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Simultaneous determination of molecular species of monoacylglycerols, diacylglycerols and triacylglycerols in human very-low-density lipoproteins by reversed-phase liquid chromatography
Abstract
The aim of the present study was to investigate the applicability of a previously developed method for the analysis of triacylglycerol molecular species to the simultaneous determination of triacylglycerols, diacylglycerols and monoacylglycerols of human very-low-density lipoproteins (VLDL). Ten elderly women were recruited for the study. Blood was obtained in fasting conditions and VLDL were isolated by ultracentrifugation. Neutral lipids were separated by solid-phase extraction and were subsequently injected on a reversed-phase HPLC system, with an elution system composed of acetone in acetonitrile. The method allowed the separation of four monoacylglycerols, 18 diacylglycerols and 24 triacylglycerols, including the resolution of positional isomers of diacylglycerols. Monoacylglycerols were composed of oleic, linoleic, palmitic and stearic acids. The major diacylglycerols were 1,2-dilinoleoyl-glycerol and 1,3-dilinoleoyl-glycerol (14.24+/-1.02 and 17.93+/-1.42%, respectively). The main triacylglycerols quantified were dioleoyl-stearoyl-glycerol (OOS), oleoyl-dipalmitoyl-glycerol (OPP), trilinoleoyl-glycerol (LLL) and linoleoyl-distearoyl-glycerol (LSS), accounting for 11.25+/-2.15, 10.14+/-2.05, 9.35+/-2.30 and 8.56+/-1.56%, respectively. An inverse relationship between polarity and fatty acid disappearance from triacylglycerols (r(2)=0.82, P<0.05) and from diacylglycerols (r(2)=0.93, P<0.01) was discovered. In conclusion, the method allowed, for the first time, the easy, rapid and simultaneous determination in a single chromatogram of triacylglycerol, diacylglycerol and monoacylglycerol molecular species of human VLDL by reversed-phase HPLC.
... In literature, methods for the extraction of MAG and DAG are reported, mainly focusing on the analysis of natural lipids ("lipidomics"), like blood and membrane lipids and animal and vegetable fats [8][9][10][11][12][13]. For sample preparation of lyophilized cells and human blood, treatment with sodium chloride solution and mixtures of chloroform/ methanol were commonly applied [8,[10][11][12][13], while milk powder was simply dissolved in a mixture of n-hexane/isopropanol [9]. ...
... In literature, methods for the extraction of MAG and DAG are reported, mainly focusing on the analysis of natural lipids ("lipidomics"), like blood and membrane lipids and animal and vegetable fats [8][9][10][11][12][13]. For sample preparation of lyophilized cells and human blood, treatment with sodium chloride solution and mixtures of chloroform/ methanol were commonly applied [8,[10][11][12][13], while milk powder was simply dissolved in a mixture of n-hexane/isopropanol [9]. Milk was extracted with a mixture of methylene chloride/methanol and the addition of sodium chloride [14]. ...
... With the intention of a short extraction procedure, LLE was evaluated first. In literature, chloroform was often mentioned for the extraction of cells, human blood, and membrane lipids [8,[10][11][12][13]32], and Fagan et al. used a solvent mixture containing methylene chloride for the extraction of lipids from milk [14]. To omit chlorinated toxic solvents, several alternative solvents were tested. ...
Mono- and diacylglycerol (MAG and DAG) emulsifiers (E 471) are widely applied to regulate techno-functional properties in different food categories, for example, in dairy products. A method for the determination of MAG and DAG in aerosol whipping cream by high-performance thin-layer chromatography with fluorescence detection (HPTLC–FLD) after derivatization with primuline was developed. For sample preparation, aerosol whipping cream was mixed with ethanol, followed by the addition of water and liquid-liquid extraction with tert-butyl methyl ether. The sample extracts were analyzed by HPTLC–FLD on silica gel LiChrospher plates with n-pentane/n-hexane/diethyl ether (22.5:22.5:55, v/v/v) as mobile phase, when interfering matrix like cholesterol and triacylglycerols were successfully separated from the E 471 food additives. For quantitation, an emulsifier with known composition was used as calibration standard and the fluorescent MAG and DAG were scanned at 366/> 400 nm. Limits of detection and quantitation of 4 and 11 mg/100 g aerosol whipping cream were obtained for both monostearin and 1,2-distearin, respectively, and allowed the reliable quantitation of MAG and DAG from E 471 far below commonly applied emulsifier amounts. Recoveries from model aerosol whipping cream with 400 mg E 471/100 g were determined in a calibration range of 200–600 mg E 471/100 g sample and ranged between 86 and 105% with relative standard deviations below 7%. In aerosol whipping creams from the German market, E 471 amounts ranged between 384 and 610 mg/100 g.
... While there are numerous normal phase HPLC techniques to separate neutral and polar lipid classes by headgroup, or lack thereof, the separation of individual lipid molecular species by HPLC relies on column interaction with the bound fatty acids. For neutral lipids such as DAG, most methods rely on a reverse phase HPLC approach, which uses octadecyl-/tricontacarbon chains (C18/C30) or silver ions bonded to a stationary phase, and a variety of non-aqueous mobile phases to separate lipids (Christie, 1988;Christie & Han, 2012;Clejan, 1998;Lee & Hajra, 1991;Lin, 2007;Nikolova-Damyanova, 2009;Perona & Ruizgutierrez, 2003). While silver ion columns are commercially available at this time, their cost, ease of use, and specialized nature make them a less attractive option (Momchilova & Nikolova-Damyanova, 2003). ...
... This includes selecting columns that are long (!25 cm) with small pore size ( 3 μm), and relatively high carbon load (!10%), as well as running the column at lower temperatures to improve sample binding. There have been many non-polar mobile phases tested for non-polar lipid molecular species separations which vary depending on sample preparation and column used (Christie & Han, 2012;Clejan, 1998;Lee & Hajra, 1991;Lin, 2007;Nikolova-Damyanova, 2009;Perona & Ruizgutierrez, 2003). Acetonitrile is often favored due to its tendency to associate with fatty acid double bonds and improve solubility. ...
Diacylglycerols (DAGs) are anabolic precursors to membrane lipid and storage triacylglycerol biosynthesis, metabolic intermediates of lipid catabolism, and potent cellular signaling molecules. The different DAG molecular species that accumulate over development or in different tissues reflect the changing aspects of cellular lipid metabolism. Consequently, an accurate determination of DAG molecular species in biological samples is essential to understand various metabolic processes and their diagnostic relevance. However, quantification of DAG molecular species in various biological samples represents a challenging task because of their low abundance, hydrophobicity, and instability. This chapter describes the most common chromatographic (TLC and HPLC) and mass spectrometry (MS) methods used to analyze DAG molecular species. In addition, we directly compared the three methods using DAG obtained by phospholipase C hydrolysis of phosphatidylcholine purified from a Nicotiana benthamiana leaf extract. We conclude that each method identified similar major molecular species, however, the exact levels of those varied mainly due to sensitivity of the technique, differences in sample preparation, and processing. This chapter provides three different methods to analyze DAG molecular species, and the discussion of the benefits and challenges of each technique will aid in choosing the right method for your analysis.
... More hydrophobic lipids are strongly retained on the octadecyl stationary phase and elutes later from the column as compare to the more polar lipids. Elution order depends not only on the carbon chain length but also on the degree of unsaturation of acyl residues (Perona and Ruiz-Gutierrez 2003). Elution order of lipids in reversed phase are presented using the coefficient equivalent chain lengths (ECL) (Chen et al. 1999) and equivalent carbon number (ECN) for fatty acid and other lipids respectively (Perona and Ruiz-Gutierrez 2003). ...
... Elution order depends not only on the carbon chain length but also on the degree of unsaturation of acyl residues (Perona and Ruiz-Gutierrez 2003). Elution order of lipids in reversed phase are presented using the coefficient equivalent chain lengths (ECL) (Chen et al. 1999) and equivalent carbon number (ECN) for fatty acid and other lipids respectively (Perona and Ruiz-Gutierrez 2003). The equivalent chain length of fatty acid can be described by the following equation: ...
Phospholipids are one of the classes of lipids, which are a major component of all cell membranes. Phosphatidylcholine (PC) is the main phospholipid present in egg yolk. The phospholipids extract from enriched egg yolk was separated using the two-dimensional high performance liquid chromatography in off-line system coupled with electrospray ionization tandem mass spectrometry (off-line HPLC-ESI-MS/MS). In the first dimension, a preparative C18 column was used, and on the second dimension, a separation was performed on N,O-dialkylphosphoramidate bonded silica column. In the analyzed egg yolk, 15 different phosphatidylcholines was found. From among the identified fatty acid of PC, a large part constitutes docosahexaenoic acid (ω-3, 22:6, DHA) and linoleic acid (ω-6, 18:2, LA).
... [72] Reverse-phase LC is based on hydrophobicity and can be used for separation and later characterization of different fatty acids based on chain length or quantity of double bonds. [73] But, the addition of a UV-absorbing moiety can be applied to simplify the detection of fatty acids by reverse-phase LC. [74] This can be performed by the reaction of fatty acids with phenyl-containing compounds to provide phenacyl or related derivatives. Compounds such as p-bromo-or p-methoxyphenacyl esters is recommended and can be used for analysis of microbial Extracted twice into 0.5 mL of hexane FAME [14,15,26] HCl (8% solution made from 9.7 mL of concentrated HCl and Heating at 80 C for 3 h 1.5 mL of 0.5 M NaCl and 1.5 mL hexane FAME [31] " Methanol (chloroform:methanol ...
Fatty acids are among the most important components of many biological systems and have been highlighted in many research fields in recent decades. In the food industry, it is important to check the amount and types of fatty acids in edible oils, beverages and other foods products, and checking the fatty acids parameters are among the quality control parameters for those products. In medical applications, investigation of fatty acids in biological samples and comparing imbalances in them can help to diagnose some diseases. On the other hand, the development of cell factories for the production of biofuels and other valuable chemicals requires the accurate analysis of fatty acids, which serve as precursors in development of those products. As a result, given all these different applications of fatty acids, rapid and accurate methods for characterization and quantification of fatty acids are essential. In recent years, various methods for the analysis of fatty acids have been proposed, which according to the specific purpose of the analysis, some of them can be used with consideration of speed, accuracy and cost. In this article, the available methods for the analysis of fatty acids are reviewed with a special emphasis on the analysis of microbial samples to pave the way for more widespread metabolic engineering research.
