C LI NI CA L RE SE AR CH A RT IC LE Raquel G. Martins,1,2,3* Luı́s G. Gonçalves,4* Nuno Cunha,5 and Maria Jo~ ao Bugalho6,7 1 Endocrinology Department, Portuguese Oncology Institute of Coimbra, 3000-075 Coimbra, Portugal; Medical Psychology Unit, Department of Clinical Neurosciences and Mental Health, Faculty of Medicine, University of Oporto, 4200-072 Porto, Portugal; 3Research Centre, Portuguese Oncology Institute of Oporto, 4200-072 Porto, Portugal; 4ITQB NOVA, Instituto de Tecnologia Quı́mica e Biológica António Xavier, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal; 5Clinical Laboratory Department, Portuguese Oncology Institute of Coimbra, 3000-075 Coimbra, Portugal; 6Endocrinology, Diabetes and Metabolism Department, CHULN-Hospital Santa Maria, 1649-035 Lisbon, Portugal; and 7Faculty of Medicine, University of Lisbon, 1649-004 Lisbon, Portugal 2 ORCiD numbers: 0000-0003-2786-3650 (R. G. Martins). Context: Metabolomic studies of pheochromocytoma and paraganglioma tissue showed a correlation between metabolomic profile and presence of SDHx mutations, especially a pronounced increase of succinate. Objective: To compare the metabolomic profile of 24-hour urine samples of SDHx mutation carriers with tumors (affected mutation carriers), without tumors (asymptomatic mutation carriers), and patients with sporadic pheochromocytomas and paragangliomas. Methods: Proton nuclear magnetic resonance spectroscopic profiling of urine samples and metabolomic analysis using pairwise comparisons were complemented by metabolite set enrichment analysis to identify meaningful patterns. Results: The urine of the affected SDHx carriers showed substantially lower levels of seven metabolites than the urine of asymptomatic mutation carriers (including, succinate and N-acetylaspartate). The urine of patients with SDHx-associated tumors presented substantially higher levels of three metabolites compared with the urine of patients without mutation; the metabolite set enrichment analysis identified gluconeogenesis, pyruvate, and aspartate metabolism as the pathways that most probably explained the differences found. N-acetylaspartate was the only metabolite the urinary levels of which were significantly different between the three groups. Conclusions: The metabolomic urine profile of the SDHx mutation carriers with tumors is different from that of asymptomatic carriers and from that of patients with sporadic neoplasms. Differences are likely to reflect the altered mitochondria energy production and pseudohypoxia signature of these tumors. The urinary levels of N-acetylaspartate and succinate contrast with those reported in tumor tissue, suggesting a defective washout process of oncometabolites in association with tumorigenesis. The role of N-acetylaspartate as a tumor marker for these tumors merits further investigation. (J Clin Endocrinol Metab 104: 5467–5477, 2019) ISSN Print 0021-972X ISSN Online 1945-7197 Printed in USA Copyright © 2019 Endocrine Society Received 11 May 2019. Accepted 17 July 2019. First Published Online 23 July 2019 doi: 10.1210/jc.2019-01101 *R.G.M. and L.G.G. are co-first authors. Abbreviations: CoA, coenzyme A; GIST, gastrointestinal stromal tumors; 1H, proton; MSEA, metabolite set enrichment analysis; NMR, nuclear magnetic resonance; PPGL, pheochromocytomas and paragangliomas. J Clin Endocrinol Metab, November 2019, 104(11):5467–5477 https://academic.oup.com/jcem 5467 Downloaded from https://academic.oup.com/jcem/article-abstract/104/11/5467/5536622 by Endocrine Society Member Access 3 user on 17 October 2019 Metabolomic Urine Profile: Searching for New Biomarkers of SDHx-Associated Pheochromocytomas and Paragangliomas 5468 Martins et al SDHx-Associated Tumors: Urine Metabolomics T substrate and the metabolite of succinate dehydrogenase. In vivo metabolomic analysis of SDHx-related tumors using proton (1H) magnetic resonance spectroscopy was also able to identify a succinate peak in the majority of SDHx mutation carriers (17, 18). Metabolomic analysis of random blood and/or urine samples showed elevated plasma succinate in one SDHB mutation carrier and in two of five SDHD mutation carriers. Increased urine succinate was observed in fewer participants, but it is not clear whether these individuals were SDHx mutation carriers (19). A role for succinate as serum biomarker in SDHx-mutated PPGL individuals was observed in another study (20). The metabolomic profile of urine samples from patients with PPGL was not performed. However, it has shown clinical utility in the identification of new biomarkers in other pathologies (21, 22). The study of biofluids using high resolution 1H nuclear magnetic resonance (NMR) spectroscopic profiling provides a global quantitative description of a wide number of endogenous metabolites (23). Because it enables a global evaluation of the intermediaries of several metabolic pathways, it might allow the highlight of those altered in a given pathological condition. Describing the metabolomic profile of SDHx patients’ biofluids might be helpful in the identification of targets for the future development of new biomarkers for PPGL. To this end, this study aims to evaluate the metabolomic profile of patients with PPGL and SDHx mutations in 24-hour urine samples, in comparison with unaffected SDHx mutation carriers and with patients with sporadic PPGL. Materials and Methods Study population The study was conducted in the three Portuguese oncology institutes (Coimbra, Lisbon, and Oporto) that follow patients with neoplasias nationwide. The sample included 76 patients: 68 SDHx mutation carriers (55 SDHB, 12 SDHD, and 1 SDHC) and 8 individuals SDHx wild-type. Only carriers of known pathogenic mutations (identified through Sanger sequencing or next generation sequencing, and multiplex ligationdependent probe amplification for deletion detection) were included. Coexistence of polymorphisms cannot be excluded. Individuals with SDHx variants of unknown significance were not eligible. Patients with questionable imaging examinations were also excluded from the analysis. Among the SDHx mutation carriers, 26 presented associated tumors (25 PPGL and 1 GIST) at the time of the 24-hour urine collection, 9 of whom had metastatic disease. The remaining SDHx mutation carriers were unaffected or were previously treated for PPGL or GIST but remained without evidence of disease at the time of urine collection; they were classified as asymptomatic mutation carriers. Among the individuals without mutation, all presented PPGL at the time of urine collection (Table 1). Participation was voluntary and data anonymity was ensured. The study received approval from the ethics committees of all three hospitals. All patients gave written informed consent. Downloaded from https://academic.oup.com/jcem/article-abstract/104/11/5467/5536622 by Endocrine Society Member Access 3 user on 17 October 2019 he pheochromocytomas and paragangliomas (PPGL) are neuroendocrine tumors with origin in the sympathetic and parasympathetic paraganglia from the autonomic nervous system. At least 40% of these rare neoplasias are associated with germline mutations (1) and this is considered as the tumor for which the contribution of genetics is the strongest (2). Among the described mutations, those of the genes coding for succinate dehydrogenase are the most frequent (3). They are broadly called SDHx mutations. Succinate dehydrogenase is a hetero-tetrameric mitochondrial enzyme complex that plays a pivotal role in cellular metabolism. It is involved in the citric acid cycle and in the mitochondrial electron transportation chain from the oxidative phosphorylation as complex II (4). Since 2000, several mutations in the genes that encode the four subunits (SDHD, SDHB, SDHC, and SDHA) or the factor needed for their activation (SDHAF2) have been described in association with the development of PPGL (5–10). The mutation penetrance is not complete and varies according to the identified