Ohira Mari, Barr Marianne, Okuyama Torayuki, Mashima Ryuichi
Department of Clinical Laboratory Medicine, National Center for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo 157-8535, Japan.
Biochemistry Department, Queen Elizabeth University Hospital, 1345 Govan Road, Govan, Glasgow G51 4TF, UK.
Mol Genet Metab Rep. 2022 Aug 26;33:100913. doi: 10.1016/j.ymgmr.2022.100913. eCollection 2022 Dec.
Lysosomal acid lipase deficiency (LAL-D) (OMIM: 278000) is a lysosomal storage disorder with two distinct disease phenotypes such as Wolman disease and cholesteryl ester storage disorder (CESD), characterized by an accumulation of endocytosed cholesterol in the body. Due to the presence of multiple lipases in DBS, previous studies measured LAL enzyme activity in the presence of Lalistat-2, an established LAL-specific inhibitor (Hamilton J Chim Clin Acta (2012) 413:1207-1210). Alternatively, a novel substrate specific for LAL has been reported very recently (Masi S. Clin Chem (2018) 64:690-696). In this study, we examined the LAL enzyme activity of a Japanese population with the LAL-specific substrate using liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based enzyme assay whether an affected individual can be identified among this population. To achieve this, we first performed assay validation using LC-MS/MS. Under our experimental setting, typically we obtained LAL enzyme activity for QC High (100% enzyme activity) as 261.9 ± 3.2 μmol/h/L ( = 5) and for QC Low as (5% enzyme activity) as 14.7 ± 0.5 μmol/h/L ( = 5). The percentage of coefficient of variation for interday assay for QC High was 9.6% ( = 4) and for QC Low was 7.9% ( = 4), respectively. Based on these results, we further examined the LAL enzyme activity of control Japanese population and that of affected individuals with Wolman disease and CESD. The averaged enzyme activity for control newborns, Wolman, and CESD was 123.9 ± 53.9 μmol/h/L ( = 131), 6.6 ± 0.9 μmol/h/L ( = 3), and 4.8 ± 0.3 μmol/h/L ( = 3), respectively. These results suggest that an LAL-D-affected individual can be readily identified by enzyme activity using LC-MS/MS-based technique.
溶酶体酸性脂肪酶缺乏症（LAL-D）（OMIM：278000）是一种溶酶体贮积病，有两种不同的疾病表型，即沃尔曼病和胆固醇酯贮积病（CESD），其特征是体内内吞胆固醇的蓄积。由于干血斑中存在多种脂肪酶，以往的研究在Lalistat-2（一种已确定的LAL特异性抑制剂）存在的情况下测量LAL酶活性（汉密尔顿J. 《临床化学学报》（2012年）413：1207 - 1210）。另外，最近报道了一种对LAL特异的新型底物（马西S. 《临床化学》（2018年）64：690 - 696）。在本研究中，我们使用基于液相色谱 - 串联质谱（LC-MS/MS）的酶测定法，用LAL特异性底物检测了日本人群的LAL酶活性，以确定在该人群中是否能识别出受影响个体。为实现这一目的，我们首先使用LC-MS/MS进行了测定验证。在我们的实验条件下，通常我们得到的QC高值（100%酶活性）的LAL酶活性为261.9±3.2μmol/h/L（n = 5），QC低值（5%酶活性）为14.7±0.5μmol/h/L（n = 5）。QC高值的日间测定变异系数百分比为9.6%（n = 4），QC低值为7.9%（n = 4）。基于这些结果，我们进一步检测了日本对照人群以及患有沃尔曼病和CESD的受影响个体的LAL酶活性。对照新生儿、沃尔曼病患者和CESD患者的平均酶活性分别为123.9±53.9μmol/h/L（n = 131）、6.6±0.9μmol/h/L（n = 3）和4.8±0.3μmol/h/L（n = 3）。这些结果表明，使用基于LC-MS/MS的技术通过酶活性可以很容易地识别出LAL-D受影响个体。
