Blanchard Sophie, Sadilek Martin, Scott C Ronald, Turecek Frantisek, Gelb Michael H
Department of Chemistry, University of Washington, Seattle, Washington 98195, USA.
Clin Chem. 2008 Dec;54(12):2067-70. doi: 10.1373/clinchem.2008.115410.
Treatments now available for mucopolysaccharidosis I require early detection for optimum therapy. Therefore, we have developed an assay appropriate for newborn screening of the activity of the relevant enzyme, alpha-L-iduronidase.
We synthesized a new alpha-L-iduronidase substrate that can be used to assay the enzyme by use of tandem mass spectrometry together with an internal standard or by fluorometry. The assay uses a dried blood spot on a newborn screening card as the enzyme source. The assay protocol uses a simple liquid-liquid extraction step before mass spectrometry. We optimized enzyme reaction conditions and procedures for the assay, including the concentration of substrate, the reaction pH, the incubation time, and mass spectrometer operation. We also assessed inter- and intraassay imprecision.
When the assay was tested on dried blood spots, the alpha-L-iduronidase activity measured for 5 patients with mucopolysaccharidosis I was well below the interval found for 10 randomly chosen newborns. Inter- and intraassay imprecision were <10%. The synthesis of the alpha-L-iduronidase substrate is practical for use on a scale needed to support newborn screening demands.
This newly developed tandem mass spectrometry assay has the potential to be adopted for newborn screening of mucopolysaccharidosis I. This assay has advantages over a previously reported assay also developed in this laboratory and has the potential to be performed in a multiplex fashion to measure several lysosomal enzymes relevant to treatable lysosomal storage diseases.
目前可用于治疗黏多糖贮积症I型的疗法需要早期检测以实现最佳治疗效果。因此，我们开发了一种适用于新生儿筛查相关酶α-L-艾杜糖醛酸酶活性的检测方法。
我们合成了一种新的α-L-艾杜糖醛酸酶底物，可通过串联质谱联用内标法或荧光法来检测该酶。该检测方法以新生儿筛查卡片上的干血斑作为酶源。检测方案在质谱分析前采用简单的液-液萃取步骤。我们优化了酶反应条件和检测程序，包括底物浓度、反应pH值、孵育时间和质谱仪操作。我们还评估了检测间和检测内的不精密度。
当在干血斑上进行该检测时，5例黏多糖贮积症I型患者测得的α-L-艾杜糖醛酸酶活性远低于10名随机选择的新生儿的检测区间。检测间和检测内的不精密度均<10%。α-L-艾杜糖醛酸酶底物的合成在满足新生儿筛查需求所需的规模上是可行的。
这种新开发的串联质谱检测方法有潜力用于黏多糖贮积症I型的新生儿筛查。该检测方法比本实验室之前报道的检测方法具有优势，并且有可能以多重方式进行，以测量与可治疗的溶酶体贮积病相关的几种溶酶体酶。
