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

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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.

cholesterol
diagnostic tools
liquid chromatography/tandem mass spectrometry
Niemann-Pick disease
oxysterols
neurodegeneration

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    Abbreviations:

    3β,5α,6β-triol

    cholestan-3β,5α,6β-triol

    7-KC

    7-ketocholesterol

    APCI

    atmospheric pressure chemical ionization

    CH-DMG2

    cholestane-5α-hydroxy-3β,6β-bis(dimethylglycinate)

    13CNMR

    carbon 13 nuclear magnetic resonance; CV, coefficient of variance

    DMAP

    4-(dimethylamino)pyridine

    DMG

    dimethylglycine

    EDC

    1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

    [2H7]3β,5α,6β-triol

    25,26,26,26,27,27,27-D7-cholestane-3β,5α,6β-triol

    [2H7]7-KC

    25,26,26,26,­27,27,27-D7-7-ketocholesterol

    1HNMR

    proton nuclear magnetic resonance

    HQC

    high quality control

    KC-DMG

    7-ketocholesteryl di­methylglycinate

    K2EDTA

    ethylenediamine-tetraacetic acid dipotassium salt

    LLOQ

    lower limit of quantification

    LQC

    low quality control

    MQC

    medium quality control

    MRM

    multiple reaction monitoring

    NPC

    Neimann Pick Type C

    PPV

    positive predictive value

    QC

    quality control

    RE

    relative error

    ROC

    receiver-operator characteristic

    ROS

    reactive oxygen species

    ULOQ

    upper limit of quantification

This work was performed in the Metabolomics Facility at Washington University. The authors received support from the Washington University Specialized Centers of Clinically Oriented Research grant P50 HL083762 (D.S.O.), Dana's Angels Research Trust (D.S.O. and N.Y.), Ara Parseghian Medical Research Foundation (D.S.O. and N.Y.), Medical Research Council, UK (A.S.), and Action Medical Research, UK (D.T.V.). This study was also supported by the intramural research program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (F.D.P.) and a Bench to Bedside award from the Office of Rare Diseases (F.D.P. and D.S.O.).

[S]

The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of one figure and three tables.