... Various methods are available for the analysis of MG, DG, TG, and FA. On the one hand, analysis by high-performance liquid chromatography (HPLC) [5][6][7][8], often coupled with detection by mass spectrometry (MS) [9][10][11][12][13][14], was applied. On the other hand, the lipid classes were analyzed after derivatization by gas chromatography (GC) with flame ionization or MS detection [15][16][17][18]. ...
Mono- and diacylglycerols (MG/DG) of fatty acids (FA), known as emulsifiers of the type E 471, are food additives used to adjust techno-functional properties of various foodstuffs. These emulsifiers, however, are not defined single compounds but comprise, in addition to MG and DG, other constituents such as FA, triacylglycerols (TG), and glycerol. Although the emulsifiers’ compositions affect techno-functional properties of the food, knowledge of the composition is scarcely available, and the emulsifiers and their dosage are generally chosen empirically. Thus, a simple and rather inexpensive method for the simultaneous determination of FA, 1-MG, 2-MG, 1,2-DG, 1,3-DG, and TG by high-performance liquid chromatography–mass spectrometry including a straightforward quantitation strategy has been developed. Reversed-phase chromatography with gradient elution offered adequate separation of 29 considered analytes within 21 peaks, while mass-selective detection provided their unequivocal identification. The quantitation strategy based on calibration just with the C16:0 representatives of each lipid class and a corresponding response factor system has proven to provide reliable results. The determined concentrations of different mixtures comprising varying compositions and concentrations of C16:0, C18:0, and C18:1 components of each lipid class deviated < 20% ( n = 351) from the respective target concentrations. Limits of decision were determined to 0.3–0.8 mg/L and limits of quantitation to 0.8–1.7 mg/L, expressed as C16:0 representatives. Application of the method to various E 471 emulsifiers provided detailed data on their chemical compositions, and calculated FA compositions matched very well those determined by common methods such as gas chromatography with flame ionization detection.
... Although much of the literature regarding NAFLD has focused on TGs and cholesterol, other classes of lipids, such as diacylglycerols (DGs) and sphingolipids, may contribute to the pathogenesis of NAFLD (11,12). Both DGs (13) and sphingolipids accumulate in steatotic human livers (11,14), and these lipids exist as constituents of circulating VLDL particles (15)(16)(17) Patients undergoing bariatric surgery represent a unique population to interrogate the relationship between hepatic fat content and the balance between uptake and disposal of fatty acids. They have a greater probability of NAFLD and surgery is scheduled on an elective basis, allowing for complex studies to be meshed with their clinical care. ...
Purpose
Nonalcoholic fatty liver disease can lead to hepatic inflammation/damage. Understanding the physiological mechanisms that contribute to excess hepatic lipid accumulation may help identify effective treatments.
Methods
We recruited 25 severely obese, non-diabetic patients scheduled for bariatric surgery. To evaluate liver export of triglyceride-fatty acids, we measured very low density lipoprotein (VLDL)-triglyceride secretion rates the day prior to surgery using an infusion of autologous [1-14C]triolein-labeled VLDL particles. Ketone body response to fasting and intra-hepatic long chain acylcarnitine concentrations were used as indices of hepatic fatty acid oxidation. We measured intraoperative hepatic uptake rates of plasma free fatty acids using a continuous infusion of [U-13C]palmitate, combined with a bolus dose of [9,10-3H]palmitate and a carefully timed liver biopsies. Total intrahepatic lipids were measured in liver biopsy samples to determine fatty liver status. The hepatic concentrations and enrichment from [U-13C]palmitate in diacylglycerols, sphingolipids, and acyl-carnitines were measured using liquid chromatography/tandem mass spectrometry.
Results
Amongst study participants with fatty liver disease, intrahepatic lipid was negatively correlated with VLDL-triglyceride secretion rates (r = -0.92, P = 0.01), but unrelated to hepatic free fatty acid uptake or indices of hepatic fatty acid oxidation. VLDL-triglyceride secretion rates were positively correlated with hepatic concentrations of saturated diacylglycerol (r = 0.46, P = 0.02) and sphingosine-1-phosphate (r = 0.44, P = 0.03).
Conclusion
We conclude that in non-diabetic, severely obese humans, excess intrahepatic lipid is associated with limited export of triglyceride in VLDL particles rather than increased uptake of systemic free fatty acids.
... The ECN value is employed to reaffirm the order of HPLC elution of various triacylglycerol molecular species [24]. As exact chromatographic peak assignment is not possible merely based on ECN values of triacylglycerols, the detailed chromatographic peak identification is achieved by the approach of logarithms of retention volumes [18,25]. ...
A long chain saturated fatty acid (SFA), behenic acid, is incorporated into the sn-1, 3 positions of triacylglycerols in palm olein (POo) and high-oleic sunflower oil (HOS) by solvent-free interesterification catalyzed by Lipozyme RM IM. The enzymatic interesterified HOS (EIE-HOS) yielded 76.5% of BOO and BOB as compared to 45.6% in POo (EIE-POo). The sn-2 position of EIE-HOS displayed 5.3 mol% of SFA which is significantly lower compared to 13.5 mol% in EIE-POo (P < 0.001). The sn-1, 3 positions of EIE-POo exhibited greater amount of behenic acid (82.0 mol%) in relation to EIE-HOS (64.0 mol%) (P < 0.001). Due to the greater variety of constitutive triacylglycerol, EIE-POo showed greater differences between onset (To) and offset temperature (Tf) in the melting endotherms (76.99 °C) as compared to EIE-HOS (68.65 °C), and may offer more intensive cooling sensation and flavor release.
... Using optimized chromatographic conditions, it is possible to separate TAGs that have the same ECN (i.e. OOO, PPP, OOP and OPP) [33,70] or TAGs that differ in double bonds position [71]. In its latest variation NARP technique allows a very good separation of a large number of TAGS [13,58,72]. ...
Vegetable oils are a dietary source of lipids that constitute an essential component of a healthy diet. The commonly used vegetable oils differ significantly for their triacylglycerol (TAG) profile. TAGs represent the principal components of oils and may contain different fatty acids (FA) esterified with glycerol leading to several positional isomers. To differentiate individual TAGs species in edible oils, advanced analysis systems and innovative methods are therefore required. TAGs can be considered as good fingerprints for quality control and many studies have been performed to develop rapid and low cost analytical methods to determinate the authenticity, origin and eventually evidence frauds or adulterations.
... More hydrophobic lipids are more strongly retained on the stationary phase and are eluted from the column later as compared with the more polar lipids. Elution in RP is dependent not only on the carbon chain length but also on the degree of unsaturation of acyl residues of the analyzed lipids (Perona and Ruiz-Gutierrez, 2003). Retention time decreases with shorter chain length and increases with the growing number of double bonds. ...
Current studies related to lipid identification and determination, or lipidomics in biological samples, are one of the most important issues in modern bioanalytical chemistry. There are many articles dedicated to specific analytical strategies used in lipidomics in various kinds of biological samples. However, in such literature, there is a lack of articles dedicated to a comprehensive review of the actual analytical methodologies used in lipidomics. The aim of this article is to characterize the lipidomics methods used in modern bioanalysis according to the methodological point of view: (1) chromatography/separation methods, (2) spectroscopic methods and (3) mass spectrometry and also hyphenated methods. In the first part, we discussed thin layer chromatography (TLC), high-pressure liquid chromatography (HPLC), gas chromatography (GC) and capillary electrophoresis (CE). The second part includes spectroscopic techniques such as Raman spectroscopy (RS), Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR). The third part is a synthetic review of mass spectrometry, matrix-assisted laser desorption/ionization (MALDI), hyphenated methods, which include liquid chromatography–mass spectrometry (LC-MS), gas chromatography–mass spectrometry (GC-MS) and also multidimensional techniques. Other aspects are the possibilities of the application of the described methods in lipidomics studies. Due to the fact that the exploration of new methods of lipidomics analysis and their applications in clinical and medical studies are still challenging for researchers working in life science, we hope that this review article will be very useful for readers.
... More hydrophobic lipids are more strongly retained on the stationary phase and are eluted from the column later as compared with the more polar lipids. Elution in RP is dependent not only on the carbon chain length but also on the degree of unsaturation of acyl residues of the analyzed lipids (Perona and Ruiz-Gutierrez, 2003). Retention time decreases with shorter chain length and increases with the growing number of double bonds. ...
Current studies related to lipid identification and determination, or lipidomics in biological samples, are one of the most important issues in modern bioanalytical chemistry. There are many articles dedicated to specific analytical strategies used in lipidomics in various kinds of biological samples. However, in such literature, there is lack of articles dedicated to a comprehensive review of the actual analytical methodologies used in lipidomics. The aim of this article is to characterize the lipidomics methods used in modern bioanalysis according to the methodological point of view: 1) chromatography/separation methods, 2) spectroscopic methods and 3) mass spectrometry and also hyphenated methods. In the first part we discussed thin layer chromatography (TLC), high-pressure liquid chromatography (HPLC), gas chromatography (GC), and capillary electrophoresis (CE). The second part includes spectroscopic techniques such as: Raman spectroscopy (RS), fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR). The third part is a synthetic review of mass spectrometry, matrix-assisted laser desorption/ionization (MALDI), hyphenated methods, which include liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and also multidimensional techniques. Other aspects are the possibilities of the application of the described methods in lipidomics studies. Due to the fact that the exploration of new methods of lipidomics analysis and their applications in clinical and medical studies are still challenging for researchers working in life science, we hope that this review article will be very useful, for readers.
... Elution order of PLs in reversed phase depends on the differences in the chain lengths and the number of double bonds of acyl residues. The elution order of lipids in reversed phase is presented using the coefficient equivalent carbon number (ECN) for fatty acid (Perona and Ruiz-Gutierrez 2003). ECN is defined as ECN = CN − 2n (where CN is the sum of the total carbon atom number of fatty acyl groups and n is the number of the sum of double bonds). ...
