Development and Application of a Peroxyl Radical Clock Approach for Measuring Both Hydrogen-Atom Transfer and Peroxyl...

The rate-determining step in free radical lipid peroxidation is the propagation of the peroxyl radical, where generally two types of reactions occur: (a) hydrogen-atom transfer (HAT) from a donor to the peroxyl radical; (b) peroxyl radical addition (PRA) to a "C═C" double bond. Peroxyl radical clocks have been used to determine the rate constants of HAT reactions (k_H), but no radical clock is available to measure the rate constants of PRA reactions (k_add). In this work, we modified the analytical approach on the linoleate-based peroxyl radical clock to enable the simultaneous measurement of both k_H and k_add. Compared to the original approach, this new approach involves the use of a strong reducing agent, LiAlH₄, to completely reduce both HAT and PRA-derived products and the relative quantitation of total linoleate oxidation products with or without reduction. The new approach was then applied to measuring the k_H and k_add values for several series of organic substrates, including para- and meta-substituted styrenes, substituted conjugated dienes, and cyclic alkenes. Furthermore, the k_H and k_add values for a variety of biologically important lipids were determined for the first time, including conjugated fatty acids, sterols, coenzyme Q10, and lipophilic vitamins, such as vitamins D₃ and A.