Linoleic Hydroperoxide Research Articles

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.

Chemical Changes of Hydroperoxy-, Epoxy-, Keto- and Hydroxy-Model Lipids under Simulated Gastric Conditions.

Chemical changes occurring in dietary lipid oxidation compounds throughout the gastrointestinal tract are practically unknown. The first site for potential chemical modifications is the stomach due to the strong acidic conditions. In this study, model lipids representative of the most abundant groups of dietary oxidation compounds were subjected to in vitro gastric conditions. Thus, methyl linoleate hydroperoxides were used as representative of the major oxidation compounds formed in food storage at low and moderate temperatures. Methyl 9,10-epoxystearate, 12-oxostearate and 12-hydroxystearate were selected as model compounds bearing the oxygenated functional groups predominantly found in oxidation compounds formed at the high temperatures of frying. Analyses were performed using gas-liquid chromatography/flame ionization detection/mass spectrometry and high performance-liquid chromatography/ultraviolet detection. Losses of methyl 9,10-epoxystearate and linoleate hydroperoxides in the ranges 17.8–58.8% and 42.3–61.7% were found, respectively, whereas methyl 12-oxostearate and methyl 12-hydroxystearate remained unaltered. Although quantitative data of the compounds formed after digestion were not obtained, methyl 9,10-dihydroxystearate was detected after digestion of methyl 9,10-epoxystearate, and some major volatiles were detected after digestion of linoleate hydroperoxides. Overall, the results showed that significant modifications of dietary oxidized lipids occurred during gastric digestion and supported that the low pH of the gastric fluid played an important role.

New Plausible Mechanism for Gastric and Colorectal Carcinogenesis: Free Radical-Mediated Acetaldehyde Generation in a Heme/Myoglobin-Linoleate-Ethanol Mixture.

Epidemiological studies have revealed that alcohol, red meat, and cooking oil (or linoleate) are risk factors for both gastric and colon cancers. A survey of the mutation spectra of the p53 tumor suppressor gene in these cancers suggested that the types of mutations and the hot spots are similar to those induced by acetaldehyde (AcAld) in an in vitro p53 mutation analysis system. Accordingly, various combinations of possible factors, components, or model compounds were reacted in an emulsion and tested for the generation of AcAld. Efficient AcAld formation was only observed with combinations of three factors, red meat homogenate (or heme/myoglobin), methyl linoleate, and ethanol, but not by any combination of the two. The generated AcAld levels (ca. 500 μM) far exceeded the minimum mutagenic concentration (40–100 μM) obtained using concentrations of meat homogenate (or heme/Mb), linoleate, and ethanol comparable to those in the stomach after an ordinary meal. A mutagenic level of AcAld (75 μM) was also generated with a physiological concentration of ethanol, heme, and linoleate in the colon. As a mechanism, linoleate hydroperoxide formation and its decomposition in the presence of myoglobin (or heme) to generate the OH radical seem to be involved in the ethanol-to-AcAld conversion.

Effect of vitamin E on low density lipoprotein oxidation at lysosomal pH

Many cholesterol-laden foam cells in atherosclerotic lesions are macrophages and much of their cholesterol is present in their lysosomes and derived from low density lipoprotein (LDL). LDL oxidation has been proposed to be involved in the pathogenesis of atherosclerosis. We have shown previously that LDL can be oxidised in the lysosomes of macrophages. α-Tocopherol has been shown to inhibit LDL oxidation in vitro, but did not protect against cardiovascular disease in large clinical trials. We have therefore investigated the effect of α-tocopherol on LDL oxidation at lysosomal pH (about pH 4.5). LDL was enriched with α-tocopherol by incubating human plasma with α-tocopherol followed by LDL isolation by ultracentrifugation. The α-tocopherol content of LDL was increased from 14.4 ± 0.2 to 24.3 ± 0.3 nmol/mg protein. LDL oxidation was assessed by measuring the formation of conjugated dienes at 234 nm and oxidised lipids (cholesteryl linoleate hydroperoxide and 7-ketocholesterol) by HPLC. As expected, LDL enriched with α-tocopherol was oxidised more slowly than control LDL by Cu2+ at pH 7.4, but was not protected against oxidation by Cu2+ or Fe3+ or a low concentration of Fe2+ at pH 4.5 (it was sometimes oxidised faster by α-tocopherol with Cu2+ or Fe3+ at pH 4.5). α-Tocopherol-enriched LDL reduced Cu2+ and Fe3+ into the more pro-oxidant Cu+ and Fe2+ faster than did control LDL at pH 4.5. These findings might help to explain why the large clinical trials of α-tocopherol did not protect against cardiovascular disease.

