|Year : 2016 | Volume
| Issue : 4 | Page : 881-886
Metabolomic profile in biliary atresia compared with cholestasis in pediatric patients: a comparative study
Ahmed A Raouf1, Mohamed A El-Gendy2, Hatem M El-Sebaay3, Ashraf Y El-Fert1, Israa M Ismail MBBCh 1
1 Department of Clinical Biochemistry, National Liver Institute, Menoufia, Egypt
2 Department of Pediatric, National Liver Institute, Menoufia, Egypt
3 Department of Medical Biochemistry, Faculty of Medicine, Menoufia University, Menoufia, Egypt
|Date of Submission||25-Oct-2014|
|Date of Acceptance||16-Dec-2014|
|Date of Web Publication||21-Mar-2017|
Israa M Ismail
Department of Clinical Biochemistry, National Liver Institute, Menoufia University, 22 Mohamed Abo-Elmaged Street, Bajour, Menoufia, 32871
Source of Support: None, Conflict of Interest: None
To compare different levels of amino acid and acyl carnitine profiles to give an insight about metabolic pathways in cholestasis and biliary atresia (BA) in pediatric patients compared with controls.
Early detection is the most effective way to improve the clinical outcome of BA. Emerging metabolomics such as amino acid and acyl carnitine provide a powerful platform for discovering new biomarkers and biochemical pathways to improve early diagnosis.
Participants and methods
This study includes 35 BA patients, 35 patients with neonatal cholestasis (NC) rather than BA, and 35 healthy controls. Liver function tests, abdominal ultrasound, and liver biopsy were performed on all participants. The amino acid and acyl carnitine profile using high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed.
Data revealed a statistically significant increase in methionine, glutamate, and citrulline (P < 0.001, P < 0.001 and P = 0.02, and P < 0.001, respectively) and a decrease in the branched-chain amino acid and fisher ratio (P = 0.02 and 0.001 and P = 0.001, respectively) in both the studied patient groups compared with the control group; there was also an increase in ornithine (P = 0.04) and a decrease in glycine (P = 0.04) amino acid in the BA group compared with the control group and an increase in arginine (P = 0.004) and aromatic amino acids (P = 0.001) and a decrease in the simplified fisher ratio (P < 0.001) in the non-BA group compared with the control group. Results also indicated a statistically significant increase in both patient groups compared with the control group regarding free carnitine (P < 0.001) and almost all studied acyl carnitines, whereas there was a significant decrease in the fisher ratio (P = 0.005) and the simplified fisher ratio (P = 0.004) and a significant increase in butyryl carnitine (C4) (P = 0.004) and octadecanoyl carnitine (C18) (P = 0.006) in the non-BA group compared with the BA group.
There is a common metabolic pathway for both BA and other causes of NC; however, there is a metabolic profile shift of amino acid and acyl carnitine in BA from other causes of NC detected by LC-MS/MS. This metabolic shift can be potentially developed into a useful diagnostic tool and can contribute towards understanding the disease mechanism.
Keywords: acyl carnitine, amino acids, biliary atresia, neonatal cholestasis
|How to cite this article:|
Raouf AA, El-Gendy MA, El-Sebaay HM, El-Fert AY, Ismail IM. Metabolomic profile in biliary atresia compared with cholestasis in pediatric patients: a comparative study. Menoufia Med J 2016;29:881-6
|How to cite this URL:|
Raouf AA, El-Gendy MA, El-Sebaay HM, El-Fert AY, Ismail IM. Metabolomic profile in biliary atresia compared with cholestasis in pediatric patients: a comparative study. Menoufia Med J [serial online] 2016 [cited 2020 Feb 17];29:881-6. Available from: http://www.mmj.eg.net/text.asp?2016/29/4/881/202522
| Introduction|| |
Cholestasis is the failure of bile to reach the duodenum, which may be caused by the pathology anywhere between the hepatocyte and the ampulla of Vater. Neonatal cholestasis (NC) is defined as the persistence of direct bilirubin more than 20% of the total serum bilirubin for more than 14 days. Cholestasis in infancy is caused by a wide range of conditions including obstructive, infectious, metabolic, and genetic causes with variable incidence .
