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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 31  |  Issue : 3  |  Page : 742-752

Study of acylcarnitine and amino acid profiles in hyperammonemia pediatric patients


1 Department of Clinical Biochemistry, National Liver Institute, Menoufia, Egypt
2 Department of Medical Biochemistry, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Genetics Unit, Pediatrics Hospital, Faculty of Medicine, Ain Shams University, Ain Shams, Egypt

Date of Submission17-Feb-2016
Date of Acceptance03-Jul-2016
Date of Web Publication31-Dec-2018

Correspondence Address:
Shimaa Abd El Sattar
Quesna, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.248715

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  Abstract 


Objective
The aim of this study was to study amino acid and acylcarnitine profiles in Egyptian pediatric patients with hyperammonemia using high-performance liquid chromatography (HPLC).
Background
Hyperammonemia is a life-threatening problem during childhood that requires prompt intervention. Emerging metabolomics such as amino acid and acylcarnitine assay provide a powerful platform for discovering new biomarkers to improve early diagnosis.
Patients and methods
A total of 110 pediatric patients were enrolled in this study: 40 patients with hyperammonemia suspected as having an inborn error of metabolism, 20 hyperammonemia patients suspected as having hepatic disorders, and 50 apparently healthy children who served as the control group. Routine laboratory investigations were carried out for all participants (blood ammonia, liver function tests, kidney function tests, and arterial blood gas analysis). Amino acid and acylcarnitine profiles were measured quantitatively for all participants using HPLC.
Results
Fourteen metabolites were supposed to be ‘potential metabolite markers’ for differentiation between patients with hyperammonemia due to inborn error of metabolism and those with hyperammonemia due to hepatic causes. Seven of these metabolites were amino acids (Gly : Ala, Leu-Ile, Leu : Phe, Met : Phe, methionine, tyrosine, and valine), and seven were acylcarnitines (C3, C3 : C2, C5-DC, C10 : C1, C16-OH, C18, and C14-carnitines).
Conclusion
Acylcarnitine and amino acid profiles detected using HPLC could be potential noninvasive diagnostic biomarkers for differentiation of hyperammonemia cases.

Keywords: acylcarnitine, high-performance liquid chromatography, hyperammonemia, inborn error of metabolism


How to cite this article:
Abd El Sattar S, Obada M, El ghobashy Y, Abou-El Nour E, Zaki O, El-Said H. Study of acylcarnitine and amino acid profiles in hyperammonemia pediatric patients. Menoufia Med J 2018;31:742-52

How to cite this URL:
Abd El Sattar S, Obada M, El ghobashy Y, Abou-El Nour E, Zaki O, El-Said H. Study of acylcarnitine and amino acid profiles in hyperammonemia pediatric patients. Menoufia Med J [serial online] 2018 [cited 2019 Jun 19];31:742-52. Available from: http://www.mmj.eg.net/text.asp?2018/31/3/742/248715




  Introduction Top


Hyperammonemia refers to a clinical condition associated with elevated ammonia levels manifested by a variety of symptoms and signs, including significant central nervous system abnormalities[1]. The causes of hyperammonemia are diverse and include liver failure, infection, medications, and inborn errors of metabolism (IEM)[2]. IEM that can cause hyperammonemia occur even less frequently and include urea cycle disorders, organic acidopathies, disorders of pyruvate metabolism, and mitochondrial fatty acid oxidation defects[3].

Free fatty acids are metabolized through β-oxidation that is mediated by l-carnitine. l-Carnitine is present in free and esterified forms in tissues and body fluids. It has multiple metabolic functions, including transport of long-chain fatty acids into the mitochondria for β-oxidation, transfer of short-chain and medium-chain acyl groups from the peroxisome to mitochondria, regulation of intracellular acyl-CoA/free CoA ratio, and export of toxic acyl residues from the mitochondria[4]. Because there is an accumulation of acylcarnitines, it is regarded as indicative of mitochondrial dysfunction and impaired cellular fatty acid metabolism[5]. Acylcarnitine analysis is now widely available as a noninvasive initial investigation in patients suspected as having underlying disorders of lipid metabolism[6].

