Comprehensive analysis of the tryptophan metabolome in urine of patients with acute intermittent porphyria

Accepted Manuscript Title: Comprehensive analysis of the tryptophan metabolome in urine of patients with acute intermittent porphyria. Authors: Alex Gomez-Gomez, Josep Marcos, Paula Aguilera, Jordi To-Figueras, Oscar J Pozo PII: DOI: Reference:

S1570-0232(17)30379-3 http://dx.doi.org/doi:10.1016/j.jchromb.2017.06.030 CHROMB 20659

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Journal of Chromatography B

Received date: Revised date: Accepted date:

6-3-2017 15-6-2017 17-6-2017

Please cite this article as: Alex Gomez-Gomez, Josep Marcos, Paula Aguilera, Jordi To-Figueras, Oscar J Pozo, Comprehensive analysis of the tryptophan metabolome in urine of patients with acute intermittent porphyria., Journal of Chromatography Bhttp://dx.doi.org/10.1016/j.jchromb.2017.06.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Comprehensive analysis of the tryptophan metabolome in urine of patients with acute intermittent porphyria.

Alex Gomez-Gomez1,2,3, Josep Marcos4, Paula Aguilera5, Jordi To-Figueras6,†,* and Oscar J Pozo1,†,*.

1

Integrative Pharmacology and Systems Neuroscience Group, IMIM, Hospital del Mar, Doctor Aiguader 88, Barcelona, Spain. 2

Programa De Recerca En Epidemiologia I Salut Pública, ISGlobal, Campus Mar, Doctor Aiguader 88, Barcelona, Spain. 3

Universitat Pompeu Fabra (CEXS-UPF), Doctor Aiguader 88, Barcelona, Spain.

4

Department of Experimental Sciences, Pompeu Fabra University, Doctor Aiguader 88, Barcelona, Spain. 5

Porphyria Unit, Dermatology Unit, Hospital Clínic, IDIBAPS, University of Barcelona, Villarrroel 170, Barcelona, Spain. 6

Biochemistry and Molecular Genetics Unit, Hospital Clinic of Barcelona, IDIBAPS, University of Barcelona, Villarroel 170, Barcelona, Spain.

†

Both authors contributed equally to the manuscript.

*Corresponding author: Óscar J Pozo e-mail: [email protected] Integrative Pharmacology and Systems Neuroscience Group, IMIM, Hospital del Mar Medical Research Institute, Doctor Aiguader 88, 08003 Barcelona, Spain Phone: 0034-933160480, Fax: 0034-933160499 Authors’ e-mails: Alex Gómez-Gómez: [email protected] Josep Marcos: [email protected] Paula Aguilera: [email protected] Jordi To-Figueras: [email protected] Oscar J. Pozo: [email protected]

Highlights:     

Targeted metabolomics is used for the study of Tryptophan metabolism in AIP patients. AIP patients show increased urinary excretion of kynurenine. AIP patients show both an elevation of kynurenine /tryptophan and a decrease of kynurenic acid/kynurenine. No differences were found in the serotonin metabolic pathway. Reduced activity of kynurenine aminotransferase and/or induction of indoleamine 2,3dioxygenase enzymes may explain these findings.

