Methods of Determining Efficacy of Therapy For Niemann-Pick C Disease And Related Disorders

Abstract

A method for identifying and screening subjects for cholesterol storage or trafficking diseases using an oxysterol as a biomarker. More particularly, subjects are screened and can be identified as having Niemann-Pick C disease, the method comprising the steps quantifying the concentration of an oxysterol in a biological sample taken from the subject and comparing the concentration of the oxysterol of the subject to a reference value of the oxysterol derived from a non-affected subject population. If the concentration of the oxysterol from the subject is higher than the reference value, the subject is identified as affected with Niemann-Pick C disease.

What is claimed is: 
     
         1 . A method of determining the concentration of 24-hydroxycholesterol in a sample, comprising:
 providing a biological sample from a subject; and   subjecting the biological sample to a gas chromatography-mass spectroscopy analysis.   
     
     
         2 . A method in accordance with  claim 1 , wherein the subjecting the biological sample to a gas chromatography-mass spectroscopy analysis comprises:
 (a) adding a known amount of an oxysterol standard to the biological sample;   (b) extracting one or more oxysterols from the biological sample; and   (c) performing a liquid chromatography-mass spectrometry procedure on the sample to quantify the 24-hydroxycholesterol.   
     
     
         3 . A method in accordance with  claim 2 , further comprising determining the relative concentrations of the 24-hydroxycholesterol and the oxysterol standard in the biological sample. 
     
     
         4 . A method in accordance with  claim 3 , wherein the determining the relative concentrations of the 24-hydroxycholesterol and the oxysterol standard comprises calculating the area under the curve obtained for the 24-hydroxycholesterol relative to the area under the curve obtained for the oxysterol standard. 
     
     
         5 . A method in accordance with  claim 1 , wherein the providing a biological sample from the subject comprises providing a neonatal screening card spotted with the biological sample from a neonatal subject. 
     
     
         6 . A method in accordance with  claim 2 , wherein the providing a biological sample from a subject comprises spotting a neonatal screening card with a biological sample from a neonatal subject. 
     
     
         7 . A method in accordance with  claim 6 , wherein the adding a known amount of an oxysterol standard comprises spotting to the neonatal screening card a known amount of an oxysterol standard. 
     
     
         8 . A method in accordance with  claim 1 , wherein the biological sample is selected from the group consisting of plasma sample, a serum sample, a blood sample, a sputum sample, a cerebrospinal fluid sample and an amniotic fluid sample. 
     
     
         9 . A method in accordance with  claim 1 , wherein the biological sample is selected from the group consisting of a plasma sample, a serum sample, and a blood sample. 
     
     
         10 . A method in accordance with  claim 1 , wherein the biological sample is a plasma sample. 
     
     
         11 . A method in accordance with  claim 1 , wherein the biological sample is a serum sample. 
     
     
         12 . A method in accordance with  claim 1 , wherein the biological sample is a blood sample. 
     
     
         13 . A method in accordance with  claim 12 , wherein the blood sample is a cord blood sample. 
     
     
         14 . A method in accordance with  claim 1 , wherein the biological sample is a cerebrospinal fluid sample. 
     
     
         15 . A method in accordance with  claim 2 , wherein the oxysterol standard is D 5 -27-hydroxycholesterol. 
     
     
         16 . A method of determining the concentration of 24-hydroxycholesterol in a sample, comprising:
 (a) providing a biological sample from the subject;   (b) adding a known quantity of an oxysterol standard to the sample;   (c) extracting oxysterols from the sample using a two-phase extraction medium;   (d) purifying the oxysterols by normal phase chromatography;   (e) derivatizing the oxysterols; and   (f) quantifying the 24-hydroxycholesterol using gas chromatography-mass spectroscopy.   
     
     
         17 . A method in accordance with  claim 16 , wherein the oxysterol standard is D 5 -27-hydroxycholesterol. 
     
     
         18 . A method in accordance with  claim 16 , wherein the two-phase extraction medium comprises chloroform and methanol. 
     
     
         19 . A method in accordance with  claim 16 , wherein the derivatizing the oxysterols comprises derivatizing the oxysterols with N,N-dimethylglycine, Girard P reagent and Pyridine:Hexamethyldisilazane:Trimethylchlorosilane. 
     
     
         20 . A method in accordance with  claim 16 , wherein the derivatizing the oxysterols comprises derivatizing the oxysterols into trimethylsilyl ethers with bis-(trimethyl-silyl)-trifluoroacetamide (BSTFA) and pyridine.