Amino acid is the simple unit after protein hydrolysis[7]. Levels of circulating amino acids mainly reflect the hepatic metabolism, as the liver is the key organ in amino acid metabolism and hence in acute and chronic liver diseases[8],[9],[10].

Hyperammonemia is a life-threatening condition that can affect patients at any age and most symptomatic patients are diagnosed at an older age. If not identified and treated rapidly, the newborn will have irreversible neurological effects. In addition, poor medication compliance reduces the efficacy of treatment. Hence, more extensive screening is imperative for ensuring early diagnosis and enhancing the treatment efficacy of IEM[11].

Metabolomics is concerned with the measurement of global sets of low molecular weight metabolites, which can be regarded as important indicators of various states of disease. By analyzing and verifying the specific biomarkers of a disease, metabolomics enables us to better understand pathological processes and substance in metabolic pathways. Compared with traditional diagnostic methods, even small changes in metabolites can help in detecting early pathologic changes more sensitively[12].

The most commonly used platforms for the detection and measurement of metabolites involve their separation using gas chromatography, liquid chromatography, or capillary electrophoresis coupled with subsequent Mass Spectrometer (MS)[13].

Liquid chromatography is currently the most commonly MS-based approach for metabolomics application because of its sensitivity and rich information content. Ultra-high-pressure liquid chromatography approach has significantly improved the chromatic resolution and reduced the limit of detection by 3–5-fold[14].

In this regard, the present study aimed to investigate the amino acid and acylcarnitine profiles in hyperammonemia pediatric patients to provide early diagnosis for the metabolic disorders, which led to better outcome using high-performance liquid chromatography (HPLC) as a sensitive and accurate assay.


  Patients and Methods Top


Study population

This study was conducted at the Chromatography Unit in Clinical Biochemistry Laboratory, National Liver Institute, Menoufia University. The protocol of the study was approved by the ethics committee of Faculty of Medicine, Menoufia University, and enrollment of the individuals in the study was conditioned by obtaining written informed consent from patients' parents or their legal guardians. The patients were selected from two different places during the period from August 2013 to August 2014: the Medical Genetic Unit, Pediatric Hospital, Ain Shams University, and inpatient hepatic wards, National Liver Institute, Menoufia University.

A total of 110 pediatric patients were enrolled in this work, comprising 60 patients with hyperammonemia who were divided according to initial provisional diagnosis into two groups.

Group I included 40 patients with clinical signs and symptoms suggestive of IEM. Their ages ranged from 2 to 84 months. Group I comprised 16 male and 24 female patients. Group II included 20 patients with clinical signs and symptoms suggestive of hepatic disorders. Their ages ranged from 1 to 72 months and included nine male and 11 female patients. In addition, a third group (group III) comprising 50 apparently healthy children matched for age and sex of the diseased groups served as the control group. Their ages ranged from 1 to 80 months. There were 24 male and 26 female patients. All patients and controls were subjected to full history taking and physical examination.

Laboratory investigations

Five milliliters of venous blood was collected from all participants and divided into two samples; one sample (3 ml) was collected in a plain vacutainer tube, left to coagulate, and centrifuged, and then the sera were used for routine laboratory investigations (liver function tests and kidney function tests) using the Beckman Coulter (Synchron CX 9 ALX) Clinical Auto analyzer (Beckman Instruments, Fullerton, California, USA). The other sample (2 ml) was collected into a tube containing an anticoagulant, either EDTA or heparin, and transported on ice, and centrifuged as soon as possible; plasma was separated within 10 min of collection and used immediately for the measurement of ammonia.

One milliliter of arterial specimen for arterial blood gas analysis was collected anaerobically in a 3 ml plastic airtight syringe containing heparin. Arterial blood gases were measured with an automated MedicaEasyLyte Analyzer microprocessor-controlled electrolyte system (Medica Corporation, 5 Oak Park Drive Bedford, MA, USA) that uses ion selective electrode.

