Improvement of a solid phase extraction method for analysis of lipid fractions in muscle foods

The most used method for muscle lipid fractionation into major lipid classes was modified for improving its separation efficiency. Extracted lipids from a masseter muscle of one Iberian pig were separated into neutral lipids (NL), free fatty acids (FFA) and polar lipids (PL) using aminopropyl minicolumns, following the extensively used method of Kaluzny et al. [1] (old method-OM-) and a method based on that, developed by Pinkart et al. [2] with some (modifications modified method–MM). Obtained lipid classes were further analysed by TLC and lipid fractions were identified. TLC evidenced the presence of a certain amount of PL in the NL fraction obtained with the OM. On the other hand, using the MM only an almost undetectable presence of PL was evidenced in the NL fraction. Fatty acid composition of NL, PL and FFA obtained with each method was studied by gas chromatography. Fatty acid profile of NL was strongly influenced by the separation method used. Thus, NL obtained using the OM showed higher amounts of saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA) and lower of monounsaturated fatty acids (MUFA) than those obtained using the MM. Moreover, NL obtained using the OM showed the presence of fatty alcohols, constituents of phospholipids (PhL) absent or present only in trace amounts in acylglycerols. This profile reflects the coelution of PL in the NL fraction. Fatty acid profile of FFA and PL fractions was also influenced by the solid phase extraction (SPE) method used, but to a lesser extent.     

Cholesterol and its fatty acid esters in native DNA preparations: lipid analysis,computer simulation of their interaction with DNA and cholesterol binding to immobilized oligodeoxyribonucleotides

Supramolecular DNA complexes were isolated from rat normal cells and murine tumors. The content of DNA-bound lipids (cholesterol and its esters) was determined. The content of cholesterol esters is higher than that of free cholesterol; the lipid content in tumor cells is higher than in normal cells. Using the molecular mechanics approach, it is demonstrated for the first time that cholesterol and its esters with stearic, oleic, linoleic, and linolenic acids bind to the DNA minor groove more strongly than with the major groove. The calculated DNA binding energies of cholesterol and its esters depend on both the number of double bonds in the fatty acid residue and on the DNA nucleotide composition. The formation of stable complexes between cholesterol molecules and d(AT)-rich oligonucleotides was demonstrated using biological microchip containing immobilized octadeoxyribonucleotides. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 9, pp. 2138–2144, September, 2005.     

DNA-bound lipids: computer modeling of DNA interaction with stearic acid and unsaturated fatty acids

It was shown for the first time by computer experiments that fatty acids are strongly bound to DNA. This is consistent with the presence of free fatty acids in the specimens of DNA-bound lipids isolated from various cells. Binding of all fatty acids to the DNA minor groove is stronger than to the major groove, which is correlated with the presence of two pools of free fatty acids isolated from DNA specimens by biochemical methods. Since DNA polymerase is also bound to the DNA minor groove, fatty acids can play an important role in the regulation mechanism of DNA replication and signal transmission. The energy of interaction of fatty acids with DNA depends on both the number of double bonds and the geometric configuration of the fatty acid and the nucleotide composition of DNA. Dependence on the bond energy in the DNA—fatty acid complex on the nucleotide composition attests to the possibility of site-specific binding of lipids to DNA. On passing from a saturated fatty acid to unsaturated acids containing one, two, or three double trans-bonds, the bond energy of DNA with the fatty acid gradually decreases. The presence of one or three double cis-bonds results in weakening of the strength of the DNA—fatty acid complexes compared to those with the saturated acid. The strongest binding between DNA and fatty acid was found for the unsaturated acid with two double cis-bonds (linoleic). This can be explained by the fact that the bent (boomerang) shape of the molecule of this acid follows the curve of the DNA helix. The pattern of variation of the energy of DNA complexes with stearic, linoleic, oleic, and linolenic acids correlates with experimental data on the melting points of these complexes: the more stable the DNA—fatty acid complex, the lower the melting point of DNA.     

