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This study was prompted by recent reports that epoxyeicosatrienoic (EET) and epoxyeicosatetraenoic (EEQ) acids accelerate tumor growth and metastasis by stimulation of angiogenesis, while eicosapentaenoic (EPA) and epoxydocosapentaenoic (EDP) acids inhibit angiogenesis, tumor growth, and metastasis. Cytochrome P450 epoxygenases convert arachidonic to EET, eicosapentaenoic acid to EEQ, and docosahexaenoic acid to EDP, which are found both in free form and esterified to glycerophosphocholine (GPC). Both free and esterified epoxy (EP) acids are also formed during lipid autoxidation. For biological activity, the GPC-EP requires hydrolysis, which we presumed could occur by sPLA

Biologically active F- and E/D-type-prostane ring isomers (F2-IP and E2/D2-IP, respectively) are produced in situ by non-enzymatic peroxidation of arachidonic acid esterified to GroPCho (PtdCho-IP) and are universally distributed in tissue lipoproteins and cell membranes. Previous work has shown that platelet-activating factor acetylhydrolases (PAF-AH) are the main endogenous PLA2 involved in degradation of PtdCho-IP. The present study shows that the PtdCho-IP are also subject to hydrolysis by group IIA, V and X secretory PLA2, which also have a wide peripheral tissue distribution. For this demonstration, we compared the LC/MS profiles of PtdCho-IP of auto-oxidized plasma lipoproteins after incubation for 1–4 h (37 °C) in the absence or presence of recombinant human sPLA2 (1–2.5 µg/ml). In the absence of exogenously added sPLA2 the total PtdCho-IP level after 4 h incubation reached 15.9, 21.6 and 8.7 nmol/mg protein of LDL, HDL and HDL3, respectively. In the presence of group V or group X sPLA2 (2.5 µg/ml), the PtdCho-IP was completely hydrolyzed in 1 h, while in the presence of group IIA sPLA2 (2.5 µg/ml) the hydrolysis was less than 25% in 4 h, although it was complete after 8–24 h incubation. This report provides the first demonstration that PtdCho-IP are readily hydrolyzed by group IIA, V and X sPLA2. A co-location of sPLA2 and the substrates in various tissues has been recorded. Thus, the initiation of interaction and production of isoprostanes in situ are highly probable.

Investigation of the diversity of human secretory phospholipases A2 (sPLA2) on the migration of human vascular smooth muscle cells (VSMC). We investigated the impact of sPLA2 IIA, V, and X and of oleic acid, linoleic acid and lysophosphatidylcholine on the migration of human VSMC. Recombinant human sPLA2’s and Boyden’s chamber method were applied. sPLA2, IIA but not V or X enhanced migration of VSMC in a dose/time dependent manner. Oleic and linoleic acids, and lysophosphatidylcholine markedly enhanced migration. These results imply that sPLA2 IIA, which is known to be present in the arterial wall in the vicinity of VSMC, as well as products of lipid hydrolysis induced by sPLA2, enhance the migration of VSMC, and thus may contribute to atherogenic process.

We have utilized the Hewlett-Packard LC/ESI-MS technology to store electronic records of total ion current profiles of molecular species of various natural and peroxidized phospholipids of native and auto-oxidized plasma lipoproteins before and after digestion with secretory phospholipase A2 (sPLA2). The present report illustrates the successful utilization of the stored data in several originally un-intended applications, including chemical, physico-chemical and enzymatic alterations of the lipoprotein lipids. The inclusion of lipid samples from enzyme and control incubations adds a dynamic aspect to multidimensional lipidomic analysis of plasma lipoproteins.

There is a worldwide demand for replacing petroleum-derived raw materials with renewable ones. Plant oils and animal fats constitute an important source of renewable lipid materials for chemical industry. Although some plants contain up to 50% of their oils as vernolic and related epoxy fatty acids, industrial extraction and production of such epoxidized acids is not practical. Therefore, epoxidized fatty acids are currently produced by chemical oxidation of unsaturated plant oils. This chapter reviews the basic chemical and biochemical reactions involved in the synthesis of the fatty acid epoxides as well as in the biochemical degradation of the fatty acid epoxides. The large-scale production of fatty acid epoxides and their incorporation into various industrial and household products, however, raises concern about the biological safety of epoxy fatty acids and their derivatives. Therefore, this chapter concludes with an extensive discussion of the biological activity of epoxy fatty acids, ranging from normal metabolism to clinical and pathological effects.

Complete resolution of hydroxyeicosatetraenoic acid (HETE) enantiomers was achieved using hydroxypropyl-γ-cycodextrin (HP-γ-CD)-modified MEKC. The optimum running conditions were determined to be utilizing a 30 mM phosphate-15 mM borate buffer (pH 9.0) containing 30 mM HP-γ-CD and 75 mM SDS as the BGE, application of +30 kV as the effective voltage, and carrying out the experiment at 15°C. The eluents were detected at 235 nm. The method was used successfully for the simultaneous separations of (S)- and (R)-enantiomers of regioisomeric 8-, 11-, 12-, and 15-HETEs. Subsequently, the optimized method was applied to evaluate the stereochemistry of 8- and 12-HETEs from the marine red algae, Gracilaria vermiculophylla and Gracilaria arcuata, respectively. The 8-HETE was found to be a mixture of 98% (R)-enantiomer and 2% (S)-enantiomer, while the 12-HETE was a mixture of 98% (S)-enantiomer and 2% (R)-enantiomer. The present study demonstrates that the HP-γ-CD-modified MEKC method is simple and sensitive and provides unambiguous information on the configuration of natural and synthetic HETEs. This article is protected by copyright. All rights reserved

Thin-layer chromatography (TLC), gas chromatography (GC), and liquid chromatography (LC) in combination with mass spectrometry (MS) have been adopted for the isolation and identification of oxolipids and for determining their functionality. TLC provides a rapid separation and access to most oxolipids as intact molecules and has recently been effectively interfaced with time-of-flight (TOF) MS (TOF-MS). GC with flame ionization (FI) (GC/FI) and electron impact (EI) MS (GC/EI-MS) has been extensively utilized in the analysis of isoprostanes and other low-molecular-weight oxolipids, although these methods require derivatization of the analytes. In contrast, LC with ultraviolet (UV) absorption (LC/UV) or evaporate light scattering detection (ELSD) (LC/ELSD) as well as electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) MS (LC/ESI-MS) or LC/APCI-MS has proven to be well suited for the analysis of intact oxolipids and their conjugates without or with minimal derivatization. Nevertheless, kit-based colorimetric and fluorescent procedures continue to serve as sensitive indicators of the presence of hydroperoxides and aldehydes.

