Quantitative Determination of Branched-Chain Amino Acids in Dried Blood Spot Samples by LC-MSMS and its Application in Diagnosis and Follow-Up of Chilean Patients with Maple Syrup Urine Disease

K Fuenzalida,V Soto,M.J Leal-Witt,V Cornejo,A Valiente,A Faundez,J.F Cabello,P Guerrero

doi:10.1590/2326-4594-jiems-2021-0005

K Fuenzalida, V Soto + Show 6 more

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https://doi.org/10.1590/2326-4594-jiems-2021-0005

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Abstract

Elevation of branched-chain amino acids (BCAAs) in biological fluids indicates a deficiency in the branched-chain ketoacid dehydrogenase complex, which causes maple syrup urine disease (MSUD). Detection of increased levels of alloisoleucine confirms the diagnosis, while routine monitoring of leucine concentration is crucial for preventing metabolic decompensation and neurological dysfunction. In the metabolic center at Universidad de Chile, we have confirmed and monitored more than fifty MSUD patients in the last 20 years. Most diagnoses were made by clinical and sibling diagnosis, as MSUD is not included in the Chilean national newborn screening program. Shortening diagnosis time has a fundamental impact on the outcome of patients, therefore we focused on implementing detection of BCAAs in dried blood spot by liquid chromatography mass spectrometry (LC-MSMS) for disease confirmation as well as for biochemical monitoring. Retrospective analysis of samples from 9 diagnosed MSUD patients were performed; BCAAs values were determined via MSMS and LC-MSMS conducted in parallel. Leucine and alloisoleucine levels were positively correlated with patient’s diagnosis age. Alloisoleucine was significatively elevated as early as 24 hr after birth. A predictable variation in BCAAs levels after nutritional intervention among diagnosed MSUD patients was found.

Highlights

Maple syrup urine disease (MSUD) is a rare autosomal disorder characterized by the accumulation of branched-chain amino acids (BCAAs) leucine (Leu), isoleucine (Ile), alloisoleucine (Allo), valine (Val) in urine, blood and spinal fluids, as a consequence of a deficiency in the branched-chain keto-acid dehydrogenase complex.[1]
Consensus about the cut-off value (COV) for Xleu and Val is not fully defined, values are independently established in each center according to local newborn screening (NBS) program experience.[6]
Since each MSUD patient was diagnosed at a different age, we evaluated whether a correlation between each BCAA and age existed

Summary

Introduction

Maple syrup urine disease (MSUD) is a rare autosomal disorder characterized by the accumulation of branched-chain amino acids (BCAAs) leucine (Leu), isoleucine (Ile), alloisoleucine (Allo), valine (Val) in urine, blood and spinal fluids, as a consequence of a deficiency in the branched-chain keto-acid dehydrogenase complex.[1]. [4] Due to favorable outcomes when disease is detected between 48 and 72 hours of birth and positive cost-effectiveness of nutritional treatment, MSUD has been included in several newborn screening (NBS) programs around the world, predominantly in developed countries.[5,6,7] MSUD screening was initially conducted by bacterial inhibition assay (BIA), which measures increased Leu concentration in dried blood spot (DBS) samples.[8] Currently, NBS by tandem mass spectrometry (MSMS) analyze total Leu levels (XLeu, sum of Leu, Ile, Allo and Hydroxyproline [OHPro]) and Val in DBS samples.[9] Consensus about the cut-off value (COV) for Xleu and Val is not fully defined, values are independently established in each center according to local NBS program experience.[6] Elevation leucine/phenylalanine (Leu/Phe)

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