Biliary atresia (BA) is progressive destruction of extrahepatic and intrahepatic bile ducts, with scarring and obliteration with a mostly unknown etiology and pathogenesis . BA constitutes 25–34% of all NC and more than 90% of obstructive cholestasis cases. It is important to be differentiated from other causes of NC as early as possible for the success of Kasai portoenterostomy. If portoenterostomy is not successful or not performed, liver transplantation is the only life-saving alternative ,.
If left untreated, these disorders produce biliary cirrhosis and eventual hepatic failure; thus, it is anticipated that several biochemical metabolic pathways would be affected through the disease progression and as it is still difficult to differentiate BA from other causes of NC due to limitations in conventional approaches, noninvasive tests have been developed to facilitate such diagnoses .
As the liver plays a central role in amino acid metabolism, it is of considerable importance to investigate the metabolic profiles in BA and in other causes of NC . Carnitine plays an important role in fatty acid oxidation and energy production. It helps in the β-oxidation of fatty acids by entering into the mitochondria. The carnitine is bound with acyl-CoA to form acyl carnitine. Experiments indicate that biliary export is a major route for acyl carnitine clearance ,.
In the previous studies, there were different amino acid and acyl carnitine metabolite alterations corresponding to the different forms of liver disease. However, there were only a few studies using liquid chromatography-mass spectrometry-based methods to investigate these alterations . Therefore, we set out to evaluate these alterations in BA and other causes of NC.
| Participants and Methods|| |
This study was conducted on 105 participants divided into the three groups: group I included 35 patients diagnosed to have BA (20 male and 15 female), their age ranging from 20 to 330 days. Group II included 35 patients with NC rather than BA (20 male and 15 female), their age ranging from 4 to 300 days, recruited from the pediatric Department, National Liver Institute, Menoufia University, in the period from July 2012 to July 2013. Diagnosis was based on clinical examination, laboratory tests, ultrasound, and liver biopsy; group III included 35 age-matched and sex-matched infants as controls. For all participants, the following were performed: full medical history and complete physical examination, abdominal ultrasonography, liver biopsy, and laboratory investigation including liver function tests and amino acid and acyl carnitine assay using high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS).
- Sampling: The blood sample for the amino acid and acyl carnitine assay was taken from the heel of the infant using a Guthrie card made of Whatman 903 filter paper purchased from GE Healthcare (Princeton, New Jersey, USA); the blood spots were then dried for 4 h on a dry, horizontal, and nonabsorbent surface at ambient temperature. In addition to this, 4 ml of venous blood and the sera were separated and used for the measurement of liver function tests.
- Laboratory methods: Liver function tests included total and direct serum bilirubin, the cholestatic enzymes alkaline phosphatase and gamma glutamyl transpeptidase, aspartate transaminase, alanine transaminase, total protein, and serum albumin were measured using the Beckman Coulter (Synchron CX 9 ALX) Clinical Auto analyzer (Beckman Instruments, Fullerton, California, USA).
- The amino acid and acyl carnitine assay was performed using high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS).
- The blood spots were analyzed for acyl carnitines and amino acids by triple-quadruple tandem mass spectrometry (ACQUITY UPLC H-Class; Waters Corporation, Milford, Massachusetts, USA) with a positive electrospray ionization probe, using MassChrom Amino acids and Acyl carnitines from a Dried Blood/Nonderivatised kit (Chromsystems Instruments & Chemicals GmbH, München, Germany).
- The assay procedure: A 3-mm dried blood spot disk was punched out of the filter card into a well of v-bottomed microtiter plate; 100 μl of the reconstituted internal standard (with 25 ml of extraction buffer provided by the kit) was added to each dried blood spot disk. Afterward, the plate was agitated and the supernatant was transferred into a new v-bottomed well plate that was sealed with protective sheet.