Mitogenesis of Vascular Smooth Muscle Cells (VSMC) plays an important role in atherogenesis. Until recently, the effect of lipid subfractions has not been clarified. Secretory phospholipases A2 (sPLA2's) hydrolyse glycerophospholipids and release pro-inflammatory lyso-lipids, oxidized and non-oxidized fatty acids and isoprostanes. They localize in the vascular wall. We hypothesized that structurally similar sPLA2's may exert different impact on VSMC. The influence of sPLA2's, IIA, V, X, HDL, LDL, and hydrolysis products was tested on mitogenesis of VSMC, i.e., the early effect on the cell membrane phospholipids, and on PGE2 and LTB4 release, i.e., late effect of Cyclooxygenase and 5-lipooxygenase activity in VSMC. Mitogenesis was significantly enhanced by HDL and LDL, and by products of sPLA2 hydrolysis. Hydrolysis of HDL or LDL enhanced mitogenic activity in order V>X>IIA. The release of PGE2 was enhanced by group X sPLA2 and by HDL hydrolyzed by groups V and X. LDL and its hydrolysis products enhanced the release of PGE2 in order X>V>IIA. The release of LTB4 was markedly increased by LDL and HDL, and by hydrolytic products of group V and X, but not group IIA sPLA2. Our study demonstrates a diverse interaction of pro-inflammatory sPLA2's with HDL and LDL affecting both mitogenesis and eicosanoid release from VSMC, therefore potentially enhancing their pro-atherogenic activity.

This study examines the feasibility that peroxidation and lipolysis of 1-O-alkyl-2,3-diacyl-sn-glycerols (DAGE) found in shark liver oil and human milk fat constitutes a potential source of dietary precursors of platelet activating factor (PAF) mimics and of gamma-hydroxybutyrate (GHB). Purified DAGE were converted into 1-O-alkyl-2-acyl-sn-glycerols by pancreatic lipase, without isomerization, and transformed into 1-O-alkyl-2-oxoacyl-sn-glycerols by mild autooxidation. The various core aldehydes without derivatization, as well as the corresponding dinitrophenylhydrazones, were characterized by chromatographic retention time and diagnostic ions by online electrospray mass spectrometry. Core aldehydes of oxidized shark liver oil yielded 23 molecular species of 1-O-alkyl-sn-glycerols with short-chain sn-2 oxoacyl groups, ranging from 4 to 13 carbons, some unsaturated. Autooxidation of human milk fat yielded 1-O-octadecyl-2-(9-oxo)nonanoyl-sn-glycerol, as the major core aldehyde. Because diradylglycerols with short fatty chains are absorbed in the intestine and react with cytidine diphosphate-choline in the enterocytes, it is concluded that formation of such PAF mimics as 1-O-alkyl-2-(omega-oxo)acyl-sn-glycerophosphocholine from unsaturated dietary DAGE is a realistic possibility. Likewise, a C4 core alcohol produced by aldol-keto reduction of a C4 core aldehyde constitutes a dietary precursor of the neuromodulator and recreational drug GHB, which has not been previously pointed out.

Phospholipids carried by very low density lipoprotein (VLDL) are hydrolysed in circulation by lipoprotein and hepatic lipases and lecithin-cholesterol acyltransferase. We have previously demonstrated [6] [J.J. Agren, A. Ravandi, A. Kuksis, G. Steiner, Structural and compositional changes in very low density lipoprotein triacylglycerols during basal lipolysis, Eur. J. Biochem. 269 (2002) 6223–6232] that the infusion of Triton WR 1339 (TWR), which inhibits these lipases, leads in 2 h to five-fold increase in VLDL triacylglycerol concentration along with major differences in the composition of their molecular species. The present study demonstrates that the accumulation of triacylglycerols is accompanied by major changes in the content of the VLDL phospholipids, of which the most significant is the enrichment of phosphatidylethanolamine (PtdEtn). This finding coincides with the enrichment in PtdEtn demonstrated in the VLDL of a hepatocytic Golgi fraction but it had not been demonstrated that the Golgi VLDL, along with its unusual phospholipid composition, can be directly transferred to plasma. Aside from providing an easy access to nascent plasma VLDL, the TWR infusion demonstrates that lipoprotein and hepatic lipases are also responsible for the degradation of plasma VLDL PtdEtn, as independently demonstrated for plasma phosphatidylcholine. Our results indicate also, with the exception of lysophosphatidylcholine, that preferential basal hydrolysis no dot lead to major differences in molecular species composition between circulating and newly secreted VLDL phospholipids. The comparison of the molecular species composition of VLDL and liver phospholipids suggests a selective secretion of PtdEtn and sphingomyelin molecular species during VLDL secretion.

This study uses normal-phase HPLC with on-line positive ion electrospray mass spectrometry (ESI-MS) to obtain quantitative compositional data on both synthetic and butterfat short-chain TAG. The product ion tandem MS of standards averaged 11.1 times lower in abundance of the ion formed by cleavage of FA from the sn-2-position for the pairs of regioisomers in the TAG classes: L/L/S-L/S/L and L/S/S-S/L/S, where L denotes long and S short acyl chain (C2-C6). The molar correction factors, determined for 42 regioisomeric pairs of short-chain TAG of 20 randomized mixture of standards, differed by 1.4-80% as the ratios varied between 0.217 and 5.847. Butterfat TAG were resolved into four fractions on short flash chromatography grade silica gel columns. Pairs of regioisomers in the TAG classes L/S/S-S/L/S with predominance of L/S/S isomers and the sole regioisomers in the TAG classes L/L(M)/S were identified by tandem MS, where M denotes either 8:0 or 10:0 acyl chain. The total proportion of L/L(M)/S isomers was estimated at 34.7 and that of L/S/S-S/L/S at 1.0 mol%, including a small proportion of S/S/S. In contrast to previous work, the present data indicate the presence of a small proportion of butyric and caproic acids in the sn-1-position. The overall distribution of the FA in the short-chain TAG of butterfat, calculated from direct MS measurements, was consistent with the results of indirect determinations based on stereospecific analyses of total butterfat.