Abstract Background: Acute intermittent porphyria (AIP) is a rare metabolic disorder due to a deficiency of porphobilinogen deaminase, the third enzyme of the heme biosynthetic pathway. This low enzymatic activity may predispose to the appearance of acute neurological attacks. Seminal studies suggested that AIP was associated with changes in tryptophan homeostasis with inconclusive results. Therefore, the aim of this study was to analyze the urinary metabolome of AIP patients focusing on tryptophan metabolism using state-of-the-art technology. Methods: This was a case-control study including a group of 25 AIP patients with active biochemical disease and increased excretion of heme-precursors and 25 healthy controls. Tryptophan and related compounds and metabolites including: large neutral amino acids (LNAAs), serotonin, kynurenine, kynurenic acid and anthranilic acid were quantified in urine by liquid chromatography tandem-mass spectrometry (LC-MS/MS). Twenty-nine biological markers (including metabolic ratios and absolute concentrations) were compared between patients and controls. Results: Significant differences were found in the tryptophan-kynurenine metabolic pathway. Compared to controls, AIP patients showed: (a) increased urinary excretion of kynurenine and anthranilic acid (P < 0.005); (b): elevation of the kynurenine /tryptophan ratio (P < 0.001) and (c): decrease of the kynurenic acid/kynurenine ratio (P = 0.001). In contrast, no differences were found in the serotonin metabolic pathway independently of the markers and ratios used. Conclusions: The results of the study demonstrate that there is an imbalance in the kynurenine metabolic pathway in AIP patients, with an increase of the kynurenine /tryptophan ratio in urine and a reduction of the kynurenic acid/kynurenine ratio. The modified ratios suggest induction of indoleamine 2,3-deoxygenase and decreased activity of kynurenine aminotransferase in the liver. The results confirm that LC-MS/MS is useful for the characterization of the urinary metabolome of hepatic porphyrias.

LIST OF ABBREVIATIONS AIP: Acute intermittent porphyria

LNAAs: large neutral amino acids LC-MS/MS: liquid chromatography tandem-mass spectrometry PBGD: porphobilinogen deaminase ALA: δ-aminolevulinic acid PBG: porphobilinogen ALAS-1: 5-aminolevulinate synthase Trp: tryptophan TPH: Tryptophan hydroxylase MAO: Monoamine oxidase IDO: Indoleamine 2,3-dioxygenase TDO: Tryptophan 2,3-dioxygenase KAT: Kynurenine aminotransferase KYNU: L-Kynurenine hydrolase KMO: Kynurenine 3-monooxygenase.

LHRH: luteinizing hormone-releasing hormone Val: valine Leu: leucine Ile: isoleucine Phe: phenylalanine Tyr: tyrosine Trp: tryptophan 5-HT: serotonin 5HIAA: 5-hydroxyindoleacetic acid Kyn: kynurenine KA: Kynurenic acid 3OHKyn 3-hydroxykynurenine XA: xanthurenic acid AA: anthranilic acid OHAA: 3-hydroxyanthranilic acid

SsC: Spearman’s correlations

Keywords acute intermittent porphyria, kynurenine, tryptophan, serotonin, metabolomics, mass spectrometry.

1. INTRODUCTION Acute intermittent porphyria (AIP) is a rare disease which is due to a deficiency of porphobilinogen deaminase (PBGD, EC 2.5.1.61), the third enzyme of the heme biosynthetic pathway [1]. Carriers of mutations within the PBGD gene are at risk of presenting acute neurovisceral attacks associated with the overproduction of heme precursors δ-aminolevulinic acid (ALA) and porphobilinogen (PBG) [2]. Acute attacks manifest as a dysfunction of the central, peripheral and autonomous nervous systems. A wide range of psychiatric manifestations are frequently presented [3-5]. Acute attacks occur due to hyperactivity of 5-aminolevulinate synthase (ALAS-1), the first enzyme of the heme biosynthetic pathway that catalyzes the formation of ALA from succinyl-CoA and glycine. ALAS-1 hyperactivity combined with partial PBGD deficiency may induce sudden accumulation of heme-precursors. Hepatic overproduction of exportation of neurotoxic ALA is the most plausible explanation for the acute crisis, however, the etiopathogenesis of most neurological manifestations of AIP remains unknown. Intravenous injection of hemin, which temporarily restores hepatic heme and inhibits ALAS-1 is the most effective treatment to reverse acute clinical symptoms [3-5]. New therapeutic approaches based on silencing overexpression of the ALAS-1 gene are currently under evaluation [6]. In the majority of AIP patients, clinical remission is not associated with a rapid decline of heme-precursors overproduction since the urinary levels of PBG/ALA remain elevated for years [7]. In some patients, the sustained deregulation of the heme-synthesis pathway is associated with recurrent neurological crises, while others may remain free of acute symptoms even while maintaining high PBG/ALA excretion. The sustained hepatic deregulation of hemesynthesis in AIP may be associated with major biochemical abnormalities. However, since AIP is a hepatic disease, the assessment of disturbances occurring in the liver is problematic if only a limited number of metabolites in blood or urine are estimated. In contrast, a comprehensive metabolomic analysis of a large number of analytes may be a useful tool. In a previous study, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to investigate the urinary steroid metabolome in AIP by simultaneously measuring 55 steroid hormones and metabolites [8]. Here, we present a follow-up study covering the metabolism of tryptophan (Trp) and related intermediates. In a seminal study published in 1961, J Price reported that patients with acute forms of porphyria exhibited abnormal metabolism after oral administration of L-Trp [9]. Subsequent studies in rodents and humans observed modifications but failed to provide conclusive results [10-11]. Moreover, it was hypothesized that enhanced plasma levels and brain uptake of Trp,

