Oxidation-specific epitopes are important targets of innate immunity.

During the oxidation of LDL, a central pathophysiological component of atherogenesis, a wide variety of chemical and physical changes occur leading to the generation of oxidation-specific neoepitopes. These epitopes are not only immunogenic, leading to adaptive humoral responses, but are also a prominent target of multiple arcs of innate immunity. The pattern recognition receptors (PRRs) of innate immunity are germ line encoded, conserved by natural selection, and bind to pathogen-associated molecular patterns (PAMPs) common on multiple structures. However, it is not intuitive as to why they should recognize oxidation-specific neoepitopes. Yet it is clear that multiple macrophage scavenger receptors, which are classic PRRs, recognize oxidation-specific epitopes, such as those found on oxidized LDL (OxLDL). Other innate proteins, such as C-reactive protein, also bind to OxLDL. Natural antibodies (NAbs), the humoral arc of innate immunity, provide a nonredundant role in the first line of defence against pathogens, but are also believed to provide important homeostatic house-keeping functions against self-antigens. Our work demonstrates that oxidation-specific epitopes, as found on OxLDL, are a major target of NAbs. In this review, we will discuss the specific example of the prototypic NAb T15/E06, which is increased in atherosclerotic mice and mediates atheroprotection, and discuss the potential role of NAbs in atherogenesis, and in inflammation in general. We also review data that oxidation-specific epitopes are generated whenever cells undergo programmed cell death, forming a common set of PAMPs recognized by oxidation-specific PRRs on macrophages, NAbs and innate proteins. We present the hypothesis that oxidation-specific epitopes on apoptotic cells exerted evolutionary pressure for the conservation of these PRRs and also serve to maintain the expansion of a substantial proportion of NAbs directed to these stress-induced self-antigens.

Regiospecific analysis of neutral ether lipids by liquid chromatography/electrospray ionization/single quadrupole mass spectrometry: validation with synthetic compounds.

A reversed-phase high-performance liquid chromatography (HPLC) method with on-line electrospray ionization/collision-induced dissociation/mass spectrometry (ESI/CID/MS) is presented for the regiospecific analysis of synthetic reference compounds of neutral ether lipids. The reference compounds were characterized by chromatographic retention times, full mass spectra, and fragmentation patterns as an aid to clarify the regiospecificity of ether lipids from natural sources. The results clearly show that single quadrupole mass spectroscopic analysis may elucidate the regiospecific structure of neutral ether lipids. Ether lipid reference compounds were characterized by five to six major ions in the positive ion mode. The 1-O-alkyl-sn-glycerols were analyzed as the diacetoyl derivative, and showed the [M - acetoyl](+) ion as an important diagnostic ion. The diagnostic ions of directly analyzed 1-O-alkyl-2-acyl-sn-glycerols and 1-O-alkyl-3-acyl-sn-glycerols were the [M - alkyl](+), [M + H - H(2)O](+) and [M + H](+) ions. Regiospecific characterization of the fatty acid position was evident from the relative ion intensities, as the sn-2 species had relatively high [M + H](+) ion intensities compared with [M + H - H(2)O](+), whereas the reverse situation characterized the sn-3 species. Furthermore, corresponding sn-2 and sn-3 species were separated by the chromatographic system. However, loss of water was promoted as fatty acid unsaturation was raised, which may complicate interpretation of the mass spectra. The diagnostic ions of directly analyzed 1-O-alkyl-2,3-diacyl-sn-glycerols were the [M - alkyl](+), [M - sn-2-acyl](+) and [M - sn-3-acyl](+) ions. Regiospecific characterization of the fatty acid identity and position was evident from the relative ion intensities, as fragmentation of the sn-2 fatty acids was preferred to the sn-3 fatty acids; however, loss of fatty acids was also promoted by higher degrees of unsaturation. Therefore, both structural and positional effects of the fatty acids affect the spectra of the neutral ether lipids. Fragmentation patterns and optimal capillary exit voltages are suggested for each neutral ether lipid class. The present study demonstrates that reversed-phase HPLC and positive ion ESI/CID/MS provide direct and unambiguous information about the configuration and identity of molecular species in neutral 1-O-alkyl-sn-glycerol classes.

