l-kynurenine, are generated from tryptophan metabolism.

Changes in kynurenine metabolites observed in ADHD children were not influenced by comorbid conditions, as shown in Table 4.
Table 4

Serum levels of l-tryptophan, selected metabolites of the kynurenine pathway, and kynurenine/tryptophan ratio in children with ADHD and at least one comorbidity

 

ADHD comorbidity (n = 53)

ADHD no comorbidity (n = 49)

p

Tryptophan (ng/ml)

9131.8 ± 2067.5

8680.4 ± 1350.9

0.2

Kynurenic acid (ng/ml)

3.1 ± 0.9

3.2 ± 0.8

0.3

Xanthurenic acid (ng/ml)

1.4 ± 0.5

1.3 ± 0.4

0.6

Anthranilic acid (ng/ml)

9.3 ± 7.3

9.8 ± 7.3

0.7

3-Hydroxyanthranilic acid (ng/ml)

4.06 ± 2.5

4.34 ± 2.6

0.6

Kynurenine (ng/ml)

445.7 ± 139.8

434.4 ± 178.05

0.7

Quinolinic acid (ng/ml)

33.2 ± 9.7

34.3 ± 10.6

0.6

Kynurenine/Tryptophan ratio

0.05 ± 0.02

0.05 ± 0.02

0.9

Data are expressed as mean ± standard deviation

ADHD attention deficit hyperactivity disorder

* Student’s t Test

We performed a Pearson’s correlation analysis between serum levels of l-tryptophan and kynurenine metabolites in children with ADHD, and either IQ or ADHD Rating Scale scores. Kynurenic acid levels positively correlated with IQ V score (p = 0.01, r = 0.31) and negatively correlated with both Total (p = 0.047, r = −0.22) and hyperactivity (p = 0.04, r = −0.23) scores of the ADHD Rating Scale. Levels of anthranilic acid showed a negative correlation with the IQ performance score (p = 0.02, r = −0.30). Levels of kynurenine positively correlated with the IQ performance score (p = 0.047, r = 0.25) and ADHD Rating Scale Inattention Score (p = 0.05, r = 0.21). No correlations were found in healthy children (Table 5).
Table 5

Pearson’s correlation analysis between serum levels of l-tryptophan and kynurenine metabolites in children with ADHD, and either IQ or ADHD Rating Scale scores

 

IQ

Performance score

IQ

Verbal

score

IQ

Total score

ADHD Rating Scale

Hyperactivity

Score

ADHD

Rating Scale

Inattention Score

ADHD

Rating Scale

Total score

Tryptophan (ng/ml)

NS

NS

NS

NS

NS

NS

Xanthurenic acid (ng/ml)

NS

NS

NS

NS

NS

NS

3-Hydroxyanthranilic acid (ng/ml)

NS

NS

NS

NS

NS

NS

Anthranilic acid (ng/ml)

p = 0.02

r = −0.30

NS

NS

NS

NS

NS

Kynurenine (ng/ml)

p = 0.047

r = 0.25

NS

NS

NS

p = 0.05

r = 0.21

NS

Kynurenic acid (ng/ml)

NS

p = 0.014

r = 0.31

NS

p = 0.04

r = −0.23

NS

p = 0.047

r = −0.22

Quinolinic acid (ng/ml)

NS

NS

NS

NS

NS

NS

ADHD attention deficit hyperactivity disorder, NS not significant

In a regression logistic model, the presence of ADHD was predicted by low anthranilic acid levels and higher tryptophan levels (Table 6).
Table 6

Multiple logistic regression analysis

Model

B

Std. error

Wald

Sig.

Exp (B)

95% CI Exp (B)

Lower limit

Upper limit

1

Anthranilic acid (ng/ml)

−0.21

0.032

42.561

0.000

0.814

0.765

0.866

Constant

3.9

0.601

44.165

0.000

54.171

  

2

Tryptophan (ng/ml)

0.000

0.000

9.863

0.002

1.000

1.000

1.001

Anthranilic acid (ng/ml)

−0.237

0.036

40.590

0.000

0.797

0.743

0.855

Constant

1.204

1.019

1.396

0.237

3.333

  
We performed ROC analysis to evaluate the diagnostic performance of anthranilic acid (Fig. 1). The AUC of anthranilic acid was 0.88 (95% CI = 0.83–0.94).
Fig. 1

Receiver operating characteristic (ROC) curves to assess the diagnostic value of anthranilic acid (ng/ml) for the prediction of ADHD

In ADHD group, an optimal anthranilic acid cut-off value of 10.4 ng/ml generated a sensitivity of 66.3%, a specificity of 96.8%, a PPV of 98.1%, and an NPV of 49.2% according to Youden’s index.

