Anthranilic Acid: A Potential Biomarker and Treatment Target for Schizophrenia

Abstract

Dysregulation of Trp-Kyn pathway is the most recent hypothesis of mechanisms of schizophrenia. In particular, over-production of kynurenic acid (KYNA), one of the three immediate downstream metabolites of kynurenine (Kyn) along tryptophan (Trp): Kyn pathway, has been considered as a new target for therapeutic intervention in schizophrenia. Up-regulation of KYNA formation was suggested to occur at the expense of down-regulated production of 3-hydroxyKyn (3-HK), the second immediate downstream metabolite of Kyn. We were interested to assess the third immediate downstream Kyn metabolite, anthranilic acid (AA). Serum AA concentrations were evaluated in schizophrenia patients and control subjects by HPLC-mass spectrometry method. We found 2-fold increase of AA and 3-fold decrease of 3-HK concentrations in serum of schizophrenia patients. Up regulated formation of AA might contribute to mechanisms of schizophrenia considering experimental evidences of AA augmentation of autoimmune processes in rat and mice; clinical findings of AA elevation in rheumatoid arthritis and type 1 diabetes, autoimmune diseases diametrical to schizophrenia; and involvement of autoimmunity in development of schizophrenia. Present data warrant further studies of AA as biological marker in, at least, a subgroup (associated with autoimmune mechanisms) of schizophrenia patients and as a new target for therapeutic intervention.

INTRODUCTION

Schizophrenia is a severe and debilitating psychiatric disorder with 1% lifetime prevalence relatively independent of geographic, cultural, and socioeconomic variables. A etiopathogenetic mechanisms of schizophrenia are not completely defined and require further investigation. The original dopaminergic hyperactivity hypothesis [1] was recently supplemented by glutamatergic hypoactivity hypothesis of aetiopathogenetic mechanisms of schizophrenia [2]. Experimental and clinical studies revealed psychotomimetic effects of glutamate receptor antagonists [2]. One of them, kynurenic acid (KYNA), is one of the three immediate downstream metabolites of kynurenine (Kyn) along tryptophan (Trp) : Kyn pathway, a major source of NAD⁺ biosynthesis in humans [3] (Figure 1). Trp conversion into Kyn (via N-formyl-Kyn) is catalyzed by indoleamine (or Trp) 2,3-dioxygenases (IDO and TDO, respectively). KYN conversion into 3-hydroxykynurenine (3-HK), catalyzed by riboflavindependent kynurenine 3-monoxygenase (KMO), is key step of formation of NAD⁺ [4]. Inhibition of KMO activity increases utilization of Kyn as a substrate for formation of KYNA, catalyzed by kynurenine aminotransferase II (KAT), and, thus, contributes to elevation of KYNA content in brains and cerebrospinal fluid of schizophrenia patients [2,5]. Besides 3-HK and KYNA, KYN is a substrate for formation of anthranilic acid (AA), catalyzed by kynureninase (KYNU) (Figure 1A). AA was detected in concentrations ranged from 0.5 to 1.8 pmol/mg protein in rat central (frontal cortex, hypothalamus, hippocampus, cerebellum, medulla, thalamus, striatum, olfactory bulb, spinal cord and retina) and peripheral organs (kidney, liver, lung, heart, pancreas, spleen, testis and adrenal gland), and serum (around 130 nM) and urine (10 nmol/mg creatinine) [6]. The same study revealed AA localization in glial cells based on a significant increase of AA content in brain tissue lesioned by intrastriatally injected quinolinate. However, we could not find in available literature reports of AA evaluation in schizophrenia patients.

Figure 1.

Proposed changes of Kyn down-stream metabolism in schizophrenia

Abbreviations: Trp: Tryptophan; Kyn : Kynurenine; KYNA : Kynurenic acid; AA : Anthranilic Acid; 3-HK : 3-HydroxyKynurenine; NAD+ : Nicotinamide Adenine Dinucleotide

Here we present preliminary results of our study of serum concentrations of downstream Kyn metabolites in schizophrenia patients

MATERIALS AND METHODS

Patients

Overnight fasting blood samples were collected from patients (three men and three women, age range from 38 to 56 years) with schizophrenia, diagnosed according to DSM-V criteria [7]. All patients were taking anti-psychotic medication: Abilify (three patients), Haloperidol (one patient), Haloperidol decanoate injections (one patient) and Saphris (one patient).

