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URL: https://dx.doi.org/DOI:10.17504/protocols.io.62thgen

Authors: Peter Havel

Group: Mouse Metabolic Phenotyping Centers

Summary: Summary: The lipid extraction method is based on work by Folch 1957. Tissue is homogenized and put into a 2:1 chloroform and methanol mix. The mix is separated into two phases by adding a salt solution. The upper phase contains any non-lipid substances and the lower phase contains the chloroform with the lipids. The upper phase is removed and a sample is taken from the bottom phase and evaporated. The sample is reconstituted with 2-propanol with a volume to bring it to the desired concentration to assay. Triglycerides are enzymatically hydrolyzed by lipase to free fatty acids and glycerol. The glycerol is phosphorylated by adenosine triphosphate (ATP) with glycerol kinase (GK) to produce glycerol-3- phosphate and adenosine diphosphate. Glycerol-3-phosphate is oxidized by dihydroxyacetone phosphate (DAP) by glycerolphosphate oxidase producing hydrogen peroxide (H²O²). In a Trinder5 type color reaction catalyzed by peroxidase, the H²O² reacts with 4-aminoantipyrine (4-AAP) and 3,5-dichloro-2- hydroxybenzene sulfonate (DHBS) to produce a red colored dye. The absorbance of this dye is proportional to the concentration of triglycerides present in the sample. Cholesterol esters are enzymatically hydrolysed by cholesterol esterase to cholesterol and free fatty acids. Free cholesterol, including that originally present, is then oxidized by cholesterol oxidase to cholest-4-en-3- one and hydrogen peroxide. The hydrogen peroxide combines with HBA and 4-aminoantipyrine to form a chromophore (quinoneimine dye) which may be quantitated at 500-550nm.

Affiliations: University of California, Davis

External URL: https://mmpc.org/shared/document.aspx?id=87&docType=Protocol

Version: 2

Publication Date: 2019

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