mutation (11). Thus, a variable number of the mutation carriers develop the associated illness, which can include pheochromocytomas or paragangliomas in the head and neck, thorax, abdomen or pelvis, and less frequently, gastrointestinal stromal tumors (GIST), renal cell carcinomas, and pituitary adenomas. Medical complications arising from these neoplasias derive from the effect of tumor mass and/or the hypersecretion of catecholamines by pheochromocytomas or functioning paragangliomas. A presymptomatic screening of the patients’ relatives who are also carriers of the SDHx mutation is recommended with the expectation of establishing an early diagnosis of the disease, which in turn, improves prognosis. Currently, the follow-up of asymptomatic carriers is based on the measurement of catecholamine metabolites (i.e., metanephrines) in blood or in 24-hour urine samples in addition to serial imaging examinations. However, the analytical studies do not allow the diagnosis of nonfunctioning tumors and imaging studies are time-consuming, relatively expensive (because of the need for cervical, thoracic, abdominal, and pelvic examinations), and lack accuracy for small tumors. These difficulties indicate the need to find new tumor markers that enable early diagnosis. Previous metabolomic studies conducted in PPGL tumor tissue showed a correlation between metabolomic profile and the presence of SDHx mutations (12–16). The profile includes mainly higher succinate and lower fumarate levels in PPGL associated with SDHx mutations, but also differences in other metabolites such as cisaconitate and isocitrate. The succinate and fumarate that are intermediates in the citric acid cycle are, respectively, the J Clin Endocrinol Metab, November 2019, 104(11):5467–5477 doi: 10.1210/jc.2019-01101 Table 1. https://academic.oup.com/jcem 5469 Participants’ Characteristics Variables SDHx Wild-Type (N 5 8) 41.6 6 15.9 61.3 6 12.7 34 (50.0) 34 (50.0) 3 (37.5) 5 (62.5) Age (y), mean 6 SD Sex, n (%) Female Male Type of mutation, n (%) SDHB SDHC SDHD State at time of urine collection, n (%) Unaffected With nonmetastatic tumor With metastatic tumor Type of tumor’s hormonal secretion, n (%) Nonfunctioning Adrenergic Noradrenergic Dopaminergic Unknown Metanephrines measurement Participants were instructed to fast for at least 8 hours, follow a diet low in catecholamine-containing products, and cease interfering medicines on the days before the blood collection. The blood samples were collected in the various hospitals after at least 30 minutes of supine rest, through a venipuncture technique into K3-EDTA and serum-separating tubes. All samples were collected and placed on ice, immediately centrifuged, aliquoted, and stored at 280°C before analyses. At the central laboratory, an online solid-phase extraction was performed using the fully automated Symbiosis™ system (Spark Holland, Emmen, Netherlands), coupled with high-performance liquid chromatography tandem quadrupole mass spectrometric detection (XLC-MS/MS, Waters XEVO TQ-MS, Waters Corp., Milford, MA), to measure metanephrine, normetanephrine, and 3-methoxytiramine in plasma samples. Tumors were classified as functioning if amines excretion was at least 1.5 times the upper limit of the normal range. NMR acquisition Participants were instructed how to collect a 24-hour urine sample properly. After reception, samples were locally frozen at a temperature of 280°C. Before NMR analysis, the urine samples were thawed at room temperature and then centrifuged for 10 minutes at 12 000g. Then, 60 mL of a 1.5 M potassium dihydrogen phosphate buffer (pH 7.4) in deuterated water with 2.9 mM 3-(trimethylsilyl)propionic-2,2,3,3-d4 and 0.05% sodium azide were added to 540 mL of urine and transferred to 5 mm NMR tubes. The spectra were acquired on a Bruker Avance II1 800 MHz (Bruker Biospin, Wissembourg, France) spectrometer equipped with a 5 mm TXI-Z H/C/N/-D probe (Bruker Biospin). All 1D 1H were acquired at 300 K using a noesygppr1d pulse program (128 scans; relaxation delay of 4 s; mixing time of 10 ms; spectral width of 16025.641 Hz; size of free induction decay was 128k points). For all the samples a JH,H-resolved (2 scans, 100 points in F1 and 8192 points in F2, relaxation delay of 2 s, spectral width of 78.113 Hz in F1 and 13368.984 in F2) was acquired to aid compound identification. 