Profiles of amino acids and biogenic amines in the plasma of Cri-du-Chat patients

Accepted Manuscript Title: Profiles of amino acids and biogenic amines in the plasma of Cri-du-Chat patients Authors: Danielle Zildeana Sousa Furtado, Fernando Brunale Vilela de Moura Leite, Cleber Nunes Barreto, Bernadete Faria, Leticia Dias Lima Jedlicka, Elisˆangela de Jesus Silva, Heron Dominguez Torres da Silva, Etelvino Jose Henriques Bechara, Nilson Antonio Assunc¸a˜ o PII: DOI: Reference:

S0731-7085(16)31368-1 http://dx.doi.org/doi:10.1016/j.jpba.2017.03.034 PBA 11160

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Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

15-12-2016 15-3-2017 17-3-2017

Please cite this article as: Danielle Zildeana Sousa Furtado, Fernando Brunale Vilela de Moura Leite, Cleber Nunes Barreto, Bernadete Faria, Leticia Dias Lima Jedlicka, Elisˆangela de Jesus Silva, Heron Dominguez Torres da Silva, Etelvino Jose Henriques Bechara, Nilson Antonio Assunc¸a˜ o, Profiles of amino acids and biogenic amines in the plasma of Cri-du-Chat patients, Journal of Pharmaceutical and Biomedical Analysishttp://dx.doi.org/10.1016/j.jpba.2017.03.034 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.

Profiles of amino acids and biogenic amines in the plasma of Cri-du-Chat patients

Danielle Zildeana Sousa Furtadoa, Fernando Brunale Vilela de Moura Leitea, Cleber Nunes Barretoa, Bernadete Fariaa, Leticia Dias Lima Jedlickaa, Elisângela de Jesus Silvab, Heron Dominguez Torres da Silvaa, Etelvino Jose Henriques Becharac and Nilson Antonio Assunçãoa*

a

Institute of Environmental, Chemical and Pharmaceutical Sciences, Federal University

of São Paulo, Diadema, SP, Brazil. b

c

A.C. Camargo Cancer Center, Sao Paulo, SP, Brazil; Institute of Chemistry, University of São Paulo, São Paulo, SP, Brazil.

* Corresponding author: Institute of Environmental, Chemical and Pharmaceutical Sciences, Federal University of São Paulo, 04039-032 Diadema, SP, Brazil. e-mail: [email protected]

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Highlights 

Evaluation of unbalanced metabolic state in CDCS patients.



Profile of metabolites was evaluated by Mass Spectrometry.



Chemometric approach was used to associations with the major metabolic pathways.

Abstract Cri-du-chat syndrome (CDCS) is a rare innate disease attributed to chromosome 5p deletion characterized by a cat-like cry, craniofacial malformation, and altered behavior of affected children. Metabolomic analysis and a chemometric approach allow description of the metabolic profile of CDCS as compared to normal subjects. In the present work, UHPLC/MS was employed to analyze blood samples withdrawn from CDCS carriers (n=18) and normal parental subjects (n=18), all aged 0-34 years, aiming to set up a representative CDCS profile constructed from 33 targeted amino acids and biogenic amines. Methionine sulfoxide (MetO) was of particular concern with respect to CDCS redox balance. Increased serotonin (3-fold), methionine sulfoxide (2-fold), and Asp levels, and a little lower Orn, citrulline, Leu, Val, Ile, Asn, Gln, Trp, Thr, His, Phe, Met, and creatinine levels were found in the plasma of CDCS patients. Nitrotyrosine and Trp did not differ in normal and CDCS individuals.The accumulated metabolites may reflect, respectively, disturbances in the redox balance, deficient purine biosynthesis, and

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altered behavior, whereas the amino acid abatement in the latter group may affect the homeostasis of the urea cycle, citric acid cycle, branched chain amino acid synthesis, Tyr and Trp metabolism and amino acid biosynthesis. The identification of enzymatic deficiencies leading to the amino acid burden in CDCS is further required for elucidating its molecular bases and eventually propose specific or mixed amino acid supplementation to newborn patients aiming to balance their metabolism.

Abbreviations 5-HT: Serotonin 5p: Chromosome 5 AcOrn: Acetyl Ornitine ADMA: Dimethylarginine asymmetric ADP: Adenosine Diphosphate AGAT: Glycine Amidinotransferase Ala: Alanine Alpha AAA: Alpha-aminoadipic acid Arg: Arginine ASL: Argininosuccinate Lyase Asn: Asparagine Asp: Aspartate ASS: Argininosuccinate Synthetase BCAA: Branched Chain Amino Acids CAPES: Coordination for the Improvement of Higher Education Personnel CAVR: Creatine Transporter CCDS: Cerebral Creatine Deficiency Syndrome CDCS: Cri-du-chat Syndrome Cit: Citrulline CPS1: Carbamoylphosphate Synthetase I ESI: Electrospray ionization FAPESP: The São Paulo Research Foundation FINEP: Brazilian Innovation Agency GAMT: Guanidinoacetate N-methyltransferase Gln: Glutamine Glu: Glutamate acid Gly: Glycine His: Histidine HPLC: High performance liquid chromatography IQ: Intelligence quotient 3

I.S.: Internal Standards IEM: Inborn errors of metabolism Ile: Isoleucine Kyn: kynurerine LDL: Low-density Lipoprotein Leu: Leucine LOD: Limits of Detection LOQ: Limits of Quantification Lys: Lysine Met: Methionine MetO: methionine sulfoxide MsrA: Methionine Sulfoxide Reductase MSUD: Maple Syrup Urine Disease NA: Neutral Amino Acids NAGS: N-acetylglutamate NAPCDC: Núcleo de Aconselhamento e Pesquisa Cri Du Chat NGO: Non-Governmental Organization N-tyr: Nitrotyrosine Orn: Ornithine OTC: Ornithine Transcarbamylase QC: Quality Control PC: Principal Component PCA: Principal Component Analysis Phe: Phenylalanine Pro: Proline RNI: Reactive Oxygen Intermediates ROS: Reactive Oxygen Species SD: Standard Deviation Ser: Serine Thr: Threonine tR: Retention time Trp/ΣNA: Ratio of free tryptophan/neutral amino acids Trp: Tryptophan Tyr: Tyrosine UPLC-MS/MS: Ultra-Performance Liquid Chromatography in tandem mass spectrometry Val: Valine

Keywords Amino

acids,

biogenic

amines,

chromosome

5,

chemometrics,

mass spectrometry.