Endogenous Labile Iron Pool-Mediated Free Radical Generation for Cancer Chemodynamic Therapy

Current chemodynamic therapy (CDT) primarily relies on the delivery of transition metal ions with Fenton activity to trigger hydroxyl radical production from hydrogen peroxide. However, administration of an excess amount of exogenous Fenton-type heavy metals may cause potential adverse effects to human health, including acute and chronic damages. Here, we present a new CDT strategy that uses intracellular labile iron pool (LIP) as the endogenous source of Fenton-reactive metals for eliciting free radical generation, and the discovery of hydroperoxides (R'OOH) as an optimal LIP-mediated chemodynamic agent against cancer. By simulating the metabolic fates of peroxo compounds within cells, R'OOH was found to have excellent free radical-producing ability in the presence of labile iron(II) and to suffer only moderate elimination by glutathione/glutathione peroxidase, which contributes to its superior chemodynamic efficacy. The LIP-initiated nontoxic-to-toxic transition of R'OOH, together with increased LIP levels in tumor cells, enabled efficient and specific CDT of cancer. Moreover, pH/labile iron(II) cascade-responsive nanomedicines comprising encapsulated methyl linoleate hydroperoxide and LIP-increasing agent in pH-sensitive polymer particles were fabricated to realize enhanced CDT. This work not only paves the way to using endogenous Fenton-type metals for cancer therapy but also offers a paradigm for the exploration of high-performance chemodynamic agents activated by intracellular LIP.

Influence of Oryzanols Concentrate on the Oxidation of Methyl Ester Linoleic Acid and Study of Their Oxidation Products

Oryzanols are frequently found in rice bran oil but almost completely removed in the neutralization step when the oil is chemically refined. In this way, oryzanols can be recovered from the soapstocks to generate a concentrate. Thereby, they could be used as antioxidants in lipids for specific purposes. In the present work the antioxidant power of oryzanols concentrate (33% purity) was studied together with pure oryzanols and butylhydroxytoulene (BHT). Methyl esters were prepared from regular sunflower oil without antioxidants to which the antioxidants before mentioned were added in an effective concentration of 3x10-3 M. The samples were oxidized in a heating block with oxygen flow and the hydroperoxides of linoleic acid methyl ester were analyzed. It was observed that all antioxidants were able to protect the sunflower oil methyl esters from oxidation with respect to methyl esters without antioxidants. Oryzanols presented a notoriously lower protection ability compared to BHT. However, the formation of the linoleic acid methyl ester hydroperoxides and their proportion, for the same oxidation stage, did not show differences between the antioxidants used. Therefore, the oxidation kinetics were similar between the different antioxidants studied.

Oxidative drying properties of a helmet pentadentate phthalocyanine-derived iron(III) complex

The structure of a hexaisoindoline iron(III) complex reported in 1962 was corrected as the one of a recently described helmet phthalocyanine-like iron(III) complex. Accordingly, its catalytic activities were compared to commercial Co(II) and Fe(II) driers towards the oxidation of bulk FAMEs through oxygen uptake measurements and by ATR-FTIR spectroscopy, as well as its ability to decompose methyl linoleate hydroperoxides. It is shown that the phthalocyanine-like iron(III) complex presents higher catalytic performances at low concentrations and under mild conditions of temperature and pressure, both on the overall oxidation process and on the decomposition steps of the drying chemical process. The efficiency of the phthalocyanine-derived iron(III) complex was confirmed by the drying of an alkyd resin emulsion and a satin white paint formulation which exhibits a very good hardness and a low yellowing of the film.