Anti-Inflammatory and Anti-Platelet Properties of Lipid Bioactives from Apple Cider By-Products

The valorization of food industry by-products as sources of bioactive compounds is at the forefront of research in functional foods and nutraceuticals. This study focuses on bioactives of apple cider by-products (ACBPs) with putative cardio-protective properties. Total lipids (TLs) were extracted from ACBPs of apple varieties that are low (ACBP1), medium (ACBP2), and high (ACBP3) in tannins and were further separated into polar lipids (PLs) and neutral lipids (NLs). The functionality of these lipid extracts and of their HPLC-derived lipid fractions/PL subclasses were assessed in vitro against human platelet aggregation induced by the thrombotic and inflammatory platelet agonists platelet-activating factor (PAF) and adenosine diphosphate (ADP). The fatty acid profile of PLs and their most bioactive lipid fractions were evaluated by GC–MS analysis. The PL extracts exhibited higher specificity against the PAF-induced platelet aggregation compared to their anti-ADP effects, while TL and NL showed lower bioactivities in all ACBPs. HPLC analysis unveiled that the most bioactive PL from all ACBPs were those in PL fraction 3 containing phosphatidylcholines (PCs). PLs from all ACBPs and their PC bioactives were rich in polyunsaturated fatty acids (PUFAs) and especially in the essential omega-6 (n-6) linoleic acid (LA) and omega-3 (n-3) alpha linolenic acid (ALA), with favorably low values of the n-6/n-3 PUFA ratio, thus providing a rationale for their higher anti-inflammatory bioactivities. Within this study, highly bioactive PL compounds with strong anti-inflammatory and anti-platelet properties were identified in ACBPs, which can be potentially utilized for producing cardio-protective functional foods and/or nutraceuticals.     

Lipid content and fatty acid composition in lemon wax

Lipids were extracted from lemon wax and fractionated into four classes on a silicic acid glass packed column by thin-layer chromatography (TLC). The free fatty acids, the fatty acid composition and the amount of each separated lipids were determined by capillary column gas chromatography (GC). Total lipids (TL) were 60 mg per 100 g raw weight and the ratio of nonpolar lipids (NPLs): glycolipids (GLs): phospholipids (PLs) was about 47:2:2. The main free fatty acids in lemon wax were hexadecanoic acid, cis-9-octadecenoic acid and cis,cis-9,12-octadecadienoic acid, while in the lipid fractions the main fatty acids were hexadecanoic acid in all the fractions, cis-cis-9,12-octadecadienoic and decanoic acids in triglyceride (TG) fraction, dodecanoic and cis-9-octadecenoic acids in diglyceride (DG) fraction and tetradecanoic, octadecanoic and cis-9-octadecenoic acids in GL and PL fractions. The ratio of unsaturated to saturated fatty acids showed a remarkable difference among these four lipid fractions. In PL and GL fractions this ratio was similar, 47.7% and 47.1% respectively, and in TG fraction it was 42.4% while in DG fraction this value was 23.5%.     

Separation of acidic and neutral lipids by aminopropyl-bonded silica gel column chromatography.

The separation of acidic and neutral lipids by aminopropyl-bonded silica gel column chromatography is presented. Total lipid extracts from Escherichia coli and human spermatozoa were loaded onto pre-packed aminopropyl-bonded silica gel columns and the lipids separated into four fractions. Non-polar lipids including cholesterol esters, triglycerides, diglycerides, monoglycerides and cholesterol, were eluted with 4 ml of isopropanol-chloroform (1:2, v/v) (fraction 1); free fatty acids were eluted with 4 ml of 2% acetic acid in diethyl ether (fraction 2); neutral polar lipids, including phosphophatidylethanolamine, phosphatidylcholine, sphingomyelin and neutral glycolipids, were eluted with 4 ml of methanol (fraction 3); and, finally, polar acidic lipids, including phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylserine, seminolipid lipid A and acidic glycosphingolipids, were eluted with 4 ml of chloroform-methanol-0.8 M sodium acetate (60:30:4.5, v/V/V) (fraction 4). The recoveries for the different lipids ranged between 89 and 98% and the intra-assay variation, expressed as the standard deviation, was less than 5%.     