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1. Introduction Aminoacidopathies encompass a group of IEM [1] characterized by enzyme failures that can disturb metabolic pathways, leading to the accumulation or deficiency of biomolecules [2]. Aminoacidopathies affect 1 in 1,000 individuals worldwide and have been classified in organic acidurias, urea cycle defects and transport defects of cycle urea intermediates [1]. Amino acid alterations in urine or blood, for instance, may denounce a primary disease or be secondarily linked to other maladies. Primary aminoacidopathies are usually autosomal recessive diseases associated with a deficient enzyme or transport deficits [3], and their symptoms display a high degree of variability from relatively benign to severe. These symptoms may include but are not limited to growth limitations, mental retardation, developmental delays, learning disabilities, seizures, lethargy, coma, vomiting, acidosis or metabolic alkalosis, sudden infant death syndrome, osteomalacia and osteoporosis. According to the natural history of the disease, the symptoms can be minimized or absent when an early diagnosis is made and treatment is started quickly [4]. An aminoacidopathy that afflicts 1:15,000 [5] to 1:50,000 individuals worldwide [6] is the CDCS. First described by Lejeune in 1963 [7], CDCS was so named because patients were found to emit cat-like mewing sounds. Later, the syndrome was found to result from a chromosomal abnormality consisting of a distal or interstitial deletion of the short arm of chromosome 5 (-5p) [8]. In general, CDCS features are attributed to abnormalities of the larynx (small, narrow and rectangular shape) and epiglottis (small and hypotonic) as well as to neurological [6], structural and functional changes that occur during embryonic development [9]. Affected individuals also have facial asymmetry, with microcephaly, ocular hypertelorism, hypotonia, upward slanting palpebral fissures,

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and cleft eyelids with epicanthal folds [10]. Other features are reported as well, such as dysplastic and low-set ears, long fingers, psychomotor retardation, congenital cardiac changes [10], respiratory infections, otitis media [11], renal disease [12], dental malocclusion, and scoliosis [13]. Children with CDCS often lack some physical skills, including verbal development [6]. Intellectual disability is also a marked feature; however, as with other aminoacidopathies, special early intervention can lead to major improvements in the condition of CDCS-affected individuals [14]. Concerning the syndrome’s metabolic processes, in 1974, Kuhner et al. [15] described an increase in proline and threonine in urine and serum in a single case of CDCS. Long thereafter, Lejeune et al. [16] observed increased plasma and urinary concentrations of aspartate and asparagine, whereas excess histidine appeared only in the urine. The increased levels of asparagine and aspartate in plasma and urine are related to impairments in the biosynthesis of purines, whereas increased excretion of histidine in the urine seems to reflect general deficiencies in amino acid metabolism. Changes consistent with clinical ketotic hyperglycinemia were also reported as well as a deficiency in the synthesis of purine nucleotides pointing to major neurotransmitter involvement in mental development. Although features and metabolic processes associated with CDCS have been documented, literature on the biochemistry of inborn CDCS is still scarce. Therefore, we decided to trace the amino acid plasmatic profile of CDCS carriers and their close relatives in an attempt to get biochemical clues that could eventually correlate their altered metabolism with physical and mental manifestations. The participants in this study were Brazilian families of patients with CDCS who were recruited by the NGO,

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NAPCDC, located in São Caetano do Sul-SP. The NAPCDC is composed of a group of 400 families from throughout Brazilian territory. The CDCS patients of the NAPCDC are of both genders, with ages ranging from newborn to adult. To unveil the fluxes of metabolic alterations in patients with CDCS, it is essential to unravel the mechanisms undermining patients’ health and social life [17]. Therefore, the present work sets out to characterize a metabolic profile of CDCS patients with the aim of identifying specific markers of the disorder that may eventually serve to monitor the evolution and consequences of the disease. In addition, the current work may offer clues for the development of therapeutic strategies including medication, dietary guidelines, or amino acid supplementation to improve the quality of life of CDCS patients.

2. Methods 2.1. Population sample CDCS patients and healthy (control) individuals belonging to 36 families were recruited by the NAPCDC. CDCS diagnosis was made by karyotyping the chromosomes in most cases and only by the clinical characteristics in others. The groups of patients and healthy relatives were distributed by gender and age of infants and adults. The control subjects included male and female siblings of the CDCS patients in this study illustrated in Supplementary File S1.

2.2. Experimental protocol 2.2.1. Sample collection

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Heparinized blood samples were collected from 4-hour fasted volunteers by a procedure approved by the Research Ethics Committee of the Federal University of São Paulo (Escola Paulista de Medicina Paulista, Number: 226594). 2.2.2. Chemicals The AbsoluteIDQ™ p180 kit (Part Number: A-WATERS-4-P180) was purchased from Biocrates Life Sciences Company (Eduard-Bodem-Gasse, Innsbruck, Austria). Preparation of standards and samples followed the manufacturer’s instructions. Three quality control vials and seven calibration standard vials were employed to analyze the following amino acids and biogenic amines: Ala, Arg, Asn, Asp, Cit, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Orn, Phe, Pro, Ser, Thr, Trp, Tyr, Val, AcOrn, ADMA, Alpha AAA, creatinine, dopa, histamine, Kyn, MetO, N-Tyr, sarcosine, 5-HT, taurine. One vial containing all the internal standards was also used. Ethanol, methanol and acetonitrile were obtained from Merck Millipore (Darmstadt, Germany), HPLC grade. Phosphate buffered saline, isopropanol, phenyl isothiocyanate, ammonium acetate, and formic acid were purchased from Sigma Aldrich (St. Louis, MO, USA). 2.2.3. Equipment Analyses were processed by a UPLC Water AQUITY UPLC System with detection by a triple quadrupole mass spectrometer and ESI source (Xevo TQ-MS, Waters, USA), using a Waters UPLC BEH C18 ACQUITI 1.7 µm 2.1 x 75 mm (No. 186 005 604) column and a Waters BEH C18 1.7 µm ACQUITI VANGUARD (No. 186003975) precolumn. 2.2.4. Standard preparation

