Second-tier test for quantification of alloisoleucine and branched-chain amino acids in dried blood spots to improve newborn screening for maple syrup urine disease (MSUD).
BACKGROUND
Newborn screening for maple syrup urine disease (MSUD) relies on finding increased concentrations of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine by tandem mass spectrometry (MS/MS). d-Alloisoleucine (allo-Ile) is the only pathognomonic marker of MSUD, but it cannot be identified by existing screening methods because it is not differentiated from isobaric amino acids. Furthermore, newborns receiving total parenteral nutrition often have increased concentrations of BCAAs. To improve the specificity of newborn screening for MSUD and to reduce the number of diet-related false-positive results, we developed a LC-MS/MS method for quantifying allo-Ile.


METHODS
Allo-Ile and other BCAAs were extracted from a 3/16-inch dried blood spot punch with methanol/H2O, dried under nitrogen, and reconstituted into mobile phase. Quantitative LC-MS/MS analysis of allo-Ile, its isomers, and isotopically labeled internal standards was achieved within 15 min. To determine a reference interval for BCAAs including allo-Ile, we analyzed 541 dried blood spots. We also measured allo-Ile in blinded samples from 16 MSUD patients and 21 controls and compared results to an HPLC method.


RESULTS
Intra- and interassay imprecision (mean CVs) for allo-Ile, leucine, isoleucine, and valine ranged from 1.8% to 7.4%, and recovery ranged from 91% to 129%. All 16 MSUD patients were correctly identified.


CONCLUSIONS
The LC-MS/MS method can reliably measure allo-Ile in dried blood spots for the diagnosis of MSUD. Applied to newborn screening as a second-tier test, it will reduce false-positive results, which produce family anxiety and increase follow-up costs. The assay also appears suitable for use in monitoring treatment of MSUD patients.
Oglesbee
Devin
D
Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Sanders
Karen A
KA
Lacey
Jean M
JM
Magera
Mark J
MJ
Casetta
Bruno
B
Strauss
Kevin A
KA
Tortorelli
Silvia
S
Rinaldo
Piero
P
Matern
Dietrich
D
D000597:Amino Acids, Branched-Chain / Q000097:blood*; D002853:Chromatography, Liquid; D006801:Humans; D007231:Infant, Newborn; D007532:Isoleucine / Q000097:blood; D008375:Maple Syrup Urine Disease / Q000175:diagnosis*; D015997:Neonatal Screening / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
D000597:Amino Acids, Branched-Chain; D007532:Isoleucine