Iron ascorbate-stimulated lipid peroxidation in vitro. Why is the method controversial?

In vitro generation of thiobarbituric acid reactive substances (TBARS) is frequently used to assess organ susceptibility to lipid peroxidation. The yield of TBARS is severalfold enhanced by an addition of iron ions with reductors or chelators such as ascorbate, NADPH, ADP or pyrophosphate. The process cannot be interpreted in a simple way, since it involves several enzymatic and nonenzymatic reactions. There are no clear interpretations of the ambiguous effects of denaturating factors and chelating agents on TBARS generation. Also controversy arises from the curvilinear relationship between the homogenate concentration and the yield of TBARS. This has been modelled in the present work by combining two functions describing the sequential reaction with two limiting steps. One of them is related to catalytic action of iron and ascorbate, while the other to an enzyme, possibly phospholipase A2, as has been suggested by some investigators. Two models should be considered since it is impossible to decide which kinetic equation should predominate in the model. Nevertheless, the model reflects kinetic properties of the process. The effects of catalyst concentration and some other modification upon the yield of TBARS were also investigated experimentally. The results of experiments and modelling showed that the analytical procedures used by investigators need standardisation as the results obtained under a variety of procedures may reflect quite different properties of the living systems.