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Metabolite and I.S. added to the plasma samples were prepared with ultrapure MilliQ water. The I.S. metabolite, the collision energy values, and the UPLC-MS/MS analyses are described in Supplementary File S2. 2.2.5. Sample preparation Plasma samples were prepared by blood centrifugation (10 min at 12,000 rpm), aliquoted into 2 mL Eppendorf tubes, and then stored in a freezer at -80 °C until use. Ten µL of plasma and 10 µL of internal standard were spotted on a 96-well ELISA plate and dried under nitrogen for 30 min. Then, 20 µL of a 5% phenyl isothiocyanate solution were added for the metabolite derivatization, and the plates were incubated for 20 min at room temperature. The filter spots were dried and extracted by the addition of 300 µL of ammonium acetate solution (5 mM) prepared in methanol by centrifuging the plate for 30 min at 450 rpm. Subsequently, the extracts were filtered in the wells of plates, which were centrifuged for 2 min at 5000 g. A 150 µL aliquot of the derivatized sample was diluted in 150 µL water and analyzed by UPLC-MS/MS. 2.2.6. UPLC-MS/MS conditions UHPLC phases A and B were 0.2% formic acid prepared, respectively, in water and in acetonitrile. The injection volume was 5 µL, the column maintained at 50°C, and the sampler at 4°C. The gradient ranged from 0% Phase B for 0.25 min, 80% B for 3.75 min, 60% B for 3.95 min, and 0% B until 5.00 min, with a flow rate of 0.90 mL min- 1. The scanning time was 5.0 min. 2.3. Data analyses The non-parametric Wilcoxon statistical test was used to compare the metabolite concentrations, adopting a significance threshold of p < 0.05. Before the test of

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comparison of averages, the dates are submitted to normality test (Kolmogorov-smirnov & lilliefors test of normality) and homogeneity of variances (Levene’s test). In addition to the mean comparison test, the PCA was also performed to compare the groups of metabolites from major metabolic pathways, with intention of identify grouping. The data were processed using the analytic software Statistica, StatSoft, Inc. (2011). STATISTICA (data analysis software system), version 10.

3. Results and discussion 3.1. Metabolomics calibration curves An HPLC calibration curve was constructed for each metabolite (Target), and both the best-fit equations and coefficients of determination (R2) were calculated and the LOD and LOQ were evaluated considering uncertainty associated with calibration curve [18] (see Supplementary File S3). Analytical curves providing greater reliability (R2 ≥ 0.98) were traced. The The tR of each metabolite, the linear range, and the MS and MS/MS ratios m/z for the main fragment, such as the LOD and LOQ for the set of metabolites were calculated, showed in Supplementary File S3. The intraday precision and recovery were determined within one day by analyzing five sample replicates at three levels for each metabolite. The inter-day precisions and recovery were determined in five different days using the same metabolites and concentrations. The RSD of intraday and inter-day precision of the method ranged from 0.01 to 10.29, and intraday revovery ranged from 90.33% to 113.66% (Supplementary File S4). Quality control at low, intermediate, and high level, using different matrices and

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conditions was quantified to evaluate robustness of the method, and to estimate its RSD, which ranged from 0.96-4.88. Plasma levels of 33 metabolites were quantified for both the CDCS patient and control groups (Table 1) in a first attempt to correlate the observed alterations with a singular metabolic pathway. With the significance threshold established at 5% and the application of PCA to data, a significant decline of the levels of the following metabolites was revealed: Arg, Asn, Cit, Gln, His, Ile, Leu, Met, Orn, Phe, Thr, Trp, Tyr, Val and creatinine. In contrast, Asp, Glu, MetO and 5-HT levels increased in the CDCS group. On the other hand, Ala, Gly, Lys, Pro, Ser, AcOrn, Adma, Alpha-AAA, dopa, histamine, Kyn, N-tyr, sarcosine and taurine were not significantly changed as compared to the two groups. Figure 1 shows the relative percentages of all analyzed metabolites when comparing the control and the CDCS groups. They can be clearly distinguished by the respective values displayed in the Y-axis. Values in the negative region represent variation of metabolites that are diminished in CDCS versus the control group; positive values, elevated metabolites in CDCS patients.

3.2.

Metabolic pathways implicated in human disorders

3.2.1 Methionine-sulfoxide (MetO) metabolism MetO is a biogenic amine produced by the reaction of Met with oxygen-derived reactive oxidants such as H2O2, 1O2, HO•, and ONOO-/ONOOH. These ROS are reportedly generated by the respiratory mitochondrial chain and other biological systems,

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which affect the cell redox balance, signaling pathways, and the innate immune response [17]. Protein Met residue oxidation to more hydrophilic MetO and consequent protein structural alterations may be counter-balanced by the action of MsrA. This is reportedly associated with neurodegenerative diseases, cancer, and the aging process [19]. However, this process can be reversed by MsrA enzyme action, which converts MetO to form Met amino acid [20], and this is most likely a mechanism that cells use to remove excess ROS/RNS consequently protecting the cells against oxidative damage [21]. MetO levels increased two fold in the CDCS group as compared to the control group, thereby raising the inference of excess ROS in Met oxidation. Accordingly, Met levels decreased by 6 ± 1% in CDCS patients. Whether these findings are related with neurological deficits in CDCS requires further investigation.

