Oxidation of squalene by singlet oxygen and free radicals results in different compositions of squalene monohydroperoxide isomers

Abstract

Oxidation of squalene (SQ) causes a decline in the nutritional value of SQ in foods, as well as an accumulation of SQ oxidation products in skin lipids which lead to adverse skin conditions. However, mechanistic insights as to how SQ is oxidized by different oxidation mechanisms have been limited, and thus effective measures towards the prevention of SQ oxidation have not been identified. In this study, we oxidized SQ by either singlet oxygen oxidation or free radical oxidation, and monitored the formation of the six SQ monohydroperoxide (SQOOH) isomers, the primary oxidation products of SQ, at the isomeric level. While singlet oxygen oxidation of SQ resulted in the formation of similar amounts of the six SQOOH isomers, free radical oxidation of SQ mainly formed two types of isomers, 2-OOH-SQ and 3-OOH-SQ. The addition of β-carotene during singlet oxygen oxidation, and the addition of α-tocopherol during free radical oxidation lead to a dose-dependent decrease in the formation of SQOOH isomers. Such results suggest that the analysis of SQOOH at the isomeric level allows for the determination of the cause of SQ oxidation in various samples, and provides a foothold for future studies concerning the prevention of SQ oxidation.

Figure 1

Chemical structures of squalene (SQ), SQ monohydroperoxide (SQOOH) isomers, 3-(1,4-epidioxy-4-methyl-1,4-dihydro-1-naphytyl) propionic acid (EP), and cumene hydroperoxide (CHP).

Figure 2

Typical normal phase LC-MS/MS chromatograms analyzing a mixture of 1 ng SQOOH isomer standards (A), SQ samples oxidized by singlet oxygen oxidation in the presence of 10 mM EP for 8 hours (B), and SQ samples oxidized by free radical oxidation via heating at 50 °C for 8 hours (C). Numbers above the peaks represent retention times (min).

Figure 3

Time-dependent changes in the amount of SQOOH isomers in SQ samples oxidized by singlet oxygen oxidation in the presence of 1, 10, or 50 mM EP (A); and samples oxidized by free radical oxidation via heating at 40 °C, 50 °C, or 60 °C (B). All samples were prepared in triplicate, and bars denote mean ± SE.

Figure 4

Infusion MS spectra of SQ samples oxidized by either singlet oxygen oxidation in the presence of 10 mM EP for 8 hours (A) or samples oxidized by free radical oxidation via heating at 50 °C for 8 hours (B).

Figure 5

Time-dependent changes in the amount of SQOOH isomers in SQ samples oxidized by singlet oxygen oxidation in the presence of 10 mM EP and 0 (control), 1, 10, 100, or 1,000 μM either β-Car (A) or α-Toc (B). All samples were prepared in triplicate, and bars denote mean ± SE.

Figure 6

Time-dependent changes in the amount of SQOOH isomers in SQ samples oxidized by free radical oxidation via heating at 50 °C in the presence of 0 (control), 0.01, 0.1, 1, or 10 nmol either β-Car (A) or α-Toc (B). All samples were prepared in triplicate, and bars denote mean ± SE.

Figure 7

Predicted pathways of the singlet oxygen oxidation of SQ (A) and free radical oxidation of SQ (B).