We identified and quantified the hydroperoxides, hydroxides, epoxides, isoprostanes, and core aldehydes of the major phospholipids as the main components of the oxophospholipids (a total of 5-25 pmol/micromol phosphatidylcholine) in a comparative study of human atheroma from selected stages of lesion development. The developmental stages examined included fatty streak, fibrous plaque, necrotic core, and calcified tissue. The lipid analyses were performed by normal-phase HPLC with on-line electrospray MS using conventional total lipid extracts. There was great variability in the proportions of the various oxidation products and a lack of a general trend. Specifically, the early oxidation products (hydroperoxides and epoxides) of the glycerophosphocholines were found at the advanced stages of the plaques in nearly the same relative abundance as the more advanced oxidation products (core aldehydes and acids). The anticipated linear accumulation of the more stable oxidation products with progressive development of the atherosclerotic plaque was not apparent. It is therefore suggested that lipid infiltration and/or local peroxidation is a continuous process characterized by the formation and destruction of both early and advanced products of lipid oxidation at all times. The process of lipid deposition appears to have been subject to both enzymatic and chemical modification of the normal tissue lipids. Clearly, the appearance of new and disproportionate old lipid species excludes randomness in any accumulation of oxidized LDL lipids in atheroma.

The molecular species of TAG core aldehydes (aldehydes still esterified to parent molecules) were detected and quantified in dietary-quality sunflowerseed oil autoxidized for 0-18 d at 60 degrees C in the dark. The analyses were performed by reversed-phase HPLC with UV (358 nm) absorption or light scattering and electrospray ionization-MS (ESI/MS) detection following preparation of the dinitrophenylhydrazone derivatives. Aldehyde production, as estimated by UV and ESI/MS, increased gradually over the 18-d period following a rapid initial destruction of the core aldehydes accumulated during storage of the commercial oil at 10 degrees C for 3 mon. The contents of hydroperoxides and hydroperoxide core aldehyde combinations were estimated to account for about 5% of total TAG, quantified as area in the chromatographic trace, after 18 d of autoxidation as estimated by an evaporative light scattering detector (ELSD). The major species of core aldehydes were tentatively identified as 9-oxononanoyl (70%)-, 12-oxo-9,10-epoxydodecenoyl (10%)-, and 13-oxo-9,11-tridecadienoyl (5%)-containing acylglycerols, plus smaller amounts of simple and mixed chain-length dialdehydes, and hydroxy and epoxy monoaldehyde-containing acylglycerols (15% of total). Quantitatively, the core aldehydes made up 2-12 g/kg of oil by UV detection and 2-9 g/kg of oil by ESI/MS detection, whereas the hydroperoxides measured in the unreduced state by HPLC with ELSD were estimated at 200 g/kg after 18 d of autoxidation. The major hydroperoxides of sunflowerseed oil were as previously identified.

Objective: High-density lipoprotein (HDL) is believed to protect against development of atherosclerosis by inhibiting the accumulation of oxidized lipids in low-density lipoprotein (LDL). Paradoxically, HDL lipid is more susceptible to oxidation than LDL lipid. In the present study, we examined the effect of oxidized phospholipids on the uptake of HDL by macrophages. Methods and results: Oxidation of HDL increased formation of phosphatidylcholine core aldehydes that was paralleled by increased covalent binding of phospholipids to HDL protein from 0.96±0.44 to 8.5±1.76 phosphorus/HDL protein (mol/mol). Incubation of apolipoprotein AI with synthetically prepared phosphatidylcholine core aldehydes, 1-palmitoyl-2-[5-oxo]valeroyl- sn -glycero-3-phosphocholine or 1-palmitoyl-2-[9-oxo] nonanoyl- sn -glycero-3-phosphocholine, significantly increased the phosphorus:apolipoprotein AI ratio from 1.1±0.5 to 7.2±2.0 and from 0.9±0.6 to 8.5±0.8, respectively. The binding and uptake of phosphatidylcholine core aldehyde–apolipoprotein AI proteoliposomes, by THP-1 macrophages, was similar to that observed for oxidized HDL and oxidized LDL. Conclusion: We conclude that oxidation of HDL increased formation of phosphatidylcholine core aldehyde–apolipoprotein AI Schiff base adducts and enhanced uptake of oxidized HDL by THP-1 macrophages.

Publisher Summary Plasma contains cholesteryl, glyceryl, and phosphoglyceryl esters of fatty acids as major lipid components. These compounds are carried in plasma and provide a source of metabolic fuel that drives all biochemical reactions required for normal cellular activity and they provide structural material for cell membranes and lipoproteins. These compounds also include molecules that signal receptor activation on the cell surface, triggering subsequent signaling cascades through various protein kinases and phospholipases. This chapter provides a brief summary of plasma lipid composition and the main chemical and biochemical methods of detecting and quantifying the major primary and secondary products of oxidation of plasma lipoproteins and presents a discussion of the physicochemical, chemical, and metabolic activities of the major individual molecular species or small groups thereof. It also provides attention to the possible involvement of oxo-lipids in metabolic syndromes and disease conditions, as well as prevention of oxidation. The nature and extent of plasma lipid autoxidation depends on the fatty acid composition of glycerolipids, glycerophospholipids, and cholesteryl esters of plasma lipo-proteins. The fatty acid composition of the lipoproteins varies with the diet and to a lesser extent with the age, sex, and general health of the individual.

