APPLICATION NOTE E184VPG-001 - VDS optilab...APPLICATION NOTE E184VPG-001 Separation of Cholesterol...
Transcript of APPLICATION NOTE E184VPG-001 - VDS optilab...APPLICATION NOTE E184VPG-001 Separation of Cholesterol...
APPLICATION NOTE
E184VPG-001
Separation of Cholesterol and its oxidation products
Abstract
Cholesterol is an important membrane lipid providing structural strength for the cell as well as
regulating membrane fluidity. As one of the main constituents of lipid rafts, it is known to influence
processes like signal transduction and membrane trafficking [1]. By partial depletion of cholesterol
from membranes and subsequently substituting it with oxycholesterols, these functions can be
studied more closely.
With this method, we are able to link cholesterol and oxycholesterol content in cell membranes to
certain observations that are made in cell culture when for example varying oxycholesterol content
in culture medium or challenging cells with stress conditions.
Keywords
• Natural products
• Sterols
• Oxysterols
• Cholesterol
• Cholesterol derivatives
Compound information
Classification Compound name
Sterol Cholesterol
Oxysterols
22(S)-hydroxycholesterol
25-hydroxycholesterol
7-beta-hydroxycholesterol
beta-epoxy-cholesterol
alpha-epoxy-cholesterol
Chromatographic conditions
Column
Particle Size, Length × inner diameter
Order number
VDSpher® PUR 100 C18-H
3 µm, 150 × 2.0 mm
N1520E184VPG
Separation mode descriptions analytical, reverse phase
Mobile Phase A: 0.1 % Formic acid,
B: Acetonitrile/Methanol (50:50 v/v)
Elution conditions Isocratic
0-25 min: 95% B
Flow rate 0.2 ml/min
Injection 10 µl
Column temperature 12°C
HPLC system Shimadzu Prominence HPLC unit
Detector ABSciex API 2000 mass spectrometer with APCI
ion source; selected ion monitoring at
m/z = 367, 369 and 385 amu
Sample and sample preparation Sample: Lipid extract from cell membranes
Preparation: cell membranes are solubilized by
detergent (e.g. Triton X) and extracted with
chloroform/methanol [2].
c = 500 ng/ml cholesterol, 50 ng/ml cholesterol
oxidation products
Chromatograms
Origin
Anna Becker, Prof. Dr. Rainer Buchholz
Friedrich-Alexander-Universität Erlangen Nürnberg
Technische Fakultät (Faculty of Engineering)
Department Chemie- und Bioingenieurwesen (Department Chemical and Biological Engineering)
Lehrstuhl für Bioverfahrenstechnik (Institute of Bioprocess Engineering)
References
“Differential regulation of cell death in head and neck cell carcinoma through alteration of
cholesterol levels in lipid rafts microdomains”
Clara Bionda, Anna Athias, Delphine Poncet, Gersende Alphonse, Amel Guezguez, Philippe Gambert,
Claire Rodriguez-Lafrasse, Dominique Ardail
Biochem. Pharmacol. 2008, 75(3) 761–772.
“A rapid method of total lipid extraction and purification”
E. G. Bligh, W. J. Dwyer
Can. J. Biochem. Physiol. 1959, 37(8), 911-917.
22(S
)-O
H
25-O
H
Chol
7b-O
H
b-E
P
a-E
P
0.00E+00
5.00E+03
1.00E+04
1.50E+04
2.00E+04
2.50E+04
3.00E+04
3.50E+04
4.00E+04
4.50E+04
0 5 10 15 20 25
Inte
nsity (
cps)
Time (min)
m/z 367 amu Intensity (cps) m/z 369 amu Intensity (cps) m/z 385 amu Intensity (cps)
VDSpher PUR C18-H 150 x 2.0 mm, 3 µm A: 0.1% formic acid, B: ACN/MeOH (50:50)Isocratic 95% B at 0.2 mL/min and 12°C for 25 min APCI-MS: Q1 MI at m/z 367.4, 369.4 and 385.4 amu
Sample: 50 ng/ml COPs, 500 ng/ml CholVinj = 10 µl
Year of application: 2016
© 2016 VDS optilab Chromatographietechnik GmbH
VDS optilab does not warrant the correctness of the application.
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