Profiling and Qualitative Assessment of Enzymatically and Thermally Oxidized Egg Yolk Phospholipids using a Two-Step High-Performance Liquid Chromatography Protocol

A two-step high-performance liquid chromatography (HPLC) method for the profiling and qualitative assessment of oxidized phospholipids (oxPL) present in foods was developed. The applicability of the investigated two-step HPLC protocol was verified for separation of enzymatically and thermally oxidized hen egg yolk phospholipids (PL) as a relevant food model. In the first step, seven individual PL classes were separated using hydrophilic interaction liquid chromatography (HILIC). The collected fractions of two main egg yolk PL classes—phosphatidylethanolamines and phosphatidylcholines—were further directed to the second step of separation aimed at profiling and qualitative assessment of their oxidized species. For this purpose, the reverse phase (RP) chromatography coupled to charged aerosol, ultraviolet detection (UV) and mass spectrometry detection were employed. A database of potential oxPL including primary (hydroperoxides) and long-chain secondary PL oxidation products (epoxides, alcohols, and ketones) as well as some of their possible combinations was created. Additionally, the results were compared with the profiles of PL hydroperoxides obtained using thin layer chromatography (TLC) with N,N-dimethyl-p-phenylenediamine visualization.     

Ultra high performance liquid chromatography–mass spectrometric analysis of oxidized free fatty acids and acylglycerols

A fast ultra high performance liquid chromatography (UHPLC)–electrospray ionization (ESI)–mass spectrometric (MS) method was developed for simultaneous analysis of free fatty acids (FFAs), monoacylglycerols (MAG), diacylglycerols (DAG), triacylglycerols (TAG), and their oxidized equivalents. Effect of elevated column temperature was studied in order to optimize the chromatography of closely eluting peaks and to reduce high back pressure formed in UHPLC. The elevated temperature enabled high flow rate, better mass transfer, and therefore more narrow peaks and better separation of the analytes. The new method was applied to the analysis of total lipid extracts of lipolysis samples prepared by an artificial digestion model in order to investigate oxidized lipids and changes in their profiles in the chyme. Over 150 compounds were identified from the extracts. The UHPLC–ESI–MS method was proved to be fast, highly selective, and sensitive. Compared to a previously used high performance LC–ESI–MS method, the new UHPLC–ESI–MS method was over five times faster and consumed one tenth of the solvents while producing comparable quantitative results. Practical applications: Edible oils and fats contain mainly TAGs, the lipolysis of which produces FFAs and MAGs with minor DAG components. These compounds are susceptible to oxidation in the stomach, and therefore the analysis of the oxidation products is important. Fast determination of FFAs and acylglycerols is also important in quality control of biodiesel. Our new method enables accurate and sensitive determination of different molecular species present in digested and processed samples with minimal sample preparation requirements. In this respect, the new method is applicable to large scale and fast screening of biological samples for lipidomic and metabolomic studies.     

Plasma HDL Reduces Nonesterified Fatty Acid Hydroperoxides Originating from Oxidized LDL: a Mechanism for Its Antioxidant Ability

The antioxidant property of plasma high-density lipoprotein (HDL) is thought to be involved in potential anti-atherogenic effects but the exact mechanism is not known. We aimed to reveal the contribution of HDL on the elimination of lipid hydroperoxides (LOOH) derived from oxidized low-density lipoprotein (LDL). Oxidized LDL prepared by copper ion-induced oxidation contained nonesterified fatty acid hydroperoxides (FFA-OOH) and lysophosphatidylcholine (lysoPtdCho), in addition to cholesteryl ester hydroperoxides (CE-OOH) and phosphatidylcholine hydroperoxides (PtdCho-OOH). A platelet-activating factor-acetylhydrolase (PAF-AH) inhibitor suppressed formation of FFA-OOH and lysoPtdCho in oxidized LDL. Among LOOH species, FFA-OOH was preferentially reduced by incubating oxidized LDL with HDL. HDL exhibited selective FFA-OOH reducing ability if it was mixed with a liposomal solution containing FFA-OOH, CE-OOH and PtdCho-OOH. Two-electron reduction of the hydroperoxy group to the hydroxy group was confirmed by the formation of 13-hydroxyoctadecadienoic acid from 13-hydroperoxyoctadecadienoic acid in HPLC analyses. This reducing effect was also found in apolipoprotein A-1 (apoA-1). FFA-OOH released from PtdCho-OOH due to PAF-AH activity in oxidized LDL undergo two-electron reduction by the reducing ability of apoA1 in HDL. This preferential reduction of FFA-OOH may participate in the mechanism of the antioxidant property of HDL.     