Triacylglycerols secreted by liver and carried by very low density lipoprotein (VLDL) are hydrolysed in circulation by lipoprotein and hepatic lipases. These enzymes have been shown to have positional and fatty acid specificity in vitro. If there were specificity in basal lipolysis in vivo, triacylglycerol compositions of circulating and newly secreted VLDL would be different. To study this we compared the composition of normal fasting VLDL triacylglycerol of Wistar rats to that obtained after blocking lipolysis by Triton WR1339, which increased plasma VLDL triacylglycerol concentration about 4.7-fold in 2 h. Analyses of molecular species of sn-1,2- and sn-2,3-diacylglycerol moieties and stereospecific triacylglycerol analysis revealed major differences between the groups in the VLDL triacylglycerol composition. In nontreated rats, the proportion of 16:0 was higher and that of 18:2n-6 lower in the sn-1 position. The proportion of 14:0 was lower in all positions and that of 18:0 was lower in the sn-1 and sn-3 positions in nontreated rats whereas the proportions of 20:4n-6, 20:5n-3, 22:5n-3 and 22:6n-3 were higher in the sn-1 and lower in the sn-2 position. These results suggest that the fatty acid of the sn-1 position is the most decisive factor in determining the sensitivity for hydrolysis of the triacylglycerol. In addition, triacylglycerol species with highly unsaturated fatty acids in the sn-2 position also favoured hydrolysis. The in vivo substrate specificity followed only partly that obtained in in vitro studies indicating that the nature of molecular association of fatty acids in natural triacylglycerol affects its susceptibility to lipolysis. To conclude, our results indicate that preferential basal lipolysis leads to major structural differences between circulating and newly secreted VLDL triacylglycerol. These differences extend beyond those anticipated from analysis of total fatty acids and constitute a previously unrecognized feature of VLDL triacylglycerol metabolism.

The lipid ester core aldehydes formed during a rapid oxidation (7.8 M tert-butyl hydroperoxide, 90 min at 37 degrees C) of the triacylglycerols of purified corn and sunflower oils were isolated as dinitrophenylhydrazones by preparative thin-layer chromatography and identified by reversed-phase high-performance liquid chromatography with on-line electrospray ionization mass spectrometry and by reference to standards. A total of 113 species of triacylglycerol core aldehydes were specifically identified, accounting for 32-53% of the 2,4-dinitrophenylhydrazine (DNPH)-reactive material of high molecular weight representing 25-33% of the total oxidation products. The major core aldehyde species (50-60% of total triacylglycerol core aldehydes) were the mono(9-oxo)nonanoyl- and mono(12-oxo)-9,10-epoxy dodecenoyl- or (12-oxo)-9-hydroxy-10,11-dodecenoyl-diacylglycerols. A significant proportion of the total (9-oxo)nonanoyl and epoxidized (12-oxo)-9,10-dodecenoyl core aldehydes was found in complex combinations with hydroperoxy or hydroxy fatty acyl groups (6-10% of total triacylglycerol core aldehydes). Identified were also di(9-oxo)nonanoylmonoacylglycerols (0.5% of total) and tri(9-oxo)nonanoylglycerols (trace). The identification of the oxoacylglycerols was consistent with the products anticipated from tert-butyl hydroperoxide oxidation of the major species of corn and sunflower oil triacylglycerols (mainly linoleoyl esters). However, the anticipated (13-oxo)-9,11-tridecadienoyl aldehyde-containing acylglycerols were absent because of further oxidation of the dienoic core aldehyde. A significant proportion of the unsaturated triacylglycerol core aldehydes contained tert-butyl groups linked to the unsaturated fatty chains via peroxide bridges (2-9%). The study demonstrates that rapid peroxidation with tert-butyl hydroperoxide constitutes an effective method for enriching natural oils and fats in triacylglycerol core aldehydes for biochemical and metabolic testing.

A new software algorithm for automatic interpretation of mass spectra of glycerolipids has been developed. The algorithm utilizes a user-specified list of parameters needed to process the spectra. The compounds in mass spectra are identified according to range of measured m/z values, after which the spectra are automatically corrected by the content of naturally occurring isotopes and ion intensities of identified compounds by response correction factors. Automatic processing of the spectra was shown to be accurate and reliable by testing with numerous spectra of glycerophospholipids obtained by liquid chromatography/electrospray ionization mass spectrometry and by comparing the results with manual interpretation of the spectra. If quantitative analysis using internal standards is performed, all the identified compounds in the sample are quantified automatically. A dilution factor may be defined for each sample and is applied to correct the alterations in sample concentration during sample preparation. Processing of several replicate spectra simultaneously produces mean results with standard deviations. The software may also be used to subtract the results of two analyses and to calculate the mean result of replicate subtractions. The algorithm was shown to save time and labor in repetitive processing of mass spectra of similar type. It may be applied to processing of spectra obtained by various mass spectrometric methods.

We recently demonstrated that exposure of synthetic mono- and diunsaturated triacylglycerols to tert-butylhydroperoxide (TBHP) leads to formation of stable adducts of the oxidizing agent and the unsaturated esters (Sjövall, O., Kuksis, A., and Kallio, H., Reversed Phase High-Performance Liquid Chromatographic Separation of tert-Butyl Hydroperoxide Oxidation Products of Unsaturated Triacylglycerols, J. Chromatogr. A 905, 119-132, 2001). In the present study we isolated and identified the TBHP adducts of corn oil triacylglycerols. The much wider range of molecular species available in the corn oil permitted us to demonstrate that the yield of the adducts varies with the degree of unsaturation of the triacylglycerol. The highest yields were obtained for the linoleate (20%, of linoleoyl-containing residual triacylglycerols) and the lowest ones for the oleate (5% of oleoyl-containing residual triacylglycerols) triacylglycerols, whereas the saturated triacylglycerols did not give TBHP adducts in readily detectable amounts. Normal-phase thin-layer chromatography along with reversed-phase high-performance liquid chromatography/mass spectrometry (LC/MS) with electrospray ionization was used to isolate and separate the major molecular species of polyunsaturated triacylglycerols and corresponding TBHP adducts. As an extreme example, the dilinoleoylmonooleoylglycerol was identified as the mono-, di-, tri-, tetra-, and penta-TBHP adduct. LC/MS with electrospray ionization at elevated capillary exit voltage (pseudo tandem mass spectrometry) was used to confirm structures of the [M-RCOOH]+ ions and the absence of TBHP adducts of [M-RCOOH]+. It is concluded that stable adduct formation is an unavoidable complication of preparation of oxotriacylglycerols by oxidation with concentrated TBHP solutions and care must be taken to resolve the adducts from the desired oxidation product.

Increased levels of plasma sterols other than cholesterol can serve as markers for abnormalities in lipid metabolism associated with clinical disease. Premature atherosclerosis and xanthomatosis occur in two rare lipid storage diseases, Cerebrotendinous xanthomatosis (CTX) and sitosterolemia. In CTX, cholestanol is present in all tissues. In sitosterolemia, dietary campesterol and sitosterol accumulate in plasma and red blood cells. Plasma accumulation of oxo-sterols is associated with inhibition of bile acid synthesis and other abnormalities in plasma lipid metabolism. Inhibition of cholesterol biosynthesis is associated with plasma appearance of precursor sterols. The increases in non-cholesterol sterols, while highly significant, represent only minor changes in plasma sterols, which require capillary gas-liquid chromatography and MS for effective detection, identification and quantification.