The paper presents the analysis of the profile composition of fatty acids in the molecules of phosphatidylcholine and phosphatidylethanolamine, by using hydrophilic interaction liquid chromatography and gas chromatography coupled with mass spectrometry. The profiles of 15 phosphatidylcholine and 8 phosphatidylethanolamine species were analyzed with a newly developed hydrophilic interaction liquid chromatography (HILIC)–electrospray ionization (ESI)–tandem mass spectroscopy (MS/MS) method, by using a new stationary bonded phase. The application of the new method in control and experimental groups of egg yolk revealed significant differences in the composition of phospholipid species containing mainly polyunsaturated fatty acids. Additionally, using GC-MS, the profile of fatty acids in four groups with different dietary supplementation of hens was analyzed and 20 fatty acids in egg yolks were determined. Monounsaturated fatty acids were found in higher amounts than saturated fatty acids and polyunsaturated fatty acids in egg yolks. Oleic acid (18:1) was the major monounsaturated fatty acid in egg yolk while palmitic acid (16:0) was the major saturated fatty acid. Linoleic acid (18:2), arachidonic acid (20:4), and docosahexaenoic acid (22:6) reached the highest levels among the polyunsaturated fatty acids.
... Endocytosis of LDL provides an D r a f t alternative mechanism to supply LA to cells including adipose tissue that circumvents the relatively poor lipolysis of LA from chylomicrons. LDL contains higher levels of LA and OA, the preferred substrate of the peroxisomes, compared to VLDL [33,34], and lower levels of saturated fats including C:16 PA. ...
“Western”, Linoleic acid (LA) and refined carbohydrate calorie rich, Alpha Linolenic acid (ALA) deficient, nutrient depleted, highly oxidised inflammatory diets, combined with; lowered antioxidant capacity, heavily refined pre-oxidised food, and excess intake of easily oxidised sugars, amplifies natures signalling systems that trigger fat storage, putting fat deposition mechanisms into overdrive, leading to activation of oxidative stress related inflammation, and related adipose tissue macrophage immune signalling, so wider immune activation, mitochondrial dysfunction, insulin resistance, diabetes, metabolic syndrome and increased occurrence of comorbid “Western” diseases. Factors contributing to the risk of obesity are complex and multiple including; genetics; epigenetics; exercise; lifestyle; declining food quality; ability to sense combined with an inbuilt programming to hunger for, seek out and consume foods normally associated with a high nutrient content, namely plant reproductive tissue related foods that in nature are generally seasonal and are rich in; LA, sucrose, glucose, fructose, carbohydrates, antioxidants and minerals, but which in industrial-foods are often pre-oxidised, antioxidant depleted, and stripped during processing of their protective conutrients. In the context of a nutrient insufficient processed pre-oxidised Western diet, the consequence of high levels of the most common plasma LA oxylipins, the HODEs, and consequential activation of PPAR gamma related peroxisomal beta-oxidation pathways, is significant fat gain. LA oxylipins, the HODEs, are associated with a wide range of “Western” diseases in addition to obesity and diabetes. ALA equivalent but less researched oxylipins are in general protective. ALA followed by LA is the preferred substrate of LOX12/15, which in the absence of the competitive presence of ALA will produce LA HODEs. Further two oxidised Omega-6 AA products activate CB1 cannabinoid receptors, helping drive hunger and fat deposition. Mechanisms that may increase hunger also exist for the plant reproductive material related products fructose and glucose. Activation by inter-meal fasting, and or exercise, of PPAR alpha peroxisomal beta oxidation pathways is obesity protective, through activation of energy production pathways, increased antioxidant capacity, and diversion of inflammatory related LA peroxisomal beta-oxidation substrate to energy rather than repair, but not options chosen by many in a “Western” 24/7/365 culture of instant, cheap, highly refined, pre-oxidised LA rich food availability. Moderating LA intake, increased ALA intake, and intermittent energy deficit stress through exercise or short term fasting, combined with a nutrient dense diet, high in antioxidants and low in pre-oxidised products, particularly in the obese, may reduce inflammation, and oxidative stress, hence direct a greater balance of resources towards energy production rather than tissue repair and fat depostion pathways, and reduce mitochondrial damage and dysfunction, so limit adipose tissue gain and comorbid medical conditions, as evidenced in the use of intermittent short term fasting treatment in diabetes. Exercise without dietary change or calorie restriction, is not an automatic route to weight loss.
... This very likely is due to the stronger interaction between the fatty acids moieties and the C30 stationary phase, while the influence of the fatty acyl chains is greater than that of the polar head group. Although a good separation of lipids can be achieved in reversed-phase chromatography, co-elution of lipids belonging to different classes in reversed-phase separations is quite commonly observed due to the fact that the mechanism of action of lipids is based on their lipophilicity, which is governed by the carbon chain length and the number of double bonds [29]. As an alternative, hydrophilic interaction liquid chromatography (HILIC) can provide a simple and effective means to separate lipids by class, offering a complementary separation technique to the reversed-phase methods [30]. ...
... Reversed-phase chromatography uses a nonpolar stationary phase and a polar mobile phase and is based on lipophilicity, where lipids within the same class separate according to carbon chain length and quantity of double bonds (Perona and Ruiz-Gutierrez, 2003;Pietiläinen et al., 2007). C 18, C 8 and octadecylsilyl RP columns have been employed using eluting solvents such as methanol, isopropanol, acetonitrile, hexane and chloroform (Medina-Gomez et al., 2007;Rainville et al., 2007;Wolf and Quinn, 2008). ...
Lipids make up a diverse subset of biomolecules that are responsible for mediating a variety of structural and functional properties as well as modulating cellular functions such as trafficking, regulation of membrane proteins and subcellular compartmentalization. In particular, phospholipids are the main constituents of biological membranes and play major roles in cellular processes like transmembrane signaling and structural dynamics. The chemical and structural variety of lipids makes analysis using a single experimental approach quite challenging. Research in the field relies on the use of multiple techniques to detect and quantify components of cellular lipidomes as well as determine structural features and cellular organization. Understanding these features can allow researchers to elucidate the biochemical mechanisms by which lipid-lipid and/or lipid-protein interactions take place within the conditions of study. Herein, we provide an overview of essential methods for the examination of lipids, including extraction methods, chromatographic techniques and approaches for mass spectrometric analysis. This article is protected by copyright. All rights reserved.
... According to an empirical rule, fatty acids and their esters, with equal "equivalent carbon number", ECN = (n C − 2 n DB ), show similar retention times in reversed-phase LC [33,34]. However, in NARP-LC, some TAGs with equal ECNs but different numbers of double bonds, n DB , can be more or less resolved on efficient columns, depending on the stationary phase properties [35][36][37][38], even separation of TAGs differing in the position(s) of double bond(s) is sometimes possible [39]. Recently, we found that the "old" types of non-endcapped C 18 columns containing residual silanol groups provide improved resolution of TAGs with equal ECNs in comparison to "better" columns with suppressed silanol activity [37]. ...
The Carotenoid S is a new C30 bonded silica stationary phase, intended for reversed-phase chromatographic applications, which is more hydrophobic and consequently shows stronger retention in comparison to conventionally used C18 stationary phases. We compared the non-polar selectivities of the columns for homologous alkylbenzenes in acetonitrile—water and methanol-water mobile phases and polar reversed-phase selectivities employing the interaction indices and the Linear Free Energy Relationship models. Further, we investigated possibilities of separations of structurally closely related compounds in the groups of phenolic acids, flavones, phthalic acids and related compounds and of acylglycerols on the new C30 column and with different types of columns for reversed-phase chromatography, including shorter alkyl C4, C8, C18 and phenyl bonded stationary phases. The C30 column has in some aspects properties similar to the non-endcapped Nova-Pak column for separation of some acylglycerols with equal equivalent carbon numbers, but enables separations of longer chain triacylglycerols in a single gradient run.
... Extensive improvement was made using the evaporative light scattering detector (ELSD) as an universal detector whose response is the function of mass of vaporised analyte but also because it is compatible with gradient elution (Marcato and Cecchin, 1996;Liu et al., 1993;Lísa et al., 2007). Several attempts have been made for the separation of acylglycerol compositions (including 1,3-and 1,2-DAG positional isomers) using either normal-phase HPLC-ELSD or reverse-phase HPLC-ELSD (RP-HPLC-ELSD) in enzymatically glycerolised olive oil (Yang and Chen, 1991), olive oil and peanut oil containing DAG as a minor component (Liu et al., 1993), standard mixtures of different acylglycerols including positional isomers of dilaurin, dimyristin, dipalmitin, distearin and diarachidin (Marcato and Cecchin, 1996), different interesterified products containing DAG as the intermediate products (Mu et al., 2000), and in human very-low-density lipoproteins comprising positional isomers of dilinolein, dipalmitin, oleoyl-palmitoyl-glycerol and linoleoyl-oleoyl-glycerol (Perona and Ruiz-Gutierrez, 2003). Nevertheless, these studies have covered identification of only few types of DAG molecular species. ...
Reversed-phase high-performance liquid chromatography method using charged aerosol detector was
developed for separation of 1,3- and 1,2(2,3)-positional isomers of palm oil- and palm kernel oil-based
diacylglycerols (PO-DAG and PKO-DAG, respectively) with different equivalent carbon numbers
(ECN) and without the need of sample derivatisation. In this method, step-wise gradient of acetone and
acetonitrile was used and a total retention time of 28 min was attained. Identification of PKO- and PODAG
molecular species was accomplished using synthetic DAG standards. Completeness of separation as
well as identification of PKO- and PO-DAG molecular species including positional isomers with different
ECN values were verified where similar elution patterns as well as the same number of identified peaks
were observed for the chromatograms of PKO-DAG and PKO-DAG standards, as well as the PO-DAG
and PO-DAG standards. Among the 1,3-DAG species and/or 1,2-DAG species, as ECN value of their
fatty acid constituents increased, their corresponding retention time always increased. However, among
the PKO- as well as PO-based synthetic DAG with the same ECN values, 1,3-DAG were always found to
elute earlier than the respective 1,2-DAG. Furthermore, some exceptional examples were observed among
the PKO- as well as PO-based synthetic DAG standards with the different ECN values where a few of
1,3-DAG with the relatively higher ECN values eluted earlier than 1,2-DAG with relatively lower ECN
values. The last two observations evidence the defect of ECN-based prediction for identification of DAG
positional isomers with the same ECN as well as for different ECN values.