3.2.2 Metabolism of branched chain amino acids The BCAA (Val, Leu, and Ile) are essential to the human organism and represent 20 to 25% of total dietary amino acids [22]. They provide nitrogen for the production of Ala and Gln, especially during prolonged fasting, and participate in the regulation of catabolic and anabolic processes [23]. BCAA reduction was observed in CDCS patients: 20 ± 6% for Val, 29 ± 7% for Leu and 16 ± 4% for Ile (Table 1), suggesting the impairment of protein syntheses [24]. These differences can be better observed using the PCA approach (Figure 2), that show the distinct grouping of groups studied. The factor of PC1 and PC2 indicates that 93% of the variance data are explained by PCA.

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Degradation of muscle BCAAs provide CoASH to the Krebs cycle intermediates via acetyl CoA and succinyl-CoA, the primary sources of NADH to the respiratory chain and ATP production [25]. They also participate in multiple signaling processes as in insulin signaling and oxidative stress [26]. Low concentrations of BCAA are observed in hyperinsulinism [27], hyperglucanonemia, catecholamines biosynthesis, starvation, and hyperammonemia [28]. Alterations of Glu levels are related to an increase of BCAA catabolism once Glu is produced from alfa-ketoglutarate, leading to decreased BCAAs and Ala, and an increase of Gln in blood and skeletal muscle [29]. CDCS patients also showed an increase in Glu (19 ± 9%) and a decrease in Gln (10 ± 1%) levels in plasma, suggesting an enzymatic failure in the conversion of Glu to Gln. High concentrations of BCAAs have been found in MSUD [30], an aminoacidopathy that results from an enzyme defect in the BCAA catabolism pathway, consequently causing accumulation of these amino acids. If untreated in infancy, accumulation of these three amino acids can cause encephalopathy and neurodegenerative problems. Treatment of MSUD is based on restricting the dietary intake of Leu, Ile, and Val [31]. Thus, it is plausible that BCAA imbalance in CDCS patients can be lessened with BCAA-rich food or dietary supplements in order to compensate for their inborn deficiency.

3.2.3 Tryptophan and serotonin metabolism Serotonin is a neurotransmitter produced from Trp oxidation and decarboxylation [32]. Approximately 90% of plasmatic Trp is bound to albumin, and 10% is freely circulating. The increase in free plasmatic Trp results from the synthesis and transport of

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serotonin [33]. The ratio of free Trp/ΣNA was evaluated in CDCS patients and found to be 37 ± 2% higher than in the control group (Figure 3).

A study [34] was reported that high levels of the ratio Trp/ΣNA stimulate the biosynthesis of 5-HT in the brain. An increase in 5-HT concentration may partially explain the anxiety and feeding alterations in the CDCS group, being that these symptoms may reflect alterations in social behavior, sleep, fatigue, appetite inhibition, aggression, anxiety, and depression [35]. Trp is an amino acid precursor of Kyn and 5-HT, whose levels are influenced by the increase of circulating fatty acids in the body [36]. Free Trp is converted to 5-OH-tryptophan, after uptake by the brain and is then decarboxylated to form 5-HT [37]. The observed 15 ± 5% decrease of Trp plasma levels in CDCS patients may be connected to the 200 ± 11% increase in serotonin in these patients. A low free Trp concentration also causes alterations in 5-HT levels though the hypothalamic system, thus impacting mood regulation [38]. Previous studies in experimental animals [39] indicated that low concentrations of Trp contribute to behavioral and cognitive impairment, corroborating with the results observed in CDCS. Furthermore, Trp has also been related to BCAAs competition for crossing the bloodbrain barrier [40] and to growth hormone activity [41].

3.2.4 Phenylalanine and tyrosine metabolism Phe is an essential amino acid that is hydroxylated by a hydroxylase yielding Tyr [42]. Deficient biosynthesis of this enzyme in phenylketonuria leads to Phe accumulation and its transamination to phenylpyruvic acid. [43]. A trend for lower concentrations of

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Phe (11 ± 2%) and Tyr (4 ± 1%) was observed in the plasma of patients with CDCS, and these concentrations could impair the production of catecholamines, as adrenalin, noradrenalin, and dopamine [44]. Alterations in neuronal dopamine are related to psychiatric disorders characterized by changes in motor activity, mood, attention deficit, hyperactivity, and fragmentation of thought processes [45]. Biogenic amines are precursors not only of hormones but also alkaloids, nucleic acids, proteins and nitrogen sources [46]. As shown by Table 1, the Tyr level in the CDCS group was decreased in comparison with the control group. Thus, the biosynthesis of catecholamine in CDCS patients might to be affected, although the DOPA level quantified in serum was not significantly different. It is necessary to evaluate the disruption of this route through analysis of the enzymes and substrates that are involved in biogenic amine metabolism, especially dopamine biosynthesis in CDCS patients.

3.2.5 Urea cycle In CDCS patients, several metabolites of the urea cycle significantly decreased in plasma, including Orn (26 ± 6%), Cit (23 ± 8%), and Arg (9 ± 3%), suggesting enzymatic failures that may cause a deficit in nitrogen metabolism or low Arg intake. The PCA (Figure 4) shows the distinct grouping of CDCS patients and control groups from levels of metabolites Arg, Cit and Orn measured. The factor of PC1 and PC2 indicates that 95% of the variance data are explained by PCA.