55 (80.9) 1 (1.5) 12 (17.6) 42 (61.8) 17 (25.0) 9 (13.2) 0 8 (100) 0 11 (42.3) 0 (0) 7 (26.9) 7 (26.9) 1 (3.8) 4 (50.0) 3 (37.5) 1 (12.5) 0 0 The spectra acquisition and processing was performed with Bruker TopSpin 3.2 (Bruker Biospin). All free induction decays were multiplied by an exponential function, followed by Fourier transformation. Spectra were manually phased, and baseline corrected. Chemical shifts were adjusted according to the chemical shift of trimethylsilylpropanoic acid at 0.00 ppm that was used as concentration reference. The metabolites present in the spectra were identified and quantified recurring to Chenomx NMR suite 8.12 (Chenomx, Edmonton, Alberta, Canada). All the metabolites were confirmed through recourse to two-dimensional NMR spectra. Samples that revealed the presence of blood or infection were discarded from the analysis. Statistical analysis In the metabolomic analysis, 58 metabolites that were present in most of the samples were considered. The metabolite concentrations that exceed 4 times the interquartile range were discarded from the analysis. Pairwise comparisons were conducted using the Wilcoxon rank-sum test to look for significant differences (P , 0.05) between the groups of samples. Correction for multiple testing was performed using the BenjaminiHochberg procedure. Metabolite set enrichment analysis The metabolite set enrichment analysis (MSEA) was performed to identify biologically meaningful patterns in the metabolite levels present in urine (www.metaboanalyst.ca) (24, 25). Urine metabolite concentrations was used as input data and the enrichment analysis was performed in the metabolic pathway associated set library. Results In the urine 1H-NMR spectra it was possible to identify and quantify 58 metabolites that were present in most of the samples. Because urine is a complex biofluid that is composed by the excreted metabolites of the body, many Downloaded from https://academic.oup.com/jcem/article-abstract/104/11/5467/5536622 by Endocrine Society Member Access 3 user on 17 October 2019 SDHx Mutation Carriers (N 5 68) Mean (mM) 1359.94 360.7 180.49 178.1 158.44 99.31 86.03 80.35 50.45 48.29 46.22 44.74 33.83 32.91 27.11 26.3 25.62 24.02 24.01 23.04 21.24 18.16 17.26 15.35 15.23 14.47 14.41 14.12 12.61 11.49 10.21 10.17 10.15 10.11 9.15 8.55 8.45 7.91 7.85 Creatinine Citrate Glycine Hippurate Taurine Creatine Trimethylamine N-oxide N-Phenylacetylglycine Acetate Dimethylamine Malonate Formate Alanine 3-Indoxylsulfate Glucose Glucuronate Xylose cis-Aconitate Lactate Threonine Galactose Allantoin 4-Hydroxyphenylacetate Fucose O-Phosphocholine Methanol Betaine Tyrosine 3-Hydroxyisobutyrate Xanthine 3-Aminoisobutyrate Trigonelline Sucrose 1,6-Anhydro-b-D-glucose Succinate Imidazole 2-Furoylglycine Dimethyl sulfone Tartrate 1214.4 324.6 119 154.4 147 65 54.8 66.4 9.6 45.5 39.9 36.6 30.2 30.1 25.2 23.5 23.3 23.9 18.9 18 17 16.6 13.5 14 11.9 13.7 11.7 12.3 10.6 7.8 7.4 6.6 7 5.2 6.7 6.7 7.4 8.2 3 Median (mM) 708.68 234.33 155.41 131.09 107.97 103.26 104.74 54.71 79.7 20.27 25.35 41.2 19.98 18.31 13.22 18.3 15.76 10.04 17.51 19.29 17.11 10.31 12.14 7.86 13.33 10.6 10.59 9.43 6.39 10.99 8.57 9.8 9.9 12.02 7.7 6.75 6.71 3.24 8.83 SD (mM) 1068.22 250.08 121.75 163.73 116.3 99.7 69.1 74.7 32.77 45.25 45.34 36.94 26.43 26.57 27.9 23.37 24.5 20.75 26.6 15.47 22.08 17.18 14.38 13.98 11.13 10.25 11.24 10.01 11.44 5.35 10.83 8.43 11.37 7.28 5.93 7.78 4.94 6.44 5.59 Mean (mM) 942 206.9 112.6 113.5 108.8 19.6 45.2 57.6 6 38.8 40.2 32.4 23.4 22.8 25.2 19.6 19.2 20 16.8 12.8 20.5 15.5 14.5 10.8 7.4 5.1 10.4 10 8.9 4.5 6.2 8 6.4 4.2 3.4 6.2 4.4 6 2.8 Median (mM) SD (mM) 519.31 160.5 70.31 148.56 63.21 145.85 60.16 49.29 57.7 34.74 26.9 23.77 14.96 12.93 12.95 13.48 21.23 8.99 29.63 10.9 11.53 9.47 7.51 9.8 10.36 14.27 6.46 6.04 8.44 2.85 10.91 5.3 9.46 9.62 6.15 6.44 3.19 3.32 6.46 SDHx Tumors (T) 1359.94 360.7 180.49 178.1 158.44 99.31 86.03 80.35 50.45 48.29 46.22 44.74 33.83 32.91 27.11 26.3 25.62 24.02 24.01 23.04 21.24 18.16 17.26 15.35 15.23 14.47 14.41 14.12 12.61 11.49 10.21 10.17 10.15 10.11 9.15 8.55 8.45 7.91 7.85 Mean (mM) 660.8 156.2 84.3 130.1 91.7 25.9 49.8 90.7 10.5 29.9 29.5 28 23.5 25.1 16.6 18.6 33.6 15.3 7.1 10 13 15.6 10.9 9.1 7.6 5.2 11.1 8.4 6 5 7.8 8.3 4.6 5.1 2.8 2.9 2.6 5 5.2 Median (mM) SD (mM) 314.86 100.47 45.77 76.31 201.85 145.46 193.03 32.95 53.73 24.02 20.31 21.68 11.01 13.97 13.66 14.62 22.46 5.45 6.53 9.55 9.88 6.16 4.07 6.25 10.37 10.75 13.84 3.87 4.44 1.78 5.68 8.37 8.6 5.41 3.78 3.21 3.38 2.56 11.57 Sporadic PPGL (S) 0.027 0.023 0.008 0.034 0.014 0.022 0.005 0.009 0.008 0.011 A vs S P Value (Continued) 0.044 T vs S P Value SDHx-Associated Tumors: Urine Metabolomics 0.020 0.021 A vs T P Value Martins et al Downloaded from https://academic.oup.com/jcem/article-abstract/104/11/5467/5536622 by Endocrine Society Member Access 3 user on 17 October 2019 Metabolite SDHx Asymptomatics (A) Table 2. Comprehensive List of the 58 Quantified Metabolite Concentrations for the SDHx Mutation Carriers Without Evidence of Disease (SDHx Carriers), the SDHx Mutation Carriers With Related Tumors (SDHx Tumors), and the Patients With Sporadic PPGL (Sporadic PPGL) 5470 J Clin Endocrinol Metab, November 2019, 104(11):5467–5477 5 5.6 6 6.2 5.3 5.9 3 5.5 5.6 3.8 4.9 4 4 3.9 3.6 3 2.8 1.6 1 0.5 4.79 4.79 4.28 3.53 3.17 2.06 1.47 0.7 Median (mM) 7.54 7.35 6.91 6.87 6.75 6.66 6.5 6.26 6.01 5.61 5.53 4.84 Mean (mM) 2.69 3.14 3.06 2.38 1.37 1.16 1.38 0.57 7.2 4.41 4.25 3.72 5.99 3.45 6.7 3.07 3.09 4.14 3.2 2.37 SD (mM) 3.8 3.85 3.99 3.05 2.52 2.21 1.21 0.74 4.7 8.6 4.87 7.39 5.8 5.99 6.44 5.32 5.63 4.75 4.7 5.43 Mean (mM) 2.9 3.4 2.1 2.7 2 1.6 0.9 0.6 3.6 7.8 4 6.7 3.6 5.7 3.4 3.6 4.2 3.6 3.6 4.3 Median (mM) SDHx Tumors (T) 2.44 1.79 3.92 1.89 1.31 1.78 0.85 0.5 3.71 6.07 2.92 4.63 6.43 2.95 6.83 4.14 4.04 4.11 3.26 3.55 SD (mM) 4.79 4.79 4.28 3.53 3.17 2.06 1.47 0.7 7.54 7.35 6.91 6.87 6.75 6.66 6.5 6.26 6.01 5.61 5.53 4.84 Mean (mM) 3.3 2.9 2.9 2.9 1.7 1.6 0.8 0.6 2.9 5.9 3.6 3.2 3.4 4 1.8 2.5 3.8 2.1 2.9 2.5 Median (mM) Sporadic PPGL (S) 3.9 2.29 2.71 1.6 0.59 0.7 0.6 0.3 1.87 4.92 4.28 3.42 1.45 2.22 0.84 1.7 2.06 1.75 2.86 3.56 SD (mM) 0.036 0.026 A vs T P Value 0.011 0.015 0.001 0.041 0.017 0.047 A vs S P Value 0.016 0.042 T vs S P Value https://academic.oup.com/jcem Downloaded from https://academic.oup.com/jcem/article-abstract/104/11/5467/5536622 by Endocrine Society Member Access 3 user on 17 October 2019 The P values for observed statistically significant differences are presented. Ascorbate N-Acetylglucosamine Choline 3-Hydroxyisovalerate Creatine phosphate 2-Hydroxyisobutyrate Benzoate N-Acetylaspartate N,N-Dimethylglycine O-Acetylcholine Maltose 4-Hydroxy3-methoxymandelate 3-Hydroxy-3-methylglutarate Carnitine trans-Aconitate Valine 5-Hydroxyindole-3-acetate Acetone Trimethylamine Fumarate Metabolite SDHx Asymptomatics (A) Table 2. Comprehensive List of the 58 Quantified Metabolite Concentrations for the SDHx Mutation Carriers Without Evidence of Disease (SDHx Carriers), the SDHx Mutation Carriers With Related Tumors (SDHx Tumors), and the Patients With Sporadic PPGL (Sporadic PPGL) (Continued) doi: 10.1210/jc.2019-01101 5471 5472 Martins et al SDHx-Associated Tumors: Urine Metabolomics samples show unique metabolites that can be the result of therapeutics, food additives, etc., that were not considered in our analysis. A comprehensive list of assignments is provided in Table 2. J Clin Endocrinol Metab, November 2019, 104(11):5467–5477 Affected SDHx carriers vs asymptomatic SDHx carriers vs patients with sporadic PPGL The metabolomic urinary profile of SDHx carriers with associated tumors was compared with that of asymptomatic Downloaded from https://academic.oup.com/jcem/article-abstract/104/11/5467/5536622 by Endocrine Society Member Access 3 user on 17 October 2019 Figure 1. Metabolites in urine that registered statistically significant different concentrations between asymptomatic SDHx mutation carriers (asymptomatic), SDHx carriers with associated tumors (SDHx tumor), and individuals with sporadic PPGL (sporadic). P . 