as an indirect consequence of hepatic heme deficiency was associated with acute attacks [1213]. The aim of this study was to analyze the urinary metabolome of AIP patients with active disease focusing on tryptophan metabolism by comprehensive target analysis of 29 markers using LC-MS/MS in AIP patients compared to healthy controls.

2. MATERIALS AND METHODS 2.1. Patients We studied 25 adult Caucasian Spanish patients with biochemically active AIP (23 women and 2 men, ranging in age from 22 to 54 years. All these patients had initially presented an acute porphyria attack, had been diagnosed with AIP and were regularly attended in the Porphyria Unit of the Hospital Clinic of Barcelona for clinical follow-up. AIP was assessed by biochemical and enzymatic analyses according to European Porphyria Initiative recommendations and external quality assessment schemes [14]. Genetic analysis of the HMBS synthase gene confirmed AIP in all cases. The patients presented a variable clinical condition with some remaining fully asymptomatic while others presented intermittent acute episodes. However, all patients had been acute symptom-free for a minimum of two months at the time of urine collection for this study. On the other hand, chronic complaints such as altered mood states, depression, fatigue or pain in the back were frequently reported. Three patients presenting frequent recurrent attacks were on a prophylactic heme-arginate regime (Normosang@; 3 mg/Kg; every 2-3 weeks). In these latter patients, the urine was collected before the heme-arginate infusions. None of the patients included in the study were receiving luteinizing hormone-releasing hormone (LHRH) agonists. Independently of the clinical status, all the patients presented increased long-term urinary excretion of the heme precursors PBG and ALA. None of the patients presented other diseases in addition to AIP. Liver function assessed by classical serum biomarkers was normal. Renal function was strictly normal in all the cases included, with two AIP patients with renal disease being excluded from the study. All AIP patients reported compliance with the dietetic and life-style recommendations aimed at minimizing the risk of new acute attacks. Twenty-five healthy volunteers (23 women and 2 men; age 25-45 years) were recruited from the laboratory staff and included in the study as controls. They all presented normal renal and liver function. All the patients were informed of the purpose of the study and provided signed consent for the collection of urine samples for a specific metabolomic study related (only) to their porphyria disease. The confidentiality of the databases and the results was explained to the participants by Drs Aguilera and To-Figueras (staff of Hospital Clínic of Barcelona) The project was assessed and approved by the Ethics Committee for Clinical Investigation (CEIC; “Comité

Ético de Investigación Clínica”) of the Hospital Clínic of Barcelona (HCB)-Fundació Clínic per Recerca Biomèdica (FCRB). Second morning urine from all patients and controls was obtained between 0900 h-1000 h in carefully controlled conditions on the hospital premises. Aliquots were immediately protected from light and frozen at -80ºC until analyses.

2.2. PBG and ALA measurements Ion-exchange chromatography using the ALA/PBG column test (Bio-Rad GmbH, Munich, Germany) was used to quantify PBG and ALA. The analysis of creatinine and liver enzymes was done by standard methods using ADVIA 2400 equipment (Siemens Medical Solutions Diagnostics, Tarrytown, NY, USA). PBG and ALA concentrations were normalized to creatinine (mmol/mol of creatinine).

