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Licensed Unlicensed Requires Authentication Published by De Gruyter January 20, 2017

Activity of the liver enzyme ornithine carbamoyltransferase (OTC) in blood: LC-MS/MS assay for non-invasive diagnosis of ornithine carbamoyltransferase deficiency

  • Jakub Krijt , Jitka Sokolová , Pavel Ješina , Lenka Dvořáková , Martin Řeboun , Katarína Brennerová , Martin Mistrík , Jiří Zeman , Tomáš Honzík and Viktor Kožich EMAIL logo

Abstract

Background:

Liver enzymes are released from hepatocytes into circulation and their activity can be measured in the blood. We examined whether the plasma activity of the liver enzyme ornithine carbamoyltransferase, determined by a novel liquid chromatography-mass spectrometry (LC-MS/MS) assay, could be utilized for the detection of OTC deficiency (OTCD), an X-linked inborn error of the urea cycle.

Methods:

The plasma ornithine carbamoyltransferase (OTC) activity was assayed in the reverse reaction using isotopically labeled citrulline-d4 as a substrate and by determination of the product, ornithine-d4, by LC-MS/MS analysis.

Results:

The plasma OTC activity in the controls was in the range of 111–658 pkat/L (n=49, median 272 pkat/L), and the activity increased linearly with serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in patients with hepatopathy. The OTC activity was subsequently determined in 32 individuals carrying mutations in the OTC gene, and OTC/ALT and OTC/AST ratios were calculated to account for the degree of hepatopathy, which is a common finding in OTCD. The OTC/ALT ratio enabled clear differentiation of OTCD hemizygotes (n=11, range 0–69×10−6) from controls (504–3440×10−6). This ratio also enabled the detection of 11 of 12 symptomatic heterozygotes (range 38–794×10−6), while this marker did not allow for reliable differentiation of asymptomatic heterozygotes (n=9) from controls.

Conclusions:

LC-MS/MS assay of plasma OTC activity enabled the detection of all hemizygous and the majority of symptomatic heterozygous OTCD patients in the tested cohort. This study demonstrates that non-invasive assay of enzymes expressed predominantly in the liver could be used as an alternative approach for diagnosing inborn errors of metabolism.

Acknowledgments

The authors would like to thank Ms. Tereza Kovačiková, MSc. and Ms. Karolína Kukačková, BSc. for technical help, and Ms. Michaela Křížková, MSc. for critical reading of the manuscript.

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: The General University Hospital in Prague received support from the Ministry of Health of the Czech Republic (grant IGA NT/14159-3/2013 and program RVO-VFN 64165) and from the Operational Program Prague Competitiveness (project reg. No.CZ.2.16/3.1.00/24012). The First Faculty of Medicine received institutional support from the Charles University in Prague (research program PRVOUK-P24/LF1/3 and research program UNCE 204011).

  3. Employment or leadership: None declared.

  4. Honorarium: None declared.

  5. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

References

1. Wróblewski F. Biochemical biopsy via body fluids. New York: Sloan-Kettering Institute for Cancer Research and Memorial Center for Cancer and Allied Diseases, 1956.Search in Google Scholar

2. Murayama H, Ikemoto M, Hamaoki M. Ornithine carbamyltransferase is a sensitive marker for alcohol-induced liver injury. Clin Chim Acta 2009;401:100–4.10.1016/j.cca.2008.11.027Search in Google Scholar

3. Tokushige K, Hashimoto E, Noto H, Yatsuji S, Tobari M, Torii N, et al. Clinical significance of serum ornithine carbamoyltransferase in patients with non-alcoholic steatohepatitis. Hepatol Res 2009;39:939–43.10.1111/j.1872-034X.2009.00530.xSearch in Google Scholar

4. Furihata T, Aizawa T, Koibuchi A, Zhu M, Yamasaki Y, Shibuya M, et al. Characterization of release profile of ornithine carbamoyltransferase from primary rat hepatocytes treated with hepatotoxic drugs: implications for its unique potential as a drug-induced liver injury biomarker. Drug Metab Pharmacokinet 2016;31:102–5.10.1016/j.dmpk.2015.11.005Search in Google Scholar

