Lipolysis Assay Kit, dual glycerol and free fatty acids detection (reagents only)

Product Description

This lipolysis assay kit provides the tool to study chemical compounds that may influence lipolysis in cultured adipocytes.

Principle of the Assay

Detection of Free Glycerol
Assessing lipolytic activity by the measurement of glycerol released into the medium. Glycerol released to the medium is phosphorylated by adenosine triphosphate (ATP) forming glycerol-1-phosphate (G-1-P) and adenosine-5’-diphosphate (ADP) in the reaction catalyzed by glycerol kinase. G-1-P is then oxidized by glycerol phosphate oxidase to dihydroxyacetone phosphate (DAP) and hydrogen peroxide (H₂O₂). A quinoneimine dye is produced by the peroxidase catalyzed coupling of 4-aminoantipyrine (4-AAP) and sodium N-ethytl-N-(3-sulfopropyl)m-anisidine (ESPA) with H₂O₂, which shows an absorbance maximum at 540nm. The increase in absorbance at 540nm is directly proportional to glycerol concentration of the sample.

Detection of Non-Esterified Fatty Acids (Free Fatty Acids; FFA)
Assessment of lipolytic activity can also be detected through a coupled reaction to measure non-Esterified fatty acids (NEFA) released by adipocytes. The initial step, carried out by acyl-CoA synthetase (ACS), produces fatty acyl-CoA thiol esters from the NEFA, ATP, Mg, and CoA in the reaction. The acyl-CoA derivatives react with oxygen in the presence of acyl-CoA oxidase (ACOD) to produce hydrogen peroxide. Hydrogen peroxide in the presence of peroxidase (POD) allows the oxidative condensation of 3-methyl-N-ethyl-N-(beta-hydroxyethyl)-aniline with 4-aminoantipyrine which forms a purple product that absorbs light at 550nm. This allows the concentration of NEFA to be determined from the optical NEFA to be determined from the optical density measured at density measured at 540 - 550nm.