MSACL 2018 US Abstract

Topic: Various OTHER

Persistently Increased Alloisoleucine in a Patient without Maple Syrup Urine Disease

Joesph Wiencek (Presenter)
University of Virginia School of Medicine

Bio: Joesph R. Wiencek received his undergraduate and graduate degrees from The Ohio State University and Cleveland State University, respectively. Following this, he completed his Clinical Chemistry Fellowship in the Department of Pathology, Microbiology and Immunology at Vanderbilt University Medical Center. Currently, Dr. Wiencek is a newly minted Assistant Professor of Pathology at the University of Virginia School of Medicine. He also serves as the Director of Biochemical Genetics, Director of Immunochemistry, and Associate Director of Clinical Chemistry for the UVA Health System. His clinical interests revolve around laboratory testing in the areas of pediatric clinical chemistry. He also has research interests in assay development and validation of LC-MS/MS methods for the measurement of clinically relevant molecules in biological fluids.

Authorship: Joesph R. Wiencek1,2, Dennis J. Dietzen3, Teresa Murray2, Sheila Dawling4, Jennifer M. Colby2, James H. Nichols2
(1) University of Virginia, Charlottesville VA (2) Vanderbilt University, Nashville TN (3) Washington University, St. Louis MO (4) Aegis Sciences Corporation, Nashville TN

Short Abstract

Quantitative analysis of plasma amino acids is a useful laboratory test in the investigation of metabolic diseases. In 1999, Schadewaldt et al. published in Clinical Chemistry the significance of alloisoleucine (allo-ile) in plasma for diagnosis of maple syrup urine disease (MSUD). Their findings indicated that the identification of allo-ile >5 μmol/L is pathognomonic for MSUD. In this study, we identified a 3-year-old patient with persistently (n=3) elevated allo-ile without clinical history or genetic confirmation of MSUD. Through the use of tandem mass spectrometry and our in-house liquid chromathography method, we were able to confirm a suspected interference.

Long Abstract


Maple Syrup Urine Disease (MSUD) (OMIM 248600) is an autosomal recessive inborn error of metabolism resulting in the deficiency of branched-chain ketoacid dehydrogenase complex. In children with MSUD, the identification of allo-ile >5 μmol/L is considered pathognomonic for MSUD (1). However, in patients with undiagnosed MSUD, there are several reported instances where individuals presented with a history of normal NBS results. These atypical cases of MSUD were diagnosed following genetic sequencing and a characteristic PAA profile seen during an intercurrent illness (2). In this study, we identified a 3-year-old female who presented to the emergency department with worsening myoclonic and atonic seizures. While admitted, nutritional assessment of PAA revealed an elevated concentration of allo-ile (13.0 μmol/L) and mildly elevated BCAAs despite unremarkable NBS and no clinical history of MSUD. The recurrent detection of allo-ile >5 μmol/L (n=3) was determined by the Waters MassTrak™ Ultraperformance® liquid chromatography Amino Acid Analyzer. Commercially available HPLC/UPLC methods require reproducible retention times as well as derivatization of the amino acid's primary or secondary (e.g. proline) amine group with a chromophore followed by either spectrophotometric or fluorescence detection (3). Due to the derivatization process, these methods are susceptible to interference from exogenous compounds containing free amine groups (3). In this case, the patient was given zonisamide, which contains a free amine in its molecular structure, for the adjunctive treatment of seizures (4).


To investigate zonisamide as a possible interferant, we designed interference studies with commercially available material as recommended by the CLSI EP7-A2 guidelines. Fresh EDTA-plasma from a healthy donor with no prescribed medications was spiked with zonisamide (1.0 mg/mL in methanol) at the lower (10 μg/mL) and upper (40 μg/mL) end of the therapeutic range. Zonisamide was also spiked (40 μg/mL) into EDTA-plasma of a known MSUD patient to determine the recovery of allo-ile and further delineate the interference.


These specimens, along with controls, were analyzed for PAA analysis by the MassTrak™ UPLC method and an LC-MS/MS method previously reported (5). The results of these experiments showed that in specimens spiked with zonisamide, allo-ile detected by the UPLC method was at concentrations diagnostic for MSUD (>5 μmol/L). However, when analyzed by LC-MS/MS, allo-ile was within the reference interval (0-2 μmol/L) or below the lower limit of quantification (LLOQ 1 ìmol/L). Additional PAA results obtained from by the UPLC method and known MSUD patient's plasma revealed a positive interference (25.8±3.1%) in the presence of 40 μg/mL of zonisamide. These results prompted a review of the laboratory information system records at our institution for all patients (n=30) with reported allo-ile results (n=300) between January 1 2014 and June 1 2017. Of these patients, 36% (n=11) had a prescription for zonisamide and an allo-ile >5 μmol/L reported with no clinical indications for MSUD.

Conclusions & Discussion

This investigation highlights the importance of LC-MS/MS in the measurement of PAA. Quantitative PAA methods that do not rely on mass spectral data will continue to be vulnerable to possible interferences from compounds with free amine groups (3). In this study, zonisamide was identified as an interferant following the detection of elevated allo-ile in a patient without clinical history or genetic confirmation of MSUD. In the future, clinical laboratories must consider PAA analysis by LC-MS/MS to avoid unnecessary and costly follow-up testing.

References & Acknowledgements:

1.)Schadewaldt P, Bodner-Leidecker A, Hammen HW, et al. Significance of L-alloisoleucine in plasma for diagnosis of maple syrup urine disease. Clin Chem 1999;45:1734-1740.

2.)Puckett RL, Lorey F, Rinaldo P, et al. Maple syrup urine disease: further evidence that newborn screening may fail to identify variant forms. Mol Genet Metab 2010;100:136-42.

3.)Peake RWA, Law T, Hoover PN et al. Improved separation and analysis of plasma amino acids by modification of the MassTrak™ AAA Ultraperformance® liquid chromatography method. Clin Chim Acta 2013;423:75-82.

4.)Patsalos PN, Berry DJ, Bourgeois BF et al. Antiepileptic drugs--best practice guidelines for therapeutic drug monitoring: a position paper by the subcommission on therapeutic drug monitoring, ILAE Commission on Therapeutic Strategies. Epilepsia 2008;49:1239-76.

5.)Dietzen DJ, Weindel AL, Carayannopoulos MO, et al. Rapid comprehensive amino acid analysis by liquid chromatography/tandem mass spectrometry: comparison to cation exchange with post-column ninhydrin detection. Rapid Commun Mass Spectrom 2008;22:3481-8.

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