5. Bondy GS, Armstrong CL, Curran IH, Barker MG, Mehta R. Retrospective evaluation of serum ornithine carbamyltransferase activity as an index of hepatotoxicity in toxicological studies with rats. Toxicol Lett 2000;114:163–71.10.1016/S0378-4274(99)00297-0Search in Google Scholar

6. Reichard H. Ornithine carbamyl transferase activity in human serum in disease of the liver and the biliary system. J Lab Clin Med 1961;57:78–87.Search in Google Scholar

7. Brusilow SW, Horowich AL. Urea cycle enzymes. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editor. The metabolic and molecular bases of inherited disease, 8th ed. New-York: McGraw-Hill, 2001:1909–63.Search in Google Scholar

8. Haberle J, Boddaert N, Burlina A, Chakrapani A, Dixon M, Huemer M, et al. Suggested guidelines for the diagnosis and management of urea cycle disorders. Orphanet J Rare Dis 2012;7:32.10.1186/1750-1172-7-32Search in Google Scholar

9. Batshaw ML, Tuchman M, Summar M, Seminara J. Members of the urea cycle disorders C. A longitudinal study of urea cycle disorders. Mol Genet Metab 2014;113:127–30.10.1016/j.ymgme.2014.08.001Search in Google Scholar

10. Brassier A, Gobin S, Arnoux JB, Valayannopoulos V, Habarou F, Kossorotoff M, et al. Long-term outcomes in Ornithine Transcarbamylase deficiency: a series of 90 patients. Orphanet J Rare Dis 2015;10:58.10.1186/s13023-015-0266-1Search in Google Scholar

11. Lichter-Konecki U, Caldovic L, Morizono H. Ornithine Transcarbamylase Deficiency. In: Pagon RA, Adam MP, Ardinger HH, Wallace SE, Amemiya A, Bean LJ, et al., editors. Gene reviews(R). Seattle, WA, 1993.Search in Google Scholar

12. Caldovic L, Abdikarim I, Narain S, Tuchman M, Morizono H. Genotype-phenotype correlations in ornithine transcarbamylase deficiency: a mutation update. J Genet Genomics 2015;42:181–94.10.1016/j.jgg.2015.04.003Search in Google Scholar

13. Laemmle A, Gallagher RC, Keogh A, Stricker T, Gautschi M, Nuoffer JM, et al. Frequency and pathophysiology of acute liver failure in ornithine transcarbamylase deficiency (OTCD). PLoS One 2016;11:e0153358.10.1371/journal.pone.0153358Search in Google Scholar

14. Batshaw ML, Msall M, Beaudet AL, Trojak J. Risk of serious illness in heterozygotes for ornithine transcarbamylase deficiency. J Pediatr 1986;108:236–41.10.1016/S0022-3476(86)80989-1Search in Google Scholar

15. Opladen T, Lindner M, Das AM, Marquardt T, Khan A, Emre SH, et al. In vivo monitoring of urea cycle activity with (13)C-acetate as a tracer of ureagenesis. Mol Genet Metab 2016;117:19–26.10.1016/j.ymgme.2015.11.007Search in Google Scholar

16. Yudkoff M, Daikhin Y, Ye X, Wilson JM, Batshaw ML. In vivo measurement of ureagenesis with stable isotopes. J Inherit Metab Dis 1998;21(Suppl 1):21–9.10.1023/A:1005345205403Search in Google Scholar

17. Yudkoff M, Ah Mew N, Daikhin Y, Horyn O, Nissim I, Nissim I, et al. Measuring in vivo ureagenesis with stable isotopes. Mol Genet Metab 2010;100(Suppl 1):S37–41.10.1016/j.ymgme.2010.02.017Search in Google Scholar

18. Wenger DA, Williams CS. Screening for lysosomal disorders. In: Hommes FA, editor. Techniques in diagnostic human biochemical genetics. New-York: Wiley-Liss, 1991:587–617.Search in Google Scholar

19. Krijt J, Kopecka J, Hnizda A, Moat S, Kluijtmans LA, Mayne P, et al. Determination of cystathionine beta-synthase activity in human plasma by LC-MS/MS: potential use in diagnosis of CBS deficiency. J Inherit Metab Dis 2011;34:49–55.10.1007/s10545-010-9178-3Search in Google Scholar