- Chromatography conditions: 10 μl of elute was injected into the LC-MS/MS system, where the flow of the 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.
| Results|| |
In the present study, regarding the amino acid profile, both the BA and the non-BA groups showed increased levels of methionine (P< 0.001), citrulline (P< 0.001), glutamate (P< 0.001, P = 0.02), and phenylalanine (P = 0.003, P = 0.01) and a decrease in valine (P = 0.04, P = 0.03), branched-chain amino acid (BCAA) (P = 0.02, P = 0.001), and the Fisher ratio (FR) (P = 0.001, P< 0.001) compared with the control group; the non-BA group showed a significant decrease in leucine/isoleucine (P = 0.002) and the simplified fisher ratio (BTR) (P< 0.001) and an increase in aromatic amino acid (AAA) (P = 0.001), tyrosine (P = 0.003), and arginine (P = 0.004), whereas the BA group showed a significant decrease in glycine (P = 0.04) and an increase in ornithine (P = 0.04) compared with the control group, with no significant alteration in the level of aspartate, alanine, and proline in the two studied patient groups compared with the control group.
In the present study, regarding the carnitines profile, both the BA and the non-BA groups showed a significant increase in free carnitine (P< 0.001) and almost all studied acyl carnitines.
On comparing the BA and the non-BA groups, there was a significant decrease in the FR (P = 0.005) and the BTR (P = 0.004), and a significant increase in butyryl carnitine (C4) (P = 0.004) and octadecanoyl carnitine (C18) (P = 0.006) in the non-BA group compared with the BA group [Table 1],[Table 2],[Table 3].
|Table 1 Statistical comparison between the three studied groups with regard to amino acids (μmmol/l)|
Click here to view
|Table 2 Statistical comparison between the three studied groups regarding the amino acid ratio|
Click here to view
|Table 3 Statistical comparison between the three studied groups with regard to acyl carnitines (μmmol/l)|
Click here to view
| Discussion|| |
The present study revealed that both the BA and the non-BA groups showed significantly increased levels of methionine, citrulline, and glutamate.
These results match those observed in earlier studies by Abukawa et al.  and Steinbach et al. , who reported that plasma levels of amino acids citrulline and methionine were significantly higher in patients with cholestatic jaundice during neonatal mass screening.
In liver disease, there is reduced metabolism of methionine and hypermethioninemia, and as the vicious circle is disrupted, methionine metabolism leads to hepatic dysfunction [12–14]. However in case of citrulline, the liver takes up the gut-derived citrulline, which limits the release of citrulline into the systemic circulation . Elevated plasma citrulline is an important biochemical marker of NC caused by citrin deficiency, which is one of the causes of NC , but glutamate plays an important role in case of liver dysfunction: as liver cells fail to get rid of toxic ammonia, glutamate detoxifies it by an alternative pathway through the formation of nontoxic glutamine from ammonia and glutamate, which takes place mainly in the brain and the muscle .
In the present study, it was found that the BA and the non-BA groups had a significant decrease in overall BCAA.
Although advances in the management of children with NC have enabled part of them to survive with their native livers, nearly all patients become cirrhotic . Hence, this could be an explanation for most of the manifestations of chronic liver disease and its metabolic impacts that have appeared in the cases in the current study.
These study results support the findings in the previous literature by Ter Borg et al. , who reported a significant decrease in the serum level of valine, isoleucine, and leucine in primary biliary cirrhosis. Also, earlier studies by Morgan et al. , Delgado Domínguez et al.  and Tajiri and Shimizu  revealed a significant decrease in the serum concentration of BCAAs in patients with chronic liver diseases.
Multiple lines of evidence have shown that the main cause of BCAA deficiency in liver cirrhosis is their consumption in the skeletal muscle for the synthesis of glutamate, which acts as a substrate for ammonia detoxification to glutamine .
In the present study, the non-BA group showed a significant increase in tyrosine and overall AAA, but phenylalanine increased significantly in both patient groups compared with the control group.
These findings matched with those of Shigematsu et al.  who showed that tyrosine was elevated in NC patients, and Ohura et al.  who reported significant hyperphenylalaninemia in NC patients. Also, Tajiri and Shimizu  revealed that concentrations of the AAAs phenylalanine and tyrosine are increased significantly in patients with liver diseases, which indicates predominantly cellular damage .
