Lipid Extraction Procedure Research Articles

Influence of two lipid extraction procedures on the peroxide value in powdered infant formulas

The influence of two extraction procedures on the peroxide value (PV) of lipid extracts of powdered infant formulas (IFs) was investigated. Thirteen commercial IFs contained in their original packages were assessed after storage at room temperature for 18 months. The extraction procedures mainly differed in the extraction solvent polarity. Extraction method 1 was one of the methods more widely applied in the industry of powdered IFs. It consisted of a single extraction with a polar solvent composed of dichloromethane : methanol (2:1, v/v). Extraction method 2 consisted of disruption of the solid matrix simply by rubbing with a mortar and pestle and subsequent extraction of the lipids released with hexane, used as a non-polar solvent. The PV was contrasted with the quantitative analysis of the non-volatile lipid oxidation compounds and tocopherols. The oxidation compounds were isolated by solid phase extraction (SPE) and analyzed by high-performance size-exclusion chromatography (HPSEC) with refraction index detection. Results showed high significant differences (P 0.05). It was found that the lower PVs in method 1 were due to reducing substances co-extracted with the lipids by the polar solvent. Therefore, the quantitative analysis of the lipid oxidation compounds by SPE–HPSEC was more reliable for evaluation of the oxidation level in powdered IFs.

Lipid composition of integral purple membrane by 1H and 31P NMR

In the purple membrane (PM) of halobacteria, lipids stabilize the trimeric arrangement of bacteriorhodopsin (BR) molecules and mediate the packing of the trimers in a regular crystalline arrangement. To date, the identification and quantification of these lipids has been based either on lipid extraction procedures or structural models. By directly solubilizing PMs from Halobacterium salinarum in aqueous detergent solutions (SDS or Triton X-100), we avoided any separation or modification steps that might modify the lipid composition or even the lipid molecules themselves. Our analysis of integral PM preparations should resolve partially conflicting literature data on the lipid composition of the PM. Using 31P and 1H NMR of detergent-solubilized but otherwise untreated samples, we found two glycolipids and 6.4 +/- 0.1 phospholipids per BR molecule, 4.4 +/- 0.1 of the latter being the phosphatidylglycerophosphate methyl ester. The only glycolipid detected was S-TGD-1. For an additional glycolipid, glycocardiolipin, that was recently identified in lipid extracts, we show that it was produced mainly during the lipid extraction procedure but also was partially dependent on the preparation of the PM suspensions.

Albumin stimulates monocyte chemotactic protein‐1 expression in rat embryonic mixed brain cells

Albumin, a blood protein absent from the adult brain in physiological situations, can be brought into contact with brain cells during development or, in adult, following breakdown of the blood-brain barrier occurring as a result of local inflammation. In the present study, we show that ovalbumin and albumin induce the release of monocyte chemotactic protein 1 (MCP-1/CCL2) from rat embryonic mixed brain cells. A short-term exposure to ovalbumin during the cell dissociation procedure is sufficient to generate MCP-1 mRNA. A comparable effect is observed when the cells are incubated for 4 hr with ovalbumin or rat albumin, while MCP-1 messengers are barely detectable following bovine albumin exposure. The amount of MCP-1 protein measured in 4 hr-supernatants of albumin-treated cells followed the same albumin-inducing pattern as that of MCP-1 mRNA, while all albumins tested induced MCP-1 protein after a 17 hr-incubation period. The albumin-induced MCP-1 production is significantly inhibited in calphostin C-treated cells, suggesting the implication of a protein kinase C-dependent signaling pathway. This MCP-1-inducing activity is maintained after a lipid extraction procedure but abolished by proteinase K or trypsin treatments of albumin. The MCP-1 secretion following albumin contact with nervous cells could thus interfere, by chemotactic gradient formation, with the brain infiltration program of blood-derived cells during development or brain injury.

Determination of phospholipids in dairy products by SPE/HPLC/ELSD

The aim of this work was to evaluate the performance of different methods for both milk lipid extraction and phospholipids separation. As far as the lipid extraction procedure is concerned, the Folch method showed a higher phospholipid recovery with respect to the Rose–Gottlieb method. Different SPE cartridges and solvent phases were tested to carry out the separation of phospholipids from fat. The yield of extraction was evaluated by isolating phospholipids from both milk fat and synthetic fat; Standard Addition Method was applied as well. The isolation of the phospholipids by SPE silica column and subsequent analysis by HPLC/ELSD was shown to be an accurate and reproducible analytical method for the determination of phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine and sphingomyelin in milk fat extracted by Folch method.

