Simultaneous determination of 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid in dried blood spots: Second-tier LC-MS/MS assay for newborn screening of propionic acidemia, methylmalonic acidemias and combined remethylation disorders

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Abstract

Background and aims

Increased propionylcarnitine levels in newborn screening are indicative for a group of potentially severe disorders including propionic acidemia (PA), methylmalonic acidemias and combined remethylation disorders (MMACBL). This alteration is relatively non-specific, resulting in the necessity of confirmation and differential diagnosis in subsequent tests. Thus, we aimed to develop a multiplex approach for concurrent determination of 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid from the same dried blood spot (DBS) as in primary screening (second-tier test). We also set out to validate the method using newborn and follow-up samples of patients with confirmed PA or MMACBL.

Methods

The assay was developed using liquid chromatography–tandem mass spectrometry and clinically validated with retrospective analysis of DBS samples from PA or MMACBL patients.

Results

Reliable determination of all three analytes in DBSs was achieved following simple and fast (<20 min) sample preparation without laborious derivatization or any additional pipetting steps. The method clearly distinguished the pathological and normal samples and differentiated between PA and MMACBL in all stored newborn specimens. Methylcitric acid was elevated in all PA samples; 3-hydroxypropionic acid was also high in most cases. Methylmalonic acid was increased in all MMACBL specimens; mostly together with methylcitric acid.

Conclusions

A liquid chromatography–tandem mass spectrometry assay allowing simultaneous determination of the biomarkers 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid in DBSs has been developed. The assay can use the same specimen as in primary screening (second-tier test) which may reduce the need for repeated blood sampling. The presented preliminary findings suggest that this method can reliably differentiate patients with PA and MMACBL in newborn screening. The validated assay is being evaluated prospectively in a pilot project for extension of the German newborn screening panel (‟Newborn screening 2020”; Newborn Screening Center, University Hospital Heidelberg).

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Most cited references 19

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The use of mass spectrometry to analyze dried blood spots.

Michel Wagner, David Tonoli, Emmanuel Varesio … (2024)

Dried blood spots (DBS) typically consist in the deposition of small volumes of capillary blood onto dedicated paper cards. Comparatively to whole blood or plasma samples, their benefits rely in the fact that sample collection is easier and that logistic aspects related to sample storage and shipment can be relatively limited, respectively, without the need of a refrigerator or dry ice. Originally, this approach has been developed in the sixties to support the analysis of phenylalanine for the detection of phenylketonuria in newborns using bacterial inhibition test. In the nineties tandem mass spectrometry was established as the detection technique for phenylalanine and tyrosine. DBS became rapidly recognized for their clinical value: they were widely implemented in pediatric settings with mass spectrometric detection, and were closely associated to the debut of newborn screening (NBS) programs, as a part of public health policies. Since then, sample collection on paper cards has been explored with various analytical techniques in other areas more or less successfully regarding large-scale applications. Moreover, in the last 5 years a regain of interest for DBS was observed and originated from the bioanalytical community to support drug development (e.g., PK studies) or therapeutic drug monitoring mainly. Those recent applications were essentially driven by improved sensitivity of triple quadrupole mass spectrometers. This review presents an overall view of all instrumental and methodological developments for DBS analysis with mass spectrometric detection, with and without separation techniques. A general introduction to DBS will describe their advantages and historical aspects of their emergence. A second section will focus on blood collection, with a strong emphasis on specific parameters that can impact quantitative analysis, including chromatographic effects, hematocrit effects, blood effects, and analyte stability. A third part of the review is dedicated to sample preparation and will consider off-line and on-line extractions; in particular, instrumental designs that have been developed so far for DBS extraction will be detailed. Flow injection analysis and applications will be discussed in section IV. The application of surface analysis mass spectrometry (DESI, paper spray, DART, APTDCI, MALDI, LDTD-APCI, and ICP) to DBS is described in section V, while applications based on separation techniques (e.g., liquid or gas chromatography) are presented in section VI. To conclude this review, the current status of DBS analysis is summarized, and future perspectives are provided.

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Acquired and inherited disorders of cobalamin and folate in children.

V. Whitehead (2006)

Cobalamin deficiency in the newborn usually results from cobalamin deficiency in the mother. Megaloblastic anaemia, pancytopenia and failure to thrive can be present, accompanied by neurological deficits if the diagnosis is delayed. Most cases of spina bifida and other neural tube defects result from maternal folate and/or cobalamin insufficiency in the periconceptual period. Polymorphisms in a number of genes involved in folate and cobalamin metabolism exacerbate the risk. Inborn errors of cobalamin metabolism affect its absorption, (intrinsic factor deficiency, Imerslund-Gräsbeck syndrome) and transport (transcobalamin deficiency) as well as its intracellular metabolism affecting adenosylcobalamin synthesis (cblA and cblB), methionine synthase function (cblE and cblG) or both (cblC, cblD and cblF). Inborn errors of folate metabolism include congenital folate malabsorption, severe methylenetetrahydrofolate reductase deficiency and formiminotransferase deficiency. The identification of disease-causing mutations in specific genes has improved our ability to diagnose many of these conditions, both before and after birth.

