ABSTRACT
BACKGROUND: Lysosphingolipids (LysoSLs) are derivatives of sphingolipids which have lost the amide-linked acyl chain. More recently, LysoSLs have been identified as storage compounds in several sphingolipidoses, including Gaucher, Fabry and Niemann-Pick diseases. To date, different methods have been developed to measure each individual lysosphingolipid in plasma. This report describes a rapid liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) assay for simultaneous quantification of several LysoSLs in plasma. METHODS: We analyzed the following compounds: hexosylsphingosine (HexSph), globotriaosylsphingosine (LysoGb3), lysosphingomyelin (LysoSM) and lysosphingomyelin-509 (LysoSM-509). The sample preparation requires only 100 µL of plasma and consists of an extraction with a mixture of MeOH/acetone/H2O (45:45:10, v/v). RESULTS: The method validation showed high sensitivity, an excellent accuracy and precision. Reference ranges were determined in healthy adult and pediatric population. The results demonstrate that the LC-MS/MS method can quantify different LysoSLs and can be used to identify patients with Fabry (LysoGb3), Gaucher and Krabbe (HexSph) diseases, prosaposine deficiency (LysoGb3 and HexSph), and Niemann-Pick disease types A/B and C (LysoSM and LysoSM-509). CONCLUSIONS: This LC-MS/MS method allows a rapid and simultaneous quantification of LysoSLs and is useful as a biochemical diagnostic tool for sphingolipidoses.
Subject(s)
Biomarkers/blood , Chromatography, Liquid/methods , Sphingolipidoses/diagnosis , Sphingolipids/blood , Tandem Mass Spectrometry/methods , Adolescent , Adult , Aged , Case-Control Studies , Child , Child, Preschool , Humans , Infant , Infant, Newborn , Middle Aged , Reference Values , Reproducibility of Results , Sphingolipidoses/blood , Young AdultABSTRACT
BACKGROUND: Niemann-Pick disease type C (NPC) is a rare lipid storage disorder characterized by progressive neurological deterioration. Diagnosing NPC is challenging as clinical signs and symptoms are variable and non-specific. Two oxysterols, cholestane-3ß,5α,6ß-triol (triol) and 7-ketocholesterol (7KC), have been proposed as biomarkers for aiding diagnosis of NPC. This study evaluated the use of triol and 7KC as biomarkers in cholestatic neonates with suspected NPC. METHODS: Plasma triol and 7KC were analysed as dimethylglycine esters using an liquid chromatography - tandem mass spectrometry (LC-MS/MS) assay in selected neonates with severe cholestasis and suspected NPC (n=7), adults with cholestasis (n=15), patients with confirmed NPC (positive controls; n=11 [one child and 10 adults]), healthy subjects (negative controls; n=40 [20 children and 20 adults]), and cholestatic adults (comparative reference; n=15). The LC-MS/MS method was subjected to a number of tests for accuracy and consistency. RESULTS: Triol and 7KC levels were substantially and significantly increased in NPC positive patients compared with healthy controls (p<0.001). However, positive results (markedly increased levels of both oxysterols) were identified in 6/7 (86%) neonates with cholestasis. Genetic testing confirmed NPC only in one neonate who had increased triol and 7KC, and increased oxysterol levels among neonates with no identified NPC gene mutations were considered likely due to biliary atresia (BA). CONCLUSIONS: While the potential of oxysterols as NPC biomarkers has been well evaluated in older patient populations (without cholestasis), our data suggest that cholestasis might represent a pitfall in oxysterol measurements intended to aid diagnosis of NPC in affected patients.