The bloodspot for amino acid and acylcarnitine assay was taken by pricking the heel of the infant or the big finger of older children using filter paper (Guthrie card made of Whatman 903) purchased from GE Healthcare (New Jersey, New York, USA). Blood spots were dried for 4 h on a dry, horizontal, nonabsorbent surface at ambient temperature and stored at −80°C until analysis using triple-quadruple tandem mass spectrometer (Acquity UPLC H-Class; Waters Corporation, Massachusetts, USA) with a positive electrospray ionization probe, using MassChrom amino acids and acylcarnitines from dried blood/nonderivatized kit (Chromsystems Instruments & Chemicals GmbH, München, Germany).

Assay procedure[15],[16]

Internal standard preparation: The lyophilized isotopically labeled internal standard was reconstituted with 25 ml of extraction buffer provided in the kit (Chromsystems Instruments and Chemicals GmbH) to produce an internal standard of certain concentrations found on the information leaflet accompanying each kit; it is used as calibration standard for each sample.

Sample preparation and assay methods: Three millimeters of dried blood spot disk was punched out of the filter card into a well of v-bottomed plate (3 mm spot is equivalent to a blood volume of ~3.1 μl); 100 μl of the reconstituted internal standard was added to each dried blood spot disk, and then the plate was sealed with a protective sheet. Afterward, the plate was agitated at 600 rpm for 20 min at ambient temperature. The supernatant was transferred into a new v-bottomed well plate and sealed with aluminum foil protective sheet. Thereafter, the samples were ready for injection. Ten microliters of the elute was injected into the LC-MS/MS system, at a 2-min interval in a flowing stream of 80% acetonitrile, where the flow of mobile phase (provided by the kit) was adjusted to 200 μl/min to be reduced to 20 μl/min at 0.25 min and up again to 600 μl/min at 1.25 min to be reduced again to 200 μl/min as the scan time of the tandem MS system has to be set at 1.25 min.

Statistical analysis

Data analysis was performed using SPSS software for Windows (version 16.00; SPSS, Inc., Chicago, Illinois, USA). Differences between patients and controls were evaluated using Student's unpaired t-test (for continuous variables) and the χ2-test (for categorical variables).

The Mann–Whitney U-test is applied for statistical comparison between two sets of data if one or both of them have a skewed distribution. A P value less than 0.05 was considered statistically significant. Principal component analysis (PCA) was used as a multivariate technique for exploratory analysis in chromatography to identify the metabolites that hold the potential to augment the metabolic biomarkers gleaned from blood spot. Receiver operating characteristic curves were drowning based on the data of PCA as a routine screening or surveillance for the causes of hyperammonemia.


  Results Top


The mean age of presentation of patients in group I was 32.63 ± 23.9 months (40% were male), 31.85 ± 21.45 months in group II (45% were male), and 34 ± 12 months in group III (48% were male). The groups were homogenous.

[Table 1] presents the demographic data of the patients (group I and group II).
Table 1: Demographic data of group I and group II

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Nonreducing sugars were not detected in group I, whereas they were detected in three patients in group II; ketones in urine were detected in 30% of patients in group I and in 25% of patients in group II. Consanguinity was noticed in 82.5% of patients in group I and in 30% of patients in group II. Presence of similar cases was 55% in group I and 10% in group II. Associated liver disease was limited to group II only.

[Table 2] presents frequency of the presenting signs and symptoms of patients in groups I and II.
Table 2: Frequency of the presenting signs and symptoms of patients and some biochemical parameters in group I and group II

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In group I, general developmental delay was the main presenting symptom (82.5%), followed by metabolic acidosis (75%) and unexplained neurological abnormalities (hypotonia 55% and seizure 47.5%). Meanwhile, in group II, jaundice was the main presenting symptom, followed by disturbed conscious level and metabolic acidosis.

[Table 3] presents the comparison between the studied groups as regards some clinical and biochemical parameters.
Table 3: Comparison between the studied groups as regards some biochemical parameters

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There was a statistically significant decrease in PO2, PCO2, and HCO3 and a statistically significant increase in anion gap in group I compared with group III.

There was a statistically significant decrease in HCO3 and PO2 in group II compared with group III.

There was no significant difference between the two patients' groups (group I and group II) as regards the studied parameters except anion gap, which showed a significant increase in group I compared with group II.

As regards ammonia, the mean serum level showed a significant increase in the patients' groups (group I and group II) compared with the control group (group III).