Variability in Associations of Phosphatidylcholine Molecular Species with Metabolic Syndrome in Mexican–American Families

Plasma lipidomic studies using high performance liquid chromatography and mass spectroscopy offer detailed insights into metabolic processes. Taking the example of the most abundant plasma lipid class (phosphatidylcholines) we used the rich phenotypic and lipidomic data from the ongoing San Antonio Family Heart Study of large extended Mexican–American families to assess the variability of association of the plasma phosphatidylcholine species with metabolic syndrome. Using robust statistical analytical methods, our study made two important observations. First, there was a wide variability in the association of phosphatidylcholine species with risk measures of metabolic syndrome. Phosphatidylcholine 40:7 was associated with a low risk while phosphatidylcholines 32:1 and 38:3 were associated with a high risk of metabolic syndrome. Second, all the odd chain phosphatidylcholines were associated with a reduced risk of metabolic syndrome implying that phosphatidylcholines derived from dairy products might be beneficial against metabolic syndrome. Our results demonstrate the value of lipid species-specific information provided by the upcoming array of lipidomic studies and open potential avenues for prevention and control of metabolic syndrome in high prevalence settings.     

Use of a liquid chromatograph-ion trap mass spectrometer for end-group characterization of various polyethoxylate materials

The separation and detection properties of various ethoxylated polymers were investigated with atmospheric pressure ionization liquid chromatography/mass spectrometry (LC/MS). Interesting chromatographic elution behavior based on functionality was noted. LC/MS using in-source CID (collision-induced dissociation) and tandem mass spectrometry (MS/MS) detection was compared for end-group identification. Excellent end-group identification was achieved when the end-group molecular weight (MW) was greater than 100 Da and the average MW of the polymer was less than 400 Da by both MS/MS and in-source CID. In cases where the end-group MW was less than 100 Da, because of the low mass cut-off in a quadrapole ion trap analyzer, in-source CID produced the only characteristic end-group ions. The use of a dual scan function allowed investigation of the oligomeric distribution followed by a narrow low-mass scan using in-source CID to generate end-group information. This approach is applicable on instruments without MS/MS capability.     

New approach to the analysis of oxidized triacylglycerols in lipoproteins

The oxidation of human LDL lipids and the structures of oxidized TAG molecules found in LDL were investigated. Pooled samples of 10 normolipidemic and 10 hyperlipidemic subjects were analyzed. For determination of the oxidation levels, the LDL baseline diene conjugation (LDL-BDC) method was used. A method based on HPLC and electrospray ionization-MS was optimized and applied to the analysis of molecular structures of oxidized TAG in LDL. Differences were found between the oxidation levels of the samples. The LDL-BDC value was 22.2 μmol/L serum in the normolipidemic group, and 88.1 μmol/L serum in the hyperlipidemic group. The amounts of oxidized TAG molecules were small. However, several species of oxidized TAG were identified. These included TAG molecules with a keto or an epoxy group attached to a FA, and TAG molecules with a FA core aldehyde. In some TAG, the keto/epoxy ratio was greater in the hyperlipidemic group compared to the normolipidemic group. The results show that our approach is applicable to research on lipid oxidation in lipoproteins.     

The Phospholipidomic Signatures of Human Blood Microparticles,Platelets and Platelet‐Derived Microparticles: a Comparative HILIC‐ESI–MS Investigation

The phospholipidomic signatures of human blood microparticles and platelets, evaluated by hydrophilic interaction liquid chromatography coupled to electrospray ionization—mass spectrometry, were compared. The phospholipidome of platelet‐derived microparticles, obtained by platelets stimulation with a mixture of Ca(II), thrombin and collagen, was also considered for the comparison. Platelets, blood microparticles and platelet‐derived microparticles displayed qualitatively similar phospholipidomes, all based on eight major phospholipid classes, namely: phosphatidylcholines, diacyl‐ and plasme(a)nyl‐phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, sphingomyelins and lyso forms of phosphatidylcholines and phosphatidylethanolamines. However, while the phospholipidomes of platelets and platelet‐derived microparticles were found to be generally similar also from a quantitative point of view, a higher relative incidence of species bearing polyunsaturated side chains, especially in phospholipid classes sharing the choline head (i.e. phosphatidylcholines and lyso‐phosphatidylcholines), was observed in the case of blood microparticles. As a further peculiar feature, never reported before, the relative abundance of lyso‐phosphatidylcholines among the eight identified phospholipid classes was found to be significantly higher in the lipid extracts of blood microparticles.     