Discussion

We examined blood levels of metabolites of the kynurenine pathway in paediatric patients affected by ADHD. An obvious limitation of our study (and in all studies measuring blood levels of kynurenine metabolites) is that the kynurenine pathway is not confined to the CNS, and the liver and other peripheral organs are an important source of blood kynurenine metabolites. Peripheral kynurenine and 3-hydroxykynurenine may cross the blood–brain barrier in significant amounts, and in the CNS, are metabolized by either KMO and kynureninase present in microglia or KATII present in astrocytes [11, 23]. In particular, astrocytes account for the biosynthesis of kynurenic acid, which is regulated by intracellular metabolic events, while 3-hydroxykynurenine and its major downstream metabolites are synthesized in microglia and other cells of monocytic origin [11]. Xanthurenic acid can also enter the brain [24]. It is generally believed that peripheral levels of kynurenine metabolites reflect CNS levels, perhaps with the exception of quinolinic acid [11, 23].

We found increased serum levels of tryptophan and kynurenine, reduced levels of kynurenic acid, anthranilic acid, and xanthurenic acid, and no changes in 3-hydroxyanthranilic acid and quinolinic acid levels in children with ADHD. These data are only partially consistent with those reported in adult ADHD patients, who showed lower levels of kynurenic acid, xanthurenic acid, and 3-hydroxyanthranilic acids, but no changes in anthranilic acid levels [18]. Possible explanations for these differences are that neurochemical modifications associated with ADHD are age dependent or that comparison of data in different studies is made difficult by a number of variables, such as comorbid conditions or drug treatments.

Interestingly, the tryptophan breakdown index (i.e., the kynurenine/tryptophan ratio) was significantly higher in children affected by ADHD, suggesting that a greater proportion of tryptophan is metabolized by IDO/TDO in ADHD. An increase of this index is definitely expected in the presence of pathology, as stated by Oades and colleagues who, in spite of this hypothesis, found a reduced breakdown index in their ADHD sample [19]. They interpret their own result as unexpected and meriting further attention in future studies. We believe that our result represents a piece of evidence contributing to clarifying this point. Data of kynurenine and its downstream metabolites are not easy to interpret. The higher levels of kynurenine associated with lower levels of kynurenic and anthranilic acids suggest that KAT and kynureninase are defective in ADHD. A defect of KAT may also explain the lower levels of xanthurenic acid found in children affected by ADHD. The lack of changes in 3-hydroxyanthanylic and quinolinic acid levels suggests that hydroxylation of kynurenine into 3-hydroxykynurenine (the precursor of 3-hydroxyantranilic acid) is not altered in ADHD. However, a reduction in 3-hydroxykynurenine levels has been reported in a small cohort of ADHD patients [19].

Kynurenic acid, quinolinic acid, and xanthurenic acid are neuroactive compounds that interact with different types of glutamate receptors. Kynurenic acid activates AMPA receptors at nanomolar concentrations, and inhibits AMPA receptors at micromolar concentrations [25]. In addition, kynurenic acid acts as a competitive antagonist at the glycine site of NMDA receptors, thereby inhibiting the activity of the NMDA-gated ion channel [26]. Owing to this mechanism, kynurenic acid exerts neuroprotective and anticonvulsant activities [27]. In contrast, quinolinic acid is an agonist of NMDA receptors and may cause seizures and excitotoxic neuronal death at pharmacological doses [23, 25]. Xanthurenic acid interacts at multiple levels with glutamatergic neurotransmission by inhibiting vesicular glutamate transporters [28] and activating mGlu2 and mGlu3 metabotropic glutamate receptors [29]. These receptors are coupled to Gi proteins and negatively modulate glutamate release from presynaptic terminals [30].

Changes in kynurenic and xanthurenic acid levels we have found in our sample of ADHD children may have an impact on the activity of NMDA and mGlu2/3 receptors, and therefore, our findings are in line with the hypothesis of an abnormal glutamatergic transmission in ADHD [31]. Glutamate plays a crucial role in cognitive processes, as well as in mechanisms of developmental plasticity that shape neuronal circuitries and network activity across the entire lifespan. During development, abnormalities in glutamatergic neurotransmission might alter the process of synaptic pruning, which refers to a progressive synaptic elimination that is completed at the time of sexual maturation [32]. Interestingly, synaptic pruning is believed to be abnormal in ADHD, and this may underlie the observed morphological changes in brain regions that are involved in cognitive functions, attention, executive functions, and emotions, such as the dorsolateral prefrontal cortex, anterior cingulate cortex, and insular cortex [33, 34].