There was a significant increase in the mean serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total and direct bilirubin and a significant decrease in the mean serum level of albumin in group II compared with the other two groups (group I and group III).

Meanwhile, the mean serum level of creatinine showed no significant difference between the three studied groups.

[Table 4] presents the analysis of acylcarnitine profile in the studied groups.
Table 4: Comparison among the studied groups as regards acylcarnitine profile analysis (μmol/l)

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In group I, there was a significant increase in (C0), C16, C2, C3, (C3: C2 ratio), C4, C5, C5-DC, C6, C10: C1 ratio, C18, and C4-DC compared with the control group (group III).

There was a significant decrease in decanoylcarnitine (C10) and 3-hydroxyhexadecanoyl-carnitine, (C16-OH) compared with controls (group III).

There was a significant increase in C16, C3: C2 ratio, C5-DC, and C10: C1 ratio and a significant decrease in C14, C16, C16-OH, and C18 compared with group II.

In group II (hyperammonemia due to hepatic cause), there was a significant increase in long-chain acyl carnitine [(C14), (C16), and (C18)], (C0), short-chain acyl carnitine [(C2), (C3), and (C4)], medium-chain acyl carnitine [(C5) and (C6)], and C4-OH compared with the control group (group III).

[Table 5] presents analysis of amino acid profile in the studied groups.
Table 5: Quantitative determination of whole blood amino acids level using high-performance liquid chromatography in the studied groups

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In group I (hyperammonemia due to IEM), the results were as follows:

  • There was a significant increase in amino acid alanine, glycine, phenylalanine and Gly: Ala, Leu: Ile, Leu: Phe, and Phe: Tyr ratios; meanwhile, there was a significant decrease in Gly: Ala ratio compared with group III.
  • There was a significant increase in amino acid (valine) and a significant decrease in amino acids (methionine and tyrosine) compared with group II.


In group II (hyperammonemia due to hepatic causes), there was a significant increase in amino acids (alanine, glycine, methionine, ornithine, phenylalanine, and glutamic acid) and Met: Phe ratio and a significant decrease in amino acids (valine) and Leu: Ile, Leu: Ala ratios compared with group III.

Univariate individual analysis of amino acids and acylcarnitines profiles of the two patients' groups (group I and group II)

Seven amino acids and ratios (methionine, tyrosine, Met: Phe, Gly: Ala were increased in group II and Leu: Ile, Leu: Phe, and valine were decreased in group II) and seven acylcarnitines species (C14, C16, C16-OH, and C18 were increased in group II and C5-DC, C10: C1, and C3: C2 were decreased in group II) could be potential biomarkers for differentiation between the two groups (P < 0.05). These markers are presented in [Table 6]. Further analysis was performed using score plots of the first two principal components for visualization of the data and establishing the differences between the two diseased groups.
Table 6: Detected significant amino acids and acylcarnitines markers for differentiation between group I and group II using principal component analysis models

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Using principal component analysis models based on high-performance liquid chromatography analysis of amino acid markers and acylcarnitines metabolites

Using PCA models based on HPLC analysis of amino acid markers and acylcarnitines metabolites, there was a good discrimination between the two diseased groups by seven amino acids markers with good separation of the samples in two different places; area under the curve (AUC) was 0.770 [Figure 1] and [Figure 2].
Figure 1: Principal component analysis (PCA) plot score 1 versus score 2 discriminating group I from group II based on high-performance liquid chromatography (HPLC) amino acid markers showing good separation of the samples in two different places.

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Figure 2: Receiver operating characteristic (ROC) curve determined using the high-performance liquid chromatography (HPLC); principal component analysis (PCA) models of amino acids showing that the area under the curve was 0.770.

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There was mild separation between the two groups by seven acylcarnitines species with moderate scattering of the samples in two different places; AUC was 0.643 [Figure 3] and [Figure 4].
Figure 3: Principal component analysis (PCA) plot score 1 versus score 2 discriminating group I from group II based on high-performance liquid chromatography (HPLC) acylcarnitines markers showing moderate scattering of the samples in two different places.

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Figure 4: Receiver operating characteristic (ROC) curve determined using the high-performance liquid chromatography (HPLC), principal component analysis (PCA) models of acylcarnitines showing that the area under the curve was 0.643.

