LC-MS/MS based assay and reference intervals in children and adolescents for oxysterols elevated in Niemann-Pick diseases.

Abstract

BACKGROUND:

Niemann-Pick type C (NP-C) is a rare progressive neurodegenerative lipid storage disorder with heterogeneous clinical presentation and challenging diagnostic procedures. Recently oxysterols have been reported to be specific biomarkers for NP-C but knowledge on the intra-individual variation and on reference intervals in children and adolescents are lacking.

METHODS:

We established a LC-MS/MS assay to measure Cholestane-3β, 5α, 6β-triol (C-triol) and 7-Ketocholesterol (7-KC) following Steglich esterification. To assess reference intervals and intra-individual variation we determined oxysterols in 148 children and adolescents from 0 to 18 years and repeat measurements in 19 of them.

RESULTS:

The reported method is linear (r>0.99), sensitive (detection limit of 0.03 ng/mL [0.07 nM] for C-triol, and 0.54 ng/mL [1.35 nM] for 7-KC) and precise, with an intra-day imprecision of 4.8% and 4.1%, and an inter-day imprecision of 7.0% and 11.0% for C-triol (28 ng/ml, 67 nM) and 7-KC (32 ng/ml, 80 nM), respectively. Recoveries for 7-KC and C-triol range between 93% and 107%. The upper reference limit obtained for C-triol is 40.4 ng/mL (95% CI: 26.4-61.7 ng/mL, 96.0 nM, 95% CI: 62.8-146.7 nM) and 75.0 ng/mL for 7-KC (95% CI: 55.5-102.5 ng/mL, 187.2 nM, 95% CI: 138.53-255.8 nM), with no age or gender dependency. Both oxysterols have a broad intra-individual variation of 46%±23% for C-triol and 52%±29% for 7-KC. Nevertheless, all Niemann-Pick patients showed increased C-triol levels including Niemann-Pick type A and B patients.

CONCLUSIONS:

The LC-MS/MS assay is a robust assay to quantify C-triol and 7-KC in plasma with well documented reference intervals in children and adolescents to screen for NP-C in the pediatric population. In addition our results suggest that especially the C-triol is a biomarker for all three Niemann-Pick diseases.

• Authors: Glynis Klinke, Marianne Rohrbach, Roberto Giugliani, Patricie Burda, Matthias R Baumgartner, Christel Tran, Matthias Gautschi, Déborah Mathis, Martin Hersberger
• Journal: Clinical biochemistry (Clin Biochem) Vol. 48 Issue 9 Pg. 596-602 (Jun 2015) ISSN: 1873-2933 [Electronic] United States
• PMID: 25819840 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Chemical References

• Biomarkers
• Cholestanols
• Ketocholesterols
• 7-ketocholesterol

Topics

• Adolescent
• Biomarkers (blood)
• Child
• Child, Preschool
• Cholestanols (blood)
• Chromatography, Liquid (methods)
• Female
• Humans
• Infant
• Infant, Newborn
• Ketocholesterols (blood)
• Limit of Detection
• Male
• Niemann-Pick Diseases (diagnosis)
• Reference Values
• Tandem Mass Spectrometry (methods)