In neonates, it has been suggested that phenylalanine hydroxylation is limited and can result in elevated phenylalanine and suboptimal tyrosine concentrations . However, the significantly higher elevation of tyrosine in the non-BA group compared with the BA group could be explained by the fact that tyrosenemia itself is a cause of NC .
The current study showed a significant decrease in the phenylalanine/tyrosine ratio in the non-BA group compared with the BA group. This could be explained by the increased tyrosine level in the non-BA group in the present study, which needs to be confirmed by a larger sample size.
The phenylalanine/tyrosine ratio expressed the conversion of phenylalanine to tyrosine, which is thought to be exclusively located in the liver .
The amino acid molar ratio, called FR, which represents the BCAA (leucine, valine, and isoleucine)/AAA (phenylalanine and tyrosine) concentration ratio is important for assessing liver metabolism, the hepatic functional reserve, and the severity of liver dysfunction ,, whereas the BTR, which represents the BCAA (leucine, valine, and isoleucine)/tyrosine concentration ratio, is proposed as a substitute for FR as an index of hepatic damage and later reported that it reflects the progression of chronic liver disease ,.
In the current study, the BA and the non-BA groups showed a significant difference between each other regarding the FR and the BTR, with the most significant decrease in the non-BA group.
The present findings seem to be consistent with earlier studies by Delgado Domínguez et al.  and Al Mardini et al.  who showed a reduction in the BCAA/AAA molar ratio in chronic liver disease, which indicated that the condition of the liver was worsening.
Also, Byrd et al.  revealed that the Fischer index was significantly decreased in BA patients compared with controls. Similar to the current results, Rutgers et al.  showed that the plasma amino acid analysis and the determination of the molar ratio may be useful in the differential diagnosis of hepatocellular and obstructive jaundice. A decrease in the molar ratio may reflect hepatocellular disease .
Regarding carnitine and acyl carnitines, both patient groups (BA and non-BA) showed a statistically significant increase in free carnitine, short-chain acyl carnitine [acetyl carnitine (C2), propionyl carnitine (C3), and butyryl carnitine (C4)] and long-chain acyl carnitine [dodecanoyl carnitine (C12), tetradecanoyl carnitine (C14), hexacanoyl carnitine (C16), and octadecanoyl carnitine(C18)].
However, medium-chain acyl carnitine in both the BA and the non-BA groups showed a statistically significant increase in isovaleryl carnitine (C5), glutaryl carnitine (C5DC), and hexanoyl carnitine (C6).
The study showed a statistically significant difference between the BA group and the non-BA group regarding butyryl carnitine (C4) and octadecanoyl carnitine (C18), with a greater increase in the non-BA group.
The current results seem to be in parallel to previous studies by Wennberg et al.  who reported that patients with liver disease had a significantly increased concentration of free and all carnitine compared with controls. Also, Lee et al.  found an elevation of free carnitine, C2-carnitine, and long-chain acyl carnitines in cholestatic patients.
In line with our study, Selimoglu et al.  reported that plasma carnitine levels were significantly increased in children with BA. Similarly, Zhao et al.  revealed a significant increase in short-chain acyl carnitines in BA patients in their study.
Hence, these results suggest that the effect of decreased bile function as it plays an important role in the elimination of long-chain acyl carnitines .
Because carnitine is synthesized by the liver, severe liver disease produces profound disturbances in whole-body carnitine metabolism. 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 patients studied ,.
Possible causes of elevated carnitine concentrations in the blood from patients with liver disease include decreased hepatic clearance and increased release from tissues .
| Conclusion|| |
This study demonstrates the possibility of metabolomics as noninvasive biomarkers for the early detection of BA and also provides new insight into the pathophysiology of BA.
There is a common metabolic pathway for BA and other causes of NC. However, differences in the metabolic pathway could be detected by differences in the metabolomics results.