Low density lipoprotein oxidation by ferritin at lysosomal pH

Oxidation of low density lipoprotein (LDL) has been proposed to be involved in the pathogenesis of atherosclerosis. We have previously shown that LDL can be oxidised by iron in lysosomes. As the iron-storage protein ferritin might enter lysosomes by autophagy, we have investigated the ability of ferritin to catalyse LDL oxidation at lysosomal pH. LDL was incubated with ferritin at 37 °C and pH 4.5 and its oxidation monitored spectrophotometrically at 234 nm by the formation of conjugated dienes and by measuring oxidised lipids by HPLC or a tri-iodide assay. Iron released from ferritin was measured using the ferrous iron chelator bathophenanthroline and by ultrafiltration followed by atomic absorption spectroscopy. LDL was oxidised effectively by ferritin (0.05–0.2 μM). The oxidation at lysosomal pH (pH 4.5) was much faster than at pH 7.4. Ferritin increased cholesteryl linoleate hydroperoxide, total lipid hydroperoxides and 7-ketocholesterol. Iron was released from ferritin at acidic pH. The iron chelators, diethylenetriaminepentaacetate and EDTA, and antioxidant N,N׳-diphenyl-p-phenylenediamine inhibited the oxidation considerably, but not entirely. The antioxidant tempol did not inhibit the initial oxidation of LDL, but inhibited its later oxidation. Cysteamine, a lysosomotropic antioxidant, inhibited the initial oxidation of LDL in a concentration-dependent manner, however, the lower concentrations exhibited a pro-oxidant effect at later times, which was diminished and then abolished as the concentration increased. These results suggest that ferritin might play a role in lysosomal LDL oxidation and that antioxidants that accumulate in lysosomes might be a novel therapy for atherosclerosis.

Singlet molecular oxygen generated by cholesterol, oleic, linoleic hydroperoxides with nitronium ion

Singlet molecular oxygen generated by cholesterol, oleic, linoleic hydroperoxides with nitronium ion

Iron‐Catalyzed Reaction of γ‐Tocopherol with Methyl Linoleate Hydroperoxides in Solutions

Abstractγ‐Tocopherol is one of the main constituents in vegetable oils and acts as an antioxidant by trapping lipid‐peroxyl radicals. This study reports reaction products of γ‐tocopherol with lipid‐peroxyl radicals formed by iron‐catalyzed decomposition of methyl linoleate hydroperoxides (MeLOOH) in toluene and methanol solutions. The products in toluene solution were tocored, methyl (8a‐dioxy‐γ‐tocopherone)‐epoxyoctadecenoates (γT‐OO‐epoxyMeL), methyl (8a‐dioxy‐γ‐tocopherone)‐octadecadienoates (γT‐OO‐MeL), γ‐tocopherol biphenyl dimer (γTBD), γ‐tocopherol diphenylether dimer (γTED), and adducts of γ‐tocopherol dimers with the MeLOOH‐derived peroxyl radicals (γTED‐OO‐epoxyMeL, γTBD‐OO‐MeL, and γTED‐OO‐MeL). The iron‐catalyzed reaction in toluene proceeded slowly, whereas the reaction in methanol was relatively fast. The reaction products in methanol were γT‐OO‐epoxyMeL and γTED‐OO‐epoxyMeL together with tocored, γTBD, and γTED. The results indicate that the iron‐catalyzed decomposition of MeLOOH in toluene produces both epoxyperoxyl and peroxyl radicals and that the decomposition in methanol yields only the epoxyperoxyl radicals. These peroxyl radicals could react with the 8a‐carbon‐centered radical of γ‐tocopherol or its dimers.

Catalytic activity of primary and secondary driers towards the oxidation and hydroperoxide decomposition steps for the chemical drying of alkyd resin

Abstract The autoxidation of fatty acid methyl esters (FAMEs) derived from colza, sunflower, linseed and tung oils and the oxidation of sunflower FAMEs in the presence of several commercial driers based on Co(II), Mn(II), Fe(II), V(IV), Sr(II), Zr(IV), Ca(II), Zn(II) have been investigated. The oxidation step was studied through oxygen uptake measurements with an automatic O 2 pressure recorder (RapidOxy ® ) and by ATR-FTIR spectroscopy. Pure methyl linoleate hydroperoxides (ROOH) were prepared by photooxidation to follow their decomposition by HPLC in the presence of the driers. Only Co(II), Mn(II) and Fe(II) as primary driers have catalytic effects both on the oxidation and on the ROOH decomposition steps. However, the Co-based drier shows the higher oxidation rate while the Fe-based drier has the greater ROOH decomposition rate. On the other hand, oxidation runs faster by increasing the amount of driers from 2.5 to 25–50 mM but an antioxidant behavior is observed at higher levels. Secondary driers show poor or no catalytic activities both on the oxidation and the ROOH decomposition steps with a conversion ranging from 5 to 9% over 24 h. Combinations of driers were finally investigated to highlight possible synergistic or antagonist effects during the oxidation and hydroperoxides decomposition steps. Synergistic effects were found only on the oxidation stage with Co(II)-Zn(II), Mn(II)-Zn(II) and Fe(II)-Ca(II), while these latter do not have influence on the decomposition step.