The effect of fatty acid chain length and unsaturation on the chromatographic behavior of triglycerides on latroscan chromarods

The chromatographic behavior of lipid classes on Chromarods with common developing solvents used for the Iatroscan system was influenced by the esterified fatty acids present in the lipid class. The R_f value of the lipid class increased with increasing chain length and increasing unsaturation of the fatty acids; the increase in R_f value per methylene carbon was about twice that per double bond. The effect was evident in both triglycerides and phosphatidylcholines. This partial separation of molecular species within a lipid class caused widening of the lipid class band on the Chromarods and can result in poorer resolution, particularly in biological samples.     

Quantitative analysis of polyenoic phospholipid molecular species by high performance liquid chromatography

The quantitative analysis of phospholipid molecular species containing polyenoic fatty acids is described. Dinitrobenzoyl derivatives of diacylglycerols prepared from phospholipids were separated into individual molecular species by reversed-phase high performance liquid chromatography (HPLC) using a combination of two solvent systems and were quantified at 254 nm. Thirty-six molecular species were resolved from the phosphatidylcholines of rat hearts, human platelets and Chinese hamster V79-R cells. The derivatives of alkenylacyl molecular species from platelet phosphatidylethanolamine were resolved concomitantly with diacyl molecular species.     

Liquid Chromatography–Light Scattering Detector–Mass Spectrometric Analysis of Digested Oxidized Rapeseed Oil

Rapeseed oil was oxidized chemically and thermally to produce two distinct oxidized oils. These oils, along with unoxidized oils, were subjected to an artificial digestion model to simulate the digestive processes in humans. Lipid digestion involves lipases that break down the intact triacylglycerol (TAG) molecules first to diacylglycerols, and eventually to sn-2-monoacylglycerols (MAG) and free fatty acids. A high performance liquid chromatography-evaporative light scattering detector-electrospray ionization-mass spectrometric (HPLC–ELSD–ESI–MS) method was developed to monitor the lipolysis and the presence of oxidized lipids. The HPLC–ELSD–ESI–MS analysis enabled the separation and detection of nearly all the lipid species present in the sample after TAG hydrolysis. The HPLC–MS analyses of digestion products revealed that oxidized triacylglycerols are hydrolyzed by the digestive enzymes in a manner similar to that of native, unoxidized molecules. Significant amounts of sn-1(3)-MAG were found in all the samples after lipolysis, however, more of these were found in unoxidized rapeseed oil samples than in the oxidized oils. Several oxidized molecules were identified with the aid of synthesized oxylipids. This novel method is scalable to small-scale preparative fractionation of oxidized lipid molecules from a complex digestion sample. Also, the fingerprint-like, diagnostic, MS profiles of oxidized oils, reference compounds, and digestion products may be a great aid in comprehensive analysis of lipid oxidation and lipolysis.     

Studies on the concentration of oxidized components of abused fats and the application of HPLC to their separation

The efficiency of five separation techniques at removing unaltered compounds from the methyl esters of oxidized fats was compared, as were concentrations of oxidized products. A batch type distribution method using acetonitrile/hexane was the most effective in concentrating the polar products and removal of palmitate and stearate from abused fats which had been hydrogenated. The level of high molecular weight material present in samples was measured indirectly by determination of the percent elutable material via gas chromatography. A high performance liquid chromatography system developed to partially separate or “profile” the oxidized products used an octadecyl bonded phase column and a linear gradient from 50% aqueous acetonitrile to 85% aqueous acetonitrile, at a rate of 5%/min. The resultant chromatograms can be useful in assessing the quality of used fats.     

Platelet-Activating Factor Quantification Using Reversed Phase Liquid Chromatography and Selected Reaction Monitoring in Negative Ion Mode

Platelet‐activating factor (PAF) is a potent biologically active phospholipid that mediates human physiological and pathophysiologic responses. PAF levels increase transiently and are typically assessed by techniques with limitations related to expense, sensitivity, pre‐analysis derivatization and interference with isobaric molecules. This study elucidates a facile, accurate liquid chromatography–mass spectrometry analytical method for PAF. In negative ion mode using electrospray ionization, collisionally‐activated dissociation analysis showed a unique product ion for acetate adducts of PAF molecular species representing the loss of methyl acetate from the polar head group and loss of a part of the acetate group from the sn‐2 position. This product ion was exploited for selected reaction monitoring of PAF molecular species following separation by reversed‐phase liquid chromatography. Standard calibration responses were determined, and this method was able to detect as low as 100 fmol of PAF. Finally, PAF molecular species were quantified in human neutrophils and monocytes.     