0.05 (ns); *P # 0.05; **P # 0.01. ns, not significant. doi: 10.1210/jc.2019-01101 5473 0.036). The MSEA analysis indicated that the pathways that are most influenced by the development of tumors in the SDHx mutation carriers are those related to catecholamine biosynthesis, tryptophan, and lipid metabolism (Fig. 2A). Restricting the analysis to individuals with tumors, the urine of patients with SDHx mutations presented higher Figure 2. MSEA of urine showing the differentially affected metabolic pathways of (A) SDHx mutation carriers with related tumors compared with SDHx mutation carriers without evidence of disease; (B) SDHx mutation carriers with related tumors compared with patients with sporadic PPGL; (C) SDHx mutation carriers without evidence of disease compared with patients with sporadic PPGL; and (D) PPGL patients with SDHB vs SDHD mutations. Downloaded from https://academic.oup.com/jcem/article-abstract/104/11/5467/5536622 by Endocrine Society Member Access 3 user on 17 October 2019 carriers and of patients with sporadic PPGL (Fig. 1; Table 2). Compared with asymptomatic SDHx carriers, the urine of affected carriers showed substantially lower levels of xanthine (P 5 0.008), methanol (P 5 0.020), citrate (P 5 0.021), succinate (P 5 0.023), choline (P 5 0.026), 2-furoyglycine (P 5 0.027), and N-acetylaspartate (P 5 https://academic.oup.com/jcem 5474 Martins et al SDHx-Associated Tumors: Urine Metabolomics Other analyses When metastatic tumors were compared with nonmetastatic PPGL in patients with SDHx mutations, only one difference emerged. Specifically, methanol levels were lower in the urine of patients with tumors who developed metastases (4.38 6 3.18 mM vs 13.36 616.83 mM, P 5 0.02). The type of SDHx mutation also influenced the metabolites present in the urine. Specifically, patients with PPGL associated with the SDHD mutation had increased levels of dimethyl sulfone (P 5 0.039) and formate (P 5 0.035) compared with affected SDHB mutation carriers (Fig. 3A). The MSEA indicated differences in gluconeogenesis, Warburg effect, citric acid cycle, and transfer of acetyl groups into mitochondria between carriers of SDHD and SDHB mutation (Fig. 2D). When we compared the urine of patients with functioning vs nonfunctioning PPGL, the only metabolite that showed a difference was dimethylglycine (P 5 0.027), an amino acid derivative produced in the metabolization of choline into glycine. Its concentrations were lower in the urine of patients with functioning Figure 3. Metabolites in urine with a substantial concentration difference (P , 0.05) between (A) affected individuals with SDHB and SDHD mutation; (B) patients with functioning tumors (Func) and nonfunctioning tumors (no Func), regardless of the existence or not of mutation. P . 0.05 (ns); *P # 0.05; **P # 0.01. ns, not significant. Downloaded from https://academic.oup.com/jcem/article-abstract/104/11/5467/5536622 by Endocrine Society Member Access 3 user on 17 October 2019 levels of N-acetylaspartate (P 5 0.016), 3-hydroxyisovalerate (P 5 0.042), and lactate (P 5 0.044) than the urine of individuals with sporadic PPGL (Fig. 1). The metabolite differences indicated that gluconeogenesis, pyruvate metabolism, ammonia recycling, porphyrin, and aspartate metabolism are differentially affected in patients with SDHx-associated neoplasias, relative to sporadic PPGL patients (Fig. 2B). Comparative analysis of urine from asymptomatic SDHx mutation carriers and from patients with sporadic PPGL revealed lower levels of a number