2.3. Standards and chemicals Reference standards of valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), serotonin (5-HT), 5-hydroxyindoleacetic acid (5HIAA), kynurenine (Kyn), Kynurenic acid (KA), 3-hydroxykynurenine (OHKyn), xanthurenic acid (XA), anthranilic acid (AA) and 3-hydroxyanthranilic acid (OHAA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The internal standards were obtained from Toronto Research Chemicals (Toronto, Canada) and Alsachim (Illkirch-Graffenstaden, France). For detailed information see reference (16). Formic acid (LC/MS grade), acetonitrile and methanol (LC gradient grade) were supplied by Merck (Darmstadt, Germany). Ammonium formate (HPLC grade) was purchased from SigmaAldrich (St. Louis, MO, USA). Ultrapure water was provided using a Milli-Q purification system (Millipore Ibérica, Barcelona, Spain).

2.4. Quantification of urinary markers Urinary markers were quantified using a method reported previously [15]. The LC-MS/MS system used to perform the analysis was an Acquity UPLC system (Waters Associates, Milford, MA, USA) coupled to a Quattro Premier triple quadrupole mass spectrometer (Waters Associates) equipped with an electrospray ionization interface. The chromatographic separation was carried out in an Acquity BEH C18 column (100 mm x 2.1 mm i.d., 1.7 µm) (Waters Associates) at a flow rate of 300 µL min-1. Water and methanol both with ammonium formate (1 mM) and formic acid (0.01 % v/v) were used as mobile phase solvents. A gradient elution was used for the chromatographic separation of the analytes. The percentage of the organic solvent linearly changed as follows: 0 min, 1%; 0.5 min, 1%; 7 min, 40%; 8.5 min, 90%; 9 min, 90%; 9.5 min, 1%; 12 min, 1%. The SRM method (including 2 ion transitions for each analyte) is described elsewhere [15]. Variability due to urine dilution was normalized measuring the urine-specific gravity of all the samples using a UG-α urine-specific gravity refractometer (Atago, Japan). Urine-specific gravity

of 1.020 allowed correction of the concentrations of the urine samples using the following equation: C1.020

=

CSAMPLE

x

(1.020

–

1)/(Specific

gravity

–

1)

2.5. Statistical analysis The data were analyzed the SPSS software (v 18.0; IBM, Armonk, New York, NY, USA. Due to the relatively low number of data analyzed, a non-parametric distribution was assumed, and the statistical analysis was carried out by non-parametric tests. The Mann-Whitney U-test was performed to compare the differences in concentrations and ratios between cases and controls. The Spearman’s correlation with a two-tailed test was performed to assess the correlations among heme precursors and the markers analyzed. Statistically significant differences were established at a p ≤ 0.05.

3. RESULTS 3.1. Urinary determination of the selected markers All the AIP cases included in the study presented increased urinary heme precursor levels. ALA ranged from 3 to 40 nmol mmol-1 creatinine (≈1 to 8-fold the upper normal limit in the healthy population, 5 nmol mmol-1 creatinine). PBG ranged from 5 to 68 nmol mmol-1 creatinine (from 6 to 85-fold the upper normal limit in the healthy population, 0.8 nmol mmol-1 creatinine). The target LC-MS/MS method included 14 analytes related to Trp (Figure 1). The chromatographic method provides the required resolution for the proper determination of some pairs of analytes, thereby allowing separation between the isobaric LNAA Leu and Ile. Additionally, this method also separates the XA from the isobaric interference caused by the isotopic contribution of the abundant Trp (Figure 2). All the analytes were correctly detected in all samples with the exception of OHKyn and OHAA which were detected in 90 % and 72 % of the samples, respectively. For statistical purposes, their concentration was assumed to be half of the limit of detection in those samples in which they were undetectable. Based on their biological origin, the analytes were divided into three different categories (see Figure 1): (a): large neutral amino acids (LNAA) competitors of Trp; (b): metabolites belonging to the serotonin pathway and (c): metabolites from the kynurenine pathway. Fifteen analytical ratios were also calculated and included as markers since they provide information about enzymatic activity. 3.2. LNAAs excretion The urinary concentrations of 6 LNAA were calculated. Table 1 shows the main results for controls and AIP patients. Control values were within the expected range for the healthy population (20). A significant decrease in Val, Leu and Ile concentrations was found in AIP patients. On the contrary, no differences were found for Trp, Phe and Tyr. The relative abundances of the different LNAAs were obtained from the quotient between one LNAA and the sum of the rest of the LNAAs showing significant differences for most of them (see Table 1 and Figure 2). Similar to the results obtained for the individual concentrations of