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.

High density lipoprotein (HDL) is rich in polyunsaturated phospholipids that are sensitive to oxidation. However, the effect of apolipoprotein A-I and paraoxonase-1 (PON-1) on phosphatidylcholine oxidation products has not been identified. We subjected native HDL, trypsinized HDL, and HDL lipid suspensions to oxidation by the peroxynitrite donor, 3-morpholinosydnonimine. HDL had a basal level of phosphatidylcholine mono- and di-hydroperoxides that increased to a greater extent in HDL, compared with either trypsinized HDL or HDL lipid alone. Phosphatidylcholine core aldehydes, which were present in small amounts, increased 10-fold during oxidation of native HDL, compared with trypsinized HDL (p = 0.004), and 4-fold compared with HDL lipid suspensions (p = 0.0021). In addition, the content of lysophosphatidylcholine increased 300% during oxidation of native HDL, but only 80 and 25%, respectively, during oxidation of trypsinized HDL and HDL lipid suspensions. Phosphatidylcholine isoprostanes accumulated in comparable amounts during the oxidation of all three preparations. Incubation of apolipoprotein A-I with 1-palmitoyl-2-linoleoyl glycerophosphocholine proteoliposomes in the presence of 3-morpholinosydnonimine or apoAI with phosphatidylcholine hydroperoxides resulted in a significant increase in phosphatidylcholine core aldehydes with no formation of lysophosphatidylcholine. We propose that apolipoprotein A-I catalyzes a one-electron oxidation of alkoxyl radicals. Purified PON-1 hydrolyzed phosphatidylcholine core aldehydes to lysophosphatidylcholine. We conclude that, upon HDL oxidation with peroxynitrite, apolipoprotein AI increases the formation of phosphatidylcholine core aldehydes that are subsequently hydrolyzed by PON1.

In the acute-phase response and in diseases with prolonged acute phases, normal HDL (NHDL) is converted into acute-phase HDL (APHDL) and becomes proinflammatory and unable to protect LDL against oxidative modification. Earlier work had demonstrated that these changes are associated with alterations in apolipoprotein composition and enzymatic activity of APHDL, but the effect of the acute-phase condition on the lipid composition of APHDL had remained obscure. The present study shows marked quantitative differences in lipid composition between NHDL and APHDL. Specifically, APHDL contained 25% less total lipid per milligram of protein. Up to 50% of cholesteryl ester in the lipid core of APHDL was replaced by triacylglycerol; however, the total phospholipid/total neutral lipid ratios were the same as in NHDL, both lipoproteins giving similar calculated lipid core radii. Furthermore, the phosphatidylcholine/sphingomyelin ratio in APHDL was nearly double that in NHDL, indicating a relative loss of sphingomyelin. A decrease was also seen in diacyl and alkenylacyl glycerophosphatidylethanolamine as well as in phosphatidylinositol of APHDL when compared with NHDL. APHDL contained proportionally more saturated and less polyunsaturated and isoprostane-containing species of phosphatidylcholine, as well as more saturated than unsaturated cholesteryl esters. APHDL also contained significantly more free fatty acids, lysophosphatidylcholine, and free cholesterol. These changes in the lipid composition of HDL are consistent with the alterations in the apoprotein composition and enzymatic activity of APHDL and indicate proinflammatory and proatherogenic roles for APHDL.

The atherogenic effects of refined, bleached and deodorized (RBD) palm oil were compared with those of randomized RBD palm oil and red palm oil. RBD palm oil contains 41.2% palmitic acid, 2.6% at the SN2 position. In randomized palm oil, 13.6% of the palmitic acid is in the SN2 position. Randomized palm oil is significantly more atherogenic for rabbits than is RBD palm oil, supporting our earlier findings that the increasing amounts of palmitic acid in the SN2 position of a fat lead to an increased atherogenic effect. Red palm oil is the oil initially obtained from the palm fruit which contains carotenes and Vitamin E that are removed during refining. Red palm oil was significantly less atherogenic than RBD palm oil supporting the hypothesis that carotenoids and Vitamin E may protect against atherosclerosis. The oils tested had similar effects on serum and liver lipids.

Extensive evidence of the deleterious biological effects of oxidized 5-cholesten-3β-ol (cholesterol) derivatives has led to great interest in their detection. We observed that known oxidized cholesterol derivatives can be rapidly quantitated by combining reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) absorption and evaporative laser light-scattering (ELSD) detection. Using a 20 × 0.46 cm C18 HPLC column and methanol/acetonitrile (60:40, vol/vol) as the mobile phase at 1.0 mL/min, 10 species of oxidized cholesterol derivative were measured by UV (205, 234, and 280 nm) while 5-cholestan-5α,6α-epoxy-3β-ol (5α-epoxycholesterol), 5-cholestan-5β,6β-epoxy-3β-ol (5β-epoxycholesterol), and 5-cholestan-3β,5α,6β-triol (cholestanetriol) were detected by only ELSD. The minimal limits of detection ranged from 100 to 500 ng depending on sterol and detector. The response was linear in the range 0–1000 or 0–2000 ng depending on detector. These oxidized cholesterol derivatives were also identified by HPLC/mass spectrometry analysis combined with UV detector. Heated tallow contained cholestanetriol, 5-cholesten-3β,7α-diol (7α-hydroxycholesterol), 5-cholesten-3β,7β-diol (7β-hydroxycholesterol), 5-cholesten-3β-ol-7-one (7-ketocholesterol), 5α- and 5β-epoxycholesterols under the developed analysis condition. Photooxidized cholesterol had cholestanetriol, 7α- and 7β-hydroxycholesterols and 3,5-cholestadien-7-one. On the other hand, 7α- and 7β-hydroxycholesterols, 7-ketocholesterol, 5α- and 5β-epoxycholesterols and 3,5-cholestadien-7-one were observed in copper-oxidized low-density lipoprotein. Thus, this developed HPLC analysis method could be applied to identification of oxidized cholesterol derivatives in food and biological specimen.