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  Discussion Top


This study highlights the importance of HPLC as a analytical technique for qualitative analyses of dried blood spot samples from neonates and infants suspected of having IEM.

The present study noticed that there was a delay in the diagnosis of cases in group I (during second year of life). This was attributed to the lack of awareness of these genetic diseases by the Egyptian physicians. Moreover, there was female sex predominance in group I, which could be due to the autosomal recessive mode of inheritance of these disorders of IEM[17].

Delayed discovery of these cases make them remain completely undiagnosed or misdiagnosed with noninheritable disorders and were subjected to recurrent metabolic crises with its concomitant irreversible damage[18]. These results are in line with the studies byEl-Mesallamy et al.[18] and El-Mesallamy et al.[19].

The present study noticed a strong parental consanguinity (82.5%) with increased number of similar cases in consanguineous families (55%) and unexplained death among these families in group I. This could be attributed to the lack of early detection of such disorders, lack of genetic follow-up, and prenatal diagnosis for most of them, as well as lack of proper genetic counseling when having a diagnosed case[20]. These findings reflect the major roles of consanguinity in the existence of these disorders in Egypt, as most IEM are autosomal recessive. This was also reported byEl-Mesallamy et al.[19] and Selim et al.[20].

The most important laboratory feature of many of the IEM during acute episodes of illness after hyperammonemia is metabolic acidosis with an increased anion gap, readily demonstrable by measurement of arterial blood gases and bicarbonate[21]. The presence of moderate-to-severe metabolic acidosis indicates that hyperammonemia is a disturbance of either organic acid metabolism or β-oxidation of fatty acids or due to congenital lactic acidosis[22].

In this regard, the present study showed that there was a significant increase in anion gap in group I compared with both groups II and III. An increased anion gap (>16) observed in many of the disorders in group I could be due to the accumulation of fixed acids such as lactic acid, ketoacids, and other organic acids[22].

Hyperammonemia is a rare, life-threatening problem during childhood that requires prompt intervention[23]. A plasma ammonia level should be obtained for any child with unexplained vomiting, lethargy, or other evidence of an encephalopathy and should be determined early with the first screening tests to introduce treatment before any brain damage[21].

Hyperammonemia was a prerequisite to be enrolled in this study; we noticed that ammonia level showed a significant increase in group II compared with group I and was significantly elevated in the patients' groups (I and II) compared with controls.

Hepatic hyperammonemia is usually seen in the setting of decompensated liver disease when the diagnosis is reasonably straightforward because of accompanying signs of chronic liver disease[24]. Liver function tests will help identify whether or not hyperammonemia is the result of hepatic dysfunction[22]. In this study, there was a statistically significant increase in the serum levels of AST, ALT, and total and direct bilirubin and a significant decrease in the serum level of albumin in group II compared with group I. These findings were attributed to the liver affection and hepatocyte injury (ALT and AST), different stages of liver diseases (bilirubin), as bilirubin acts as markers of biliary function and cholestasis, and decreased liver function following hepatocellular disease (albumin)[25],[26]. However, a low serum albumin concentration is a late finding in liver disease; when it is present it suggests chronic disease[27].

A breakthrough in the technology, particularly the use of MS/MS has allowed for the development of fast and inexpensive protocols to quantify amino, organic, and fatty acids disorders from a single dried blood spot[17].

Acylcarnitine profile analysis is performed for the biochemical screening of disorders of fatty acid oxidation and organic acid metabolism[28]. The conditions revealed by acylcarnitine analysis have in common the accumulation of C2–C18 acyl-CoA species, which are substrates for one of several carnitine acyl-CoA transferases expressed in different intracellular compartments[29].

The current study detected a significant increase in 3-hydroxyhexadecanoyl-carnitine (C16-OH) and of long-chain acylcarnitine [tetradecanoylcarnitine (C14), hexadecanoylcarnitine (C16), and octadecanoylcarnitine (C18)] in group II compared with group I. These results are in parallel with previous studies by Wennberg et al.[30], who found that patients with liver disease had a significant increase in all carnitine concentrations, and the study by Mihalik et al.[31], who reported that long-chain acylcarnitines increased in liver diseases.

