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Intracellular in vitro probe acylcarnitine assay for identifying deficiencies of carnitine transporter and carnitine palmitoyltransferase-1

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Abstract

Mitochondrial fatty acid oxidation (FAO) disorders are caused by defects in one of the FAO enzymes that regulates cellular uptake of fatty acids and free carnitine. An in vitro probe acylcarnitine (IVP) assay using cultured cells and tandem mass spectrometry is a tool to diagnose enzyme defects linked to most FAO disorders. Extracellular acylcarnitine (AC) profiling detects carnitine palmitoyltransferase-2, carnitine acylcarnitine translocase, and other FAO deficiencies. However, the diagnosis of primary carnitine deficiency (PCD) or carnitine palmitoyltransferase-1 (CPT1) deficiency using the conventional IVP assay has been hampered by the presence of a large amount of free carnitine (C0), a key molecule deregulated by these deficiencies. In the present study, we developed a novel IVP assay for the diagnosis of PCD and CPT1 deficiency by analyzing intracellular ACs. When exogenous C0 was reduced, intracellular C0 and total AC in these deficiencies showed specific profiles clearly distinguishable from other FAO disorders and control cells. Also, the ratio of intracellular to extracellular C0 levels showed a significant difference in cells with these deficiencies compared with control. Hence, intracellular AC profiling using the IVP assay under reduced C0 conditions is a useful method for diagnosing PCD or CPT1 deficiency.

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Acknowledgments

We thank all the attending physicians for providing clinical information regarding each patient. We are also grateful to Y. Ito, M. Furui, T. Esumi, and N. Tomita for their technical assistance. This work was supported by a Grant-in-Aid for scientific research from the Japan Society for the Promotion of Science (J.P., and S.Y.) and a Grant from the Ministry of Education, Science, Technology, Sports and Culture of Japan and the Ministry of Health, Labour and Welfare of Japan (S.Y).

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Authors and Affiliations

  1. Department of Pediatrics, Shimane University School of Medicine, 89-1 Enya, Izumo, Shimane, 693-8501, Japan

    Jamiyan Purevsuren, Hironori Kobayashi, Yuki Hasegawa, Kenji Yamada, Tomoo Takahashi, Seiji Fukuda & Seiji Yamaguchi

  2. Division of Metabolism, Chiba Children’s Hospital, Chiba, 266-0007, Japan

    Masaki Takayanagi

  3. Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu, Gifu, 501-1194, Japan

    Toshiyuki Fukao

  4. Medical Information Sciences Division, United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu, Gifu, 501-1194, Japan

    Toshiyuki Fukao

  5. Medical Genetics Laboratory, National Center for Maternal and Child Health, Khuvisgalchdyn street, Bayangol district, Ulaanbaatar, 210624, Mongolia

    Jamiyan Purevsuren

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  1. Jamiyan Purevsuren
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  2. Hironori Kobayashi
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  3. Yuki Hasegawa
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  4. Kenji Yamada
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  5. Tomoo Takahashi
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  6. Masaki Takayanagi
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  7. Toshiyuki Fukao
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  8. Seiji Fukuda
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  9. Seiji Yamaguchi
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Correspondence to Seiji Yamaguchi.

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Purevsuren, J., Kobayashi, H., Hasegawa, Y. et al. Intracellular in vitro probe acylcarnitine assay for identifying deficiencies of carnitine transporter and carnitine palmitoyltransferase-1. Anal Bioanal Chem 405, 1345–1351 (2013). https://doi.org/10.1007/s00216-012-6532-3

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  • DOI: https://doi.org/10.1007/s00216-012-6532-3

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