A highly sensitive high-performance liquid chromatographic method was developed for the separation of enantiomeric 1,2- (S configuration) and 2,3-diacyl-sn-glycerols (R configuration) and regioisomeric 1,3-diacyl-sn-glycerols. For this purpose the diacylglycerols were converted to their 2-anthrylurethanes and subjected to chiral-phase HPLC with fluorescence detection. Satisfactory resolution of the enantiomers and regioisomers was achieved on a (R)-1-(1-naphthyl)ethylamine polymeric phase, using a mixture of n-hexane–dichloromethane–ethanol (150:10:1, v/v) as the mobile phase. The formation of various hydrogen bonding, dipole–dipole stacking and charge transfer complexes between the urethane derivatives and the stationary phase was thought to contribute to the enantiomer separation. The detection limit of 2-anthrylurethanes was 1 fmol when the signal-to-noise ratio was 3:1.

Tallow and lard are used as representative animal fats in experimental diets. We have compared their respective atherogenic properties by feeding each fat (14%) as part of a semi-purified diet containing 0.5% cholesterol. The lard containing diet was significantly ( P 2 position, whereas only approximately 15% of the 16:0 of tallow is in that position. The fats were randomized so that every component fatty acid was present at each triglyceride carbon to approximately one third of its total concentration. Randomized tallow and lard had 8.5% and 7.6% of their component 16:0 at the SN 2 position, respectively. The atherogenicity of diets containing either randomized lard or tallow was virtually the same and significantly lower than the atherogenicity of the diet containing native lard. This study confirms earlier studies from our laboratory that suggest that the level of 16:0 at the SN 2 position of a triglyceride influences its atherogenicity.

Of the ovarian follicles that develop during reproductive life, more than 99% do not ovulate and are eliminated from the ovary by follicular atresia. Atresia is achieved by the self destruction of thecal and granulosa cells that comprise the follicle, by the process of apoptosis. The objective of this study was to determine if activation of the Fas receptor could enact apoptosis of thecal cells, and to explore the signal transduction pathway involved. Primary cultures of thecal/interstitial cells isolated from immature rat ovaries were treated with anti-Fas monoclonal antibody (anti-Fas mAb) (2.5 microg/ml). Morphological changes indicative of apoptosis, such as, condensation of chromatin, nucleoplasmic segmentation and formation of apoptotic bodies, were observed by fluorescence microscopy following nucleic acid staining with Hoechst 33342 dye and propidium iodide. DNA analysis of cells after 10 h of treatment with anti-Fas mAb showed that DNA had been cleaved into fragments that were multiples of 180-300 bp in length; biochemical evidence of apoptosis. The sphingomyelin (N-acylsphingosine-1-phosphocholine, SM) pathway that is initiated by the hydrolysis of SM to ceramide (Cer) has been shown previously to be activated by the Fas ligand/receptor system in a number of different cell types. It was therefore possible that the intracellular transduction of Fas receptor activation of thecal/interstitial cells could also involve the SM-Cer pathway. Hence, we have measured the SM levels in control and treated thecal/interstitial cells. Extracts of untreated thecal/interstitial cells contained six major species of SM identified as d18:1/16:0 (sphingosine base/fatty acid), d18:1/18:0, d18:1/20:0, d18:1/22:0, d18:1/24:1, d18:1/24:0 by normal phase high performance liquid chromatography interfaced with electrospray mass spectrometry. Treatment with anti-Fas mAb (2.5 microg/ml) for 30 min caused significant hydrolysis of only two of the SM species, d18:1/16:0 and d18:1/24:1. The involvement of ceramide, the central lipid in this phospholipid second messenger system, was tested using the synthetic cell permeable Cer analog (N-acetyl-N-sphingosine, C2-Cer). C2-Cer (10 microM). This analog induced both morphological and biochemical changes in thecal/interstitial cells, that were characteristic of apoptosis, and the same as those induced by anti-Fas mAb. C2-dihydroceramide (10 microM), an inactive analog of C2-Cer, failed to induce apoptosis of thecal/interstitial cells. In conclusion, the sphingomyelin-ceramide cycle that can lead to cell suicide by apoptosis is functional and activated through the Fas ligand/receptor signal transduction pathway, not only in the immune system, but also in thecal/interstitial cells of the ovarian follicle.

Earlier studies have suggested that the presence of palmitic acid in the SN 2 position of a triglyceride increases its atherogenic potential. Cottonseed oil contains about 24% palmitic acid of which only 2% is present at SN 2 . After randomization one third of the palmitic acid of cottonseed oil is at SN 2 . Rabbits were fed a semipurified diet containing 14% native or randomized cottonseed oil and 0.10% cholesterol. After 90 days the rabbits fed the randomized cottonseed oil exhibited significantly more severe atherosclerosis than those fed the native cottonseed oil. Plasma lipid levels were similar in the two groups. The increased atherogenicity of the randomized cottonseed oil may reflect the increased absorbability of fats with palmitic acid at the SN 2 position.

Using chiral-phase high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry (ESI/MS), we have redetermined the stereochemical configuration of some natural and synthetic phosphatidylglycerols (PG). For this purpose, the synthetic and natural PG were converted to their bis-3,5-dinitrophenylurethanes (DNPU), which were separated by HPLC using two columns having chiral phases of opposite configuration, (R)-(+)- and (S)-(-)-1-(1-naphthyl)ethylamine polymers. The molecular species were identified by on-line negative-ion ESI/MS. Absolute configurations of the resolved peaks were assigned by comparison with the elution order of the corresponding 1(3)-monoacyl-sn-glycerol enantiomers as bis-DNPU derivatives on the same column. The results clearly showed that the PG from cabbage leaf lipids and soybean phospholipids consisted of single R,S isomers (1,2-diacyl-sn-glycero-3-phospho-1'-sn-glycerols), despite the presence of nonstereospecific phospholipase D in the tissues. On the other hand, the PG derived from egg yolk phosphatidylcholine and glycerol by transphosphatidylation with cabbage phospholipase D was a mixture of 45% R,S isomers (1, 2-diacyl-sn-glycero-3-phospho-1'-sn-glycerols) and 55% R,R isomers (1,2-diacyl-sn-glycero-3-phospho-3'-sn-glycerols). The PG from Escherichia coli lipids was a mixture of 89% R,S and 11% R,R isomers. The present study demonstrates that chiral-phase HPLC and negative-ion ESI/MS provide direct and unambiguous information about the configuration, identity, and quantity of molecular species in natural and synthetic PG.