... RP- HPLC is used to separate individual molecular species of a particular phospholipid class (Bunger and Pison, 1995 ). The sample will be separated based on a partition mechanism and will elute based on lipophilicity owing to the combined chain length and number of double bonds present in the fatty acids side chains (Perona and Ruiz-Gutierrez, 2003). Shorter chains will have longer retention times (Pacetti et al., 2004). ...
Phospholipids are important constituents of all living cell membranes. Lipidomics is a rapidly growing field that provides insight as to how specific phospholipids play roles in normal physiological and disease states. There are many analytical methods available for the qualitative and quantitative determination of phospholipids. This review provides a summary of the methods that were historically used such as thin layer chromatography, gas chromatography and high-performance liquid chromatography. In addition, an introduction to applications of interfacing these traditional chromatographic techniques with mass spectrometry is provided.
... Such systems separate lipids according to the number, geometry and position of the double bonds within FA residues and to the regiospecific distribution of FAs chains. TAGs can be detected using a refractive index [56,57], flame ionisation [52,58], UV [59,60], evaporative light-scattering detectors [54,61] or mass spectrometric detectors, which can provide conclusive information on the identity of TAGs. Both atmospheric pressure chemical ionisation (APCI) [45][46][47]49,62,63] and electrospray ionisation (ESI) [53,[64][65][66][67][68] mass spectrometry have been used. ...
Triacylglycerols (TAGs) from the fat body of several bumblebee species (Bombus lucorum, B. terrestris, B. lapidarius, B. hypnorum, B. hortorum, and B. confusus) were studied using chromatographic techniques. Semi-preparative thin-layer chromatography was used to isolate the TAGs from the tissue extract. Gas chromatography (GC) enabled us to identify the fatty acids (FAs) that form bumblebee TAGs and to quantify their relative proportions. The TAGs were subsequently analysed by high-performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry. Two chromatographic systems, including non-aqueous reversed-phase chromatography and silver ion chromatography on cation exchange resin in silver (I) ionic form, were optimised and their performance compared. The most abundant fatty acids in bumblebees TAGs contained 18 or 16 carbon atoms; oleic acid predominated in most samples. TAGs were found to be a complex mixture of isomers; some of them, e.g. OLnO, PLnO, PoPoO, PoPoP, POO, or OOO (where Po is palmitoleic, P is palmitic, Ln is linolenic, and O is oleic acid) were abundant in particular species. The composition of both FAs and TAGs was found to be species-specific. Only minor differences were found among specimens of the same species.
Lipidomics refers to the large-scale study of pathways and networks of cellular lipids in biological systems. A lipidomic analysis often involves the identification and quantification of the thousands of cellular lipid molecular species within a complex biological sample and therefore requires a well optimized method for lipid profiling. In this chapter, the methods for lipidomic analysis, including sample collection and preparation, lipid derivatization and separation, mass spectrometric identification of lipids, data processing and interpretation, and quality control, are overviewed.
Nuclear magnetic resonance (NMR) spectroscopy offers reproducible quantitative analysis and structural identification of metabolites in various complex biological samples, such as biofluids (plasma, serum, and urine), cells, tissue extracts, and even intact organs. Therefore, NMR-based metabolomics, a mainstream metabolomic platform, has been extensively applied in many research fields, including pharmacology, toxicology, pathophysiology, nutritional intervention, disease diagnosis/prognosis, and microbiology. In particular, NMR-based metabolomics has been successfully used for cancer research to investigate cancer metabolism and identify biomarker and therapeutic targets. This chapter highlights the innovations and challenges of NMR-based metabolomics platform and its applications in cancer research.
Milk fat is a complex material that exists in the form of discrete lipid globules within the milk serum. The composition of milk fat can vary because of physiological and environmental factors, and these variations contribute to the lipid’s functional and flavour properties. Sensitive and discriminating analytical tools are required to quantify accurately both the major (fatty acids in the form of triacylglycerols) and minor (diacylglycerols, monoacylglycerols, free fatty acids, phospholipids, membranous proteins, cholesterol, etc.) components. This chapter provides an overview of the different chromatographic and spectroscopic methods used in industry and research to examine the polar and non-polar components of milk fat. Topics include lipid extraction, the proximate determination of fat content, and the analysis of fatty acids, lipid class, lipid species, milk fat globule membranous material, and headspace volatile flavour components. Advances in column specificity, instrumentation, and methodology for optimum separation and resolution are presented and limitations discussed.
We examined the effects of virgin olive oil (VOO) triacylglycerols (TGs) on the lipid composition of human very low-density lipoprotein (VLDL).
Cholesteryl esters (CE), triacylglycerols (TG), free cholesterol (FC), monoacylglycerols
(MG) and phospholipids (PL) were simultaneously determined by high-performance liquid
chromatography (HPLC) coupled to a light-scattering detector (ELSD) and in Iberian pig fat
from adipose tissue and Masseter muscle. The precision of the HPLC system was excellent,
showing RSD values of repeatability lower than 5.27% for detector response and 0.12% for
retention times. TG were the major lipid class detected, accounting for nearly 80% in the
muscle and 86% in adipose tissue. The fatty acid compositions of total lipids, TG and PL in
the tissues studied were characterized for high concentrations of oleic (18:1, n-9), palmitic
(16:0) and stearic (18:0) acids. We conclude that the method reported here is suitable for rapid
and precise determination of lipid classes in Iberian pig fat and may be suitable for the
analysis of the sensory quality of this and other meat products.
Key words: Iberian pig, lipid classes, HPLC, triacylglycerol, cholesterol
The complexity of natural triacylglycerols (TAGs) in various edible oils is prodigious due to the hundreds of set is of TAG compositions, which makes the identification of TAGs quite difficult. In this investigation, the off-line 2D system coupling of nonaqueous RP and silver-ion HPLC with atmospheric pressure chemical ionization MS detection has been applied to the identification and quantification of TAGs in peanut oil. The method was successful in the separation of a high number of TAG solutes, and the TAG structures were evaluated by analyzing their atmospheric pressure chemical ionization mass spectra information. HPLC and MS conditions have been optimized and the fragmentation mechanisms of isomers have been validated. In addition, an internal standard approach has been developed for TAG quantification. Then this system was applied in peanut oil samples and there was a total of 48 TAGs including regioisomers that have been determined and quantified.
Lipids are the most diverse class of metabolites in mammalian physiology and dysregulation of lipid metabolism is linked to various diseases. Alterations in acylglycerols, a major class of lipids in plasma and adipose tissue, are involved in the pathogenesis of obesity and type 2 diabetes. Therefore, determination of acylglycerols is important to depict and unravel cellular mechanisms related to pathological outcomes, and specific molecular species of acylglycerols might be promising biomarker candidates. The variety of acylglycerols can be characterized in different ways. Enzymatic assays enable the determination of total tri- or diacylglycerols showing a possible relation to diseases, but they do not allow clarification of molecular mechanism. While gas chromatography can provide an overview of the fatty acid composition of total or separated lipids, a very detailed description of the individual molecular acylglycerol species is possible via liquid chromatography, particularly when combined with mass spectrometry. This review describes the determination of acylglycerols considering recent developments, with a focus on mammalian serum/plasma and tissue.
The use of HPLC-MS to separate and identify the feruloylated acylglycerols formed during the transesterification of ethyl ferulate with TAG was examined. Novozym 435 (Candida antarctica lipase B)-catalyzed transesterifications of ethyl ferulate and soybean oil resulted in a mixture of feruloylated MAG, DAG, and TAG and diferuloylated DAG and TAG. These feruloylated acylglycerols have recently garnered much interest as cosmeceutical ingredients. The ratio of the various feruloylated acylglycerol species in the resultant oils is presumed to affect the oil's cosmetic efficacy as well as its physical (formulation) properties. Thus, it was desirable to develop an analytical method to separate, identify, and quantify the individual feruloylated acylglycerols to determine their relative ratios. The feruloylated acylglycerols were successfully separated and identified by HPLC-MS using a phenyl-hexyl reversed-phase column developed with a water/methanol/1-butanol gradient. The chromatograms of the feruloylated acylglycerols from soybean oil were convoluted by myriad fatty acids; therefore, feruloylated acylglycerols from triolein were studied as a model reaction. Hydrolysis of the feruloylated acylglycerols from triolein catalyzed by Lipase PS-C “Amano” I (Burkholderia cepacia), which showed no hydrolysis reactivity toward ethyl ferulate, allowed for the chromatographic assignment of the feruloyl acylglycerol positional isomers.
We examined the effects of virgin olive oil (VOO) triacylglycerols (TGs) on the lipid composition of human very low-density lipoprotein (VLDL).
Twenty-one normocholesterolemic, normotensive, non-diabetic elderly subjects were recruited for the study. Two VOOs (VOO1 and VOO2) of the same variety, with an equivalent composition in minor components and differing only in the oleic and linoleic acid concentrations, were administered for 4 wk each to assess the effect of their TG molecular species compositions. Blood was collected after an overnight fast, VLDLs were isolated by ultracentrifugation, and lipid classes, TG molecular species, and TG fatty acid composition were determined.
Dietary VOOs significantly differed in TG molecular species composition. VOO1 represented larger amounts of triolein (P < 0.01), whereas VOO2 was significantly enriched with dilinoleoyl-oleoyl-glycerol, linoleoyl-dioleoyl-glycerol, and linoleoyl-oleoyl-palmitoyl-glycerol (P < 0.01). For VLDL, intake of VOO1 caused an increase of total TG (P < 0.01) due mainly to increases in triolein and linoleoyl-dioleoyl-glycerol. Conversely, VOO2 increased VLDL cholesteryl esters (P < 0.01) and TG rich in arachidonic acid (P < 0.01). Conclusions: The different TG molecular species compositions of dietary oils may be an independent determinant of the lipid composition of VLDL in elderly people and therefore may play a role in regulating lipoprotein metabolism in these subjects.
