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Catalog Item
Lipid extraction, separation, quantification
Common Name(s): Lipid Analysis

CenterVanderbilt University School of Medicine
Center CoreAnalytical Resources Core
Test No.V3071
Test PriceInquire for Pricing. Analytical services are provided as part of in vivo projects.
DescriptionTotal plasma cholesterol and triglyceride are measured by standard enzymatic
assays. HDL cholesterol is measured with the enzymatic method after
precipitation of VLDL and LDL using dextran sulfate and Mg++. From these data
LDL cholesterol is calculated using the Friedewald equation, as long as
triglyceride levels are below 400 mg/dl. Investigators may request a total
plasma lipid profile or specific plasma lipid measurements. Plasma FFAs are
analyzed with a commercially available enzymatic kit from Wako Life Sciences.
Free fatty acids are extracted from plasma using heptane/isopropanol and
separated from other lipid classes by thin layer chromatography. The FFAs are
methylated and analyzed by gas chromatography. Data: (1) total FFAs present
(mg/ml plasma); (2) percent distribution of individual fatty acids in the FFA
fraction. Tissue/Cell Lipid Analyses (Phospholipid, Diglycerides, Free Fatty
Acids, Triglycerides, and Cholesterol Esters)
Lipids are extracted by the Folch-Lees method. Internal standards are added, and
the lipid classes are separated by thin layer chromatography. The appropriate
band(s) are scraped from the TLC plate, and the fatty acids from the lipid ester
classes are methylated and analyzed by gas chromatography. Results: (1) total
amount of lipid class present; (2) fatty acid profiles of the lipid classes
presented as percent of total fatty acids. Total unesterified cholesterol is
analyzed in the lipid extract by gas chromatography after addition of
appropriate internal standard. An aliquot of the lipid extract is dried and
saponified at 80 C in 1 N KOH in 90% methanol for 1 hour. The non-saponifiable
sterol is extracted into hexane, concentrated under nitrogen, solubilized in
carbon disulfide and analyzed by gas chromatography.
Lipoprotein fractions are separated from 100 microliters of plasma (or serum) by
gel filtration column chromatography. Approximately 40 fractions (0.5 ml) are
collected and the amount of triglyceride and cholesterol in each fraction is
determined using microtiter plate, enzyme-based assays. Profiles of triglyceride
and cholesterol are constructed. Calibration of the column with purified
lipoprotein fractions permits quantitation of each lipid in various lipoprotein
classes.
DataData will be collected for this catalog item
Keywordsfatty acids, lipids, metabolism, obesity
Tissuesadipose tissue



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