20. Stiburkova B, Krijt J, Vyletal P, Bartl J, Gerhatova E, Korinek M, et al. Novel mutations in xanthine dehydrogenase/oxidase cause severe hypouricemia: biochemical and molecular genetic analysis in two Czech families with xanthinuria type I. Clin Chim Acta 2012;413:93–9.10.1016/j.cca.2011.08.038Search in Google Scholar

21. Wolf B, Secor McVoy J. A sensitive radioassay for biotinidase activity: deficient activity in tissues of serum biotinidase-deficient individuals. Clin Chim Acta 1983;135:275–81.10.1016/0009-8981(83)90286-3Search in Google Scholar

22. Alcaide P, Krijt J, Ruiz-Sala P, Jesina P, Ugarte M, Kozich V, et al. Enzymatic diagnosis of homocystinuria by determination of cystathionine-ss-synthase activity in plasma using LC-MS/MS. Clin Chim Acta 2015;438:261–5.10.1016/j.cca.2014.09.009Search in Google Scholar

23. Bagrel A, Museur G, Siest G. Colorimetric measurement of ornithine carbamoyl transferase activity in plasma, and results for a supposedly healthy population. Clin Chem 1975;21:1716–20.10.1093/clinchem/21.12.1716Search in Google Scholar

24. Peltenburg HG, Janssen MA, Soeters PB, Flendrig JG, Hermens WT. Measurement of ornithine carbamyl transferase (OCT) in plasma by means of enzymatic determination of ammonia. Clin Chim Acta 1991;203:395–402.10.1016/0009-8981(91)90314-3Search in Google Scholar

25. Clayson KJ, Fine JS, Strandjord PE. A more sensitive automated method for determination of ornithine carbamoyltransferase activity in human serum. Clin Chem 1975;21:754–7.10.1093/clinchem/21.6.754Search in Google Scholar

26. Gray RG, Black JA, Lyons VH, Pollitt RJ. Ornithine transcarbamylase deficiency: enzyme studies on a further case and a method of diagnosis using plasma enzyme ratios. Pediatr Res 1976;10:918–23.10.1203/00006450-197611000-00003Search in Google Scholar PubMed

27. Colnot S, Perret C. Liver zonation. In: Monga SP, editor. Molecular pathology of liver diseases, 1 ed. New York, NY, USA: Springer US, 2011:7–16.10.1007/978-1-4419-7107-4_2Search in Google Scholar

28. Yorifuji T, Muroi J, Uematsu A, Tanaka K, Kiwaki K, Endo F, et al. X-inactivation pattern in the liver of a manifesting female with ornithine transcarbamylase (OTC) deficiency. Clin Genet 1998;54:349–53.10.1034/j.1399-0004.1998.5440415.xSearch in Google Scholar PubMed

29. Storkanova G, Vlaskova H, Chuzhanova N, Zeman J, Stranecky V, Majer F, et al. Ornithine carbamoyltransferase deficiency: molecular characterization of 29 families. Clin Genet 2013;84:552–9.10.1111/cge.12085Search in Google Scholar PubMed

30. de Hoon B, Monkhorst K, Riegman P, Laven JS, Gribnau J. Buccal swab as a reliable predictor for X inactivation ratio in inaccessible tissues. J Med Genet 2015;52:784–90.10.1136/jmedgenet-2015-103194Search in Google Scholar PubMed PubMed Central

31. The Human Gene Mutation Database. http://www.hgmd.cf.ac.uk.Search in Google Scholar

32. Kolker S, Cazorla AG, Valayannopoulos V, Lund AM, Burlina AB, Sykut-Cegielska J, et al. The phenotypic spectrum of organic acidurias and urea cycle disorders. Part 1: the initial presentation. J Inherit Metab Dis 2015;38:1041–57.10.1007/s10545-015-9839-3Search in Google Scholar PubMed


Supplemental Material:

The online version of this article offers supplementary material (DOI: https://doi.org/10.1515/cclm-2016-0715).


Received: 2016-8-10
Accepted: 2016-11-30
Published Online: 2017-1-20
Published in Print: 2017-7-26

©2017 Walter de Gruyter GmbH, Berlin/Boston

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