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The urea cycle encompasses enzymatic reactions that occur both in the cytosol and in the mitochondrial matrix of cells. Urea cycle disorders are caused by deficiencies in one or more of the following enzymes: NAGS, CPS1, OTC, ASS and ASL [47]. The investigation of these enzymes in CDCS could perhaps clarify the altered pattern found in the patients and support Arg supplementation to alleviate the syndrome. Arg plays crucial roles in protein and enzyme syntheses, nitric oxide production, urea excretion, and hormone secretion (prolactin, insulin and growth hormone) [48], in addition to being associated with intestinal malabsorption syndromes when at low levels [49]. 3.2.6 Creatinine metabolism Creatinine, an important indicator of renal function, is a metabolite produced from the degradation of phosphocreatine in the muscle, which primarily acts as a phosphate donor to ADP [50]. In the present work, the obtained PCA (Figure 5) distinguished the CDCS patients from the control group from dates of concentrations of Gly, Arg, and creatinine plasmatic. The factor of PC1 and PC2 indicates that 84% of the variance data are explained by PCA. The creatinine pathway is related to reactions with Gly and Arg, which are the substrates for the production of guanidinoacetate by enzymatic action of AGAT. Subsequently, the GAMT converts guanidinoacetate into creatine, which undergoes creatine kinase-catalyzed phosphorylation to creatine phosphate, whose spontaneous degradation yields creatinine. No significant Gly levels in plasma of CDCS patients were found; contrarily, the Arg and creatinine plasma levels were found to be lower by 9 ± 3% and 36 ± 9%, respectively. These data suggest that the urea and creatinine pathway

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enzymes are affected in CDCS once Arg is used for the biosynthesis of creatine and participates in the urea cycle. These results may be discussed in the light of the low muscle mass reported in CDCS patients. Alterations in creatinine levels are present in CCDS, which is defined by inborn errors in creatine metabolism resulting from enzymatic (AGAT and GAMT) failures and a deficiency in the CAVR [51]. Some clinical features of CCDS are similar to those of CDCS patients, particularly intellectual disability, behavioral disorders and speech delay. CCDS is treated by supplementation with creatine monohydrate and ornithine [51]; however, creatine supplementation alone is not sufficient to improve CAVR deficiency. In contrast, supplementation with Arg and Gly has been reported to increase muscle mass, motor ability, and personal, social and IQ skills [51]. It is tempting to propose that the treatment of CDCS-related creatinine deficiency could be based on dietary supplementation with Arg, Orn and Cit, both to improve the disruptions involving the urea cycle described above and creatinine biosynthesis.

3.2.7 Histidine metabolism Histidine is an essential amino acid in infancy that is converted into the neurotransmitter histamine by the enzyme histidine decarboxylase. Histamine synthesis occurs in the central nervous system of mammals, or more precisely, in the tuberomammillary nucleus of the hypothalamus. The neurons found in this region are responsible for various functions such as appetite, hormonal secretion, sleep/wake regulation, thermoregulation, and control of the cardiovascular system [52]. Inside mast

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cells and basophils, histamine is produced slowly and accounts for 90% of the body's stock. Additionally, His participates in the biosynthesis of purinergic bases and has antiinflammatory, antioxidant and anti-secretory activity [53]. Patients with CDCS were shown here to be deficient (12% ± 2%) in plasmatic His as compared to the control group. A low concentration of His has been associated with protein-energy wasting, inflammation and oxidative stress [54] by virtue of its quenching effect of highly reactive singlet oxygen and hydroxyl radical [55]. In addition, His protects cholesterol from oxidation by singlet oxygen [57] and accumulation of LDL [58]. On the other hand, increased His levels in histidinemia, a rare autosomal recessive metabolic disorder resulting from a deficiency of the enzyme histidinase [59], can be overcome with a low His diet [59]. Therefore, taking into account that His is a non-essential amino acid in adults, His deficiency found in older CDCS patients could be compensated by His intake to attain normal levels in the blood.

3.2.8 Aspartate metabolism Aspartic acid is a non-essential amino acid constituent of proteins and plays an important role as a bioactive metabolite. Besides being used in protein synthesis, the ASP also presents bioenergetic functions in nitrogen and energy metabolism [60]. The plasmatic Asp levels in CDCS patients were increased (33 ± 10%) in comparison with the control group. These results corroborate with the findings of Lejeune [16], who observed increased plasmatic concentrations of asparagine and aspartate in patients with CDCS and

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concluded that increases in these amino acids are associated with deficient purine biosynthesis [16].

4. Conclusion The plasmatic metabolite profile of patients with CDCS is characterized and correlated with different metabolic pathways. The observed increase of MetO, Asp, Glu and 5-HT levels might cause disturbances of the following metabolic pathways, respectively: oxidative stress processes, purine biosynthesis, the citric acid cycle and serotonergic biosynthesis. In contrast, the decrease of Asn, Cit, Gln, His, Ile, Leu, Met, Orn, Phe, Thr, Trp, Tyr, Val and creatinine levels in plasma can reflect disorders of the urea cycle, citric acid cycle, branched chain amino acid synthesis, tyrosine metabolism and amino acid biosynthesis. It is alluring to propose that the observed unbalanced metabolic state in CDCS patients can be compensated with amino acid supplementation beginning after birth. This study is expected to provide clues for the clarification of the network of deficient enzymes and disturbed metabolic pathways that underlie the adverse physical and neuropsychiatric picture of CDCS patients. Altogether, these data give rise to the speculation that restriction or supplementation of certain amino acids in early CDCS infancy may ameliorate patient health and improve quality of life.

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Funding This work was supported by the FAPESP through grants (2013/21455-6, 2013/07763-0 and 2012/02514-9) as well as the CAPES for scholarship and the FINEP for equipment

. Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethics approval and consent to participate The Trial was approved by the the Research Ethics Committee of the Federal University of São Paulo (Escola Paulista de Medicina Paulista, Number: 226594). All participants signed written informed consent before any trial related intervention.

Acknowledgements We would like to especially thank the Brazilian Association for Research and Support of the families of Cri-du-chat in Brazil by providing the samples.

References

[1] L.E. Rosenberg, Diagnosis and management of inherited aminoacidopathies in the newborn and the unborn, Clin. Endocrinol. Metab. 3 (1974) 145–152. [2] L. Waber, Inborn errors of metabolism, Pediatr. Ann. 19 (1990) 105–109, 112–113, 117–118.