The FR, the simplified FR, C4, and C18 could be used as metabolomics markers differentiating BA from non-BA causes of NC.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
| References|| |
Donia AE, Ibrahim SM, Kader MS, Saleh AM, El-Hakim MS, El-Shorbagy MS, et al.
Predictive value of assessment of different modalities in the diagnosis of infantile cholestasis. J Int Med Res 2010; 38
Małgorzata R, Piotr C, Krystyna C, Joann CK, Aleksandra M, Diana K, Joanna P. Biliary atresia in children with aberrations involving chromosome 11q. J Pediatr Gastroenterol Nutr 2014; 58
Ghoneim E, Sira M, Abd Elaziz M, Khalil F, Sultan M, Mahmoud A. Diagnostic value of hepaticintercellular adhesion molecule-1 expression in Egyptian infants with biliary atresia and other forms of neonatal cholestasis. Hepatol Res 2011; 41
Elkholy MR, Elshazly HM. Role of three-dimensional multidetector computed tomography angiography of hepatic vessels in the evaluation of living donors. Menoufia Med J 2014; 27
Hartley JL, Davenport M, Kelly DA. Biliary atresia. Lancet 2009; 374
Ohura T, Kobayashi K, Tazawa Y, Abukawa D, Sakamoto O, Tsuchiya S, Saheki T. Clinical pictures of 75 patients with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). J Inherit Metab Dis 2007; 30
Zhang A, Sun H, Wang X. Serum metabolomics as a novel diagnostic approach for disease: a systematic review. Anal Bioanal Chem 2012; 404
Zhao D, Han L, He Z, Zhang J, Zhang Y. Identification of the plasma metabolomics as early diagnostic markers between biliary atresia and neonatal hepatitis syndrome. PLoS One 2014; 9
Krahenbuhl S, Brass EP, Hoppel CL. Decreased carnitine biosynthesis in rats with secondary biliary cirrhosis. Hepatology 2000; 31
Abukawa D, Ohura T, Iinuma K, Tazawa Y. An undescribed subset of neonatal intrahepatic cholestasis associated with multiple hyperaminoacidemia. Hepatol Res 2001; 21
Steinbach M, Clark RH, Kelleher AS, Flores C, White R, Chace DH, Spitzer AR. Demographic and nutritional factors associated with prolonged cholestatic jaundice in the premature infant. J Perinatol 2008; 28
Shelton CM, Clark AJ, Storm MC, Helms RA. Plasma amino acid concentrations in 108 children receiving a pediatric amino acid formulation as part of parenteral nutrition. J Pediatr Pharmacol Ther 2010; 15
Dever JT, Elfarra AA. The biochemical and toxicological significance of hypermethionemia: new insights and clinical relevance. Expert Opin Drug Metab Toxicol 2010; 6
Kaore SN, Amane HS, Kaore NM. Citrulline: pharmacological perspectives and its role as an emerging biomarker in future. Fundam Clin Pharmacol 2013; 27
Ngu HL, Zabedah MY, Kobayashi K. Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) in three Malay children. Malays J Pathol 2010; 32
Brosnan JT, Man KC, Hall DE, Colbourne SA, Brosnan ME. Interorgan metabolism of amino acids in streptozotocin-diabetic ketoacidotic rat. Am J Physiol 1983; 244
Dejong CH, van de Poll MC, Soeters PB, Jalan R, Olde Damink SW. Aromatic amino acid metabolism during liver failure. J Nutr 2007; 137
(Suppl 1): 1579S–1585S.
Ter Borg PC, Fekkes D, Vrolijk JM, van Buuren HR. The relation between plasma tyrosine concentration and fatigue in primary biliary cirrhosis and primary sclerosing cholangitis. BMC Gastroenterol 2005; 5
Morgan MY, Marshall AW, Milsom JP, Sherlock S. Plasma amino-acid patterns in liver disease. Gut 1982; 23
Delgado Domínguez MA, Ruza Tarrío FJ, Hernanz Macías A, Madero Jarabo R, González Ojeda V, García García S, de la Oliva Senovilla P. Amino acid changes in blood of children with severe liver disease. The evaluation of differences according to distinct physiopathology. An Esp Pediatr 1998; 48
Tajiri K, Shimizu Y. Branched-chain amino acids in liver diseases. World J Gastroenterol 2013; 19
Shigematsu Y, Hirano S, Hata I, Tanaka Y, Sudo M, Sakura N, et al.