Healthy Subjects (Controls)

There were 12 subjects (6 females and 6 males, age range from 32 to 64 years). Study was approved by Tufts Medical Center IRB.

Assessment of kynurenine metabolites

Serum samples were stored at −50°C until analysis. AA, Trp, Kyn, KYNA and 3-HK concentrations were analyzed by HPLC:mass spectrometry method [8].

Statistical Analysis

Results are presented as mean ± standard error (Trp and Kyn in μM and AA, KYNA and 3-HK in nM). Statistical significance was assessed by Mann-Whitney test, two-tailed.

RESULTS AND DISCUSSION

Serum concentrations of Kyn and its metabolites

Assessment of peripheral AA is an appropriate approach in clinical studies considering that AA, in difference with KYNA, easily penetrates blood-brain barrier (via passive diffusion), and, therefore, peripherally produced AA might contribute significantly to it brain pools and act on targets in brain [9].

AA concentrations were increased (approximately two-fold) in schizophrenia patients in comparison with control subjects. AA increase was robust: all controls had AA serum concentrations lower than schizophrenia patients] (Table 1).

Table 1.

Down-stream kynurenine metabolites in serum of schizophrenia patients.

	Schizophrenia* (n=6)	Control* (n=12)	P (Mann-Whitney test, two-tailed)
Tryptophan (μM)	77.88±7.53	68.90± 2.49	ns
Kynurenine (μM)	2.06±0.27	1.76± 0.09	ns
Kyn × 100 : Trp	2.64±0.21	2.56±0.35	ns
Anthranilic acid (nM)	111.43±7.56	21.65±5.99	0.0001
AA : Kyn	60.89±11.98	12.22±3.22	0.0001
3-HK (nM)	6.25±0.05	19.55± 3.14	0.03
3-HK : Kyn	3.14±1.34	11.58±1.3	0.0003
KYNA (nM)	36.43±3.99	35.78± 3.59	ns
KYNA : Kyn	18.33±1.47	19.76±1.69	Ns

^*mean±standard error

Abbreviations: Kyn : Kynurenine; Trp : Tryptophan; AA : Anthranilic Acid; 3-HK : 3-HydroxyKynurenine; KYNA : Kynurenic acid.

3-HK concentrations were approximately three-fold lower in schizophrenia patients than in controls.

There was no statistically significant difference between Trp, Kyn and KYNA concentrations in serum of schizophrenia patients and controls.

End product: substrate ratios of Trp: Kyn metabolic pathway

In clinical studies, evaluation of end product: substrate ratios are used for assessment of activities of enzymes, catalyzing corresponding metabolic reactions [10].

AA: Kyn ratio was higher (1.6 fold) while 3-HK: Kyn ratio was lower (3.7 fold) in patients than in controls.

KYNA: KYN ratio did not differ between patients and controls (Table 1).

Kyn: Trp ratio, a clinical index of IDO and TDO activity, did not differ between patients and controls. It is noteworthy that elevated serum AA concentrations might restrict utilization of Kyn: Trp ratio for assessment of IDO/TDO activity considering that AA is not only metabolite of Trp but a substrate of Trp formation by bacteria, e.g., by intestinal microbiome [11].