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[3] P.E. Karam, M.-Z. Habbal, M.A. Mikati, G.E. Zaatari, N.K. Cortas, R.T. Daher, Diagnostic challenges of aminoacidopathies and organic acidemias in a developing country: A twelve-year experience, Clin. Biochem. 46 (2013) 1787–1792. doi:10.1016/j.clinbiochem.2013.08.009. [4] C.G. Barbosa, N.S. Gonçalves, E.J.H. Bechara, N.A. Assunção, Potential Diagnostic Assay for Cystinuria by Capillary Electrophoresis Coupled to Mass Spectrometry, J. Braz. Chem. Soc. (2013). doi:10.5935/0103-5053.20130085. [5] M. Higurashi, M. Oda, K. Iijima, S. Iijima, T. Takeshita, N. Watanabe, K. Yoneyama, Livebirth prevalence and follow-up of malformation syndromes in 27,472 newborns, Brain Dev. 12 (1990) 770–773. doi:10.1016/S03877604(12)80004-0. [6] E. Niebuhr, The Cri du Chat syndrome: epidemiology, cytogenetics, and clinical features, Hum. Genet. 44 (1978) 227–275. [7] J. Lejeune, J. Lafourcade, R. Berger, J. Vialatte, M. Boeswillwald, P. Seringe, R. Turpin, [3 Cases of partial deletion of the short arm of a 5 chromosome], Comptes Rendus Hebd. Séances Académie Sci. 257 (1963) 3098–3102. [8] J. Lejeune, J. Lafourcade, R. Berger, R. Turpin, [Familial segregation of a 5-13 translocation determining partial monosomy and a trisomy of the short arm of the 5 chromosome: “Cat cry” disease and its “Receprocal”], Comptes Rendus Hebd. Séances Académie Sci. 258 (1964) 5767–5770. [9] I. Kjaer, E. Niebuhr, Studies of the cranial base in 23 patients with cri-du-chat syndrome suggest a cranial developmental field involved in the condition, Am. J. Med. Genet. 82 (1999) 6–14. [10] A. Basinko, M.L. Giovannucci Uzielli, G. Scarselli, M. Priolo, G. Timpani, M. De Braekeleer, Clinical and molecular cytogenetic studies in ring chromosome 5: Report of a child with congenital abnormalities, Eur. J. Med. Genet. 55 (2012) 112– 116. doi:10.1016/j.ejmg.2011.11.005. [11] K.M. Cornish, G. Cross, A. Green, L. Willatt, J.M. Bradshaw, A neuropsychological-genetic profile of atypical cri du chat syndrome: implications for prognosis, J. Med. Genet. 36 (1999) 567–570. [12] M.S.R. Collins, J. Eaton-Evans, Growth study of cri du chat syndrome, Arch. Dis. Child. 85 (2001) 337–338. [13] P. Cerruti Mainardi, Cri du Chat syndrome, Orphanet J. Rare Dis. 1 (2006) 33. doi:10.1186/1750-1172-1-33. [14] L.E. Wilkins, J.A. Brown, B. Wolf, Psychomotor development in 65 home-reared children with cri-du-chat syndrome, J. Pediatr. 97 (1980) 401–405. [15] U. Kühner, M. Büsse, G. Buchinger, Cir-du-chat syndrome with an increased level of proline and threonine, Z. Für Kinderheilkd. 117 (1974) 259–264. [16] J. Lejeune, M.O. Rethoré, M. Peeters, M.C. de Blois, D. Rabier, P. Parvy, J. Bardet, P. Kamoun, [Cri-du-chat disease: plasma and urinary amino acids], Ann. Génétique. 33 (1990) 16–20. [17] E.J.H. Bechara, F. Dutra, V.E.S. Cardoso, A. Sartori, K.P.K. Olympio, C.A.A. Penatti, A. Adhikari, N.A. Assunção, The dual face of endogenous α-aminoketones: Pro-oxidizing metabolic weapons, Comp. Biochem. Physiol. Part C Toxicol. Pharmacol. 146 (2007) 88–110. doi:10.1016/j.cbpc.2006.07.004.

21

[18] A. Hubaux, G. Vos, Decision and detection limits for calibration curves, Anal. Chem. 42 (1970) 849–855. doi:10.1021/ac60290a013. [19] A. Drazic, J. Winter, The physiological role of reversible methionine oxidation, Biochim. Biophys. Acta BBA - Proteins Proteomics. 1844 (2014) 1367–1382. doi:10.1016/j.bbapap.2014.01.001. [20] H. Weissbach, F. Etienne, T. Hoshi, S.H. Heinemann, W.T. Lowther, B. Matthews, G. St. John, C. Nathan, N. Brot, Peptide Methionine Sulfoxide Reductase: Structure, Mechanism of Action, and Biological Function, Arch. Biochem. Biophys. 397 (2002) 172–178. doi:10.1006/abbi.2001.2664. [21] R.L. Levine, L. Mosoni, B.S. Berlett, E.R. Stadtman, Methionine residues as endogenous antioxidants in proteins, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 15036–15040. [22] M.M. Trintinalia, A.N.L. Alves, L. Fernandes, E.J.H. Bechara, N.A. Assunção, Potential Diagnostic of Branched-Chain Ketoaciduria by HPLC-DAD, J. Braz. Chem. Soc. (2014). doi:10.5935/0103-5053.20140156. [23] J.T. Brosnan, M.E. Brosnan, Branched-chain amino acids: enzyme and substrate regulation, J. Nutr. 136 (2006) 207S–211S. [24] P.J. Garlick, I. Grant, Amino acid infusion increases the sensitivity of muscle protein synthesis in vivo to insulin. Effect of branched-chain amino acids, Biochem. J. 254 (1988) 579–584. [25] A. Valerio, G. D’Antona, E. Nisoli, Branched-chain amino acids, mitochondrial biogenesis, and healthspan: an evolutionary perspective, Aging. 3 (2011) 464–78. [26] J.S.A. Mattick, K. Kamisoglu, M.G. Ierapetritou, I.P. Androulakis, F. Berthiaume, Branched-chain amino acid supplementation: impact on signaling and relevance to critical illness, Wiley Interdiscip. Rev. Syst. Biol. Med. 5 (2013) 449–460. doi:10.1002/wsbm.1219. [27] J.L. Chaussain, P. Georges, D. Gendrel, M. Donnadieu, J.C. Job, Serum branchedchain amino acids in the diagnosis of hyperinsulinism in infancy, J. Pediatr. 97 (1980) 923–926. [28] H. Leweling, R. Breitkreutz, F. Behne, U. Staedt, J.P. Striebel, E. Holm, Hyperammonemia-induced depletion of glutamate and branched-chain amino acids in muscle and plasma, J. Hepatol. 25 (1996) 756–762. [29] M. Holecek, L. Sprongl, M. Tichý, Effect of hyperammonemia on leucine and protein metabolism in rats, Metabolism. 49 (2000) 1330–1334. doi:10.1053/meta.2000.9531. [30] J. Dancis, M. Levitz, R.G. Westall, Maple syrup urine disease: branched-chain ketoaciduria, Pediatrics. 25 (1960) 72–79. [31] C.R. Scriver, C.L. Clow, S. Mackenzie, E. Delvin, Thiamine-responsive maplesyrup-urine disease, The Lancet. 297 (1971) 310–312. [32] R. Meeusen, K. De Meirleir, Exercise and brain neurotransmission, Sports Med. Auckl. NZ. 20 (1995) 160–188. [33] J.D. Fernstrom, Dietary amino acids and brain function, J. Am. Diet. Assoc. 94 (1994) 71–77. [34] J.D. Fernstrom, R.J. Wurtman, Brain serotonin content: physiological regulation by plasma neutral amino acids, Science. 178 (1972) 414–416.