Lipid oxidation in fish oil enriched mayonnaise: calcium disodium ethylenediaminetetraacetate, but not gallic acid, strongly inhibited oxidative deterioration.

The antioxidative effects of gallic acid, EDTA, and extra emulsifier Panodan DATEM TR in mayonnaise enriched with 16% fish oil were investigated. EDTA reduced the formation of free radicals, lipid hydroperoxides, volatiles, and fishy and rancid off-flavors. The antioxidative effect of EDTA was attributed to its ability to chelate free metal ions and iron from egg yolk located at the oil-water interface. Gallic acid reduced the levels of both free radicals and lipid hydroperoxides but promoted slightly the oxidative flavor deterioration in mayonnaise and influenced the profile of volatiles. Gallic acid may therefore promote the decomposition of lipid hydroperoxides to volatile oxidation products. Addition of extra emulsifier reduced the lipid hydroperoxide levels but did not influence the level of free radicals or the oxidative flavor deterioration in mayonnaisse; however, it appeared to alter the profile of volatiles. The effect of the emulsifier on the physical structure and rheological properties depended on the presence of antioxidants.

Dynamic headspace gas chromatography/mass spectrometry characterization of volatiles produced in fish oil enriched mayonnaise during storage.

Protection against lipid oxidation and formation of unpleasant fishy and rancid off-flavors in oil-in-water food emulsions, such as fish oil enriched mayonnaise, is difficult to achieve. Volatile profiles from stored mayonnaises with different oil phase compositions were collected using a developed dynamic headspace sampling technique, in which interfering acetic acid was removed in situ with potassium hydroxide, and subsequently 148 volatiles were characterized and monitored by gas chromatography/mass spectrometry. Multivariate statistics showed correlation between the concentration of 62 volatiles and the fish oil and storage parameters, indicating the formation of lipid oxidation products, which impose fishy off-flavors. Further verification was obtained by gas chromatography/olfactometry, by which, among 78 odors, cis-4-heptenal and trans,cis-2,4-heptadienal were detected as distinct fishy notes. In total, 27 volatiles, including 1-penten-3-one, cis-2-penten-1-ol, cis-3-hexenal, cis-4-heptenal, 1-octen-3-one, 1,cis-5-octadien-3-one, 1-octen-3-ol, trans,cis-2, 4-heptadienal, and trans,cis-2,6-nonadienal, were suggested to contribute to the developed unpleasant fishy and rancid off-flavors.

Determination of neutral lipid hydroperoxides by size exclusion HPLC with fluorometric detection. Application to fish oil enriched mayonnaises during storage.

A fast (12 min) high-performance size exclusion chromatography (HPSEC) method for the separation of neutral lipid class hydroperoxides in the oil phases from fish oil enriched mayonnaises was developed. Detection and quantification were performed using the postcolumn fluorometric (FL) diphenyl-1-pyrenylphosphine oxidation principle. The reproducibilities judged by intra- and inter-assay variations were 0.64 and 7.2%, respectively. The HPSEC-FL method was applied to assess the effect of supplementations with emulsifier, gallic acid, and EDTA on the oxidative processes in the mayonnaises during storage. Substantial amounts of hydroperoxy triacylglycerols (TAGOOH) and cholesterol esters (CEOOH), together with traces of TAGOOH-dimers, were detected. All supplementations significantly decreased the levels of TAGOOH and to a lesser degree CEOOH. Supplementations with EDTA and gallic acid resulted in constant and slightly increasing levels of TAGOOH, respectively, thus affecting the oxidation mechanisms seen in reference mayonnaise. The emulsifier Panodan TR DATEM reduced the levels of TAGOOH as compared to the appropriate controls.