Triacylglycerol-rich lipoproteins (TRL), comprising chylomicrons (CM) and very-low-density lipoproteins (VLDL), have been associated with cardiovascular disease. The lipid class content in the remnant particles of these lipoproteins is a determinant for the accumulation of lipids in macrophages and their transformation into foam cells. We have optimized a method for the simultaneous determination of cholesteryl esters (CE), triacylglycerols (TG), free cholesterol (FC), monoacylglycerols (MG), and phospholipids (PL) by HPLC coupled to a light-scattering detector (ELSD). A diol column and a ternary gradient of hexane, 2-propanol, and methanol were applied to CM and VLDL of human origin (n = 10), with excellent precision in terms of repeatability of peak areas and retention times. All peaks were baseline resolved although the resolution of CE and TG was compromised for the sake of simplicity of the solvent gradient. The ELSD response was fitted to second-order equations, with correlation coefficients (r2) higher than 0.999 for a wide range of concentrations (0.25-10 microg of lipid injected). TG were the major lipid class detected in human TRL, accounting for 62% in CM obtained 2 h after the oil intake. In addition we recorded a depletion of TG and CE in CM obtained 2 h after the oil intake of about 60%. We conclude that the method reported here is suitable for a rapid and precise determination of lipid classes in human TRL and, therefore, may be a useful tool for investigations on the atherogenicity of these lipoproteins.
The lipid composition of very-low-density lipoprotein (VLDL) in plasma is crucial for human health. A pre-requisite for the alteration of VLDL composition is a co-ordinated understanding of the complex interactions in VLDL assembly. In order to determine the potential effects of changes in substrate availability on VLDL lipid composition, we constructed, parameterized and evaluated a mechanistic mathematical model of the biosynthesis of triglycerides, phospholipids, and cholesterol esters and the assembly of VLDL in human hepatocytes. Using published data on human liver metabolism, the model was also used to provide insight into the complex process of lipid metabolism and to estimate the affinities of different liver enzymes for different fatty acids (FA). For example, we found that Delta6-desaturase is 19 times more selective for C18:3n-3 than C18:2n-6, stearoyl-CoA-desaturase is 2.7 times more selective for C18:0 than C16:0, Delta5-desaturase desaturates C20:4n-3 preferentially over C20:3n-6 and FA elongase preferentially elongates C18:3n-6. The model was also used to predict the plasma free fatty acid (FFA) composition required to generate a prescribed change in plasma lipoprotein FA composition. Furthermore, the model was tested against a published human feeding trial that investigated the effect of changes in dietary FA composition on human plasma lipid FA composition. The model is a useful tool for predicting the effect of changes in plasma FFA composition on plasma lipoprotein lipid FA composition.
The main constituents of plant oils are complex mixtures of TGs differing in acyl chain lengths, number and positions of double bonds, and regioisomerism. A non-aqueous reversed-phase HPLC method with acetonitrile-2-propanol gradient and 30 + 15 cm NovaPak C18 columns makes possible an unambiguous identification of the highest number of TGs ever reported for these oils, based on positive-ion APCI mass spectra. A new approach to TG quantitation is based on the use of response factors with three typical detection techniques for that purpose (APCI-MS, evaporative light-scattering detection, and UV at 205 nm). Response factors of 23 single-acid TGs (saturated TGs from C7 to C22, 7 unsaturated TGs), 4 mixed-acid TGs, diolein and monoolein are calculated from their calibration curves and related to OOO. Due to differences between saturated and unsaturated acyl chains, the use of response factors significantly improves the quantitation of TGs. 133 TGs containing 22 fatty acids with 8-25 carbon atoms and 0-3 double bonds are identified and quantified in 9 plant oils (walnut, hazelnut, cashew nut, almond, poppy seed, yellow melon, mango, fig, date) using HPLC/APCI-MS with a response factor approach. Average parameters and relative fatty acid concentrations are calculated with both HPLC/APCI-MS and GC/ FID.
Edible conifer seeds can serve as a source of triacylglycerols (TGs) with unusual Delta5 unsaturated polymethylene interrupted fatty acids (UPIFAs), such as cis-5,9-octadecadienoic (taxoleic), cis-5,9,12-octadecatrienoic (pinolenic), cis-5,11-eicosadienoic (keteleeronic) and cis-5,11,14-eicosatrienoic acids (sciadonic). Conifer seed oils from European Larch (Larix decidua), Norway Spruce (Picea abies) and European Silver Fir (Abies alba) have been analyzed by non-aqueous reversed-phase high-performance liquid chromatography (NARP-HPLC) with atmospheric pressure chemical ionisation (APCI)-MS detection. The influence of different positions of double bonds in Delta5-UPIFAs on the retention and fragmentation behavior is described and used for the successful identification of TGs in each oil. TGs containing Delta5-UPIFAs have a higher retention in comparison with common TGs found in plant oils with single methylene interrupted Delta6(9)-FAs and also significantly changed relative abundances of fragment ions in APCI mass spectra. Results obtained from HPLC/MS analyses are supported by validated GC/FID analyses of fatty acid methyl esters after the transesterification. The total content of Delta5-UPIFAs is about 32% for European Larch, 27% for Norway Spruce and 20% for European Silver Fir. In total, 20 FAs with acyl chain lengths from 16 to 24 carbon atoms and from 0 to 3 double bonds have been identified in 64 triacylglycerols from 3 conifer seed oils.
Quantitative analysis of triacylglycerols (TGs) in plant oils and animal fats by normalization of peak areas can lead to erroneous results due to the large response differences with common HPLC detectors between the various TGs. The charged aerosol detector (CAD), that generates an almost universal response for non-volatile compounds, was combined with non-aqueous reversed-phase HPLC (NARP-HPLC) to develop a simple quantitative method, without need for RFs, for the analysis of complex natural TG mixtures from plant oils. Two 25 cm Hypersil ODS columns, connected in series, and a mobile phase gradient composed of acetonitrile, 2-propanol and hexane were used. Mobile phase compensation was applied, by mixing of the column effluent with the inversed gradient delivered by a second HPLC pump, for the suppression of the response dependency of the analytes on the mobile phase composition. Calibration curves of 16 saturated (from C7:0 to C22:0) and 3 unsaturated (C18:1, C18:2, C18:3) single-acid TG standards were measured and their RFs were compared with a previously described method using atmospheric pressure chemical ionization-mass spectrometry (APCI-MS). The variation in response of the most common TGs (containing fatty acid chains from 12 to 19 carbons) could be reduced to less than 5% making the combination of NARP-HPLC with CAD and mobile phase compensation an adequate tool for fast quantitative analysis of TGs in common plant oils.
Optimized non-aqueous reversed-phase high-performance liquid chromatography method using acetonitrile-2-propanol gradient elution and the column coupling in the total length of 45 cm has been applied for the high resolution separation of plant oils important in food industry, dietetics and cosmetics. Positive-ion atmospheric pressure chemical ionization mass spectrometry is used for the unambiguous identification and also the reliable quantitation with the response factors approach. Based on the precise determination of individual triacyglycerol concentrations, the calculation of average parameters important in the nutrition is performed, i.e. average carbon number, average double bond number, relative concentrations of essential, saturated, monounsaturated and polyunsaturated fatty acids. Results are reported in the form of both chromatographic fingerprints and tables containing relative concentrations for all triacylglycerols and fatty acids in individual samples. In total, 264 triacylglycerols consisting of 28 fatty acids with the alkyl chain length from 6 to 26 carbon atoms and 0 to 4 double bonds have been identified in 26 industrial important plant oils.
Lipoprotein lipase (LPL), the major lipolytic enzyme involved in the conversion of triglyceride-rich lipoproteins to remnants, was found to compete with binding of activated alpha 2-macroglobulin (alpha 2M*) to the low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor. Bovine milk LPL displaced both 125I-labeled alpha 2M* and 39-kDa alpha 2M receptor-associated protein (RAP) from the surface of cultured mutant fibroblasts lacking LDL receptors with apparent KI values at 4 degrees C of 6.8 and 30 nM, respectively. Furthermore, LPL inhibited the cellular degradation of 125I-alpha 2M* at 37 degrees C. Because both alpha 2M* and RAP interact with LRP, these data suggest that LPL binds specifically to this receptor. This was further supported by observing that an immunoaffinity-isolated polyclonal antibody against LRP blocked cellular degradation of 125I-LPL in a dose-dependent manner. In addition, 125I-LPL bound to highly purified LRP in a solid-phase assay with a KD of 18 nM, and this binding could be partially displaced with alpha 2M* (KI = 7 nM) and RAP (KI = 3 nM). Taken together, these data establish that LPL binds with high affinity to LRP and undergoes LRP-mediated cellular uptake. The implication of these findings for lipoprotein catabolism in vivo may be important if LRP binding is preserved when LPL is attached to lipoproteins. If so, LPL might facilitate LRP-mediated clearance of lipoproteins.
We have compared the molecular species composition of the glycerolipids of rat liver and rat plasma very low density lipoproteins (VLDL). There were differences in the stereospecific distribution of the fatty acids in the triacylglycerols (TG) of the liver and of VLDL. While chiral and reversed phase chromatography with mass spectrometry (LC/MS) revealed great similarities in positional distribution and molecular association of the fatty acids between the sn-1,2-diacylglycerol (DG) moieties of the VLDL and liver TG, the corresponding sn-2,3-DG were distinctly different. The free hepatic sn-1,2-DG and the sn-1,2-DG moiety contained within hepatic phosphatidic acid showed a maximum 60% homology to the sn-1,2-DG contained within the TG of the liver and of VLDL. By contrast, the smaller pool of hepatic free sn-2,3-DG was nearly identical to the sn-2,3-DG moiety contained in the TG of the liver. These differences between hepatic and VLDL TG indicate that direct transfer of hepatic triacylglycerols is not a major mechanism of VLDL TG formation. On the other hand, the results suggest that stored hepatic TG are largely hydrolyzed to sn-1,2-DG and then reesterified to TG before being secreted as VLDL TG. Although an involvement of 2-monoacylglycerol pathway could not be excluded, it probably plays a minor role in VLDL TG formation. Our data suggest that a minimum of 60% of the VLDL TG could have been derived via hydrolysis to DG and reesterification, and a maximum of 40% could have originated via the conventional phosphatidic acid pathway.