Phosphatidylcholine dimers can be easily misinterpreted as cardiolipins in complex lipid mixtures: a matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometric study of lipids from hepatocytes

The liver is an important organ that is particularly involved in the lipid metabolism of the organism. Thus, high interest is nowadays focused on the lipid composition of the liver and particularly the liver parenchymal cells, the hepatocytes. Hepatocytes contain common phospholipids (PL) such as phosphatidylcholines, ‐ethanolamines and ‐inositols, for instance, that can be easily analyzed by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) even without previous separation of the PL mixture. However, in addition to common PL, hepatocytes possess also significant amounts of cardiolipin (CLP). The MS analysis of this PL is quite challenging because it (a) has a higher mass than common lipids and (b) possesses a higher negative charge. We will show here that caution is required if CLP is analyzed directly from the total lipid extract because PC dimers may be interpreted as cardiolipins if the positive ion MALDI mass spectra are analyzed. Copyright © 2011 John Wiley & Sons, Ltd.     

Protein-lipid complexes of cotton kernels and the products of their processing

The composition and lipid content of the lipoprotein complexes of cotton kernels (I), meal (II), and isolate (III) have been investigated. It has been shown that with the aid of an acidic alcohol-containing extractant it is possible to isolate up to 99% of the strongly bound lipids (SBLs) of their total mass. In the composition of the strongly bound lipids (I–III) have been found neutral lipids comparable with the free lipids of the kernel; of polar lipids, glycolipids were present. It was found that an acid extractant extracts polyphenolic compounds together with the SBLs.Institute of Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 466–472, July–August, 1990.     

Comparative analysis of lipophilic compounds in eggs of organically raised ISA Brown and Araucana hens

Hens?? eggs represent a rich source of important nutrients, including lipids and carotenoids. The lipid composition of hens?? eggs is influenced by genetic factors, age, and diet. The aim of this study was to compare the fatty acids, cholesterol, and carotenoids content of the egg yolk of ISA Brown and Araucana hens grown in free-range housing systems. Fatty acids and cholesterol were analysed by GC-FID and GC-MS and carotenoids were quantified by RP-HPLC-PDA. The Araucana egg yolk has a higher lipid content and higher egg-to-albumen ratio than the ISA Brown yolk, while the total cholesterol, carotenoids content and profile are not significantly different. The lipids of the Araucana egg yolk have a higher content of mono-unsaturated fatty acids (MUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) and a better n-6/n-3 ratio than the ISA Brown egg yolk lipids. The major carotenoids were lutein and zeaxanthin, which account for more than 83 % in egg yolk. Eggs of both breeds, when raised organically, represent very good sources of highly bio-available lutein and zeaxanthin, pigments which are related to lower risk of age-related macular degeneration. We report for the first time on the fatty acids composition in lipid fractions and the profile and content of carotenoids of the Araucana egg yolk.     

Lipids from leaves ofEphedra equizetina

The content and composition by class and fatty acid of neutral (NL), glyco- (GL), and phospholipids (PL) in leaves ofEphedra equizetina Bunge (Ephedraceae) are determined. The acid composition of NL, GL, and PL includes saturated 12∶0–32∶0 acids and unsaturated 15∶1, 16∶1, 18∶1, 18∶2, and 18∶3 acids. Unsaponified components of the total lipids also contained biologically active substances such as α-tocopherol, carotenoids, high-molecular-weight fatty alcohols, triterpenes, and sterols. Academician S. Yu. Yunusov Institute of the Chemistry of Plant Substances. Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (371) 120 64 75. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 718–721, November–December, 1999.     

Lipid lowering effects of high linoleate and high alpha-linolenate diets in rats and mice. Consequence of long-term feedings.