22

[35] C. Bell, J. Abrams, D. Nutt, Tryptophan depletion and its implications for psychiatry, Br. J. Psychiatry J. Ment. Sci. 178 (2001) 399–405. [36] D. Huffman, T. Altena, T. Mawhinney, T. Thomas, Effect of n-3 fatty acids on free tryptophan and exercise fatigue, Eur. J. Appl. Physiol. 92 (2004). doi:10.1007/s00421-004-1069-6. [37] R.S. Jones, Tryptamine: a neuromodulator or neurotransmitter in mammalian brain?, Prog. Neurobiol. 19 (1982) 117–139. [38] T. Jenkins, J. Nguyen, K. Polglaze, P. Bertrand, Influence of Tryptophan and Serotonin on Mood and Cognition with a Possible Role of the Gut-Brain Axis, Nutrients. 8 (2016) 56. doi:10.3390/nu8010056. [39] T. Ardis, M. Cahir, J. Elliott, R. Bell, G. Reynolds, S. Cooper, Effect of acute tryptophan depletion on noradrenaline and dopamine in the rat brain, J. Psychopharmacol. (Oxf.). 23 (2009) 51–55. doi:10.1177/0269881108089597. [40] J.M. Davis, Carbohydrates, branched-chain amino acids, and endurance: the central fatigue hypothesis, Int. J. Sport Nutr. 5 Suppl (1995) S29-38. [41] D.M. Richard, M.A. Dawes, C.W. Mathias, A. Acheson, N. Hill-Kapturczak, D.M. Dougherty, L-Tryptophan: Basic Metabolic Functions, Behavioral Research and Therapeutic Indications, Int. J. Tryptophan Res. IJTR. 2 (2009) 45–60. [42] S.E. Hufton, I.G. Jennings, R.G. Cotton, Structure and function of the aromatic amino acid hydroxylases., Biochem. J. 311 (1995) 353–366. [43] J. Zschocke, Phenylketonuria mutations in Europe, Hum. Mutat. 21 (2003) 345–356. doi:10.1002/humu.10192. [44] A.N. Fonteh, R.J. Harrington, A. Tsai, P. Liao, M.G. Harrington, Free amino acid and dipeptide changes in the body fluids from Alzheimer’s disease subjects, Amino Acids. 32 (2007) 213–224. doi:10.1007/s00726-006-0409-8. [45] S. Kapur, J. John Mann, Role of the dopaminergic system in depression, Biol. Psychiatry. 32 (1992) 1–17. doi:10.1016/0006-3223(92)90137-O. [46] M.H. Silla Santos, Biogenic amines: their importance in foods, Int. J. Food Microbiol. 29 (1996) 213–231. [47] B. Wilcken, Problems in the management of urea cycle disorders, Mol. Genet. Metab. 81 Suppl 1 (2004) S86-91. doi:10.1016/j.ymgme.2003.10.016. [48] C. Nieves, B. Langkamp-Henken, Arginine and immunity: a unique perspective, Biomed. Pharmacother. Bioméd. Pharmacothérapie. 56 (2002) 471–482. [49] N.E. Flynn, C.J. Meininger, T.E. Haynes, G. Wu, The metabolic basis of arginine nutrition and pharmacotherapy, Biomed. Pharmacother. Bioméd. Pharmacothérapie. 56 (2002) 427–438. [50] H.E. Sauberlich, R.P. Dowdy, J.H. Skala, Laboratory tests for the assessment of nutritional status, CRC Crit. Rev. Clin. Lab. Sci. 4 (1973) 215–340. [51] S. Mercimek-Mahmutoglu, G.S. Salomons, Creatine Deficiency Syndromes, in: R.A. Pagon, M.P. Adam, H.H. Ardinger, S.E. Wallace, A. Amemiya, L.J. Bean, T.D. Bird, C.-T. Fong, H.C. Mefford, R.J. Smith, K. Stephens (Eds.), GeneReviews(®), University of Washington, Seattle, Seattle (WA), 1993. http://www.ncbi.nlm.nih.gov/books/NBK3794/ (accessed May 31, 2016). [52] M.M. Thakkar, Histamine in the regulation of wakefulness, Sleep Med. Rev. 15 (2011) 65–74. doi:10.1016/j.smrv.2010.06.004.