The main finding of the present communication is a robust and dramatic increase of AA concentrations in serum of schizophrenia patients. To the best of our knowledge, this is the first observation of increased AA concentrations in schizophrenia patients. Our results are in agreement with previously reported AA elevation in rat plasma in social isolation rearing model of schizophrenia [12]. It is unlikely that AA elevation depends on the use of anti-psychotic medication, considering that chronic administration of anti-psychotic drug risperidone did not affect Trp: Kyn metabolism in rat brain [5]. Elevation of AA concentrations could be caused by up-regulation of enzyme (KYNU), catalyzing conversion of Kyn into AA. Our finding of increased AA: Kyn ratio suggests up-regulation of KYNU activity. However, direct assessment of brain KYNU activity did not reveal its up-regulation in schizophrenia patients [5]. In the same vein, diet deficient of vitamin B6, a cofactor of KYNU, did not decrease urine AA excretion in baboons [13]. The other cause of AA elevation might be an increased formation of Kyn, a substrate for AA synthesis, from Trp, catalyzed by IDO/TDO, and/or on decreased conversion of Kyn into KYNA or 3-HK. We did not find increased Kyn: Trp (index of IDO/TDO activity) or decreased KYNA : Kyn ratio (index of KAT activity) in schizophrenia patients in agreement with observation of no changes of KAT activity in Broadmann areas of schizophrenia patients [5]. We did find decreased 3-HK : Kyn ratio (index of KMO activity) and drastic decrease of 3-HK serum concentrations in schizophrenia patients in agreement with previously reported reduction in KMO gene expression and KMO enzyme activity and 3-HK decrease in Broadmann areas of schizophrenia patients [5,14]. Administration of diet deficient of riboflavin, a KMO cofactor, increased AA (12-fold) and decreased 3-HK (10-fold) urine excretion in baboons suggesting that increased availability of substrate, Kyn, due to inhibition of KMO, up-regulates AA formation [13]. Effect of KMO inhibition on AA formation might be further supported by observation of increased AA excretion in riboflavin deficient rats [15] and AA elevation in brain, liver and plasma KMO(−/−) mice [16].

Therefore, the most likely cause of AA increase in schizophrenia is a shift of downstream Kyn metabolism from formation of 3-HK towards production of AA due to inhibition of KMO, an enzyme catalyzing Kyn conversion into 3-HK (Figure 1B).

Common features of schizophrenia with autoimmune diseases suggest that, at least, some group of schizophrenia could be an autoimmune disease itself [17]. Experimental study revealed that AA significantly enhanced the development of adjuvant-induced model of rheumatoid arthritis in rats and increased antibody formation to sheep erythrocytes in mice [18]. Protection against autoimmune-induced apoptosis is mediated by Trp degradation mediated by up-regulation of IDO activity [19]. AA might prevent Trp depletion by serving as a substrate for Trp formation by bacteria, e.g., intestinal microbiome, [11], and by inhibition of sodium-coupled transporter of uncharged solutes (e.g., Trp) across mammalian cell membranes [20].

Clinical studies reported drastic elevation of AA in organ-specific autoimmune disorders, rheumatoid arthritis [21] and Type 1 diabetes (T1D) [22]. AA elevation in schizophrenia, rheumatoid arthritis, and T1D suggests that AA contributes to common autoimmune etiology of these disorders. Inverse correlations between schizophrenia with rheumatoid arthritis [23] and T1D [24] suggests that these conditions are diametrically related, i.e., increased risk for one set of diseases commonly engenders decreased risk for another [25]. Diametrical diseases reflect evolutionary-genetic tradeoffs centrally mediating the expression of human adaptations and influencing the prevalence and forms of human maladaptation manifest in disease [25]. Considering that Trp: Kyn pathway is evolutionary conserved (and was described in flies, worms, bacteria, mammals and humans), it might be interesting to explore possible role of AA (and other Kyn metabolites), in mechanisms of diametrical disorders.

CONCLUSION

Preliminary report from our studies reveals a drastic and robust elevation of AA serum concentrations in schizophrenia patients. Dysregulation of Trp - Kyn pathway is the most recent hypothesis of mechanisms of schizophrenia. However, possible involvement of AA in mechanisms of schizophrenia has not been considered so far. Present data warrant further studies of AA as biological marker of, at least, a subgroup of schizophrenia patients (e.g., associated with autoimmune mechanisms) and as a new target for therapeutic intervention in schizophrenia.

ABBREVIATIONS

Trp

Tryptophan

Kyn

Kynurenine

KYNA

Kynurenic acid

AA

Anthranilic Acid

3-HK

3-HydroxyKynurenine

IDO

Indoleamine 2,3-dioxygenase

TDO

Tryptophan 2,3-dioxygenase

KMO

Kynurenine 3-Monoxygenase

KAT

Kynurenine Amino Transferase II

KYNU

Kynureninase

NAD⁺

Nicotinamide Adenine Dinucleotide