Inhibition of Hydroperoxy‐, Keto‐ and Hydroxy‐FAME by Alpha‐ and Delta‐Tocopherol at Rancimat Conditions

AbstractThe effects of α‐ and δ‐tocopherol on inhibition of hydroperoxides, keto and hydroxy compounds under Rancimat conditions, i.e. 100 °C and air bubbling, were studied in samples of fatty acid methyl esters (FAME) obtained from high linoleic (HL) and high oleic (HO) sunflower oils. Primary hydroperoxides from methyl linoleate and methyl oleate and secondary keto and hydroxy compounds derived from methyl linoleate hydroperoxides were analyzed by HPLC–UV‐ELS. Different tocopherol concentrations, namely, 10, 50, 100, 500 and 1000 mg/kg, were tested. Irrespective of the lipid substrate and the initial concentration of tocopherol, results showed that the content of hydroperoxides accumulated during the induction period was remarkably higher in the samples containing δ‐tocopherol. The relative concentrations of oleate hydroperoxides in the HO samples were also higher in the presence of δ‐tocopherol. α‐Tocopherol was more effective in inhibiting hydroperoxides at low levels, with 100 mg/kg as optimal concentration, while δ‐tocopherol displayed optimal protection at 1000 mg/kg. Under the oxidation conditions applied, neither α‐ nor δ‐tocopherol showed a protective effect on hydroperoxide decomposition at any level assayed. Formation of keto‐ and hydroxy‐dienes was more related to the concentration of their hydroperoxide precursors. Furthermore, both tocopherols gave rise to increased concentrations of ketodienes at 500 and 1000 mg/kg compared to the controls. Such an effect was more pronounced for α‐tocopherol and in the HL samples.

Prooxidant Activity of Polar Lipid Oxidation Products in Bulk Oil and Oil‐in‐Water Emulsion

AbstractLipid oxidation products can arise when oils are subjected to high temperature and exposed to oxygen. Many of these oxidation products have higher polarity than the original triacylglycerols due to the incorporation of oxygen. These polar oxidation products could have a negative impact on oxidative stability by acting as prooxidants. In this study, the influence of polar lipid oxidation products on the oxidative stability of bulk oils and oil‐in‐water emulsions was investigated. Polar compounds were isolated from used frying oil by silica gel column chromatography. They were added to bulk stripped corn oil (with/without reverse micelles formed by dioleoylphosphatidylcholine, DOPC) and oil‐in‐water (O/W) emulsion to evaluate their prooxidative activity. Polar compounds increased lipid oxidation in bulk oil with and without DOPC. The presence of DOPC reverse micelles decreased the prooxidant activity of the polar oxidation products. On the other hand, there was no significant effect of the polar compounds on oxidation of O/W emulsions. To gain a better understanding of the polar compounds responsible for the prooxidant effect, linoleic acid and linoleic hydroperoxide were added into bulk oil at the same concentration as those in the polar fraction of the frying oil. However, they did not show the same prooxidative activity compared to oil with the polar fraction.

Antioxidant and antiproliferative activities of phenolics isolated from fruits of Himalayan yellow raspberry (Rubus ellipticus).

Yellow Himalayan raspberry, a wild edible fruit, was analyzed for phenolic contents, and antioxidant, antibacterial and antiproliferative activities. Phenolics were extracted using 80% aqueous solvents containing methanol, acidic methanol, acetone and acidic acetone. Our analysis revealed that the acidic acetone extracts recovered the highest level of total phenolics (899mg GAE/100g FW) and flavonoids (433.5mg CE/100g FW). Free radical scavenging activities (DPPH, ABTS, superoxide and linoleate hydroperoxide radicals) and ferric reducing activity were highest in the acetone and acidic acetone extracts. No metal chelating or antibacterial activity was detected in any of the extracts. Acetone and methanol extracts showed potent antiproliferative activity against human cervical cancer cells (C33A) with an EC50 of inhibition at 5.04 and 4. 9mg/ml fruit concentration respectively, while showing no cytotoxicity to normal PBMCs cells. Therefore, the present study concluded that the yellow Himalayan raspberry is a potent source of phytochemicals having super antioxidant and potent antiproliferative activities.

