title: 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.affiliation: Division of Clinical Chemistry and Biochemistry, Children's Research Center, University Children's Hospital Zurich, Switzerland; Zurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland.
authors.forename: Glynis
authors.initials: G
authors.lastname: Klinke
authors.affiliation: Division of Metabolism, Children's Research Center, University Children's Hospital Zurich, Switzerland.
authors.forename: Marianne
authors.initials: M
authors.lastname: Rohrbach
authors.affiliation: Medical Genetics Service, HCPA, Porto Alegre, Brazil; Department of Genetics, UFRGS, Porto Alegre, Brazil.
authors.forename: Roberto
authors.initials: R
authors.lastname: Giugliani
authors.affiliation: Division of Metabolism, Children's Research Center, University Children's Hospital Zurich, Switzerland.
authors.forename: Patricie
authors.initials: P
authors.lastname: Burda
authors.affiliation: Zurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland; Division of Metabolism, Children's Research Center, University Children's Hospital Zurich, Switzerland.
authors.forename: Matthias R
authors.initials: MR
authors.lastname: Baumgartner
authors.affiliation: Center for Molecular Diseases, Lausanne University Hospital, Lausanne, Switzerland.
authors.forename: Christel
authors.initials: C
authors.lastname: Tran
authors.affiliation: Paediatric Endocrinology, Diabetology and Metabolism, University Children's Hospital Bern, Switzerland; University Institute of Clinical Chemistry, Inselspital Bern, Switzerland.
authors.forename: Matthias
authors.initials: M
authors.lastname: Gautschi
authors.affiliation: Division of Clinical Chemistry and Biochemistry, Children's Research Center, University Children's Hospital Zurich, Switzerland.
authors.forename: Déborah
authors.initials: D
authors.lastname: Mathis
authors.affiliation: Division of Clinical Chemistry and Biochemistry, Children's Research Center, University Children's Hospital Zurich, Switzerland; Zurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland. Electronic address: martin.hersberger@kispi.uzh.ch.
authors.forename: Martin
authors.initials: M
authors.lastname: Hersberger
mesh_terms: D000293:Adolescent; D015415:Biomarkers / Q000097:blood; D002648:Child; D002675:Child, Preschool; D002777:Cholestanols / Q000097:blood*; D002853:Chromatography, Liquid / Q000379:methods*; D005260:Female; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D007653:Ketocholesterols / Q000097:blood*; D057230:Limit of Detection; D008297:Male; D009542:Niemann-Pick Diseases / Q000175:diagnosis*; D012016:Reference Values; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D015415:Biomarkers; D002777:Cholestanols; D007653:Ketocholesterols; C003001:7-ketocholesterol
keywords: Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS); Niemann–Pick; Oxysterols; Reference intervals; Steglich esterification