A sensitive and specific LC-MS/MS method for rapid diagnosis of Niemann-Pick C1 disease from human plasma

Anneliese O Speak,Forbes D Porter,Daniel S Ory,Xuntian Jiang,Jessie Zhang,Danielle Taylor Te Vruchte,Nicole M Yanjanin,Hideji Fujiwara,Jean E Schaffer,Dennis J Dietzen,David E Scherrer,Frances M Platt,Rohini Sidhu

doi:10.1194/jlr.d015735

Abstract

Niemann-Pick type C1 (NPC1) disease is a rare, progressively fatal neurodegenerative disease for which there are no FDA-approved therapies. A major barrier to developing new therapies for this disorder has been the lack of a sensitive and noninvasive diagnostic test. Recently, we demonstrated that two cholesterol oxidation products, specifically cholestane-3β,5α,6β-triol (3β,5α,6β-triol) and 7-ketocholesterol (7-KC), were markedly increased in the plasma of human NPC1 subjects, suggesting a role for these oxysterols in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials. In the present study, we describe the development of a sensitive and specific LC-MS/MS method for quantifying 3β,5α,6β-triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. We show that dimethylglycine derivatization successfully enhanced the ionization and fragmentation of 3β,5α,6β-triol and 7-KC for mass spectrometric detection of the oxysterol species in human plasma. The oxysterol dimethylglycinates were resolved with high sensitivity and selectivity, and enabled accurate quantification of 3β,5α,6β-triol and 7-KC concentrations in human plasma. The LC-MS/MS assay was able to discriminate with high sensitivity and specificity between control and NPC1 subjects, and offers for the first time a noninvasive, rapid, and highly sensitive method for diagnosis of NPC1 disease.

Highlights

Niemann-Pick type C1 (NPC1) disease is a rare, progressively fatal neurodegenerative disease for which there are no FDA-approved therapies
These findings indicate that 3␤,5␣,6␤-triol and 7-KC are NPC1 disease-specific biochemical markers and suggest a role for these markers in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials
We describe a noninvasive, rapid, and highly sensitive method for diagnosis of NPC1 disease and use this assay to discriminate with high sensitivity and specificity between control and NPC1 subjects

Summary

Introduction

Niemann-Pick type C1 (NPC1) disease is a rare, progressively fatal neurodegenerative disease for which there are no FDA-approved therapies. In the NPC1 mouse model, the oxidative stress is accompanied by elevated ROS and nonenzymatic oxidation of cholesterol in multiple tissues [11,12,13] These cholesterol oxidation products, cholestane-3␤,5␣,6␤-triol (3␤,5␣,6␤-triol) and 7-ketocholesterol (7-KC), were markedly increased in the plasma of all human NPC1 subjects studied [13]. The 3␤,5␣,6␤-triol and 7-KC markers were not increased in the plasma of other neurodegenerative or lysosomal storage diseases, correlated with severity and age of onset of disease, and, in a NPC1 disease model, were reduced in response to therapy [13] These findings indicate that 3␤,5␣,6␤-triol and 7-KC are NPC1 disease-specific biochemical markers and suggest a role for these markers in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials

Objectives

Methods

Conclusion