An improved assay for platelet-activating factor using HPLC-tandem mass spectrometry

We describe an improved assay for platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) using HPLC-tandem mass spectrometry (LC-MS/MS). The present method can readily detect as little as 1 pg (1.9 fmol) of PAF, a significant improvement over previously described LC-MS/MS methods, and gives a linear response up to 1,000 pg of PAF. Our method also overcomes the artifacts from isobaric lipids that have limited the usefulness of certain existing LC-MS/MS assays for PAF. In the course of these studies, we detected three novel lipid species in human neutrophils. One of the novel lipids appears to be a new molecular species of PAF, and the other two have chromatographic and mass spectrometric properties consistent with stearoyl-formyl-glycerophosphocholine and oleoyl-formyl-glycerophosphocholine. These observations identify previously unknown potential interferences in the measurement of PAF by LC-MS/MS. Moreover, our data suggest that the previously described palmitoyl-formyl-glycerophosphocholine is not unique but rather is a member of a new and poorly understood family of formylated lipids.

Automated GC–MS analysis of raw biological samples: Application to fatty acid profiling of aquatic micro-organisms

A direct thermal desorption (DTD) interface was applied to profile the fatty acid composition of whole/intact aquatic micro-organisms using trimethylsulfonium hydroxide (TMSH) as derivatization reagent in a fully automated fashion without any sample treatment. DTD was used to release cellular free and esterified fatty acids, after conversion into their methyl esters, from the very limited amounts of cells available in plankton community analysis using fluorescence-activated cell sorting of mixed (natural) microbial/algal populations. The liner of the GC injector is used as a sample-and-reaction container with the aid of the DTD interface. The fatty acids are converted into their methyl esters after an incubation (hydrolysis) time of 30 min with TMSH. After transportation into the injector, the liner is heated to effect thermally assisted methylation. Simultaneously, the fatty acid methyl esters (FAMEs) are introduced from the liner into the capillary GC column. On the basis of peak intensities, the fatty acid compositions, thus obtained, were shown to be closely similar to those of a conventional lipid extraction and methylation procedure. The method was applied to some common freshwater algae, the green algae Scenedesmus acutus, the diatom Asterionella formosa and the filamentous cyanobacterium, Limnothrix sp. strain MRI. Using this novel method, poly-unsaturated fatty acids were methylated into their corresponding methyl esters without isomerization/pyrolytic side-reactions. The present method has been used to perform large series of analyses of algal and microbial cells. Up to 18 samples could be analysed per day. In order to reach this sample throughput, preparation of the ( n + 1)th sample was performed simultaneously with GC analysis of the nth sample.

A simplified method for analysis of polyunsaturated fatty acids

BackgroundAnalysis of fatty acid composition of biological materials is a common task in lipid research. Conventionally, preparation of samples for fatty acid analysis by gas chromatography involves two separate procedures: lipid extraction and methylation. This conventional method is complicated, tedious and time consuming. Development of a rapid and simple method for lipid analysis is warranted.ResultsWe simplified the conventional method by combining the extraction and methylation into a single step (omitting the procedure of prior extraction). Various biological samples including cultured cells, animal tissues and human specimens have been tested using the new method. Statistical analysis indicates that the recovery of long chain fatty acids from tissue samples by the simplified method is significantly higher than that by the traditional method, but there is no difference in relative fatty acid composition between the two methods. This simplified method can significantly save time and materials, and reduce the potentials of sample loss and contamination.ConclusionThe lipid extraction procedure prior to methylation employed conventionally in lipid analysis can be omitted without affecting the recovery of long chain (≥ 18 C) fatty acids and their composition. The simplified method is rapid, easy-to-use, suitable for analysis of total long chain polyunsaturated fatty acid contents (e.g. n-6 and n-3 fatty acids) in various biological samples, especially when the number of samples to be analyzed is large and/or the specimen size is small.

Determination of docosahexaenoic acid in milk using affinity solid-phase purification with argentous ions and modified thin layer chromatography

This paper reports a simple method for docosahexaenoic acid determination in bovine milk. The method consists of three steps: (1) one-step DHA methylation in bovine milk without lipid extraction, (2) concentration of DHA methyl ester by AgNO3-modified silica gel column chromatography, and (3) detection of DHA methyl ester using spot focused-high performance thin layer chromatography. DHA was directly methylated in bovine milk and selectively concentrated in accordance with its intrinsic affinity to argentous ions. Purified DHA methyl ester was developed on newly designed thin layer chromatographic plates with focusing slits and determined densitometrically using conventional software, following visualisation with iodine staining. In addition to the densitometrical analysis, it is also possible for practical, semiquantitative DHA determination to be visually performed on site in manufacturing industries. Unlike conventional methods, the method described needs neither lipid extraction procedures that use a large amount of organic solvent, nor a gas chromatograph system.