Newborn mass screening and selective screening using electrospray tandem mass spectrometry in Japan. J Chromatogr B Analyt Technol Biomed Life Sci 2002; 776
Ohura T, Kobayashi K, Abukawa D, Tazawa Y, Aikawa J, Sakamoto O, et al.
A novel inborn error of metabolism detected by elevated methionine and/or galactose in newborn screening: neonatal intrahepatic cholestasis caused by citrin deficiency. Eur J Pediatr 2003; 162
Mukherjee S, Das SK, Vaidyanathan K, Vasudevan DM. Consequences of alcohol consumption on neurotransmitters – an overview. Curr Neurovasc Res 2008; 5
Soeters PB, Fischer JE. Insulin, glucagon, amino acid imbalance, and hepatic encephalopathy. Lancet 1976; 2
Fischer JE, Rosen HM, Ebeid AM, James JH, Keane JM, Soeters PB. The effect of normalization of plasma amino acids on hepatic encephalopathy in man. Surgery 1976; 80
Holecek M. Branched-chain amino acids and ammonia metabolism in liver disease: therapeutic implications. Nutrition 2013; 29
Azuma Y, Maekawa M, Kuwabara Y, Nakajima T, Taniguchi K, Kanno T. Determination of branched-chain amino acids and tyrosine in serum of patients with various hepatic diseases, and its clinical usefulness. Clin Chem 1989; 35
Al Mardini H, Douglass A, Record C. Amino acid challenge in patients with cirrhosis and control subjects: ammonia, plasma amino acid and EEG changes. Metab Brain Dis 2006; 21
Byrd J, Wiltfang. A, Rodeck B, Latta. A, Burdelski M, Brodehl J. The plasma amino acid profile and its relationships to standard quantities of liver function in infants and children with extra hepaticbiliary atresia and pre terminal liver cirrhosis. Eur J Clin Chem Biochem 1993; 31
Rutgers C, Stradley RP, Rogers WA. Plasma amino acid analysis in dogs with experimentally induced hepatocellular and obstructive jaundice. Am J Vet Res 1987; 48
Duro D, Fitzgibbons S, Valim C, Yang CF, Zurakowski D, Dolan M, et al.
C]Methionine breath test to assess intestinal failure-associated liver disease. Pediatr Res 2010; 68
Wennberg A, Hyltander A, Sjöberg A, Arfvidsson B, Sandström R, Wickström K. Prevalence of carnitine depletion in critically ill patients with under nutrition. Metabolism 1992; 41
Lee NC, Chien YH, Kobayashi K, Saheki T, Chen HL, Chiu PC, et al
. Time course of acyl carnitine elevation in neonatal intrahepatic cholestasis caused by citrin deficiency. J Inherit Metab Dis 2006; 29
Selimoglu MA, Aydogdu S, Yagci RV, Huseyinov A. Plasma and liver carnitine status of children with chronic liver disease and cirrhosis. Pediatr Int 2001; 43
Chace DH, Diperna JC, Mitchell BL, Sgroi B, Hofman LF, Naylor EW. Electrospray tandem mass spectrometry for analysis of acyl carnitines in dried postmortem blood specimens collected at autopsy from infants with unexplained cause of death. Clin Chem 2001; 47
Rudman D, Sewell CW, Ansley JD. Deficiency of carnitine in cachectic cirrhotic patients. J Clin Invest 1977; 60
Fuller RK, Hoppel CL. Elevated plasma carnitine in hepatic cirrhosis. Hepatology 1983; 3
Palombo JD, Borum PR, Jenkins RL, Trey C, Bistrian BR. Blood carnitine status after orthotopic liver transplantation in patients with end-stage liver disease. Am J Clin Nutr 1989; 50
[Table 1], [Table 2], [Table 3]