It is difficult to predict in which direction a reduction in kynurenic acid levels influences excitatory neurotransmission. Lower levels of kynurenic acid may limit endogenous inhibition of NMDA receptors, thereby facilitating receptor activation by the endogenous glutamate in the ADHD brain. However, at least in the prefrontal cortex, NMDA receptors are predominantly expressed by GABAergic interneurons, as shown by the evidence that systemic administration of NMDA receptor antagonists enhances the firing rate of glutamatergic pyramidal neurons [35, 36]. In ADHD, we expect that the reduction in kynurenic acid levels reinforces NMDA-mediated excitation of GABAergic interneurons, thereby restraining the activity of pyramidal neurons. Of note, genetic and animal studies suggest that a dysregulation of NMDA receptors is involved in the pathophysiology of ADHD [17, 37], and anti-ADHD drugs, such as methylphenidate and atomoxetine, cause changes in the expression or function of NMDA receptors [38]. An interesting question is whether these changes in kynurenine metabolites are specific for ADHD or not. ADHD is frequently comorbid with other diagnoses and it has been proposed that the association between ADHD and certain comorbidities (especially conduct problems) may represent a distinct nosographic entity [39]. For this reason, we compared levels of kynurenine metabolites in ADHD subjects with and without comorbidities. No significant differences were found in our sample. This provides preliminary evidence that ADHD is associated with specific neurobiological features even in the presence of comorbidities.

Furthermore, in favour of an “ADHD specificity” of our findings, a different scenario is found in patients affected by schizophrenia, in which levels of the NMDA receptor antagonist, kynurenic acid, are increased, and levels of the NMDA receptor agonist, quinolinic acid, are reduced because of a defective conversion of kynurenine into 3-hydroxykynurenine [14, 40]. The resulting hypoactivity of NMDA receptors in GABAergic interneurons is expected to enhance the overall firing rate of pyramidal neurons and to severely impair neuronal synchronization and network oscillations in schizophrenia [41]. Where ADHD and schizophrenia converge is in the reduction of xanthurenic acid levels, which, however, is more prominent in patients affected by schizophrenia (compare our data with data reported by Fazio et al. [14]). The reduction of xanthurenic acid levels raises the interesting hypothesis that the function of mGlu2 and/or mGlu3 receptors is abnormal in ADHD. This hypothesis warrants further investigation, because mGlu2 receptor ligands are under clinical development for the treatment of psychiatric disorders [42].

Perhaps, the most striking finding of our study is the large reduction of anthranilic acid, which was associated with a two-fold increase in kynurenine levels. Again, this strongly suggests that the activity of kynureninase—the enzyme that converts kynurenine into anthranilic acid—is defective in ADHD. The biological relevance of this finding is unknown, because neither kynurenine nor anthranilic acid is so far considered as neuroactive compounds. However, the large reduction in the ratio between anthranilic acid and kynurenine levels we have found in children affected by ADHD suggests that this ratio can be investigated as a potential peripheral biomarker of ADHD. This requires further studies with large cohorts of ADHD patients in which the influence of age and drug treatment on the ratio between anthranilic acid and kynurenine should be specifically investigated.

Notes

Compliance with ethical standards

Conflict of interest

MelaniaEvangelisti—Reports no disclosures. JoleRabasco—Reports no disclosures. Renato Donfrancesco—Reports no disclosures. Pietro De Rossi—Reports no disclosures. LuanaLionetto—Reports no disclosures. MatildeCapi—Reports no disclosures. Gabriele Sani—Reports no disclosures. Maurizio Simmaco—Reports no disclosures. Ferdinando Nicoletti—Reports no disclosures. Maria Pia Villa—Reports no disclosures.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Melania Evangelisti
    • 1
  • Pietro De Rossi
    • 1
    • 3
    • 7
  • Jole Rabasco
    • 1
  • Renato Donfrancesco
    • 2
  • Luana Lionetto
    • 4
  • Matilde Capi
    • 4
  • Gabriele Sani
    • 1
    • 3
    • 7
  • Maurizio Simmaco
    • 1
  • Ferdinando Nicoletti
    • 5
    • 6
  • Maria Pia Villa
    • 1
  1. 1.NESMOS Department, School of Medicine and Psychology, Sant’Andrea HospitalSapienza UniversityRomeItaly
  2. 2.S. Pertini HospitalASL RM/BRomeItaly
  3. 3.I.R.C.C.S. Fondazione Santa LuciaRomeItaly
  4. 4.Experimental Immunology Laboratory, Biochemistry LaboratoryIDI-IRCCS FLMMRomeItaly
  5. 5.I.R.C.C.S. NeuromedPozzilliItaly
  6. 6.Department of Physiology and PharmacologySapienza UniversityRomeItaly
  7. 7.Department of Neurology and PsychiatrySapienza University of RomeRomeItaly

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