Oglesbee Devin, Sanders Karen A, Lacey Jean M, Magera Mark J, Casetta Bruno, Strauss Kevin A, Tortorelli Silvia, Rinaldo Piero, Matern Dietrich
Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Clin Chem. 2008 Mar;54(3):542-9. doi: 10.1373/clinchem.2007.098434. Epub 2008 Jan 4.
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.
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.
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.
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.
枫糖尿症（MSUD）的新生儿筛查依赖于通过串联质谱（MS/MS）检测支链氨基酸（BCAA）亮氨酸、异亮氨酸和缬氨酸浓度的升高。d-别异亮氨酸（allo-Ile）是MSUD唯一的病理标志性标志物，但由于它无法与等压氨基酸区分开来，现有筛查方法无法识别。此外，接受全胃肠外营养的新生儿往往BCAA浓度升高。为提高MSUD新生儿筛查的特异性并减少与饮食相关的假阳性结果数量，我们开发了一种用于定量allo-Ile的液相色谱-串联质谱（LC-MS/MS）方法。
用甲醇/水从直径3/16英寸的干血斑冲孔中提取allo-Ile和其他BCAA，氮气吹干后再用流动相复溶。在15分钟内完成对allo-Ile及其异构体和同位素标记内标的定量LC-MS/MS分析。为确定包括allo-Ile在内的BCAA的参考区间，我们分析了541份干血斑。我们还检测了16例MSUD患者和21例对照的盲样中的allo-Ile，并将结果与一种高效液相色谱（HPLC）方法进行比较。
allo-Ile、亮氨酸、异亮氨酸和缬氨酸的批内和批间不精密度（平均变异系数）在1.8%至7.4%之间，回收率在91%至129%之间。所有16例MSUD患者均被正确识别。
LC-MS/MS方法能够可靠地检测干血斑中的allo-Ile以诊断MSUD。作为二级检测应用于新生儿筛查，它将减少假阳性结果，这些结果会引起家庭焦虑并增加后续成本。该检测方法似乎也适用于监测MSUD患者的治疗情况。