High pressure liquid chromatographic separation of molecular species of phosphatidic acid dimethyl esters derived from phosphatidylcholine

A majority of the individual molecular species of phosphatidic acid dimethyl esters derived from multispecies egg yolk and soybean phosphatidylcholines have been separated by reverse-phase high pressure liquid chromatography. Two Partisil-10 ODS columns connected in tandem and the eluents acetonitrile or methanol/water (95∶5) were used for molecular species resolution, based on total fatty acyl carbon number and degree of unsaturation.     

Lipidome of Atherosclerotic Plaques from Hypercholesterolemic Rabbits

The cellular, macromolecular and neutral lipid composition of the atherosclerotic plaque has been extensively characterized. However, a comprehensive lipidomic analysis of the major lipid classes within atherosclerotic lesions has not been reported. The objective of this study was to produce a detailed framework of the lipids that comprise the atherosclerotic lesion of a widely used pre-clinical model of plaque progression. Male New Zealand White rabbits were administered regular chow supplemented with 0.5% cholesterol (HC) for 12 weeks to induce hypercholesterolemia and atherosclerosis. Our lipidomic analyses of plaques isolated from rabbits fed the HC diet, using ultra-performance liquid chromatography (UPLC) and high-resolution mass spectrometry, detected most of the major lipid classes including: Cholesteryl esters, triacylglycerols, phosphatidylcholines, sphingomyelins, diacylglycerols, fatty acids, phosphatidylserines, lysophosphatidylcholines, ceramides, phosphatidylglycerols, phosphatidylinositols and phosphatidylethanolamines. Given that cholesteryl esters, triacylglycerols and phosphatidylcholines comprise greater than 75% of total plasma lipids, we directed particular attention towards the qualitative and quantitative assessment of the fatty acid composition of these lipids. We additionally found that sphingomyelins were relatively abundant lipid class within lesions, and compared the abundance of sphingomyelins to their precursor phosphatidylcholines. The studies presented here are the first approach to a comprehensive characterization of the atherosclerotic plaque lipidome.     

Characterization of N-acylphosphatidylethanolamine and acylphosphatidylglycerol in oats

Two polar lipid classes, both with three acyl groups, were isolated from an extract of oats and characterized by nuclear magnetic resonance spectroscopy, electrospray mass spectrometry (MS), and electron ionization MS (EIMS). Distortionless enhancement by polarization transfer (DEPT) and the two-dimensional correlation experiments 1H-detected heteronuclear multiple quantum coherence spectroscopy, heteronuclear multiple bond correlation spectroscopy, double quantum filtered correlation spectroscopy, and total correlation spectroscopy provided sufficient information for determination of the structure of the two lipid classes. The polar lipid classes were found to be N-acylphosphatidylethanolamine [1,2-diacyl-sn-glycero-3-phospho-(N-acyl)-1'-ethanolamine; N-acyl-PE] and acylphosphatidylglycerol [1,2-diacyl-sn-glycero-3-phospho-(3'-acyl)-1'-sn-glycerol]. High-performance liquid chromatography with electrospray ionization MS (HPLC-ESMS) and with electrospray ionization tandem MS (HPLC-MS/MS) were utilized for the separation and subsequent determination of molecular species. With HPLC-ESMS, ions of deprotonated molecules were obtained and with HPLC-MS/MS carboxylate ions (representing acyl groups) were obtained as well as other structurally significant ions. Fifty molecular species of N-acylphosphatidylethanolamine and 24 molecular species of acylphosphatidylglycerol were found, with a molecular mass range of 924-1032 Da and 959-1035 Da, respectively. Identification of the fatty acid isomers, as picolinyl ester derivatives, was done with gas chromatography with EIMS. Three isomers of 16:1 fatty acids were found in N-acyl-PE, and their double bond positions were determined to 6, 9, and 11 with a relative abundance of 4:10:1.     

