Glycerol undergoes phosphorylation through the enzymatic action of glycerol kinase (GK), utilizing adenosine-5μ-triphosphate (ATP) to produce glycerol-1-phosphate (G-1-P) and adenosine-5μ-diphosphate (ADP). Subsequently, glycerol-1-phosphate (G-1-P) is oxidized by glycerol phosphate oxidase (GPO) to yield dihydroxyacetone phosphate (DAP) and hydrogen peroxide (H2O2). This process involves peroxidase (POD), which catalyzes the coupling of hydrogen peroxide (H2O2) with 4-aminoantipyrine (4-AAP) and sodium N-ethyl-N-(3-sulfopropyl) m-anisidine (ESPA) to generate a quinoneimine dye exhibiting an absorbance maximum at 540 nm.The observed increase in absorbance at 540 nm correlates directly with the concentration of free glycerol present in the sample. This method provides a reliable means for quantifying free glycerol levels, enabling accurate assessment of biochemical samples.