Bovine milk lipoprotein lipase (LPL) induced binding, uptake, and degradation of 125I-labeled normal human triglyceride-rich lipoproteins by cultured mutant fibroblasts lacking LDL receptors. The induction was dose-dependent and occurred whether LPL and 125I-lipoproteins were added to incubation media simultaneously or LPL was allowed to bind to cell surfaces, and unbound LPL was removed by washing prior to the assay. Lipolytic modification of lipoproteins did not appear to be necessary for increased catabolism because the effect of LPL was not prevented by inhibitors of LPL's enzymatic activity, p-nitrophenyl N-dodecylcarbamate or phenylmethylsulfonyl fluoride. However, the effect was abolished by boiling LPL prior to the assay suggesting that major structural features of LPL were required. Also, LPL-induced binding to cells was blocked by an anti-LPL monoclonal antibody but not by antibodies that are known to block apolipoprotein E- or B-100-mediated binding to low density lipoprotein (LDL) receptors. This indicates that LPL itself mediated 125I-lipoprotein binding to cells. Cellular degradation of 125I-lipoproteins was partially or completely blocked by two previously described ligands for the LDL receptor-related protein/alpha 2-macroglobulin receptor (LRP): activated alpha 2-macroglobulin (alpha 2M*), and the 39-kDa receptor-associated protein. These data implicated LRP as mediating LPL-induced lipoprotein degradation and were confirmed by showing that LPL's effects were prevented by an immunoaffinity-isolated polyclonal antibody against LRP. Furthermore, LPL promoted binding of 125I-lipoproteins to highly purified LRP in a solid-phase assay. Heparin or heparinase treatment of cells markedly decreased LPL-induced binding, uptake, and degradation of lipoproteins, but had no effect on catabolism of alpha 2M*. Thus, cell-surface proteoglycans were obligatory participants in the effects of LPL but were not required for LRP-mediated catabolism of alpha 2M*. Taken together, these in vitro findings establish that through interaction with cell-surface proteoglycans, LPL induces catabolism of normal human triglyceride-rich lipoproteins via LRP.
Chylomicrons are lipoproteins synthesized exclusively by the intestine to transport dietary fat and fat-soluble vitamins. Synthesis of apoB48, a translational product of the apob gene, is required for the assembly of chylomicrons. The apob gene transcription in the intestine results in 14 and 7 kb mRNAs. These mRNAs are post-transcriptionally edited creating a stop codon. The edited mRNAs chylomicrons from the shorter apoB48 peptide remains to be elucidated. In addition, the roles of proteins involved in the assembly pathway, e.g. apobec-1, MTP and apoA-IV, needs to be studied. Cloning of enzymes involved in the intestinal biosynthesis of triglycerides will be crucial to fully appreciate the assembly of chylomicrons. There is a need for cell culture and transgenic animal models that can be used for intestinal lipoprotein assembly. The catabolism of chylomicrons is far more complex and efficient than the catabolism of VLDL. Even though the major steps involved in the catabolism of chylomicrons are now known, the determinants for apolipoprotein exchange, processing of remnants in the space of Disse, as well as the mechanism of uptake of these particles by extra-hepatic tissue needs further exploration.
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.
Apoliprotein (apo) B-100-containing very low density lipoprotein (VLDL) particles secreted from the liver accumulate in plasma during alimentary lipemia. To determine whether changes of VLDL composition occur in the postprandial state that may render these lipoproteins more atherogenic, apoE, C-I, C-II, and C-III, and lipids (triglycerides, phospholipids, and cholesterol) were measured in Svedberg flotation (Sf) 60-400 (large) and Sf 20-60 (small) VLDL before and after an oral fat load. Ten normotriglyceridemic (NTG) and three hypertriglyceridemic (HTG) healthy men were given a fat-rich mixed meal (1,000 kCal with 60.2 E% from fat). Triglyceride-rich lipoproteins were isolated by density gradient ultracentrifugation from plasma samples obtained before (fasting) and at 2-h intervals after the meal. VLDL was then separated from chylomicrons and their remnants by immunoaffinity chromatography using monoclonal antibodies 4G3 and 5E11, recognizing apoB-100, but not apoB-48 epitopes. Large and small VLDL isolated from the NTG group were enriched with apoE and C-I, and cholesterol, but depleted of apoC-II in the postprandial state, whereas the apoC-III, triglyceride, and phospholipid contents were essentially unchanged. The compositional changes of VLDL in HTG subjects were similar but more pronounced compared with NTG subjects. We conclude that postprandial lipemia in healthy men induces transient compositional alterations of VLDL that link these lipoprotein species to the formation of atherosclerosis.
The size of cholesterol-rich lipoprotein particles is a strong determinant of whether they may be deposited in the arterial wall and by this become potentially atherogenic. This study deals with the in vivo transformation of larger-sized chylomicrons and chylomicron remnants to smaller-sized remnants. Twelve healthy men aged 22 to 45 years were given a fatty meal to which retinyl palmitate (RP) had been added. Plasmapheresis was performed 4 1/2 h after meal intake to isolate approximately 400 ml plasma. The RP-rich plasma was re-injected to the subject 24 h later. The RP content was determined in whole plasma and in Svedberg flotation rate fractions (Sf) > 400, Sf 60-400 and Sf 20-60. A compartmental model was developed for the kinetic analysis. Lipoprotein fractions showed minimal signs of aggregation, thus arguing for well-preserved postprandial lipoproteins. Approximately a fourth [23% (4-68%)] of the RP-containing lipoproteins in the Sf > 400 pool was converted to smaller species (Sf 60-400). Conversion of material from the Sf 60-400 to the Sf 20-60 fraction could not be detected. In a second study a large bolus dose of a triglyceride emulsion (Intralipid) was injected to subjects shortly after the RP-labeled plasma to investigate the endothelial binding of the chylomicron/chylomicron remnants. RP material in the Sf > 400 fraction rapidly returned to plasma, arguing for margination of chylomicrons, whereas the corresponding effect was minimal in the Sf 60-400 and Sf 20-60 fractions. The formation of small chylomicron remnants from the larger chylomicron/chylomicron remnant species is limited and large chylomicron/chylomicron remnants are not evenly distributed in plasma, rather they show signs of being marginated to the vascular endothelium.
This work was undertaken to determine the effect of diets enriched with olive oil or high oleic sunflower oil on very low density lipoprotein (VLDL) triacylglycerol composition of healthy human subjects. Both oils contain a similar proportion of monounsaturated fatty acids (MUFA) but differ in their triacylglycerol composition. All 22 human subjects initially consumed a low fat, high carbohydrate diet as recommended by the National Cholesterol Education Program (NCEP-I). They then consumed the two experimental oils (40% dietary energy) in a crossover design. The olive oil and high oleic sunflower oil diets resulted in significant increases in palmitoleic (55%, P < 0.05), oleic (27%, P < 0.01) and eicosenoic (>100%, P < 0.001) acids of VLDL triacylglycerols, whereas there was a significant decrease in linoleic acid (38%, P < 0.001). In addition, the high oleic sunflower oil diet increased the content of stearic acid (60%, P < 0.05) and total saturated fatty acids (14%, P < 0.05). Both MUFA-rich diets significantly (P < 0.01) decreased the content of sn-glycerol-palmitate-linoleate-oleate, sn-glycerol-palmitoleate-dioleate and sn-glycerol-palmitate-dilinoleate in VLDL with regard to the NCEP-I diet, whereas they increased the content of sn-glycerol-trioleate (>100%, P < 0.001 after the olive oil diet; 80%, P < 0.05 after the high oleic sunflower oil diet). Intake of olive oil, in particular, significantly decreased the content of sn-glycerol-tripalmitate (36%, P < 0.01) and increased the content of dioleoyl-containing triacylglycerols. MUFA (P < 0.01) and arachidonic acid (P < 0.001) tended to be rich in the sn-2 position of VLDL triacylglycerols during the periods of consuming the olive oil or high oleic sunflower oil diets. In addition, olive oil, but not high oleic sunflower oil, further contributed to VLDL triacylglycerols that contained alpha-linolenic and docosahexaenoic acids acylated in the sn-2 position. These data suggest that differences in the composition of VLDL triacylglycerols may be of major importance in explaining the beneficial effects of dietary olive oil in reducing the atherogenic risk profile in healthy subjects.
To establish whether the ingestion of diets enriched with olive oil or high-oleic sunflower oil may produce changes in the composition of VLDL triacylglycerols from hypertensive patients. It could be relevant for the uptake and metabolism of triacylglycerol-derived metabolites by extrahepatic tissues.
Patients were assigned to the diets in a random-order sequence.
The participants were 24 hypertensive patients recruited from a religious community.
The study was conducted over two four week periods with a four week washout period between both MUFA diets.
Dietary olive oil kept in balance the content of saturated fatty acids and decreased the content of arachidonic acid in VLDL triacylglycerols. HOSO diet reduced the content of palmitic acid and increased the content of linoleic acid. There was also a decrease in trioleate-glycerol and an increase in tripalmitate-glycerol of VLDL after the MUFA diets, but these effects were more pronounced in the HOSO group. Intake of olive oil decreased the content of disaturated triacylglycerols and increased the content of dioleate-containing triacylglycerols. A decrease in palmitate-dioleate-glycerol after dietary HOSO was observed. Olive oil (but not HOSO) promoted the presence of long-chain PUFA of n-3 family at the sn-2 position of VLDL triacylglycerols.
Our data indicate that olive oil and HOSO, providing a similar concentration of MUFA (oleic acid), differ in the formation of VLDL triacylglycerols in hypertensive patients.
The aims of the present study were to evaluate the effect of a meal rich in virgin olive oil on triacylglycerol composition of human postprandial triacylglycerol-rich lipoproteins (fraction Sf > 400), and to assess the role of the triacylglycerol molecular species concentration and polarity on lipoprotein clearance. Fasting (0 h) and postprandial blood samples were collected hourly for 7 h from eight healthy normolipidemic subjects after the ingestion of the meal. Plasma and lipoprotein triacylglycerol concentrations increased quickly over fasting values and peaked twice at 2 and 6 h during the 7-h postprandial period. The triacylglycerols in the lipoprotein fraction at 2 h generally reflected the composition of the olive oil, however, the proportions of the individualmolecular species were altered by the processes leading to their formation. Among the major triacylglycerols, the proportion of triolein (OOO; 43.6%) decreased (P < 0.05), palmitoyl-dioleoyl-glycerol (POO; 31. 1%) and stearoyl-dioleoyl-glycerol (SOO; 2.1%) were maintained and linoleoyl-dioleoyl-glycerol (LOO; 11.4%) and palmitoyl-oleoyl-linoleoyl-glycerol (POL; 4.6%) significantly increased (P < 0.05) compared with the composition of the triacylglycerols in the olive oil. Smaller amounts of endogenous triacylglycerol (0.8%), mainly constituted of the saturated myristic (14:0)and palmitic (16:0) fatty acids, were also identified. Analysis of total fatty acids suggested the presence of molecular species composed of long-chain polyunsaturated fatty acids of the (n-3) family, docosapentaenoic acid, [22:5(n-3)] and docosahexaenoic acid (DHA), [22:6(n-3)] and of the (n-6) family [arachidonic acid, [20:4(n-6)]. The fastest conversion of lipoproteins to remnants occurred from 2 to 4 h and was directly related to the concentration of the triacylglycerols in the lipoprotein particle (r = 0.9969, P < 0.05) and not with its polarity (r = 0.1769, P > 0.05). The rates of clearance were significantly different among the major triacylglycerols (OOO, POO, OOL and POL) (P < 0.05) and among the latter ones and PLL (palmitoyl-dilinoleoyl-glycerol, POS (palmitoyl-oleoyl-stearoyl-glycerol) and OLL (oleoyl-dilinoleoyl-glycerol) (P < 0.01). OOO was removed faster and was followed by POO, OOL, POL, PPO (dipalmitoyl-oleoyl-glycerol), SOO, PLL, POS and OLL.
