Fujiwaki Takehisa, Tasaka Masaru, Yamaguchi Seiji
Department of Pediatrics, Shimane University School of Medicine, 89-1 Enya-cho, Izumo 693-8501, Japan.
J Chromatogr B Analyt Technol Biomed Life Sci. 2008 Jul 15;870(2):170-6. doi: 10.1016/j.jchromb.2008.05.013. Epub 2008 May 16.
Niemann-Pick disease types A and C, and Gaucher disease are glycolipid storage disorders characterized by the systemic deposition of glycosphingolipids, i.e., sphingomyelin in Niemann-Pick disease types A and C tissues and glucosylceramide in Gaucher disease ones, respectively. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS), we analyzed the sphingolipids in liver and spleen specimens from patients with Niemann-Pick disease types A and C, and Gaucher disease. Crude lipids were extracted from tissue containing 5mg protein with chloroform and methanol. After mild alkaline treatment of the crude lipids, a sphingolipid fraction was prepared and analyzed by MALDI-TOF/MS. The results were as follows: (a) ion peaks with m/z values corresponding to different sphingomyelin and ceramide monohexoside (CMH) species were clearly detected. (b) With sphingosylphosphorylcholine as the internal standard for quantification of sphingomyelin and CMH, the relative peak heights of sphingomyelin and CMH were calculated and plotted versus their contents. The relative peak heights of sphingomyelin and CMH showed linearity between 50 and 1500 ng sphingomyelin content, and between 5 and 150 ng CMH content, respectively. (c) Quantitative analysis revealed the accumulation of sphingomyelin in the liver and spleen specimens from the patients with Niemann-Pick disease types A and C. Striking accumulation of CMH was also detected in the liver and spleen specimens from the patients with Gaucher disease. This investigation indicated that accumulated sphingomyelin and CMH in small amounts of tissues from sphingolipidosis patients can be detected quantatively with the MALDI-TOF/MS method. This method will be useful not only for the diagnosis but also for biochemical pathophysiology evaluation of patients with various sphingolipidosis.
A型和C型尼曼-匹克病以及戈谢病是糖脂贮积病，其特征分别是糖鞘脂在全身沉积，即A型和C型尼曼-匹克病组织中的鞘磷脂沉积以及戈谢病组织中的葡糖神经酰胺沉积。我们使用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF/MS）分析了A型和C型尼曼-匹克病患者以及戈谢病患者肝脏和脾脏标本中的鞘脂。用氯仿和甲醇从含5mg蛋白质的组织中提取粗脂质。对粗脂质进行温和的碱性处理后，制备鞘脂组分并通过MALDI-TOF/MS进行分析。结果如下：（a）清晰检测到了质荷比（m/z）值对应于不同鞘磷脂和单己糖神经酰胺（CMH）种类的离子峰。（b）以鞘氨醇磷酸胆碱作为鞘磷脂和CMH定量的内标，计算鞘磷脂和CMH的相对峰高，并将其相对于它们的含量作图。鞘磷脂和CMH的相对峰高分别在鞘磷脂含量50至1500 ng以及CMH含量5至150 ng之间呈线性关系。（c）定量分析显示，A型和C型尼曼-匹克病患者肝脏和脾脏标本中鞘磷脂蓄积。在戈谢病患者的肝脏和脾脏标本中也检测到CMH显著蓄积。这项研究表明，使用MALDI-TOF/MS方法可以定量检测鞘脂贮积症患者少量组织中蓄积的鞘磷脂和CMH。该方法不仅对诊断有用，而且对各种鞘脂贮积症患者的生化病理生理学评估也有用。