of metabolites in the latter (Fig. 1): N-acetylaspartate (P 5 0.001), cisaconitate (P 5 0.005), creatinine (P 5 0.008), lactate (P 5 0.009), citrate (P 5 0.011), 5-hydroxyindole-3acetate (P 5 0.011), 3-hydroxyisobutyrate (P 5 0.014), O-acetylcholine (P 5 0.015), 3-hydroxyisovalerate (P 5 0.017), xanthine (P 5 0.022), imidazole (P 5 0.034), 2hydroxyisobutyrate (P 5 0.041), and ascorbate (P 5 0.047). The metabolite differences could result from alterations in mitochondria pathways, such as acetyl groups transference, glucose metabolism, and citric acid cycle (Fig. 2C). J Clin Endocrinol Metab, November 2019, 104(11):5467–5477 doi: 10.1210/jc.2019-01101 Discussion Our results indicate that the metabolomic urine profile of SDHx mutation carriers with associated tumors is different from that of asymptomatic carriers. Seven metabolites presented lower levels in the former than in the latter carriers. These findings, and the MSEA, suggest the presence of alterations in the pathways related to the PPGL catecholamine biosynthesis. Unlike the metabolomic profile of tumoral tissue, in which the oncometabolite succinate characteristically presents high levels (13–16), in the urine, its levels were lower for SDHx mutation carriers with associated tumors than for SDHx asymptomatic carriers. The increased retention and accumulation of succinate in tumor cells, and its role as an oncometabolite (26), might be associated with its deficient elimination and low excretion in urine. Larger studies measuring this metabolite in plasma can help to clarify these findings and to establish the relevance of succinate as a tumor marker for this disease. Similar to succinate, the citrate levels were lower in SDHx carriers with associated tumors than in asymptomatic mutation carriers. Citrate is also a metabolite of the citric acid cycle, and this finding is consistent with the changes observed in tumor tissue, in which citrate levels in SDHxassociated tumors were lower than in sporadic tumors (15). Other metabolites were also different in affected vs asymptomatic carriers. Patients with tumors always presented lower levels of such metabolites, suggesting a washout problem in association with tumorigenesis. Reinforcing this hypothesis, all the statistically significant differences between patients with disease (sporadic or associated with SDHx) and asymptomatic SDHx carriers showed lower levels in the former group. The metabolomic urinary profile of patients with SDHx-associated and sporadic PPGL was also different. Lactate levels, for example, were higher in patients with SDHx-related tumors than in patients with sporadic 5475 PPGL. This finding underscores the preference for anaerobic pathways, in line with the known pseudohypoxia signature of these tumors (27). The fact that lactate levels were also higher in asymptomatic SDHx mutation carriers than in patients with sporadic tumors provides support for the occurrence of pseudohypoxia even in unaffected SDHx mutation carriers. The MSEA analysis suggested that gluconeogenesis, pyruvate metabolism, ammonia recycling, porphyrin, and aspartate metabolism most probably explain the differences found between SDHx-associated and sporadic PPGL patients. Gluconeogenesis, the process of generating glucose from carbon substrates (as pyruvate) other than carbohydrates, has shown to be upregulated in cancer (28). Lussey-Lepoutre et al. (29) previously demonstrated that SDH-mutated tumors presented altered metabolism of pyruvate, which is the glycolytic entry point into the citric acid cycle. The alteration consisted of an increased production of aspartate, which worked as an alternative pathway enabling the truncated citric acid cycle to continue and upon which the cells become dependent for proliferation and survival. Synthesized from aspartate and acetyl-coenzyme A (CoA; an intermediary of the citric acid cycle, derived from pyruvate) (30), N-acetylaspartate was the only metabolite the urinary levels of which registered