31P NMR quantification and monophasic solvent purification of human and bovine lens phospholipids.

Most lipid extraction procedures [Folch, J., Lees, M., and Sloane-Stanley, G.H. (1957) A Simple Method for the Isolation and Purification of Total Lipids from Animal Tissues, J. Biol. Chem. 226, 497-509; Bligh, E.G., and Dyer, W.J. (1959) A Rapid Method of Total Lipid Extraction and Purification, Can. J. Biochem. Physiol. 37, 911-917] employ biphasic solvent mixtures designed to dissolve the lipids in an organic phase and remove impurities in an aqueous phase. However, when applying these protocols to biological matrices such as that of the ocular lens, the formation of an emulsion layer between the organic and aqueous phases causes poor reproducibility in extraction yields and gives only a small amount of the lipid-containing chloroform phase. In this study, we quantified phospholipids at each step of the Folch et al. extraction protocol and compared the yield of human and bovine lens phospholipids obtained by the Folch-based approach and a novel monophasic methanol extraction method designed to circumvent the problems associated with biphasic extraction protocols. A monophasic methanol extraction coupled with 31P NMR spectroscopy was found to be the simplest, quickest, and most effective method for quantifying the phospholipid content of the lens.

Development in lipid analysis: some new extraction techniques and in situ transesterification.

The development of methods for analyzing fatty acids that provide rapid and reliable results is currently in great demand. Recently, different lipid extraction procedures such as microwave or supercritical fluid extraction have heen researched. Both procedures avoid the use of large volumes of solvents and provide rapid lipid isolations. Only a few papers have reported work on microwave extraction, hut many studies ahout supercritical fluid extraction have heen carried out and have heen gaining acceptance within the scientific community. Avoiding the lipid isolation step, hy synthesizing fatty acid esters hy simultaneous lipid extraction and derivatization through in situ reactions, has also heen proposed. The saving of time and reagents is significant. Owing to the differences among the procedures, some knowledge of their characteristics is essential in order to improve methods and achieve reliable and accurate results. Clearly, results depend on factors such as the type of catalysis selected, the use of nonpolar solvents, heating applied during the synthesis, and the degree of suitability of the procedure chosen for the particular features of each sample.

Morphological Transformation of C3H/M2 Mouse Fibroblasts by Extracts of Human Mammary Lipid

Mammary lipid may act as a reservoir for genotoxins. Mammary lipid extracts (MLEs), obtained from eight UK women (21–41 years) undergoing reduction mammoplasty, were examined for their abilities to morphologically transform C3H/M2 mouse fibroblasts. Resultant transformation rates were 0.27, 0.33, 0.07, 0.29, 0.21, 0.00, 0.07, and 0.13 transformed foci/treated dish, respectively. Although the lipid-extraction procedure used was originally designed to extract heterocyclic aromatic amines (HAAs), liquid chromatography/mass spectroscopy (LC/MS) with selective ion monitoring has failed to detect HAAs in any of the lipid extracts so far examined. Genotoxicities were also assessed inS. typhimuriumTA98 and in metabolically competent human (MCL-5) cells by the micronucleus and by the alkaline single-cell gel (“comet”) assays. The MLEs induced bacterial mutagenicity rates ranging from 0 to 498 revertants/plate/g-lipid equivalent and micronucleus-formation rates from 0 to 20 micronuclei/500 binucleate cells/g-lipid. Median comet tail lengths (induced with MLEs of 8.0 g-lipid equivalent) ranged from 6.0 to 74.0 μm. The results demonstrate the presence of as-yet-unidentified transforming agents in mammary lipid.

Development of a routine analysis method for liposome encapsulated recombinant interleukin-2

This paper describes the development of an isocratic reversed-phase high-performance liquid chromatographic method for the routine analysis of recombinant interleukin-2 (rIL-2) in liposome samples. The chromatographic system employed a C 4 column maintained at 30°C eluted with 52.5% (w/w) acetonitrile in water, containing 100 m M NaClO 4 and 10 m M HClO 4. To remove phospholipid interference the chromatographic method was combined with a lipid-extraction procedure. No significant loss of rIL-2 was noted upon inclusion of this extraction step. The protein eluted from the column with a capacity factor ( k′) of 5.8. The method was validated for robustness, linearity, precision and reproducibility. It was shown that the method was linear over a sample concentration range of 1–100 μg/ml. Upon assessment of the intra-day and inter-day precision, the relative standard deviations (RSD) were within the range of the methodical error (approximately 5%), except at the lower concentration of 10 μg/ml, where the intra-day RSD was relatively high (17.8%). The recovery of rIL-2 upon liposome preparation and subsequent analysis of the samples was in the range 94±9%. The results indicate that the method is suitable for routine quantitation of rIL-2 in liposomal samples.