It has been demonstrated that triacylglycerol (TAG) mobilization from adipose tissue is selective and depends on fatty acid (FA) chain length, unsaturation and positional isomerism. The present study was performed to determine the influence of dietary fat on the composition of TAG stored in rat perirenal and subcutaneous adipose tissues. These results may provide information on the susceptibility of stored TAG to hydrolysis and further mobilization, and may help to establish an interrelationship between dietary composition and the FA efflux from adipose tissue. TAG molecular species and FA composition were determined by HPLC and GLC respectively. No significant differences were found in either FA or TAG composition between perirenal and subcutaneous adipose depots. The major FA in the dietary fats were present in the adipose tissues of the animals; in most cases, in similar proportions. However, differences were found between dietary and adipose tissue content of minor FA, which suggests that dietary FA composition is altered between ingestion and deposition in adipose tissue. The TAG molecular species of rat adipose tissue were enriched with the FA characteristic of each dietary fat. Dietary sunflower oil was responsible for enrichment with the most polar TAG. This finding may suggest easier mobilization of stored TAG. In conclusion, the process of fatty acid and TAG deposition in rat adipose tissue is selective, and depends on the composition of the diet.
Monounsaturated oils, virgin olive oil (VOO) and high oleic sunflower oil (HOSO) are suggested to have selective physiologic effects on humans in the fasting state. The aim of the study was to evaluate whether two oils with equal amounts of oleic acid but with different compositions of minor fatty acids and triacylglycerol molecular species (TAG) could produce different triacylglycerol-rich lipoprotein (TRL)-TAG responses in the postprandial state. Eight normolipidemic men consumed the following three meals in random order on separate occasions with 2 wk between meals: control meal, control meal plus VOO and control meal plus HOSO. Plasma total TAG and TRL-TAG were measured hourly for 7 h after ingestion. TAG and sn-2 positional fatty acids within TAG were analyzed in the TRL fraction. Plasma total TAG concentrations in response to the dietary oils did not differ. However, TRL triglyceridemia was significantly lower after VOO intake (P < 0.05). The molecular species in the TRL fraction returned toward basal levels more quickly (P < 0.05) after VOO than HOSO intake. 2-Positional fatty acid analysis demonstrated higher proportions of stearic and palmitic acids and a lower proportion of oleic acid (P < 0.05) in TRL-TAG derived from HOSO. This study shows that VOO intake results in attenuated postprandial TAG concentration and faster TRL-TAG disappearance from blood compared with HOSO, suggesting that the oleic acid content may not be the main factor affecting TAG metabolism. Minor fatty acids such as linoleic acid and the 2-positional distribution of saturated stearic and palmitic acids into the TAG molecule may be important determinants of postprandial lipemia in normolipidemic men.
Rat liver triacylglycerol species are known to respond differentially to metabolic and nutritional manipulation but changes in triacylglycerols containing 20–22 carbon polyunsaturates have been difficult to determine. Liver total triacylglycerols were separated by high performance liquid chromatography into subclasses to determine whether their remodelling by fasting induced changes consistent with reacylation causing enrichment by long-chain polyunsaturated fatty acids. Triacylglycerol subclasses and their fatty acid composition were quantitatively determined. The fasting-induced changes in liver triacylglycerols were characterized by the appearance of new subclasses containing 20–22 carbon polyunsaturates at 18–30% but relative and quantitative depletion of more saturated subclasses. Previous studies have not been able to identify whether changes in the polyunsaturated fatty acid content of triacylglycerols during fasting were mainly a function of depletion of saturates and monounsaturates or whether extensive reacylation of subclasses/species specifically enriched in polyunsaturated fatty acids also occurred; our present data suggest that the latter is a significant component of the fasting-induced triacylglycerol remodelling process.
The nutritional benefits attributed to fish oils have been the basis for the study of the structural composition of Sardine oil triacylglycerols (TAGs). TAGs were separated reversed-phase high performance liquid chromatography (RP-HPLC) with the result of 65 chromatographic peaks resolved. The problem of identification was avoided by the use of two more chromatographic techniques. Separation of the sardine oil TAGs into fractions by silver-ion thin layer chromatography (TLC) and its subsequent fatty acid analysis by gas chromatography allowed identification of 59 of the 65 chromatographic peaks. From those peaks, the major was trimyristin (MMM), with 8.22 % of the total. Dioleoyl-acyl-glycerol (OPO, OOE), dipalmitoyl-acyl-glycerol (PPO, PPPo), dipalmitoleoyl-acyl.glycerol (PoPoO) and dieicosapentaenoyl-acyl-glycerol (EEP) species were found in important amounts.
Triacylglycerols from Idiazabal cheese fat were analysed by high-performance liquid chromatography (LC) with a non-linear
gradient of acetone in acetonitrile and a light-scattering detector. Molecular species of triacylglycerols were predicted
by a simple and a multiple linear regression analysis of logk of the LC peaks and molecular variables such as equivalent carbon number of the possible triacylglycerol, chain length and
number of double bonds of each fatty acid of the triacylglycerol. Predicted results were confirmed by gas chromatographic
analysis of the fatty acids in the LC peaks. The main triacylglycerols of Idiazabal cheese fat contained butyric acid, butyroyl-dipalmitin,
butyroyl-myristoyl-palmitin and butyroyl-palmitoyl-olein. The most abundant triacylglycerols were those with even partition
numbers of 36, 34 and 38.
Rats fed with a fat-free or an olive oil-rich diet were employed to compare the response of two chromatographic techniques in the determination of rat liver triglyceride (TG) molecular species composition. Gas–liquid chromatography (GLC) on polarizable liquid phase and reversed-phase high-performance liquid chromatography (RP-HPLC) have been commonly employed for TG analysis, obtaining a similar number of chromatographic peaks when used for animal tissue TG determination. In the present study similar results were achieved with regard to most relevant chromatographic peaks, however, important differences were found in the content of minor TGs. Indeed, RP-HPLC permitted separation of long chain polyunsaturated fatty acids, which were not detected by GLC, while the latter technique reported a higher number of myristoyl-containing TG species. RP-HPLC analysis reported a greater number of TGs, with more similarity to a random composition, made up from the liver fatty acid composition. Therefore, it was concluded that utilization of both techniques would be helpful for liver TG analysis as the use of only one of them does not provide a complete profile of liver TGs. Nevertheless RP-HPLC seems to be more useful for this purpose since revealed a more extensive profile.
Reversed-phase high-performance liquid chromatography (HPLC) methods were developed for the separation of molecular species of 45 synthetic triacylglycerols and diacylglycerols. These methods used linear gradients of methanol-isopropanol and UV detection at 205 nm as well as radioactive flow detection, which we add for metabolic studies. The elution orders of triacylglycerols and diacylglycerols depend on the polarity of their fatty acid constituents with elution time increasing as polarity decreases. The elution orders of triacylglycerols depending on their fatty acid constituents were as follows: ricinoliec acid<linolenic acid<palmitoleic acid<myristic acid<palmitelaidic acid<linoleic acid<linolelaidic acid<oleic acid<palmitic acid<elaidic acid<petroselinic acid<petroselaidic acid<stearic acid, while the elution orders of diacylglycerols were: palmitic acid<oleic acid<stearic acid. For both classes of glycerides, elution corresponded closely with chain length, degree of unsaturation and presence of polar groups and they were similar to the elution orders of fatty acids on an aqueous C18 HPLC which we reported recently: Other structural features also affect eltion order, as triacylglycerol containing a cis-fatty acid elutes slightly earlier than its isomer containing a trans-fatty acid. Additionally, higher polarity in the sn-2 position causes earlier elution; Diacylglycerol with a hydroxy group at sn-2 position of the glycerol backbone and triacylglycerol with a polar group on the fatty acid chain at sn-2 position elute slightly earlier than their respective sn-1(3) isomers.
Although the literature on epidemiological associations between plasma triglyceride and CHD is not completely consistent, trends do emerge from the studies described here. First, the majority of observational studies demonstrate a significant univariate relation, although the results of case-control and cross-sectional studies are more uniform than those from prospective study designs. In many but not all studies, triglyceride remains a significant predictor of CHD in multivariate statistical analyses after controlling for TC or LDL-C. Perhaps the least consistent result is that the triglyceride association does not persist in some analyses controlling for HDL-C, while in other studies, the association remains significant. Although most studies have been conducted in men, the studies providing data on women, normocholesterolemic subjects, and diabetic subjects have generally found triglyceride to be, at the very least, a univariate risk factor. The results of intervention trials differ considerably, but no such study to date has been specifically designed to evaluate triglyceride-lowering effects on primary prevention of CHD. Important statistical properties must be taken into consideration in evaluating triglyceride as a risk factor for CHD. The large variability of triglyceride measurements and the correlation of triglyceride values with other lipid measures appears to result in the underestimation of the association between triglyceride and disease in multivariate analyses. Finally, individual genetic susceptibility may play an important role in the relation between plasma triglyceride levels and CHD. For example, risk of CHD clearly varies among the well-established familial forms of hypertriglyceridemia. A predominance of small, dense, LDL particles (LDL subclass pattern B) also appears to be a genetic trait associated with both increased risk of MI and increases in plasma triglyceride levels.
