First Experience with a Second-Tier LC-MS/MS Assay for Newborn Screening of Propionic Acidemia, Methylmalonic Acidemias and Combined Remethylation Disorders Glynis Klinke (Presenter) University of Heidelberg Presenter Bio: Currently working as post-doc on LC-MS/MS method establishment in the field of biomarker analysis of neurological impairment in the context of inborn errors of metabolism at the Metabolic Centre of Heidelberg, Germany. I mainly gained my experience during my PhD in the Division of Clinical Chemistry and Biochemistry at the University Children’s Hospital Zurich, Switzerland working on the Oxysterol Signature as putative biomarker in Niemann-Pick Type C and Inflammatory Bowel Diseases. I obtained my Master with specialization on the analysis of natural plant products for pharmacy purposes and my Bachelor degree in Chemistry-Biology at the University of Strasbourg, France.

Authors: Péter Monostori1*, Glynis Klinke1*, Sylvia Richter1, Ákos Baráth2, Ralph Fingerhut3, Matthias R. Baumgartner3, Stefan Kölker1, Georg F. Hoffmann1, Gwendolyn Gramer1*, Jürgen G. Okun1*
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

Increased propionylcarnitine levels in newborn screening are indicative for disorders such as propionic acidemia (PA), methylmalonic acidemia and combined remethylation disorders (MMACBL). Elevated propionylcarnitine occurs relatively frequently and requires a differential diagnosis. Thus, we aimed to develop a second-tier assay for concurrent determination of 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid. The assay was developed using liquid chromatography coupled to tandem mass spectrometry, and clinically validated with retrospective analysis of DBS samples from PA or MMACBL patients. All three analytes were determined by this simple and fast assay, allowing a more specific and reliable identification of PA, MMACBL in stored newborn samples.

Introduction

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 & Discussion

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).

For more information, please refer to Monostori P, Klinke G, Richter S, Baráth Á, Fingerhut R, Baumgartner MR, et al. (2017) 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. PLoS ONE 12(9): e0184897. doi:10.1371/journal.pone.0184897

The authors thank the Dietmar-Hopp foundation for financial support.