Diets high in linoleate (safflower oil) or high in alpha-linolenate (perilla oil) were fed to rats for 11 months, and the effects of the diets on plasma and tissue lipids were compared. The plasma levels of total cholesterol (Cho), phospholipids (PL) and triacylglycerol (TG) were significantly lower in the high alpha-linolenate group than in the high linoleate group, the differences being more than 30% in the levels of total Cho and TG. The diets had differential effects on the lipid contents of major tissues: the TG level in muscle was higher but both the TG level in depot fat and the PL level in muscle were lower in the high alpha-linolenate group than in the high linoleate group. In order to clarify whether or not the hypolipidemic effect of the high alpha-linolenate diet was due to changes in the distribution of lipids among tissues, whole body lipids were estimated in mice fed these diets for 5 months. The whole body Cho content was significantly lower, by 28%, in the high alpha-linolenate group compared with the high linoleate group, but the total lipid content, PL and neutral lipids were similar between the groups. Our results indicate that the high alpha-linolenate diet has a more potent cholesterol lowering effect in plasma and body tissue than the high linoleate diet; interestingly, whole body TG levels are similar but tissue distributions of TG are different between the two dietary groups.     

Softness of atherogenic lipoproteins: a comparison of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) using elastic incoherent neutron scattering (EINS)

Apolipoprotein B100 (apoB100)-containing plasma lipoproteins (LDL and VLDL) supply tissues and cells with cholesterol and fat. During lipolytic conversion from VLDL to LDL the size and chemical composition of the particles change, but the apoB100 molecule remains bound to the lipids and regulates the receptor mediated uptake. The molecular physical parameters which control lipoprotein remodeling and enable particle stabilization by apoB100 are largely unknown. Here, we have compared the molecular dynamics and elasticities of VLDL and LDL derived by elastic neutron scattering temperature scans. We have determined thermal motions, dynamical transitions, and molecular fluctuations, which reflect the temperature-dependent motional coupling between lipid and protein. Our results revealed that lipoprotein particles are extremely soft and flexible. We found substantial differences in the molecular resiliences of lipoproteins, especially at higher temperatures. These discrepancies not only can be explained in terms of lipid composition and mobility but also suggest that apoB100 displays different dynamics dependent on the lipoprotein it is bound to. Hence, we suppose that the inherent conformational flexibility of apoB100 permits particle stabilization upon lipid exchange, whereas the dynamic coupling between protein and lipids might be a key determinant for lipoprotein conversion and atherogenicity.     

Neutral Lipids of Ruta graveolens L. Grown in vivo and in vitro

The composition of neutral lipids (NL) and fatty acids of acyl-containing components in differentiated tissues ofRuta graveolensL. (Rutaceae) grown in vivo and in vitro is studied. The NL of in vitro cultures differ from NL of the intact plant by a larger content of esters of fatty acids with alcohols, fewer high-molecular-weight alkanes, alkylbenzenes, free alkanols, and linolenic acid     

Electrostatic interactions between model mitochondrial membranes

Lipids are very diverse in both their respective structures and functions; and cells exquisitely control membrane composition. One intriguing issue is the specific role of lipids in modulating the physical properties of membranes. Cardiolipin (CL) is a unique four-tailed, doubly negatively charged lipid found predominately within the inner mitochondrial membrane, and is thought to be influential in determining the inner mitochondrial membrane potential and permeability. To determine the role of cardiolipin in modulating the charge properties of membranes, this study investigated the electrostatic interactions between mixed cardiolipin and phosphatidylcholine bilayers as a function of cardiolipin concentration. For physiologically relevant concentrations of cardiolipin, the surface charge density of the membrane was found to increase linearly with increasing concentration of cardiolipin. However, only a fraction of the cardiolipin molecules predicted to carry a charge from pK-values were ionized. Clearly environmental factors, beyond that of pH, play a role in determining the charge of bilayers containing cardiolipin.     