Combined lipid/DNA extraction method for environmental samples

Previously, separate methods have been developed for the extraction and purification of lipids and DNA from soils and sediments. This paper describes a new method for the isolation of both lipids and DNA from the same environmental sample. This combined method is based on the Bligh and Dyer lipid extraction technique. Upon phase separation, lipids partition into the organic phase and DNA partitions into the aqueous phase. DNA extraction and recovery from the solid phase is necessary under certain conditions. Preliminary experiments performed with 32P-labeled DNA in the absence of soil showed that greater than 98% of the total DNA was present in the aqueous phase after the modified Bligh and Dyer extraction. Analysis of the DNA by polyacrylamide gel electrophoresis and autoradiography demonstrated that no degradation of DNA occurred during the lipid extraction procedure. Lipid extraction of lyophilized cells showed that DNA was released from Pseudomonas putida and Bacillus subtilis cells corresponding to 26±5 and 14±4% of the theoretical DNA yield, respectively. The combined lipid/DNA extraction method was applied to both lyophilized cells and wet cells added to soil. Analysis by DNA:DNA hybridization showed that approx. 40–50% of the DNA from cells added to soil was recovered after lipid extraction relative to samples treated only with conventional DNA extraction. Estimation of cell number per gram soil based on either lipid or DNA analysis showed good agreement with actual numbers added based on plate counts of the inocula. DNA extracts from samples which had been lipid-extracted also had lower amounts of humic material. Although some DNA was not recovered after lipid extraction, that which was recovered was of sufficiently high quality for hybridization analysis. This method shows utility for the co-recovery of both lipids and DNA from a single sample; this is particularly useful when a small sample size is all that is available or procurable.

A rapid method for extraction of total lipids from whey protein concentrates and separation of lipid classes with solid phase extraction

AbstractA modified procedure for extraction of total lipids from whey protein concentrates was developed such that stable emulsion with extracting solvents was avoided and the solvent system remained monophasic. Nonlipid contaminants from the extract were removed using gel filtration instead of traditional aqueous washing to prevent any loss of polar lipids. The extraction of total lipids by the modified procedure was complete and comparable with a reference procedure. Traditional thin‐layer chromatography is tedious and more qualitative than quantitative for lipid class separation. Total lipids were further separated into free fatty acids, phospholipids, cholesterol ester, triacylglycerol, cholesterol, diacylglycerol, and monoacylglycerol, using modified solid phase extraction procedure. Columns with 2 g amino propyl packing allowed separation of up to 80 mg of total lipids into lipid classes gravimetrically. The values for anhydrous milk fat for all lipid classes agreed with those in the literature. Separation of total lipids into lipid classes with solid phase extraction is easy, quantitative, and can also be performed on a preparative scale.

The occurrence of hopanoid lipids inBradyrhizobiumbacteria

Lipid extraction procedures followed by GLC and GLC-MS analysis were used to investigate the triterpenoid content in Bradyrhizobium and Rhizobium bacteria. Unlike the tested strains of Rhizobium bacteria, a range of triterpenoids e.g., squalene and different classes of hopanoid derivatives were detected in bacteria from all Bradyrhizobium strains investigated (different strains from Bradyrhizobium japonicum, Bradyrhizobium elkanii as well as Bradyrhizobium sp.). Furthermore, related compounds were identified from some hopanoid lipids (e.g., diplopterol) that carried an additional methyl group in their molecular structure. The hopanoid content was high in some strains and accounted for more than 40% of the total lipid fraction (e.g., in strains Bradyrhizobium japonicum USDA 110 and USDA 31), while other strains contained only about a tenth of that amount (e.g., Bradyrhizobium japonicum ATCC 10324 and Bradyrhizobium sp. (Lupinus) ATCC 10319).

