Lipoprotein oxidation and measurement of hydroperoxide formation in a single microtitre plate

Abstract

Wallin B, Camejo G. Lipoprotein oxidation and measurement of hydroperoxide formation in a single microtitre plate. Scand J Clin Lab Invest 1994; 54: 341-6.

The oxidative modification of lipoproteins is of clinical importance because of potential contribution to atherogenesis [1, 2, 3]. An early step in the complex process of oxidation is the peroxidation of polyunsaturated fatty acids. We describe here a method for the Cu(II)-catalyzed oxidation of human low density lipoproteins with the subsequent analysis of hydroperoxides formation in a single microtitre plate. The procedure includes a modification of an iodo-metric peroxide assay for test tubes using a commercially available reagent. The microtitre plate method correlated well with the test tube procedure (r = 0.99) and showed comparable sensitivity and reproducibility. It was sensitive down to 0.5nmol hydroperoxides/well and linear up to at least 20nmolwell⁻¹. The method can handle several hundreds of samples a day with considerably less labour than the test tube procedure. It was well suited to monitor the kinetics of lipoprotein oxidation. The method was also used to test the potency of antioxidants, however, some antioxidants may interfere with the iodometric reaction and should be tested in the assay before use.

Key Words:

• antioxidants
• dienes
• iodometric assay
• kinetics of hydroperoxide formation
• low density lipoproteins
• TBARS