We have prepared Schiff base adducts of the core aldehydes of phosphatidylcholine and aminophospholipids, free amino acids, and myoglobin. The Schiff bases of the ethanolamine and serine glycerophospholipids were obtained by reacting sn-1-palmitoyl(stearoyl)-2-[9-oxo]nonanoyl-glycerophosphocholine (PC-Ald) with a twofold excess of the aminophospholipid in chloroform/methanol 2:1 (vol/vol) for 18 h at room temperature. The Schiff bases of the amino acids and myoglobin were obtained by reacting the aldehyde with an excess of isoleucine, valine, lysine, methyl ester lysine and myoglobin in aqueous methanol for 18 h at room temperature. Prior to isolation, the Schiff bases were reduced with sodium cyanoborohydride in methanol for 30 min at 4 degrees C. The reaction products were characterized by normal-phase high-performance liquid chromatography and on-line mass spectrometry with electrospray ionization. The amino acids and aminophospholipids yielded single adducts. A double adduct was obtained for myoglobin, which theoretically could have accepted up to 23 PC-Ald groups. The yields of the products ranged from 12 to 44% for the aminophospholipids and from 15-57% for the amino acids, while the Schiff base of the myoglobin was estimated at 5% level. The new compounds are used as reference standards for the detection of high molecular weight Schiff bases in lipid extracts of natural products.

Accumulation of the insoluble lipid-protein complex, ceroid, is a characteristic of atherosclerotic plaques. To determine whether deficient processing of cholesteryl esters in oxidized (ox) low density lipoprotein (LDL) contributes to ceroid formation, we studied the hydrolysis of internalized [3H] cholesteryl linoleate (CL) in oxLDL by mouse peritoneal macrophages (MPM). The hydrolysis by MPM of [3H]CL incorporated into oxLDL or LDL did not differ, suggesting that products of lipid and/or apoB oxidation had no impact on the lysosomal hydrolysis of [3H]CL. To evaluate the hydrolysis of oxCL by MPM, we subjected extensively ox[3H]CL to fractionation by TLC. The predominant fraction (D) consisted of sterols and oxysterols esterified to scission products of oxidized fatty acids containing terminal carbonyl groups, i.e., lipid core aldehydes. The extent of hydrolysis of [3H]-fraction D by MPM cultures, as well as by MPM extracts at pH 4.0, was significantly reduced when compared to the hydrolysis of intact [3H]CL. Fraction D also formed complexes with serum proteins, and the purified core aldehyde, cholesteryl 9-oxononanoate reacted with epsilon-amino group of lysines. Finally, several cholesteryl ester aldehydes were detected in lipid extracts of human atheroma. These results suggest that decomposition products of extensively oxidized cholesteryl linoleate that are also present in atherosclerotic lesions, are not adequately degraded by mouse peritoneal macrophage lysosomes and could interact with proteins to form ceroid.

We have determined the changes in the lipid structure of the adipocyte plasma membranes of rats receiving lard or fish oil in their diet. For this purpose, mature Wistar rats were fed 20% (w/w) lard or fish oil diets for 22 days, when the plasma membranes of the epididymal and perirenal adipocytes were prepared. Detailed analysis of the membrane lipids by chromatographic methods showed that dietary fat exerted a major effect on the lipid class and molecular species composition of the phospholipids. As a result of fish oil feeding, significant increases in the 20:5( n −3), 22:5( n −3) and 22:6( n −3) were detected in all glycerophospholipid classes, while the 18:1( n −9) and 18:2( n −6) and to a lesser extent 20:4( n −6) decreased. Incorporation of n −3 fatty acids increased the phosphatidylcholine/sphingomyelin ratio without changing the total phospholipid or free cholesterol content of the membrane. Fish oil feeding also caused a marked increase in the proportion of 24:1 in sphingomyelins, which occurred mainly at the expense of 18:0 and 24:0. New n −3 fatty acid-containing species appeared in the choline and ethanolamine glycerophospholipids, when compared to membrane lipids from lard-fed rats. Membranes from fish oil fed rats also had moderately higher levels of ether lipids. Few differences were seen between the membranes of the epididymal and perirenal adipocytes. It is concluded that dietary fish oils modify the lipid structure of rat adipocyte plasma membranes by increasing the ratio of phosphatidylcholine to sphingomyelin and by increasing the proportion of molecular species with polyunsaturated fatty acids, which would be anticipated to increase the fluidity of the lipid bilayer of adipocyte plasma membranes.

A detailed comparison of the structures of plasma very low density lipoprotein (VLDL) and liver triacylglycerols (TG) (Yang et al. 1995. J. Lipid Res. 36: 125-136) has demonstrated that a minimum of 60% of the secreted TG could have been derived from partial lipolysis and reesterification of stored TG and a maximum of 40% could have been derived from direct secretion of newly made TG. To investigate the processes involved in the transfer of TG to VLDL in vivo, we have determined the distribution of deuterium among the molecular species of the liver-TG and VLDL-TG during the infusion of perdeuterated ethanol along with fructose or glucose and during the provision of either glucose or fructose in the drinking water for 2 weeks. The deuterium labeling (percent excess and percent replacement) of the total fatty acids was determined by GC/MS of the methyl esters while the labeling of the glycerol and the glycerol plus fatty acids of the enantiomeric diacylglycerol moieties of TG was determined by LC/MS with on-line mass spectrometry. Supplementation of the diet for 2 weeks with either glucose and fructose stimulated the synthesis of TG containing new fatty acids and glycerol. The proportion of the newly made to preexisting TG differed in VLDL from that in the liver. The 2H % replacement in glycerol and in total fatty acids was greater in VLDL-TG than in the liver-TG. On the basis of the mass isotopomer distribution analysis it was estimated that a maximum of 30% of the VLDL-TG could have been derived directly from TG that was made de novo and did not equilibrate with the liver-TG stores. The transfer of the stored TG to VLDL was best accounted for by a degradation to 2-monoacylglycerols and resynthesis via the 2-monoacylglycerol pathway with addition of an excess of newly synthesized fatty acids to the resynthesized TG.