Lipid peroxidation in low density lipoproteins from human plasma and egg yolk promotes accumulation of 1-acyl analogues of platelet-activating factor-like lipids

Oxidative modification of low density lipoprotein (LDL) is known to be a key event for induction of atherosclerosis. However, there has been little progress in structural elucidation of oxidized lipids, especially oxidatively fragmented phospholipids retaining a glycerol backbone. In this study, we found that LDL derived from egg yolk has no platelet-activating factor (PAF) acetylhydrolase activity, and that prolonged incubation of egg yolk LDL with Cu²⁺ resulted in the formation of various PAF-like lipids: 1-acyl type phosphatidylcholines with ansn-2-short-chain dicarboxylate or monocarboxylate group. Only a very small amount of the PAF-like lipid having ansn-2-short-chain monocarboxylate group was detected by gas chromatography-mass spectrometry in Cu²⁺-oxidized LDL from human plasma with high PAF-acetylhydrolase activity, which has been reported to hydrolyze PAF-like lipids to lysophosphatidylcholines. Preincubation of plasma LDL with diisopropyl fluorophosphate dose-dependently inhibited PAF-acetylhydrolase activity, resulting in accumulation of the PAF-like lipids when the LDL was oxidized with Cu²⁺. As well as PAF and lysophosphatidylcholines, several PAF-like lipids were found to inhibit [³H]thymidine incorporation into cultured vascular smooth muscle cells derived from rat aorta. The possible formation of PAF-like lipids by lipid peroxidation in LDL is discussed as well as its possible significance for induction of atherosclerosis.     

Characterization of α- and γ-linolenic acid oils by reversed-phase high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

Triacylglycerols of oils rich in α- and/or γ-linolenic acids were analyzed by reversed-phase high-performance liquid chromatography (HPLC) coupled with atmospheric pressure chemical ionization mass spectrometry [(APCI)MS]. The (APCI)MS spectra of most triacylglycerols exhibited [M + H]⁺ and [M - RCOO]⁺ ions, which defined the molecular weight and the molecular association of fatty acyl residues, respectively. Reversed-phase HPLC resulted in, at least, partial separation of triacylglycerols containing α- and/or γ-linolenic acid moieties. Molecules containing α-linolenic acid residues exhibited a relatively weaker retention by the stationary phase than the corresponding molecules containing γ-linolenic acid residues. Good separation of triacylglycerols of cloudberry seed oil, evening primrose oil, borage oil, and black-currant seed oil was achieved. The molecular species identification of separated components was based on the (APCI)MS data as well as on the elution properties of triacylglycerols on reversed-phase HPLC. This study demonstrated the efficiency of HPLC-(APCI)MS in determining the molecular species compositions of triacylglycerols in complex natural mixtures. Good quality mass spectra could be extracted even from minor chromatographic peaks representing 0.2% or less of the total triacylglycerols.     

氧化型低密度脂蛋白的制备及其对血管平滑肌细胞增殖能力的影响

目的制备氧化型低密度脂蛋白(ox-LDL),并检测其对血管平滑肌细胞(SMC)增殖能力的影响。方法肝素沉淀法提取LDL,Cu2+氧化法制备ox-LDL,MTT法测定其对Wistar大鼠血管SMC增殖能力的影响。结果LDL完全被氧化成ox-LDL SDS-PAGE显示,LDL相对分子质量降低;200mg/Lox-LDL可刺激体外培养的SMC增殖能力明显增强;ox-LDL浓度不低于600mg/L时,SMC增殖能力明显下降。结论成功制备了ox-LDL,一定浓度的ox-LDL可促进SMCs的增殖。     

Oxidized LDL Is Strictly Limited to Hyperthyroidism Irrespective of Fat Feeding in Female Sprague Dawley Rats

Metabolic dysfunctions might play a crucial role in the pathophysiology of thyroid dysfunctions. This study aimed to investigate the impact of a controlled diet (normal versus high fat feeding) on hypothyroid and hyperthyroid Sprague Dawley rats. Female Sprague Dawley rats (n = 66) were grouped into normal diet (n = 30) and high-fat diet (n = 36) groups and subdivided into controls, hypothyroid and hyperthyroid groups, induced through propylthiouracil or triiodothyronine (T3) treatment, respectively. After 12 weeks of treatment metabolic parameters, such as oxidized LDL (oxLDL), malondialdehyde (MDA), 4-hydroxynonenal (HNE), the lipid profile, body weight and food intake parameters were analyzed. Successfully induced thyroid dysfunctions were shown by T3 levels, both under normal and high fat diet. Thyroid dysfunctions were accompanied by changes in calorie intake and body weight as well as in the lipid profile. In detail, hypothyroid rats showed significantly decreased oxLDL levels, whereas hyperthyroid rats showed significantly increased oxLDL levels. These effects were seen under high fat diet and were less pronounced with normal feeding. Taken together, we showed for the first time in female SD rats that only hyper-, but not hypothyroidism, is associated with high atherogenic oxidized LDL irrespective of normal or high-fat diet in Sprague Dawley rats.