the LNAAs, AIP patients showed lower relative Val, Leu and Ile excretion. Additionally, a significant increase was also observed in the relative excretion of Trp and Phe. Only the relative excretion of Tyr was found to have a p > 0.05, showing a trend to a decrease of Tyr in AIP patients (p = 0.076).

3.3. Serotonin pathway. Five markers belonging to the serotonin pathway were included in the present study, including the urinary concentrations of 5-HT and 5HIAA. Additionally, three ratios were also evaluated. The values of 5-HT/Trp and 5HIAA/5-HT were used to estimate the activity of the TPH and MAO enzymes, respectively. On the other hand, 5HIAA/Trp provided information about the whole serotonin enzymatic pathway. Table 2 shows the results related to the serotonin pathway. Similar concentrations were found for 5-HT and 5HIAA in both controls and AIP cases. No differences were found in the evaluation of either TPH or MAO. In addition, similar values were obtained in the evaluation of the whole metabolic pathway.

3.4. Kynurenine pathway. Fourteen markers belonging to the kynurenine pathway were evaluated. The urinary concentrations of Kyn and 6 metabolites were obtained. Additionally, 5 ratios were studied providing an estimation of the activity of relevant enzymes. Kyn/Trp provides information about IDO/TDO enzymes, KA/Kyn and XA/OHKyn about KAT, OHKyn/Kyn about KMO and AA/Kyn and OHAA/OHKyn about KYNU (Table 3). Several steps of the Kyn pathway were found to be altered in AIP patients. Thus, AIP patients showed significantly higher urinary concentrations of Kyn and AA, with the mean concentrations of both compounds being around 2-fold higher in AIP patients. No significant differences were found between the two groups with respect to the remaining metabolites (KA, OHKyn, XA and OHAA). Figure 3 shows the chromatograms obtained for Kyn and KA in cases and controls.

The study of the selected ratios of the Kyn pathway showed significant differences between cases and controls (Table 3). The 2-fold increase of the Kyn/Trp ratio (P < 0.001) in the AIP group was similar to that observed for urinary Kyn. Moreover, significant differences were observed in the step that converts Kyn into KA towards the action of the KAT enzyme. KA/Kyn (P: 0.001) values were around 2-fold lower in AIP patients than in controls (Figure 4). Remarkably, despite the clear increase in Kyn concentrations, no statistical differences were found in other ratios related to Kyn degradation (OHKyn/Kyn, AA/Kyn and XA/OHKyn).

3.5. Correlations of markers versus heme precursors.

Spearman’s correlations (SsC) between the selected markers and heme precursors (ALA and PBG) were performed in AIP patients. Regarding LNAA, the Spearman correlation study showed a positive correlation between Trp/LNAAs with PBG (0.528, p: 0.007) and ALA (0.429, p: 0.033), whereas negative correlations were found between Tyr/LNAAs and both PBG (-0.605, p: 0.001) and ALA (-0,443, p: 0.027). Despite the alterations observed in the excretion of LNAAs, there were no further significant correlations between this group of analytes and heme precursors. In the case of the 5-HT pathway, none of the markers studied showed a correlation with heme precursors. Finally, although no correlation was found between Kyn and heme precursors, some of the steps belonging to the Kyn pathway presented significant correlations with ALA. That was the case of AA and the OHAA/AA ratio. AA presented a positive correlation with ALA (SsC 0.447, p: 0.025) while OHAA/AA had a negative correlation (SsC -0.516, p: 0.012). Kyn/Trp showed a tendency to a negative correlation with ALA (SsC -0.392, p: 0.052). No correlation was found between markers of the Kyn pathway and PBG.

