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Simple, Fast, and Simultaneous Detection of Plasma Total Homocysteine, Methylmalonic Acid, Methionine, and 2-Methylcitric Acid Using Liquid Chromatography and Mass Spectrometry (LC/MS/MS)

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JIMD Reports - Volume 10

Part of the book series: JIMD Reports ((JIMD,volume 10))

Abstract

Cobalamin (Vitamin B12) plays an essential role both in the conversion of methylmalonyl-CoA to succinyl-CoA and in the synthesis of methionine (Met) from homocysteine (Hcy). Elevations of total homocysteine (tHcy), Met, methylmalonic acid (MMA), and 2-methylcitric acid (2MCA) are indicative of disorders in these related pathways, and can clinically present as methylmalonic acidemia, cobalamin defects or deficiency, propionic acidemia, homocystinuria, and hypermethioninemia. We have developed a fast, sensitive, and simple method for the simultaneous detection of plasma tHcy, MMA, Met, and 2MCA using liquid chromatography mass spectrometry (LC/MS/MS). All analytes were directly determined without the need of derivatization. Both positive and negative modes were used to achieve the best sensitivity and specificity. The two stereo isomers of 2MCA (2S, 3S) and (2R, 3S) were successfully separated and were designated as 2MCA1 and 2MCA2. The assays were linear up to a concentration of 800 μMol/l for tHcy, 2,000 μMol/l for Met, 80 μMol/l for MMA, 40 μMol/l for 2MCA1, and 40 μMol/l for 2MCA2 (80 μMol/l for total 2MCA), respectively. The recovery was between 84.42 % and 120.05 %. The intra-assay coefficient of variations (CVs) ranged from 2.1 % to 6.9 % (n = 20), and the inter-assay CVs ranged from 2.7 % to 11.6 % (n = 20). Reference intervals were established and verified (n = 125). A total of 15 patients with variable disorders in related pathway were successfully confirmed. The assay can be performed either in diagnostic laboratories or as second-tier, follow-up test in newborn screening laboratories.

A fast, sensitive, and simple LC/MS/MS method was developed successfully for the simultaneous detection of plasma total homocysteine, methylmalonic acid, methionine, and 2-methylcitric acid for diagnosis of disorders in related pathways.

Competing interests: None declared

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Abbreviations

2MCA:

2-methylcitric acid

Cbl:

Cobalamin

GC/MS:

Gas chromatography–mass spectrometry

Hcy:

Homocysteine

HPLC:

High performance liquid chromatography

LC/MS/MS:

Liquid chromatography-mass spectrometry

Met:

Methionine

MMA:

Methylmalonic acid

MTHFR:

Methylenetetrahydrofolate reductase

SA:

Succinic acid

TCEP-HCL:

Tris 2-carboxyethyl phosphine hydrochloride

tHcy:

Total homocysteine

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Acknowledgments

The authors would like to thank Dr. Larry Sweetman (Baylor Research Institute, Dallas, TX) for providing the 2MCA standard; Dr. Robert Allen (Division of Hematology, Department of Medicine, University of Colorado Health Sciences Center, Denver) for parallel testing of 2MCA on GC/MS; and the technologists in Biochemical Genetics and Special Chemistry, Department of Pathology and Laboratory Medicine, Children’s Hospital Los Angeles for analyzing controls and patient’s samples.

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Authors and Affiliations

  1. Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, US

    Xiaowei Fu & Paul K. Pattengale

  2. Department of Pathology and Laboratory Medicine, Children’s Hospital Los Angeles, 4650 Sunset Boulevard MS#32, Los Angeles, CA, 90027, US

    Xiaowei Fu, Yan-Kang Xu, Penny Chan & Paul K. Pattengale

Authors
  1. Xiaowei Fu
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  2. Yan-Kang Xu
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  3. Penny Chan
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  4. Paul K. Pattengale
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Correspondence to Xiaowei Fu .

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Editors and Affiliations

  1. Sektionen für Humangenetik und Klinische, Medizinische Universität Innsbruck, Schöpfstr. 41, Innsbruck, A-6020, Austria

    Johannes Zschocke

  2. Washington State University, Clinical Pharmacology Unit, Division of Health Sciences, E. Spokane Falls Blvd. 412, Spokane, 99210-1495, Washington, USA

    K Michael Gibson

  3. , Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom

    Garry Brown

  4. Medical Center, Department of Pediatrics, Radboud University Nijmegen, Geert Grooteplein 9, Nijmegen, 6500 HB, Netherlands

    Eva Morava

  5. Heidelberg University Hospital, Center for Child and Adolescent Medicine, Im Neuenheimer Feld 430, Heidelberg, 69120, Germany

    Verena Peters

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Communicated by: Viktor Kožich

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Fu, X., Xu, YK., Chan, P., Pattengale, P.K. (2013). Simple, Fast, and Simultaneous Detection of Plasma Total Homocysteine, Methylmalonic Acid, Methionine, and 2-Methylcitric Acid Using Liquid Chromatography and Mass Spectrometry (LC/MS/MS). In: Zschocke, J., Gibson, K., Brown, G., Morava, E., Peters, V. (eds) JIMD Reports - Volume 10. JIMD Reports, vol 10. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8904_2012_205

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  • DOI: https://doi.org/10.1007/8904_2012_205

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  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-642-37333-6

  • Online ISBN: 978-3-642-37334-3

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