Fluorescence quenching of ethidium ion by porphyrin cations and quaternary ammonium surfactants in the presence of DNA

Fluorescence quenching of free and DNA-bound ethidium bromide (EB) by a number of quaternary ammonium and other compounds was studied. For free EB or bound EB at lower DNA concentration the fluorescence quenching follows the Stern–Volmer equation and at higher DNA concentration follows an exponential model. At least at low quencher concentrations the quenching efficiency varies with DNA or NaCl concentrations and is about 100 times greater for bound than free EB. The quenching pathways may involve energy transfer and conformational loosening or distortion of the DNA helix in addition to possible electron transfer.     

Influence of Reduced Temperature on Lipid Composition of <Emphasis Type="Italic">Viburnum opulus</Emphasis> Seeds

The influence of reduced temperature (frost) on the lipid composition of Viburnum opulus seeds was studied. Low temperatures changed lipid metabolism by forming hydroxyglycerides in NL, lowering significantly the GL and PL contents, and changing their class composition. The structures of hydroxyacids in the hydroxyglycerides were established. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 523–528, November–December, 2005.     

Incorporation of3H2O into lipids of adult male rat lung after castration

The aim of this study was to determine whether testosterone regulates the rate of lipid synthesis in lung. Rats were either sham-operated controls (Co) or castrated (Ca)·[³H] H₂O was administered in vivo, twenty-one days after castration. The animals were sacrificed 1h later to ensure that the newly synthesized lipids in the lung had been labeled. The radioactivity incorporated in the different lipid fractions that had been separated by TLC was counted. The results are expressed in ng³H incorporated/h/mg lipids. We observed that the incorporation of³H in total lipids, phospholipids and free cholesterol increased, while trygliceride and esterified cholesterol did not change in castrated rats in relation to the control. These results suggest that the rate of lipid synthesis in the lung is regulated, directly or indirectly by androgens.     

Quantification of brain lipids by FTIR spectroscopy and partial least squares regression

Brain tissue is characterized by high lipid content. Its content decreases and the lipid composition changes during transformation from normal brain tissue to tumors. Therefore, the analysis of brain lipids might complement the existing diagnostic tools to determine the tumor type and tumor grade. Objective of this work is to extract lipids from gray matter and white matter of porcine brain tissue, record infrared (IR) spectra of these extracts and develop a quantification model for the main lipids based on partial least squares (PLS) regression. IR spectra of the pure lipids cholesterol, cholesterol ester, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, galactocerebroside and sulfatide were used as references. Two lipid mixtures were prepared for training and validation of the quantification model. The composition of lipid extracts that were predicted by the PLS regression of IR spectra was compared with lipid quantification by thin layer chromatography.     

Laser time-resolved fluorescence study of the interaction between anthracyclines and cardiolipin

The molecular interaction between cardiolipin vesicles and two representative anthracyclines, daunomycin and 5-iminodaunomycin, has been studied at pH 7.1 by laser time-resolved fluorescence, for a cardiolipin-to-anthracycline ratio r ranging from 0.02 to 5. The fluorescence lifetime of daunomycin is 1.03 ns. For r = 0.3 - 5 a longer-lived transient (1.91 - 1.49 ns) is present and originates from the excitation of daunomycin bound on a single phosphate group of cardiolipin. At r = 0.3 two lifetimes are observed, the second one being due, partially, to free daunomycin and bound drug molecules embedded in the lipid bilayer. The fastest-decaying species is present for r = 0.5 - 2.0 and identified as two adjacent, stacked-up daunomycin molecules bound onto the two phosphate groups of the cardiolipin. In the case of 5-iminodaunomycin, a less cardiotoxic analogue, three-exponential decay is never observed and a fast-decaying component, pi approximately 0.2 ns, is already present at low r and vanishes for r greater than 0.5. The constancy of the lifetimes of the longer-lived species may originate from the reorientation of the bound drug from the hydrophilic to the lipid domain.