The occurrence of hopanoid lipids in Bradyrhizobium bacteria

Lipid extraction procedures followed by GLC and GLC-MS analysis were used to investigate the triterpenoid content in Bradyrhizobium and Rhizobium bacteria. Unlike the tested strains of Rhizobium bacteria, a range of triterpenoids e.g., squalene and different classes of hopanoid derivatives were detected in bacteria from all Bradyrhizobium strains investigated (different strains from Bradyrhizobium japonicum, Bradyrhizobium elkanii as well as Bradyrhizobium sp.). Furthermore, related compounds were identified from some hopanoid lipids (e.g., diplopterol) that carried an additional methyl group in their molecular structure. The hopanoid content was high in some strains and accounted for more than 40% of the total lipid fraction (e.g., in strains Bradyrhizobium japonicum USDA 110 and USDA 31), while other strains contained only about a tenth of that amount (e.g., Bradyrhizobium japonicum ATCC 10324 and Bradyrhizobium sp. ( Lupinus) ATCC 10319).

The effect of choline and myo-inositol on liver and carcass fat levels in aerobically trained rats.

Choline and myo-inositol are dietary supplements ingested under the premise that they facilitate the burning of stored fat. Choline and myo-inositol have been shown to prevent abnormal or excessive liver accumulation of cholesterol and triglycerides in choline and myoinositol deficient rats. The current study was designed to determine whether the consumption of choline and myoinositol by non-deficient aerobically trained rats affects the percent liver and carcass fat. Nineteen rats were trained aerobically for ten weeks then randomly assigned to an experimental group fed choline and myo-inositol mixed with their chow, or a control group fed only chow. Rats were sacrificed after 24 more days of aerobic training. Percent carcass and liver fat were determined by a lipid extraction procedure. There was a significant difference in the percent liver fat between groups, with the experimental group having less fat (6.69 +/- 2.23 vs 9.22 +/- 2.91 percent fat; r = 0.05). Percent carcass fat was not significantly different. There was a significant difference in the amount of weight gained during the 24 days of treatment, with the experimental group gaining less weight (5.1 +/- 9.2 vs 11.8 +/- 3.1 g; r < 0.05). The lack of an effect on percent carcass fat indicates that choline plus myoinositol supplements do not reduce adipose tissue mass but can inhibit weight gain while decreasing liver fat.

QUANTIFICATION AND DISTRIBUTION OF LIPID IN ARCHAEOLOGICAL CERAMICS: IMPLICATIONS FOR SAMPLING POTSHERDS FOR ORGANIC RESIDUE ANALYSIS AND THE CLASSIFICATION OF VESSEL USE

Organic residue analysis has been performed on 62 reconstructed vessels from a single archaeological site (Rounds, Northamptonshire, U.K.). In order to establish regions of lipid accumulation within a vessel, sherds were sampled from different parts of a vessel, for example base, body and rim, and submitted to lipid extraction procedures. The techniques of high‐temperature gas chromatography (GC) and GC/mass spectrometry (GC/MS) were then used to analyse the sherd lipid extracts. The quantitative results obtained show differential accumulation and preservation of lipid in various parts of the same vessel. This latter observation has serious implications for the sampling of potsherds for organic residue analysis. Furthermore, the amount of absorbed lipid varies quite considerably between vessel types. On this basis, a new method is proposed for classifying vessel use by comparing the concentration of lipid present in different parts of individual vessels.

Identification of meat treated with ionizing radiation by capillary gas chromatographic determination of radiolytically produced hydrocarbons

When triglycerides or fatty acids are irradiated, some of the major stable product are hydrocarbons formed from the loss of CO 2 and CH 3 COOH in various free-radical reactions. A capillary gas chromatographic procedure has been developed to monitor the presence of these radiolytically generated hydrocarbons in meats treated with ionizing radiation. Several lipid extraction procedures for isolating the radiolytically generated hydrocarbons from the irradiated meat were compared. The radiolytically generated hydrocarbons were separated from the extracted lipids on a Florisil column and determined by capillary gas chromatography. The yield of these radiolytically generated hydrocarbons was linear with absorbed dose

脂質簡易抽出法併用による魚肉脂質の酸化度評価

Assessment of fish muscle lipid oxidation was performed by using a color difference meter subsequent to a novel rapid lipid extraction procedure, which was achieved by applying (internally cross-linked) starch-grafted-polyacrylate. The color stimulus ΔX ΔT ΔZ values of 5% chloroform solution of the extracted lipid were borne out to be suitable independent variables in assessing the oxidation rate especially for the early stage of oxidation. This approach was practically viable for ordinary muscles without liposoluble pigments such as those of sardine and arabesque greenling, but could not be applied to fish which contain liposoluble pigments e. g. carotenoids. A fragrance meter equipped with synthetic bilayer lipid was applicable to monitor the early off-flavor of fish oil which is impossible to detect with a hydrogen flame ionization detector of gas chromatograph.