differences among the three groups analyzed. The lowest levels were observed in patients with sporadic PPGL followed by intermediate levels observed in individuals with SDHx-associated tumors; the highest levels corresponded to SDHx asymptomatic carriers. These results concerning urinary levels of N-acetylaspartate contrast, once again, with those reported in previous studies using tumor tissue, which documented lower N-acetylaspartate levels in SDHx tumors, compared with sporadic tumors (12). N-acetylaspartate has been classically associated with central nervous system disturbances (30). Our study reinforces its relevance also for this disease of peripheral nervous system, as recently suggested (12). Its synthesis in mitochondria depends on respiratory chain and oxidative phosphorylation (31), and a strong positive correlation was shown between N-acetylaspartate content and ATP/ADP/AMP contents in PPGL (12). In a recent review about this metabolite, the authors hypothesized that N-acetylaspartate might complement citrate, delivering acetyl-CoA to the cytosol (30). Our findings support the relation of this metabolite with the development of neoplasms associated with SDHx mutations. Future studies based, for example, on plasma analysis, are needed for a better understanding of the present findings and for the establishment of the potential utility of Nacetylaspartate as a biomarker. Ammonia recycling is another pathway highlighted in MSEA that enables Downloaded from https://academic.oup.com/jcem/article-abstract/104/11/5467/5536622 by Endocrine Society Member Access 3 user on 17 October 2019 PPGL. Interestingly, the levels of vanillylmandelic acid (also known as 4-hydroxy-3-methoxymandelate), a product of the catecholamine catabolism, were higher (P 5 0.022) in the urine of patients with functioning PPGL (Fig. 3B). The MSEA indicated that one of the pathways that was altered in the functioning PPGL was catecholamines biosynthesis, but phospholipid synthesis, citric acid cycle, and tyrosine metabolism were also altered. Analysis by type of hormonal secretion (nonfunctioning, adrenergic, noradrenergic, dopaminergic) did not show differences in metabolites concentrations. This can be explained by the reduced number of samples of each type. https://academic.oup.com/jcem 5476 Martins et al SDHx-Associated Tumors: Urine Metabolomics Acknowledgments The authors would like to thank all the clinicians from the involved hospitals who contributed to this study, allowing access to the patients (Helder Sim~ oes, Maria Jo~ ao Matos, Isabel Torres, Valeriano Leite, Fernando Rodrigues, Manuel R. Teixeira); all those who contributed to sample collection and storage (Celeste Fontoura, Nuno Gonçalves, Susana Prazeres); Professor Irene Carvalho who proofread the manuscript; and the study participants for their contribution to this research. The NMR data were acquired at CERMAX (Centro de Ressonância Magnética António Xavier). Financial Support: R.G.M. received a Dr. Rocha Alves research grant from the Núcleo Regional do Centro da Liga Portuguesa Contra o Cancro. L.G.G. had a post-doc grant, SFRH/BPD/111100/2015, awarded by FCT - “Fundaç~ ao para a Ciência e a Tecnologia.” The use of NMR facility was funded by project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular), by FEDER through COMPETE2020 - POCI and by national funds through FCT“Fundaç~ ao para a Ciência e a Tecnologia.” Additional Information Correspondence and Reprint Requests: Raquel G. Martins, MD, MsC, Departamento de Neurociências Clı́nicas e Saúde Mental/Unidade de Psicologia Médica, Faculdade de Medicina da Universidade do Porto, Al. Prof. Hernâni Monteiro, 4200-319 Porto, Portugal. E-mail: martins.raquel@hotmail.com. Disclosure Summary: The authors have nothing to disclose. Data Availability: The datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request. References and Notes 1. Favier J, Amar L, Gimenez-Roqueplo AP. 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