4. DISCUSSION The LC-MS/MS metabolomic study allowed comprehensive estimation of three Trp-related pathways: (a) balance of LNAAs; (b): Trp metabolism via the sertotonin pathway and (c): Trp metabolism via the kynurenine pathway. The determination of a large number of analytes represents a substantial advantage over studies focused on a limited number of metabolites [9-13]. Therefore, despite the pioneer finding of abnormal Trp metabolism in AIP patients, the analysis of the metabolic ratios in the present study allowed the elucidation of the specific enzymatic deficiencies involved. An imbalance was observed in the urinary LNAAs in AIP patients. Thus, whereas relative concentrations of branched-chain amino acids (Leu, Ile and Val) were found to be lower in AIP patients, Trp/LNAAs and Phe/LNAAs (both aromatic amino acids) showed higher values in AIP patients than in controls. Even if dietary and nutritional habits of AIP patients could account for some of these urinary changes, a follow-up study in plasma is required to fully characterize these observations. The analysis of the 5-HT pathway did not reveal significant changes in either urinary concentrations (5-HT and 5HIAA) or the selected ratios (5-HT/Trp, 5HIAA/5-HT and 5HIAA/Trp), suggesting that both TPH and MAO activities were not altered in our samples. Thus, our results do not confirm any impairment of the serotoninergic route of Trp metabolism, as had been reported in rodent models of chemically-induced porphyria [16, 17]. However, we did not study samples of patients who attended the emergency room with clinical symptoms of an acute neurological attack, therefore we cannot discard a different imbalance in this clinical situation. On the other hand, we detected significant alterations of the Kyn pathway with increased urinary concentrations of Kyn and its metabolite AA. This increased concentration of urinary

Kyn would confirm the results of the seminal study which described an abnormal increase of urinary Kyn in AIP patients after a Trp-loading test [9]. However, this test was inconclusive since Trp loading itself could, in some conditions, induce TDO activity, and therefore, increase the Kyn levels. In contrast, our results using state-of-the art LC-MS/MS instruments of high sensitivity unambiguously show that the basal urinary concentration of Kyn is increased in AIP patients. Strikingly, the increase of Kyn concentration was not associated with an increase of either its precursor (Trp) or its main metabolite (KA). Thus, AIP patients showed an increase in the Kyn/Trp ratio and a decrease in the KA/Kyn ratio, suggesting either a deficiency in the enzymatic step that leads to the formation of KA from Kyn or an induction of the conversion of Trp to Kyn. An excess of Kyn could originated from an overproduction from Trp. The conversion Trp  Kyn is catalysed by indoleamine 2,3-dioxygenase (IDO, formerly known as tryptophan pyrrolase) and tryptophan 2,3-dioxygenase (TDO) (Figure 1). Although both enzymes catalyse the same reaction, they show a number of differences. TDO is mainly expressed in the liver and is induced by tryptophan while IDO is ubiquitously expressed and regulated by complex immunological signals [18]. Both IDO and TDO are heme-proteins and it has been hypothesized that heme-deficiency in the liver of AIP patients decreases TDO activity, thus enhancing plasma levels and subsequent brain uptake of Trp. In turn, this increases 5-HT synthesis within the brain, thus contributing to acute neurological/psychiatric manifestations [19]. However, our results suggest an activation of IDO rather than decreased activity, an effect that was already anticipated in rodent models of porphyria [20]. IDO is induced by hepatic oxidative stress and pro-inflammatory cytokines to the point that a Kyn/Trp ratio in used as a plasma biomarker of inflammation in different diseases [21]. There are no conclusive data, regarding oxidative stress and the hepatic pro-inflammatory status of AIP patients, but a recent study using plasma biomarkers reported low-grade systemic inflammation in AIP [22]. On the other hand, an increase in Kyn might be explained by a decrease in Kyn metabolism. Kyn can be transformed into KA (via KAT), OHKyn (via KMO) and AA (via KYNU; Figure 1). Our results indicate that neither KMO nor KYNU are modified in AIP patients. On the other hand, according to our results the conversion the main pathway Kyn  KA is significantly modified, and this could also explain Kyn hepatic accumulation and increased urinary excretion. This step is catalyzed by KAT (Figure 1) that uses pyridoxal 5’-phosphate (PLP; vitamin B6) as a co-factor. ALAS-1 also uses PLP as a cofactor, thus ALAS-1 hyperactivity in AIP could decrease hepatic PLP stores and therefore reduce KAT activity. There are no definitive studies addressing vitamin B6 status in this kind of patients, despite seminal experiments reporting that AIP was associated with low values of plasma PLP [23]. A recent study reported that the hydroxykynurenine/xanthurenic acid ratio (HK/XA, a functional marker of PLP) was increased in a group of AIP patients, thus suggesting impaired vitamin B6 status [24].