Six oils of marine, algal, and microbial origin were analyzed for stereospecific distribution of component fatty acids. The general procedure involved preparation of sn-1,2-(2,3)-diacylglycerols by partial deacylation with ethylmagnesium bromide or pancreatic lipase, separation of X-1,3- and sn-1,2(2,3)-diacylglycerols by borate thin-layer chromatography, resolution of the sn-1,2- and sn-2,3-enantiomers by chiral phase high-performance liquid chromatography following preparation of dinitrophenylurethane derivatives, and determination of the fatty acid composition by gas chromatography. Unexpected complications arose during a stereospecific analysis of triacylglycerols containing over 33% of either 20:4 or 22:6 fatty acids. The sn-1,2(2,3)-diacylglycerols made up of two long-chain polyunsaturated acids migrated with the X-1,3-diacylglycerols and required separate chiral phase resolution. Furthermore, the enzymatic method yielded sn-1,2(2,3)-diacylglycerols, overrepresenting the polyenoic species due to their relative resistance to lipolysis, but prolonged digestion yielded correct composition for the 2-monoacylglycerols. The final positional distribution of the fatty acids was established by pooling and normalizing the data from subfractions obtained by normal- and chiral-phase separation of diacylglycerols. The molecular species of X-1,3-, sn-1,2- and sn-2,3-diacylglycerol dinitrophenylurethanes were identified by chiral-phase liquid chromatography/mass spectrometry with electrospray ionization, which demonstrated a preferential association of the paired long-chain acids with the sn-1,2- and sn-2,3-diacylglycerol isomers.

Unsaturated triacylglycerols (TG) and choline (PC) and ethanolamine (PE) phosphatides of known structure were subjected to ozonization and reduction with triphenylphosphine to yield the corresponding lipid ester core aldehydes. Mono- and di-C 9 aldehyde palmitoylglycerols were prepared from oleoyldipalmitoyl and oleoyllinoleoylpalmitoyl glycerols, respectively, while egg yolk PC and PE provided the mono-C 5 and mono-C 9 aldehydes of palmitoyl-and stearoyl glycerophospholipids. The aldehydes were isolated in the free form and as the dinitrophenylhydrazone (DNPH) derivatives by thin-layer chromatography (TLC). The intermediate ozonides, free aldehydes and hydrazones were identified by reversed phase high performance liquid chromatography (HPLC) with on-line negative ion thermospray and normal phase HPLC with on-line positive ion electrospray mass spectrometry (LC-MS). The synthetic aldehydes were used as carriers during isolation from natural sources and as reference compounds in quantitative analyses

Lipid extracts of natural sources contain a large number of lipid classes and molecular species. Completely reproducible samples are obtained only with great care and skill. Analytical methods other than chromatography and/or mass spectrometry are of little use for resolution and identification of lipid molecules even in simple mixtures. The analytical information desired governs the selection of the chromatographic and mass spectrometric method, which determine the sample preparation and derivative needed. Usually a combination of chromatographic methods is necessary to identify specific species of lipids. The recent development of soft ionization techniques, that are readily interfaced with mass spectrometers, have greatly simplified the sample preparation and have largely eliminated the need for derivatization. Because these techniques require expensive equipment and dedicated operators, the methods selected must be consistent with the true analytical needs and the available resources. Although personal preference cannot be eliminated entirely, the general strategies outlined below should help to reduce the number of possibilities facing a lipid analyst to a few practical choices.

We have isolated the core aldehydes (aldehydes still bound to parent molecules) of phosphatidylcholine (PC) and cholesteryl esters (CE) from copper-catalyzed peroxidation of human plasma low (LDL) and high (HDL) density lipoproteins. The aldehydes were isolated by extraction with acidified chloroform-methanol containing 2,4-dinitrophenylhydrazine. The 2,4-dinitrophenylhydrazone (DNPH) derivatives formed were resolved by reversed phase high performance liquid chromatography (HPLC) and identified by on-line quadrupole mass spectrometry (LC/MS). The major PC core aldehydes from oxidized LDL and HDL were identified as 1-palmitoyl-(1-stearoyl) 2-(9-oxononanoyl)-, 1-palmitoyl-(1-stearoyl) 2-(8-oxooctanoyl)-, and 1-palmitoyl-(1-stearoyl) 2-(5-oxovaleroyl)-sn-glycerols after phospholipase C digestion of the DNPH derivatives of the phospholipids. The major aldehydes from peroxidation of cholesteryl esters were the 9-oxononanoyl, 8-oxooctanoyl, and 5-oxovaleroyl esters of cholesterol and 7-ketocholesterol. The core aldehydes were estimated to account for a minimum of 1-2% of the consumed linoleate and arachidonate esters. A relatively smaller yield of the PC core aldehydes from LDL compared to HDL was attributed to the presence of greater amounts of phospholipases in LDL than in HDL. More comparable yields of PC core aldehydes were obtained in the presence of phenylmethylsulfonylfluoride, which inhibits phospholipases. We conclude that peroxidation of LDL and HDL results in formation of detectable amounts of cholesteryl and glycerophospholipid esters containing aldehyde functions. The yield of PC aldehydes varies with the activity of the platelet activating factor (PAF) acetyl hydrolase.

Bacterial adhesion to mucosal surfaces is an important pathogenic mechanism for Helicobacter-induced gastritis. The aims of this study were to compare binding of selected Helicobacter mustelae and Helicobacter pylori strains to lipids extracted from HEp-2, Chinese hamster ovary, human embryonic lung cells, and ferret gastrointestinal tissues as well as to intact tissue culture cells and to analyze the fatty acids of the receptor.Thin-layer chromatography overlay binding and a receptor-based immunoassay detected adhesion of bacteria to commercial lipids and to individual species within the lipid extracts. H. mustelae binding to tissue culture cells was performed by whole cell bacterial adhesion assay.H. mustelae and H. pylori both bound to phosphatidylethanolamine and lysophosphatidylethanolamine. Adhesion of H. mustelae to intact eukaryotic cells correlated with the amount of phosphatidylethanolamine. Binding of helicobacters was greater to lipids derived from ferret antrum compared with colon (P0.05). Biochemical analysis suggested that heterogeneity in fatty acid composition of phosphatidylethanolamine could influence the degree of Helicobacter binding.Adhesion of Helicobacter strains correlates with the quantity of phosphatidylethanolamine present in the epithelial cell and with the differences in the fatty acid profile of the lipid.