23

[53] J.W. Peterson, I. Boldogh, V.L. Popov, S.S. Saini, A.K. Chopra, Anti-inflammatory and antisecretory potential of histidine in Salmonella-challenged mouse small intestine, Lab. Investig. J. Tech. Methods Pathol. 78 (1998) 523–534. [54] M. Watanabe, M.E. Suliman, A.R. Qureshi, E. Garcia-Lopez, P. Bárány, O. Heimbürger, P. Stenvinkel, B. Lindholm, Consequences of low plasma histidine in chronic kidney disease patients: associations with inflammation, oxidative stress, and mortality, Am. J. Clin. Nutr. 87 (2008) 1860–1866. [55] O.I. Pisarenko, Mechanisms of myocardial protection by amino acids: facts and hypotheses, Clin. Exp. Pharmacol. Physiol. 23 (1996) 627–633. [56] C.M. Mano, F.M. Prado, J. Massari, G.E. Ronsein, G.R. Martinez, S. Miyamoto, J. Cadet, H. Sies, M.H.G. Medeiros, E.J.H. Bechara, P. Di Mascio, Excited singlet molecular O2 (1Δg) is generated enzymatically from excited carbonyls in the dark, Sci. Rep. 4 (2014). doi:10.1038/srep05938. [57] P.S. Peres, A. Valerio, S.M.S.C. Cadena, S.M.B. Winnischofer, A.C. Scalfo, P. Di Mascio, G.R. Martinez, Glutathione modifies the oxidation products of 2′deoxyguanosine by singlet molecular oxygen, Arch. Biochem. Biophys. 586 (2015) 33–44. doi:10.1016/j.abb.2015.09.020. [58] N. Kalant, S. McCormick, Inhibition by serum components of oxidation and collagen-binding of low-density lipoprotein, Biochim. Biophys. Acta. 1128 (1992) 211–219. [59] R.G. Taylor, H.L. Levy, R.R. McInnes, Histidase and histidinemia. Clinical and molecular considerations, Mol. Biol. Med. 8 (1991) 101–116. [60] L.D. Stegink, Absorption, utilization, and safety of aspartic acid, J. Toxicol. Environ. Health. 2 (1976) 215–242. doi:10.1080/15287397609529428.

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Fig. 1 Difference of variation (percentage) of each metabolite between CDCS patients and control group. Arg (1), Asn (2), Asp (3), Cit (4), Leu (5), Ile (6) Val (7), Gly (8) Ala (9) Gln (10), Glu (11), His (12), Orn (13), Met (14), Phe (15), Tyr (16), Pro (17), Ser (18), Thr (19), AcOrn (20), Adma (21), Alpha AAA (22), Creatinine (23), Dopa (24), histamine (25), MetO (26), Kyn (27), N-tyr (28), sarcosine (29), 5-HT (30) taurine (31), Trp (32) and Lys (33). * Variation of metabolites significant.

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Fig. 2 Principal Component Analysis of metabolites Val, Leu and Ile in CDCS patients ( ) and control ( ) group.

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Fig. 3 Box Plot of Trp (n = 36)/ΣNA (n = 36) of CDCS pateints and the control group. NA: Neutral amino acids. ºOutliers. Σ: Plus

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Fig. 4 Principal Component Analysis of metabolites Arg, Orn and Cit in the plasma of CDCS patients ( ) and the control ( ) group.

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Fig. 5 Principal Component Analysis of the metabolites Gly, Arg and creatinine found in the plasma of CDCS patients ( ) and the control ( ) group.

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Table 1. Comparison of metabolite levels in plasma of CDCS patients and control group. Metabolites Ala AcOrn Adma Alpha-aaa Dopa Gly Histamine Kyn Lys N-Tyr Pro Sarcosine Ser Taurine Asp Glu 5-HT MetO Arg Asn Cit Creatinine Gln His Ile Leu Met Orn Phe Thr Tpr Tyr Val

Control Mean (µmol L-1) 366.50 ± 2.85 2.90 ± 0.01 0.50 ± 0.03 1.10 ± 0.07 0.30 ± 0.01 261.90 ± 1.32 0.70 ± 0.01 2.70 ± 0.03 219.30 ± 10.00 0.10 ± 0.01 187.90 ± 1.07 20.00 ± 0.33 127.90 ± 0.94 72.40 ± 0.36 7.10 ± 0.85 66.00 ± 1.49 0.10 ± 0.01 0.20 ± 0.03 73.80 ± 0.26 52.10 ± 1.10 33.50 ± 0.60 58.60 ± 3.80 555.60 ± 3.08 88.20 ± 0.34 65.90 ± 0.38 133.10 ± 3.23 25.60 ± 0.19 91.30 ± 0.31 61.30 ± 0.81 137.30 ± 1.11 66.80 ± 0.24 69.90 ± 0.28 240.70 ± 0.62

CDCS patients Comparison ↔

Mean (µmol L-1) 324.90 ± 1.37

↔ ↔ ↔ ↔ ↔ ↔ ↔ ↔ ↔ ↔ ↔ ↔ ↔ ↑ ↑ ↑ ↑ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

2.80 ± 0.02 0.50 ± 0.03 1.00 ± 0.07 0.30 ± 0.01 275.40 ± 1.13 0.70 ± 0.01 2.50 ± 0.02 200.50 ± 14.00 0.10 ± 0.01 226.00 ± 1.26 18.00 ± 0.29 122.20 ± 0.91 76.50 ± 0.41 10.60 ± 1.31 79.10 ± 2.00 0.30 ± 0.01 0.40 ± 0.03 67.50 ± 0.41 43.00 ± 1.47 25.60 ± 0.51 37.50 ± 3.39 497.20 ± 4.86 77.40 ± 0.39 55.30 ± 0.37 94.80 ± 2.57 24.00 ± 0.19 67.30 ± 0.36 54.50 ± 0.82 119.40 ± 0.97 56.90 ± 2.79 67.00 ± 0.39 192.50 ± 0.69

N = 36, mean ± standard error; ↑ significant increase as compared to the control group; ↓ significant decrease as compared to the control group; ↔ no significant differences (Wilcoxon test). 30