DOI 10.1515/jpem-2012-0343 a

Na Lin ,Jun Ye b,WenjuanQiub,Lianshu Han b,HuiwenZhangbandXuefanGuc, *Application of liquid chromatography-tandem mass

spectrometry in the diagnosis and follow-up of maple syrup urine disease in a Chinese population

Abstract

Background: Maple syrup urine disease (MSUD) is an

inherited disorder caused by a deficiency of the mitochon-drial branched-chain keto acid dehydrogenase complex. We investigated whether liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a more reliable and accurate method than MS/MS in the diagnosis and man-agement of patients with MSUD in a Chinese population. Methods: A total of 370 dried blood spots (DBS) from

healthy neonates, 44 DBS specimens from phenylketon-uria neonates, and 38 DBS samples from 10 MSUD patients were retrospectively tested using the LC-MS/MS method. The results were compared with those obtained by the MS/MS method.

Results: The reference intervals of branched-chain amino

acids (BCAAs) and alloiosleucine (Allo-Ile) were esti-mated for both sexes. In classic MSUD patients, Allo-Ile was markedly elevated (average of 136 μ mol/L, which was significantly higher than the normal value, <5μmol/L).The averages of BCAAs were also markedly elevated con-tinually during the treatment.

Conclusions: The application of the LC-MS/MS method

in the measurement of Allo-Ile and BCAAs in DBS is more useful for diagnosing and managing classic MSUD than the MS/MS method. Keywords: alloisoleucine; branched chain amino acid;

dried blood spots; LC-MS/MS; maple syrup urine disease.

N. Lin conceived the study and wrote the first draft of the paper. b J. Ye, W. Qiu, L. Han, and H. Zhang participated in the analysis and interpretation of data, and drafting of the manuscript. c

X.F. Gu contributed to the conception, supervision, and revision of

the manuscript. *Corresponding author: Xuefan Gu, Shanghai Institute for Pediatric Research, Department of Pediatric Endocrinology and Genetic

Metabolism, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China, Phone: +86-21-25076454, Fax: +86-21-65791316, E-mail: gu_xf53@yahoo.com.cn Na Lin, Jun Ye, Wenjuan Qiu, Lianshu Han, and Huiwen Zhang: Shanghai Institute for Pediatric Research , Department of Pediatric Endocrinology and Genetic Metabolism, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai ,China a

Introduction

aple syrup urine disease (MSUD) is an autosomal reces-M

sive disorder caused by a deficiency in the branched-chain keto acid dehydrogenase (BCKDH) complex. The estimated worldwide incidence is about 1 in 185,000 ( 1, 2) . The deficiency of BCKDH results in the accumu-lation of the branched-chain amino acids (BCAAs) iso-leucine (Ile), leucine (Leu), valine (Val), and alloisoleu-cine (Allo-Ile) in blood, urine, and spinal fluid (3 – 5) . An elevated concentration of Leu in particular is associated with neurotoxicity. High concentrations of Ile and its metabolites generate a characteristic smell in urine that is reminiscent of maple syrup, from which the name of the disease was derived. The clinical manifestations of classic MSUD include poor feeding, vomiting, lethargy, metabolic acidosis, and encephalopathy, which can pro-gress to seizure, coma, and death if left untreated. Early diagnosis and treatment by means of dietary intervention are crucial for the normal development of MSUD patients to prevent the brain damage that causes intellectual dis-ability (6) . The concentration of Leu in blood was meas-ured by bacterial inhibition assay for newborn screening (NBS) of MSUD until the 1990s (7) . With the widespread application of tandem mass spectrometry (MS/MS), this technology was introduced into NBS in 1995 (8) . Using the MS/MS method, Val and the total concentration of Leu, Ile, Allo-Ile, and hydroxyproline (Xle) were measured for NBS for MSUD. Compared with the bacterial inhibition assay, the MS/MS method is more sensitive and has been used in NBS for MSUD worldwide since the 2000s. In addi-tion, many other metabolic diseases can also be simulta-neously detected. The elevated concentrations of BCAAs are considered indicative of MSUD. However, BCAA eleva-tions are not very specific, as the existing NBS method has failed to differentiate isobaric amino acids such as Leu, Ile, Xle, and Allo-Ile, the latter of which is a pathogno-monic marker for MSUD. The total concentration of these isomers in newborns that receive total parenteral nutri-tion or have other metabolic disorders can be elevated and result in a false-positive outcome. As the major neurotoxic