35.3 Triglycerides Estimation
Triglycerides are esters of glycerol possessing usually three different fatty acids. The
widely distributed fats and oils in plants and animals are chemically triglycerides,
and they contribute about 95% of adipose tissue. Triglycerides are transported in
plasma, mostly in the form of chylomicrons and VLDL.
35.3.1 Principle
Lipases split triglycerides into glycerol and fatty acids. Glycerol kinase hydrolyzes
glycerol in the presence of ATP to form glycerol-3-phosphate and ADP. Glycerol-3-
phosphate oxidase enzyme splits glycerol-3-phosphate in the presence of O 2 to
dihydroacetone phosphate and H 2 O 2. Peroxide enzyme splits H 2 O 2 to water and
oxygen. The oxygen thus formed will oxidize 4-chlorophenol in the presence of
4-aminoantipyrine to form red quinone imine, which is measured at 520 nm and
compared with that of triglyceride standard.
35.3.2 Reagents
1.Buffer solution
PIPES buffer (50 mM, pH 7.5)
4-chlorophenol (5 mM)
Magnesium ions (4.7 mM)
ATP (1 mM)
Lipases (1 U/ml)
Peroxidase (0.5 U/ml)
Glycerol kinase (0.4 U/ml)
Sodium azide (0.05%)
2.Enzyme reagent
4-amineantipyrine (0.4 mM)
Glycerol-3-phosphate oxidase (1.5 U/ml)
Sodium azide (0.095%)
35.3 Triglycerides Estimation 141