In conclusion, a convergence of factors (low-grade inflammation/oxidative stress due to overproduction of PBG/ALA and low PLP status due to ALAS-1 hyperactivity) may account for the anomalies in the Kyn pathway levels reported in the present study.,The clinical consequences of this metabolic disturbance need to be further evaluated. Up-regulation of the tryptophan-kynurenine pathway is associated with depression and other psychiatric diseases [25]. Therefore abnormal Trp metabolism may contribute to some of the common clinical features found in AIP patients. However, further studies including follow-up analysis in plasma and the inclusion of patients with acute porphyria symptoms are needed.

Conflicts of interest None

Funding This work was supported by a grant from Instituto de Salud Carlos III FEDER (PI14/00147). Support from the Generalitat de Catalunya (2014SGR692 to the research team) is also acknowledged. Spanish Health National System is acknowledged for O. J. Pozo contract (MS10/00576).

Authors' Contributions AGG carried out the LC-MS/MS analysis, the statistical analysis and drafted the manuscript. JM participated in the analysis of the data and helped in the interpretation of the results. PA collected the samples and performed the analysis of the heme precursors. JTF and OP conceived the study and participated in its design and coordination and helped to draft the manuscript. All the authors have read and approved the final manuscript.

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FIGURE LEGENDS

Figure 1. Tryptophan metabolism. TPH: Tryptophan hydroxylase, MAO: Monoamine oxidase, IDO: Indoleamine 2,3-dioxygenase, TDO: Tryptophan 2,3-dioxygenase, KAT: Kynurenine aminotransferase, KYNU: L-Kynurenine hydrolase and KMO: Kynurenine 3-monooxygenase. Color code: Red: decreased in AIP patients, Green: unaltered in AIP patients. Yellow: Increased in AIP patients. Figure 2. Total ion current chromatograms for a solvent standard (containing 50 ng/mL of 5HT, OHKyn, AA and OHAA; 500 ng/mL of Val, Leu, Kyn and XA; 2500 ng/mL of Tyr Ile, Trp, 5HIAA and Phe; and 10000 ng/mL of KA) showing the chromatographic separation achieved by the analytes from the selected metabolic pathways. Figure 3. Relative urinary excretion of each LNAA compared to the remaining LNAAs. Figure 4. LC-MS/MS chromatograms of Trp, Kyn and KA for (a) a control subject and (b) an AIP patient. Boxplots showing the differences between the (c) Kyn/Trp and (d) KA/Kyn ratios.

Figure 1.