Free Radical Oxidation of Polyunsaturated Lipids: New Mechanistic Insights and the Development of Peroxyl Radical Clocks

The peroxidation of lipids in biological membranes has been implicated in both the onset and development of most degenerative diseases. The primary products of this autoxidation process are usually lipid hydroperoxides. They form as a consequence of a free radical chain reaction: lipid peroxyl radicals propagate the chain by rate-limiting H-atom abstraction from another lipid. Studies of the mechanism of lipid peroxidation are a specific part of a wider effort to understand the more general phenomenon of hydrocarbon autoxidation, which dates back some 70 years. However, the autoxidation of lipids is generally much more complicated than that of other hydrocarbons because of additional reaction pathways afforded by a variety of uniquely positioned unsaturated bonds. Indeed, polyunsaturation is an important aspect of many of the most relevant of physiological lipids, such as linoleate and arachidonate. In this Account, we present our current understanding of the mechanism of unsaturated lipid peroxidation, effectively updating our Account on the same topic published 25 years ago. Our more recent work has, in large part, been stimulated by the discovery of the nonconjugated linoleate hydroperoxide as a product under certain autoxidation conditions. The identification of this long-elusive bis-allylic hydroperoxide prompted our kinetic characterization of the reaction leading to its formation. The product distributions obtained from autoxidations of newly synthesized model compounds, which vary in either the substitution of the bis-allylic moiety or the configuration of the double bonds, have provided key insights into the overall mechanism. These insights have in turn been reinforced by the results of theoretical calculations. The picture that emerges is one wherein the delocalized carbon-centered radicals, which arise as intermediates in these reactions, first associate with dioxygen to form pre-reaction complexes. These complexes then collapse through transition state structures that maximize the orbital interactions between the delocalized radical SOMO and dioxygen. The energies of these transition states are influenced by steric effects; thus, there are distinct changes in product distribution in the autoxidation of dienes having different substitution patterns. The radical-dioxygen complexes are also intermediates in the isomerization of allylperoxyl and pentadienylperoxyls, helping explain the high regio- and stereochemical fidelity of these processes. We have taken advantage of the rapid fragmentation of nonconjugated peroxyl radicals to develop a powerful peroxyl radical clock methodology, which can be used to determine rate constants for reactions of peroxyl radicals with molecules having rate constants ranging from 1 to 10(7) M(-1) s(-1). We can make use of this methodology to address various questions, both fundamental and applied, relating to lipid peroxidation and its inhibition by radical-trapping antioxidants.

High-Throughput Assay for Detection of Soybean Lipoxygenase-1

A high-throughput assay was developed to detect soybean lipoxygenase 1 (LOX-1) using a multilabel plate reader. The assay was also adapted to a single cell fluorometer. Fluorescein is degraded by linoleic hydroperoxide produced from soybean lipoxygenase and linoleic acid. The decrease in fluorescence is measured over time, and the area-under-the-curve (AUC) is used to quantify the LOX-1 content of soybean extract. A dose-dependent response is seen with varied dilutions of pure LOX enzyme or soybean extracts. Percent recovery was between 97% and 108%, and relative standard deviation was 4.3%. Advantages of the assay include the reduced preparation time of samples and reduced use of reagents in the high-throughput assay. Multiple samples can be measured in a single run with a multilabel plate reader.

Novel Fluorescent Probe for Analysis of Hydroperoxides Based on Boron Dipyrromethane Fluorophore

A new reagent, N-[2-(diphenylphosphino)ethyl]-4-(1,3,5,7-tetramethyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-8-yl)benzamide (DPPEA-BODIPY), was designed and synthesized for analysis of hydroperoxides. DPPEA-BODIPY fluoresces at low levels in the visible range (lambda(ex)/lambda(em) = 502 nm/515 nm) and reacts with hydroperoxides to produce DPPEA-BODIPY oxide, which fluoresces at high levels. The fluorescence intensity of the reaction mixture was observed to be linearly related to the methyl linoleate hydroperoxide (MeLOOH) concentration.

