Unveiling an unexpected superoxide-mediated photooxidation mechanism of squalene monohydroperoxides to squalene hydroperoxy cyclic peroxides through ESR and LC-MS/MS analyses.

Lipid cyclic peroxides are a rarely reported and documented class of compounds in the human organism. Recently, we reported the formation of squalene (SQ) hydroperoxy cyclic peroxides derived from SQ monohydroperoxide isomers (SQ-OOHs) for the first time. Notably, we successfully detected and quantified cis-2-OOH-3-(1,2-dioxane)-SQ in the human skin. Nevertheless, the underlying mechanism governing the formation of these compounds remained elusive. Therefore, in the current study, we set to determine the reaction's mechanism. To this end, a comprehensive analysis of the precise conditions involved in the onset and propagation of this conversion was carried out by oxidizing total SQ-OOHs under different conditions, including singlet oxygen (1O2), thermal, and photoinduced oxidations monitored by quantifying the generated 2-OOH-3-(1,2-dioxane)-SQ using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Radical intermediates were thoroughly investigated using Electron Spin Resonance (ESR) with the aid of spin traps and radical references. Moreover, calculations of SQ-OOHs' electrostatic charges were performed on Spartan 18 software. We found that the reaction is ideally induced and favored under photooxidation in the presence of 3O2 in hexane, and that superoxide radical (O2•-) is the first key intermediate in this mechanism, whereas peroxyl radicals were the major species observed throughout the oxidation. Chemical calculations provided an explanation for the targeting of tertiary SQ-OOHs by this reaction and gave further evidence on the proposed heterolytic cleavage initiating the reaction. The novel oxidation mechanism suggested herein offers new insights into understanding lipid secondary oxidation and is a promising finding for further studying lipid cyclic peroxides in general.

The inhibition of interaction with serum albumin enhances the physiological activity of curcumin by increasing its cellular uptake.

Based on the free drug hypothesis, we hypothesized that food compounds that bind stronger to BSA than CUR inhibit the binding between BSA and CUR, and that this results in an increase of the cellular uptake and physiological activities of CUR. To verify this hypothesis, food compounds that bind stronger to BSA than CUR were identified. When THP-1 monocytes were co-treated with the identified compounds (e.g., piperine) and CUR, cell viability significantly decreased, suggesting that the physiological activity of CUR was enhanced. Also, when THP-1 macrophages were co-treated with CUR and the identified compounds following LPS + IFNγ treatment, the decrement of TNF-α was higher compared to treatment with CUR only. Furthermore, the cellular uptake of CUR was increased during this co-treatment. Such results verify our hypothesis, and provide insights into the development of ways to enhance the physiological activities of various food compounds via focusing on their interaction with albumin.

Novel Photoinduced Squalene Cyclic Peroxide Identified, Detected, and Quantified in Human Skin Surface Lipids.

Skin surface lipids (SSLs) form the first barrier that protects the human organism from external stressors, disruption of the homeostasis of SSLs can result in severe skin abnormalities. One of the main causes of this disruption is oxidative stress that is primarily due to SSLs oxidation. Squalene (SQ), the most abundant lipid among SSLs, was shown to first undergo singlet molecular oxygen (1O2) oxidation to yield 6 SQ-monohydroperoxide (SQ-OOH) isomers as the primary oxidation products. However, due to the instability and lability of hydroperoxides, we found that when total SQ-OOH isomers are further photooxidized, they form a unique higher molecular weight secondary oxidation product. To generate the compound, we photooxidized total SQ-OOH isomers in the presence of ground state molecular oxygen (3O2), after its isolation and purification, we studied its structure using MS/MS, NMR, derivatization reactions, and chemical calculations. The compound was identified as 2-OOH-3-(1,2-dioxane)-SQ. Photooxidation of individual SQ-OOH isomers revealed that 6-OOH-SQ is the precursor of 2-OOH-3-(1,2-dioxane)-SQ and indicated the possibility of the formation of similar cyclic peroxides from each isomer following the same photoinduced chain reaction mechanism. An HPLC-MS/MS method was developed for the analysis of 2-OOH-3-(1,2-dioxane)-SQ and its presence on the skin was confirmed in SSLs of six healthy individuals. Its quantity on the skin correlated directly to that of SQ and was not inversely proportional to its precursor, indicating the possibility of its accumulation on the skin surface and the constant regeneration of 6-OOH-SQ from SQ's oxidation. In general, research on lipid cyclic peroxides in the human organism is very limited, and especially on the skin. This study shows for the first time the identification and presence of a novel SQ cyclic peroxide "2-OOH-3-(1,2-dioxane)-SQ" in SSLs, shedding light on the importance of further studying its effect and role on the skin.

Investigation of Lipoproteins Oxidation Mechanisms by the Analysis of Lipid Hydroperoxide Isomers.

The continuous formation and accumulation of oxidized lipids (e.g., lipid hydroperoxides (LOOH)) which are present even in plasma lipoproteins of healthy subjects, are ultimately considered to be linked to various diseases. Because lipid peroxidation mechanisms (i.e., radical, singlet oxygen, and enzymatic oxidation) can be suppressed by certain proper antioxidants (e.g., radical oxidation is efficiently suppressed by tocopherol), in order to suppress lipid peroxidation successfully, the determination of the peroxidation mechanism involved in the formation of LOOH is deemed crucial. In this study, to determine the peroxidation mechanisms of plasma lipoproteins of healthy subjects, we develop novel analytical methods using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine hydroperoxide (PC 16:0/18:2;OOH) and cholesteryl linoleate hydroperoxide (CE 18:2;OOH) isomers. Using the newly developed methods, these PC 16:0/18:2;OOH and CE 18:2;OOH isomers in the low-density lipoprotein (LDL) and high-density lipoprotein (HDL) of healthy subjects are analyzed. Consequently, it is found that predominant PC 16:0/18:2;OOH and CE 18:2;OOH isomers in LDL and HDL are PC 16:0/18:2;9OOH, PC 16:0/18:2;13OOH, CE 18:2;9OOH, and CE 18:2;13OOH, which means that PC and CE in LDL and HDL are mainly oxidized by radical and/or enzymatic oxidation. In conclusion, the insights about the oxidation mechanisms shown in this study would be useful for a more effective suppression of oxidative stress in the human organism.