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
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
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