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National academy of clinical biochemistry laboratory medicine practice guidelines: follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry; executive summary.

C Dietzen, W Rhead, Francesco Coco … (2009)

Almost all newborns in the US are screened at birth for multiple inborn errors of metabolism using tandem mass spectrometry. Screening tests are designed to be sufficiently sensitive so that cases are not missed. The NACB recognized a need for standard guidelines for laboratory confirmation of a positive newborn screen such that all babies would benefit from equal and optimal follow-up by confirmatory testing. A committee was formed to review available data pertaining to confirmatory testing. The committee evaluated previously published guidelines, published methodological and clinical studies, clinical case reports, and expert opinion to support optimal confirmatory testing. Grading was based on guidelines adopted from criteria derived from the US Preventive Services Task Force and on the strength of recommendations and the quality of the evidence. Three primary methods of analyte measurement were evaluated for confirmatory testing including measurement of amino acids, organic acids, and carnitine esters. The committee graded the evidence for diagnostic utility of each test for the screened conditions. Ample data and experience were available to make strong recommendations for the practice of analyzing amino acids, organic acids, and acylcarnitines. Likewise, strong recommendations were made for the follow-up test menu for many disorders, particularly those with highest prevalence. Fewer data exist to determine the impact of newborn screening on patient outcomes in all but a few disorders. The guidelines also provide an assessment of developing technology that will fuel a refinement of current practice and ultimate expansion of the diseases detectable by tandem mass spectrometry. Guidelines are provided for optimal follow-up testing for positive newborn screens using tandem mass spectrometry. The committee regards these tests as reliable and currently optimal for follow-up testing. .

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Author and article information

Contributors

Péter Monostori:

ORCID: http://orcid.org/0000-0003-3591-6054

Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – original draft

Glynis Klinke: Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – original draft

Sylvia Richter: Role: Data curationRole: InvestigationRole: MethodologyRole: ValidationRole: Writing – original draft

Ákos Baráth: Role: Data curationRole: InvestigationRole: Writing – review & editing

Ralph Fingerhut: Role: Data curationRole: InvestigationRole: Writing – review & editing

Matthias R. Baumgartner: Role: SupervisionRole: Writing – review & editing

Stefan Kölker: Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: Writing – review & editing

Georg F. Hoffmann: Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – review & editing

Gwendolyn Gramer: Role: ConceptualizationRole: Data curationRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: SupervisionRole: ValidationRole: Writing – original draft

Jürgen G. Okun: Role: ConceptualizationRole: Data curationRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: Writing – original draft

Mohana Krishna Reddy Mudiam: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, CA USA )

ISSN (Electronic): 1932-6203

Publication date (Electronic): 15 September 2017

Publication date Collection: 2017

Volume: 12

Issue: 9

Electronic Location Identifier: e0184897

Affiliations

[1 ] Department of General Pediatrics, Division of Neuropediatrics and Metabolic Medicine, Center for Pediatric and Adolescent Medicine, University Hospital Heidelberg, Heidelberg, Germany

[2 ] Department of Pediatrics, University of Szeged, Szeged, Hungary

[3 ] Children’s Research Center, Division of Metabolism, University Children’s Hospital Zurich, Zurich, Switzerland

Indian Institute of Chemical Technology, INDIA

Author notes

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: monostoripeter@ 123456gmail.com

Author information

Péter Monostori http://orcid.org/0000-0003-3591-6054

Article

Publisher ID: PONE-D-17-20815

DOI: 10.1371/journal.pone.0184897

PMC ID: 5600371

PubMed ID: 28915261

SO-VID: 847e19c6-888a-46e4-9a63-bac3f5b2c37a

License:

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 31 May 2017

Date accepted : 1 September 2017

Page count

Figures: 4, Tables: 5, Pages: 16

Funding

Funded by: Dietmar Hopp Foundation

Award ID: „Newborn screening 2020” pilot study

This work and the pilot study "Newborn screening 2020” at the Newborn Screening Center of the University Hospital Heidelberg are generously supported by the Dietmar Hopp Foundation, St. Leon Rot ( http://dietmar-hopp-stiftung.de). We acknowledge the financial support of the Deutsche Forschungsgemeinschaft and Ruprecht-Karls-Universität Heidelberg within the funding programme Open Access Publishing. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Simultaneous determination of 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid in dried blood spots: Second-tier LC-MS/MS assay for newborn screening of propionic acidemia, methylmalonic acidemias and combined remethylation disorders

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Abstract

Background and aims

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Related collections

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Most cited references 19

The use of mass spectrometry to analyze dried blood spots.

Acquired and inherited disorders of cobalamin and folate in children.

National academy of clinical biochemistry laboratory medicine practice guidelines: follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry; executive summary.

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