Figure 2

1 0.9

**

LNAAs ratios excretion Control

0.8

AIP

0.7

Ratios

0.6

**

0.5 0.4 0.3

**

0.2

*

0.1

**

0 Trp/LNAA

Figure 3

Tyr/LNAA

Phe/LNAA

Val/LNAA

Leu/LNAA

Ile/LNAA

3.45 171662

100

%

%

100

Trp

0 3.40

3.60

0

Time 2.50

3.00

4.01 49367

100

%

0

KA

Time 2.00

2.50

3.00

4.01 49668

%

2.00

3.60

%

%

2.57 538

3.40 2.57 1183

100

0

4.00

0

Time 4.50

4.00

(a)

(b)

(c)

(d)

Figure 4

Time 3.20

100

KYN

0

Time 3.20

100

3.45 173511

Time 4.50

TABLES Table 1. Median corrected LNAA concentrations (in ng/mL) (Q1; Q3) and p values. Median LNAAs ratios (Q1; Q3) and p values. Corrected concentrations (ng/mL) Median (Q1; Q3) Control Val 3354 (2879; 4167) Leu 4412 (3789; 5007) Ile 1594 (1443; 1981) Trp 13184 (10001; 16104) Phe 6273 (5149; 7703) Tyr 11843 (9578; 15626) Ratios Median (Q1; Q3) Control Trp/LNAA 0.45 (0.39; 0.53) Tyr/LNAA 0.43 (0.35; 0.49) Phe/LNAA 0.30 (0.26; 0.32) Val/LNAA 0.09 (0.07; 0.11) Leu/LNAA 0.11 (0.10; 0.12) Ile/LNAA 0.041 (0.036; 0.046)

AIP 2421 (2058; 3302) 2856 (1719; 4111) 1155 (862; 1506) 13602 (9118; 21512) 5913 (4941; 8209) 10871 (6785; 14780)

AIP 0.64 (0.43; 0.80) 0.39 (0.9; 0.43) 0.37 (0.31; 0.40) 0.07 (0.06; 0.09) 0.08 (0.06; 0.11) 0.031 (0.026; 0.039)

p value 0.001** 0.001** 0.001** 0.503 0.793 0.233

p value 0.002** 0.076 <0.001** 0.017* <0.001** 0.006**

Table 2. Serotonin pathway concentrations (in ng/mL) and ratios (Q1; Q3) and p values. Corrected concentrations (ng/mL) Median (Q1; Q3) Control AIP 5-HT 125 (98; 160) 150 (94; 227) 5HIAA 4000 (3297; 5629) 5014 (3612; 7601) Ratios Enzyme involved Median (Q1; Q3) TPH 5-HT/Trp 5HIAA/5-HT MAO 5HIAA/Trp -

Control 0.009 (0.007; 0.013) 31 (28; 38) 0.31 (0.21; 0.45)

p value 0.337 0.240

p value AIP 0.011 (0.007; 0.012) 0.839 37 (27; 43) 0.443 0.41 (0.22; 0.69) 0.367

Table 3. Kynurenine pathway concentrations (in ng/mL) and ratios (Q1; Q3) and p values. Corrected concentrations (ng/mL)

Trp Kyn KA OHKyn XA AA OHAA Ratios

Median (Q1; Q3) Control 13184 (10001; 16104) 641 (269; 858) 3605 (3188; 4543) 181 (57; 256) 824 (651; 1169) 42 (30; 61) 281 (212; 1041) Enzyme involved

Kyn/Trp KA/Kyn OHKyn/Kyn AA/Kyn OHAA/AA XA/OHKyn

IDO/TDO KAT KMO KYNU KAT

AIP 13602 (9118; 21512) 1041 (509; 2128) 4407 (3001; 5425) 250 (51; 778) 762 (476; 1040) 109 (79; 168) 285 (147; 1064) Median (Q1; Q3) Control 0.04 (0.03; 0.06) 6.2 (4.5; 13.4) 0.29 (0.16; 0.36) 0.089 (0.040; 0.164) 6.0 (2.8; 29.3) 4.9 (3.2; 10.8)

p value 0.503 0.004** 0.443 0.270 0.327 <0.001** 0.792

AIP 0.09 (0.06; 0.11) 3.9 (2.6; 5.9) 0.20 (0.11; 0.38) 0.11 (0.04; 0.21) 4.0 (1.4; 11.8) 3.9 (1.1; 12.8)

p value <0.001** 0.001** 0.327 0.528 0.121 0.190