Intramolecular rearrangement of linolenate peroxyl radicals in lipoxygenase reactions at lower oxygen content

Variation of tissue oxygen content is thought to be a possible factor in determining the structural diversity of hydroperoxy fatty acids. In the present study, we evaluated the structural diversity of intermediate carbon-centered radicals at lower oxygen content. When the buffered solution (pH 7.4) containing 1.0 mM alpha-linolenic acid, 1.0 muM soybean 15-lipoxygenase, and 1.0 mM nitroxyl radical [3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (CmDeltaP)], which selectively traps carbon-centered radicals, was incubated in a sealed vial, the generation of linolenate hydroperoxide was completed within 1 min. In the subsequent reaction at lower oxygen content, the production of the [LnA-H+O(2)].-CmDeltaP adduct was ascertained by liquid chromatography tandem mass spectrometry with precursor ion scanning. Furthermore, HPLC analysis with photodiode array detection showed that the adduct exhibits an absorption maximum at 278 nm, indicating a conjugated triene moiety. On the basis of these facts, the structure of the adduct was speculated to be C(2)H(5)-CH(CmDeltaP)-CH = CH-CH = CH-CH = CH-CH(OOH) -C(7)H(14)-COOH. We proposed a possible reaction pathway as follows: a linolenate 9-peroxyl radical generated in the lipoxygenase reaction might be converted into C(2)H(5)-.CH-CH = CH-CH = CH-CH = CH-CH(OOH) -C(7)H(14)-COOH through an intramolecular rearrangement. This intermediate radical may give rise to hydroperoxy fatty acids with structural diversity.

Occurrence of Conjugated Linolenic Acids in Purified Soybean Oil

AbstractA high‐performance liquid chromatographic (HPLC) method is described for the determination of conjugated linoleic acids (CLA) and conjugated linolenic acids (CLN). Methyl esters prepared from purified lipid fractions of soybean oil were analyzed using an HPLC system equipped with photodiode‐array detector to detect peaks having maximum absorption around 233 and 275 nm. These peaks were concentrated by AgNO3‐silicic acid column chromatography and reversed‐phase HPLC. The structural analysis, of dimethyloxazoline (DMOX) derivatized methyl esters, using gas chromatography–mass spectrometry (GC–MS) showed the occurrence of 9,11‐ and 10,12‐CLA and 8,10,13‐, 8,10,12‐, and 9,11,13‐CLN. The comparison of these conjugated fatty acids with authentic isomers by HPLC revealed the presence of isomeric mixtures of CLA [cis (c),trans(t) or t,c and t,t] and CLN (c,t,t or t,t,c and t,t,t). Traces of 9,11‐ and 10,12‐CLA (c,t or t,c) were found in crude oil. CLN isomers (8,10,12‐18:3 and 9,11,13‐18:3) were found to be forming during the bleaching phase of soybean oil processing. 8,10,13‐CLN and 9,11‐ and 10,12‐CLA (t,t) were only found in soybean oil after the deodorization step. CLN contents in commercial soybean oil varied from 387 to 1,316 mg/kg oil. A decreased level of bleaching earth and temperature resulted in a reduced CLN content. It is possible that CLN would be derived from the linoleate hydroperoxides formed during the processing and storage of soybean oil.

Detection of cholesteryl ester hydroperoxide isomers using gas chromatography–mass spectrometry combined with thin-layer chromatography blotting

Oxidative modification of low-density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. During the oxidation of LDL, cholesteryl esters, the major lipid components in LDL, are oxidized to cholesteryl ester hydroperoxides (CEOOH). The isomers of CEOOH may reflect the reactive species that initiate the peroxidation reaction. In the current study, a novel analytical method for the determination of CEOOH isomers, especially cholesteryl linoleate hydroperoxide isomers, was developed using the combination of two chromatographic techniques: (i) thin-layer chromatography blotting with diphenyl-1-pyrenylphosphine (DPPP) fluorescent detection (DPPP–TLC blotting) and (ii) gas chromatography–electron ionization–mass spectrometry (GC–EI–MS). CEOOH was applied to DPPP–TLC blotting, the obtained DPPP-derived fluorescent spots containing cholesteryl ester hydroxides were extracted and derivatized (hydrogenation, transmethylation, and trimethylsilylation), and the formed methyl ester/trimethylsilylether derivatives of hydroxyoctadecenoic acid were then analyzed by GC–EI–MS. The CEOOH isomers were determined by selected ion monitoring of isomer-specific fragment ions originated from the alpha-cleavage of the trimethylsilyloxyl group. Using these two chromatographic techniques, we were able to detect isomeric CEOOH in the oxidized human LDL. Our results indicated that GC–EI–MS analysis combined with DPPP–TLC blot is a specific method for analyzing cholesteryl ester hydroperoxide isomers in biological samples such as oxidized LDL.