Carnitine is synthesized by the liver, and hence severe liver disease produces profound disturbances in whole-body carnitine metabolism. Possible causes of elevated carnitine concentrations in liver disease may include impaired hepatic uptake and/or excessive release from peripheral and hepatic tissues. Furthermore, muscle contains a significant quantity of carnitine for fatty acid transport and loss of muscle mass may promote carnitine release into the blood[32]. However, studies conducted to determine the carnitine status of liver disease patients yielded conflicting results. This in part may be due to the varying severity and etiology of liver disease in the studied patients[33].

In addition to the primary acylcarnitine markers, calculation of ratios is useful in the interpretation of abnormal results; this study noticed that the ratio of the propionyl to the acetyl carnitine (C3 : C2), glutarylcarnitine (C5-DC), and decenoylcarnitine (C10 : C1) were significantly elevated in group I compared with group II. Some IEMs showed an elevation of specific ratios, such as propionic academia or methylmalonicacidemia, which had elevated C3 : C2, and others such as glutaricacidemia, which showed elevated C5-DC[20]. The diagnosis of such conditions of group I is almost totally a laboratory process, of which acylcarnitine analysis is a key factor[34].

As regards amino acid profiles, there was a statistically significant increase in glycine/alanine ratio (Gly : Ala), methionine/phenylalanine ratio (Met : Phe), methionine/tyrosine in group II compared with group I.

Dever and Elfarra[35]noticed that there is a significant hypermethionemia in liver disease, which could be explained by reduced metabolism of methionine, decreased formation of methionine end products, and hypermethioninemia and a vicious circle occurs as disrupted methionine metabolism leads to hepatic dysfunction, which is caused by aberrant methyl group flux[35],[36].

In line with our study, Tajiri and Shimizu[37] and Holecek[38] revealed that concentrations of the aromatic amino acids phenylalanine and tyrosine are increased significantly in patients with liver diseases as a result of impaired hepatic metabolism and portal systemic hunting of blood[38]. The presence of a catabolic state and increased gluconeogenesis could also contribute to the abnormal aminoacid pattern[37].

Meanwhile, group II showed a significant decrease in overall branched chain amino acids (BCAA) valine, leucine, and isoleucine compared with group I. These results are supported by previous literature as Tajiri and Shimizu[37], which detected a significant decrease in serum concentration of BCAAs in patients with chronic liver diseases. Multiple lines of evidence have shown that the main cause of the BCAA deficiency in liver cirrhosis is their consumption in skeletal muscle for synthesis of glutamate, which acts as a substrate for ammonia detoxification to glutamine[38]. Unfortunately, most previous studies on amino acid have not differentiated patients on the etiology of their liver disease and hence comparisons may have some difficulties.

From the previous point of view, chemometric tools such as PCA were used, which are generally proposed to display and explore analysis purposes for variables in the metabolome, whereas univariate statistical tests such as Student's t-test are used to identify the relevant variables[39].

PCA detected 14 metabolites deemed as ‘potential marker metabolites’ for differentiation of the two patients' groups (group I and group II); some detected in higher quantities and some in lower quantities in the samples. Seven of these compounds were amino acids (Gly : Ala, Leu-Ile, Leu : Phe, Met : Phe, methionine, tyrosine, and valine) and seven were acylcarnitine species (C3, C3 : C2, C5-DC, C10 : C1, C16-OH, C18 and C14-carnitines) for the separation of the hyperammonemia due to IEM from the hyperammonemia due to hepatic causes. Data obtained from the receiver operating characteristic curves based on PCA scores showed varied diagnostic power of the different metabolities among the two diseased groups, as AUC of acylcarnitines species was 0.643, whereas that of amino acids was 0.770. Thus, by this metabolomic study, HPLC-based metabolite analysis combined with a multivariate statistical technique could be able to detect metabolic profile variations between the hyperammonemia groups.


  Conclusion Top


This work detected 14 metabolites of amino acids and acylcarnitines, which constructed a noninvasive diagnostic model for differentiation of different causes of hyperammonemia in pediatric patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
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  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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