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