A new diagnostic assay for glycogen storage disease type II in mixed leukocytes
A new diagnostic assay for glycogen storage disease type II in mixed leukocytes
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DOI：
10.1016/j.ymgme.2005.10.016
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发表时间：
2006-05-01
影响因子：
3.8
通讯作者：
Reuser, AJJ
中科院分区：
文献类型：
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作者：
Okumiya, T;Keulemans, JLM;Reuser, AJJ
我们建立了一种使用混合白细胞诊断糖原储存病II型（Pompe病或酸性麦芽糖酶缺乏症）的新方法。该方法以糖原和4-methylumbelliferyl- α - d -glucopyranoside （4mu - α Glc）为底物测定溶酶体酸性α -葡萄糖苷酶（acid α - Glu）活性，并加入阿卡波糖以消除不相关的α -葡萄糖苷酶（主要是麦芽糖酶-葡萄糖淀粉酶）的干扰。结果表明，3.0 μ mol/L的阿卡波糖在pH为4.0时完全抑制麦芽糖淀粉酶活性，但对溶酶体酸- Glu活性的抑制作用小于5%。我们用这种方法测定了25例II型糖原储存病患者（2例婴儿和23例晚发病例）、1例GAA2/GAA2纯合子和30名健康受试者混合白细胞中酸α - Glu活性。在以糖原为底物的测定中，阿卡波糖的加入在患者和对照范围之间建立了明确的分离。在以4mu - α Glc为底物的测定中，这两个范围完全分离，但尽管使用了阿卡波糖，但仍然非常接近。病人和正常范围的分离是通过采取碳糖抑制比未抑制活性显著改善。用糖原作为底物时，GAA2/GAA2纯合子被正确诊断为4MU-aGlc，但被误诊为患者。我们得出结论，在pH为4.0的糖原和4mu - α - Glc底物中加入3.0 μ mol/L的阿卡波糖，可以特异性测量混合白细胞中溶酶体酸α - Glu活性，从而可靠地诊断该标本的糖原储存病II型。(c) 2005爱思唯尔公司版权所有。
We have established a new method for the enzymatic diagnosis of glycogen storage disease type II (Pompe disease or acid maltase deficiency) using mixed leukocytes. The method employs glycogen and 4-methylumbelliferyl-alpha-D-glucopyranoside (4MU-alpha Glc) as substrates for measuring the lysosomal acid alpha-glucosidase (acid alpha Glu) activity, and incorporates acarbose to eliminate the interference of unrelated alpha-glucosidases (predominantly maltase-glucoamylase). It is shown that 3.0 mu mol/L acarbose completely inhibits the maltase-glucoamylase activity at pH 4.0, but the lysosomal acid alpha Glu activity by less than 5%. With this method, we determined the acid alpha Glu activity in mixed leukocytes from 25 patients with glycogen storage disease type II (2 infantile and 23 late-onset cases), one GAA2/GAA2 homozygote and 30 healthy subjects. In the assay with glycogen as substrate, the addition of acarbose created a clear separation between the patient and the control ranges. In the assay with 4MU-alpha Glc as substrate, the two ranges were fully separated but remained very close despite the use of acarbose. The separation of the patient and normal ranges was improved considerably by taking the ratio of acarbose-inhibited over uninhibited activity. A GAA2/GAA2 homozygote was correctly diagnosed with 4MU-aGlc but misdiagnosed as patient when glycogen was used as substrate. We conclude that the inclusion of 3.0 mu mol/L acarbose in the assays with glycogen and 4MU-alpha Glc substrates at pH 4.0 allows for the specific measurement of lysosomal acid alpha Glu activity in mixed leukocytes, thus enabling a reliable diagnosis of glycogen storage disease type II in this specimen. (c) 2005 Elsevier Inc. All rights reserved.