A total of 116 molecular species of triglycerides were identified in milk fat using a combination of HPLC and GLC. Triglyceride composition was predicted from the random composition, which was calculated on the basis of the mole fractions of the main fatty acids making up the total triglyceride fraction. The qualitative composition of the milk fat was similar in cows', ewes' and goats' milk. In all three milks the partition number of the main triglycerides was 46, but the proportions of the triglycerides with partition numbers of 34, 38, 42, and 48 exhibited substantial differences among the milks of the three species.
This review will deal with the occurrence, regulation and physiological role of the enzyme, including its relation with cholesterol metabolism.
A novel method is described for determining serum triglycerides, in which an enzymatic hydrolysis replaces the more commonly used saponification procedure. Under the conditions of the assay, the enzymatic hydrolysis can be completed in less than 10 min by the combined action of a microbial lipase and a protease. The authors demonstrated complete hydrolysis of triglycerides by thin layer chromatography of the reaction products, by recovery of glycerol from sera of known triglycerides content, and by comparison of triglyceride assays on a number of sera assayed by their method vs. the AutoAnalyzer procedure. The hydrolysis is directly coupled to the enzymatic determination of glycerol, and is followed through absorbance changes at 340 nm. The assay is simple, rapid, and requires only 50 μl or less of sample. Because the enzymes used do not release glycerol from other compounds in serum, the hydrolysis can be considered specific for triglycerides.
An enzymatic method is described for determination of total serum cholesterol by use of a single aqueous reagent. The method requires no prior treatment of sample and the calibration curve is linear to 600 mg/dl. Cholesterol esters are hydrolized to free cholesterol by cholesterol ester hydrolase (EC 3.1.1.13). The free cholesterol produced is oxidized by cholesterol oxidase to cholest 4 en 3 one with the simultaneous production of hydrogen peroxide, which oxidatively couples with 4 aminoantipyrine and phenol in the presence of peroxidase to yield a chromogen with maximum absorption at 500 nm. The method is reproducible, and the results correlate well with those obtained by automated Liebermann Burchard procedures and the method of Abell et al. The present method affords better specificity than those previously reported and has excellent precision.
The aortic accumulation of chylomicrons, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and albumin were compared in normal New Zealand White rabbits.
Lipoproteins and albumin were labelled with radioiodinated tyramine cellobiose (TC) to avoid potential oxidative modification of lipoproteins and as a marker of intracellular degradation. In preliminary experiments it was established that TC labelling did not alter the kinetic properties of lipoproteins in vivo. Importantly, radiolabelled apolipoproteins did not transfer significantly between plasma lipoproteins. Therefore, aortic radioactivity following infusion of TC-radiolabelled lipoproteins was considered to be indicative of lipoprotein accumulation.
In conscious rabbits, net aortic accumulation of chylomicrons or their remnants was similar to those of LDL, HDL and albumin up to 2 h after infusion, despite rapid clearance from plasma. When accumulation was calculated on the basis of mean arterial exposure to allow for the differences in plasma clearance, the accumulation of aortic chylomicrons/remnants was substantially greater than that of LDL, HDL or albumin. Qualitatively similar results were obtained in rabbits that were functionally eviscerated to slow clearance of chylomicron remnants. Chylomicrons/remnants did not appear to efflux from aortic tissue as rapidly as did LDL or other plasma lipoproteins. Autoradiographic analysis showed that the primary site of lipoprotein accumulation was within medial smooth muscle cells.
Our data demonstrate that chylomicrons/remnants accumulate in arterial blood vessels more rapidly than does LDL, suggesting that dietary lipoproteins may be directly involved in the pathogenesis of atherosclerosis.
The fatty acid composition of human very-low-density lipoproteins (VLDL) was studied in a population from western Andalusia with a diet in which the fat content came mainly from olive oil. The lipid composition of VLDL, including the fatty acid composition of the phospholipids and triacylglycerols, was examined by capillary gas chromatography. Twenty-five peaks were resolved, ranging in chain length from 14 to 24 carbon atoms, including geometric and positional isomers. The major fatty acids present in phospholipids were 16:0, 18:0, 18:1(n - 9) and 18:2(n - 6), and in triacylglycerols were 18:1(n - 9), 16:0 and 18:2(n - 6). The major triacylglycerol was POO, followed by PLO and OOO. MLP, PPS and LLL were absent. The presence of a large amount of OOO in this fraction demonstrates that the triacylglycerol composition of the VLDL depends on the type of diet consumed.
Using chiral phase high-performance liquid chromatography of diacylglycerols, we have redetermined the ratios of 1,2-/2,3-diacyl-sn-glycerols resulting from acylation of 2-monoacylglycerols by membrane bound and solubilized triacylglycerol synthetase of rat intestinal mucosa. With 2-oleoyl[-3H]glycerol as the acyl acceptor and oleoyl-CoA as the acyl donor, 97-98% of the diacylglycerol product was 1,2(2,3)-dioleoyl-sn-glycerol, 90% of which was the sn-1,2- and 10% the sn-2,3-enantiomer. The remaining diacylglycerol (less than 3%) was the sn-1,3-isomer. The overall yield of acylation products was 70%, of which 60% were diacylglycerols and 40% triacylglycerols. With 2-oleylglycerol ether as the acyl acceptor and [1-14C]oleoyl-CoA as the acyl donor, 90% of the diradylglycerol was 1-oleoyl-2-oleyl-sn-glycerol and 10% was the 2-oleyl-3-oleoyl-sn-glycerol. The diradylglycerols made up 96% and the triradylglycerols 4% of the radioactive product. With 1-palmitoyl-sn-glycerol as the acyl acceptor and [1-14C]oleoyl-CoA as the acyl donor, the predominant reaction product was 1-palmitoyl-3-oleoyl-sn-glycerol. The 3-palmitoyl-sn-glycerol was not a suitable acyl acceptor. Both 1,2- and 2,3-diacyl-sn-glycerols were substrates for diacylglycerol acyltransferase as neither isomer was favored when 1,2-dioleoyl-rac-[2-3H]glycerol was used as the acyl acceptor. There was a marked decrease in the acylation of the 1(3)-oleoyl-2-oleyl-sn-glycerol to the 1,3-dioleoyl-2-oleyl-sn-glycerol. It is concluded that neither monoacylglycerol nor diacylglycerol acyltransferase exhibit absolute stereospecificity for acylglycerols as fatty acid acceptors.
The lipolysis of chylomicrons derived from palm, olive, corn or fish oil (enriched in saturated, monounsaturated, n - 6 polyunsaturated and n - 3 polyunsaturated fatty acids, respectively) by rat post-heparin lipoprotein lipase in vitro was compared by measuring the release of [3H]oleate from their triacylglycerol. Chylomicrons derived from corn oil were lipolysed more rapidly than the other types in the first 20 min of the reaction, but after 120 min the total amount of triacylglycerol hydrolysed was similar with all types of chylomicrons used. The rate of lipolysis of the different types of chylomicrons also showed different dependencies on the substrate concentration. The highest Vmax values were obtained when the chylomicrons were derived from olive and corn oil and the lowest when they were derived from palm oil, while olive oil chylomicrons gave the highest Km and palm oil chylomicrons the lowest. These results indicate that differential metabolism of chylomicrons of different fatty acid composition by lipoprotein lipase may play a part in the differential rates of clearance from the blood of lipid of dietary origin demonstrated in earlier work from our laboratory.
To identify the substrate specificity of lipoprotein lipase (LPL) for triacylglycerol-rich lipoproteins with monoacid-rich triacylglycerols, monoacid-rich lipoproteins were prepared and kinetic parameters of LPL were characterized. Male broiler chickens were fed 8 g/100 g fat diets differing only in the fat source: palm oil (tripalmitin-rich), olive oil (triolein-rich), safflower oil (trilinolein-rich) and linseed oil (trilinolenin-rich). After diets were fed for 3 d, chickens were starved for 2 d and then force-fed emulsions containing one of the monoacid-triacylglycerols: tripalmitin, triolein, trilinolein or trilinolenin. The triacylglycerols in chylomicrons and very low density lipoprotein (VLDL) of chickens force-fed tripalmitin, triolein or trilinolein contained the corresponding acid at more than 70% of total acids. Linolenic acid was incorporated into chylomicrons and VLDL to a lower extent (51.2 and 57.2%, respectively) in chickens force-fed trilinolein. Major apolipoproteins and lipid compositions were not significantly different among all lipoproteins isolated from chickens fed the different fats. Vmax of LPL was significantly higher (P < 0.05) for palmitic acid-rich chylomicrons and VLDL and decreased with increasing chain length and unsaturation of monoacid: 16:0>18:1>18:2>18:3. The electron spin resonance analysis, order parameter (S), decreased with monoacid chain length and unsaturation. In addition, the Vmax of LPL increased linearly (P < 0.01, r = 0. 912) with an increase in the palmitic acid content of the lipoprotein triacylglycerols. These findings suggest that lipoprotein catalysis by LPL is modulated by the palmitic acid content of the lipoprotein triacylglycerol, which affects the fluidity of lipoproteins.
Elevated plasma triacylglycerol (TG; triglyceride) concentrations, especially in the postprandial state, have been associated with an increased risk of coronary heart disease (CHD). Postprandial lipemia represents a complex series of reactions which occur following the ingestion of a meal containing fat and is associated with a number of adverse metabolic events including the production of atherogenic chylomicron remnants, the formation of the highly atherogenic small dense low density lipoprotein particles, a reduction in the concentration of the cardioprotective high density lipoprotein fraction and the activation of coagulation factor VII. Fish oils are a rich source of the long-chain n-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid and docosahexaenoic acid. Long chain n-3 PUFA are effective hypotriglyceridemic agents, lowering both fasting and postprandial TG concentrations. There is a large body of evidence which shows that n-3 PUFA reduces plasma TG concentrations through reduced endogenous very low density lipoprotein production. This in turn may account for the reduced postprandial lipemic response following n-3 PUFA supplementation. However, this does not preclude a contribution of enhanced chylomicron clearance, which may be mediated through altered chylomicron size, structure or chemical composition, or altered lipoprotein lipase metabolism in terms of enzyme concentration, activity, or affinity for chylomicrons. However the precise biochemical nature of this effect remains to be established. The reduction of postprandial plasma TG concentrations by n-3 PUFA may partly explain why n-3 PUFA intake is inversely related to CHD mortality.
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