Silicic Acid Column Research Articles

Chromatographic Separation and Quantitation of Sphingolipids from the Central Nervous System or Any Other Biological Tissue.

Chromatographic separation and purification of an individual lipid to homogeneity have long been introduced. Using this concept, a more precise method has been developed to identify and characterize the sphingolipid composition(s) using a small amount (30mg) of biological sample. Sphingolipids (lipids containing sphingosine or dihydrosphingosine) are well-known regulators of the central nervous system development and play a critical role in neurodegenerative diseases. Introducing a silicic acid column chromatography, sphingolipid components have been separated to individual fractions such as ceramide, glucosyl/galactosylceramide, other neutral and acidic glycosphingolipids, including (dihydro)sphingosine and psychosine; as well as phospholipids from which individual components are quantified employing a single or combination of other advanced chromatography procedures such as thin-layer chromatography, gas chromatography-mass spectrometry, and high-performance liquid chromatography-mass spectrometry.

Potential of Lentibacillus sp. NS12IITR for production of lipids with enriched branched-chain fatty acids for improving biodiesel properties along with hydrocarbon co-production.

Hypersaline environment is inhabited by array of microbes which have the potential to produce industrially important products. This study explored biomass and lipid production potential of the halophilic bacterium, strain NS12IITR which was isolated from Sambhar Lake, Rajasthan. Sequencing and phylogenetic analysis revealed that the bacterium belonged to genus Lentibacillus. The salient feature of the isolate is its ability to accumulate total cellular lipid up to 18.9 ± 0.45% of dry cell weight. In addition, trans-esterification of extracted lipid yielded 77.6 ± 5.56% of total esters as methyl ester of branched-chain fatty acids (BCFAs). To assess the nature of extracted lipid, lipid sample was fractionated on the silicic acid column, which demonstrated that 49.03 ± 1.35% of the total lipids was neutral in nature. Trans-esterification of the neutral lipid fraction yielded 60.62 ± 4.88% of total esters as methyl ester of BCFAs. Methyl esters of BCFAs were present in trans-esterified products of neutral as well as polar lipid fractions. Furthermore, the isolate produced hydrocarbons both extracellularly (C10-C30) and intra-cellularly (C15-C28). The concentration of extracellular hydrocarbon (21.11 ± 0.78mg/L) synthesized by strain NS12IITR is in close agreement with the yield reported from other hydrocarbon producing bacteria. This is hereby a first report on the co-production of lipids and hydrocarbon from a halophilic bacterium. The production of neutral lipid with high percentage of BCFAs and co-production of hydrocarbons makes the isolate NS12IITR a potential claimant for biofuel production.

The dietary effect of milk sphingomyelin on the lipid metabolism of obese/diabetic KK-A(y) mice and wild-type C57BL/6J mice.

Purified milk sphingomyelin (SM) was obtained from lipid concentrated butter serum (LC-BS) by successive separations involving solvent fractionation, selective saponification, and silicic acid column chromatography. The SM obtained was given to obese/diabetic KK-A(y) mice and wild-type C57BL/6J mice. SM supplementation significantly increased fecal lipids paralleled with a decrease in non-HDL cholesterol levels in the serum and neutral lipids and in cholesterol levels in the livers of KK-A(y) mice. The reduction of liver lipid levels also resulted in a decrease in the total fatty acid content of the KK-A(y) mice livers, while n-3 fatty acids derived from the conversion of α-linolenic acid (18:3n-3) increased due to SM supplementation. In contrast to the KK-A(y) mice, little change in the serum and liver lipids was observed in wild-type C57BL/6J mice. The present study suggests that SM may be effective only in subjects with metabolic disorders.

A Teaching Laboratory for Comprehensive Lipid Characterization from Food Samples

Traditional and state-of-the-art techniques were combined to probe for various lipid classes from egg yolk and avocado qualitatively and quantitatively. A total lipid extract was isolated using liquid–liquid extraction. An aliquot of the total lipid extract was subjected to transesterification to form volatile fatty acid methyl esters suitable for gas chromatography to quantify fatty acyl residues in both food samples. Another total lipid aliquot was separated into two lipid fractions of higher and lower polarity using silicic acid column chromatography. All fractions were analyzed using thin-layer chromatography and were checked for cholesterol using the Liebermann-Burchard test. In addition, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using 2,5-dihydroxybenzoic acid and 4-chloro-α-cyanocinnamic acid matrixes was performed to analyze the composition of separated lipid fractions. Egg yolk exhibited phosphatidylcholines and phosphatidylethanolamines in the more polar fraction and cholesterol in the less polar one. Avocado contained detectable amounts of the nonpolar triglyceride triolein. The experiments can be split between three 3-h laboratory periods to allow upper-division students to gain exposure to both lipid theory and hands-on experimental lipid analysis.

Investigation Some Physicochemical Properties, Lipids, Glycerides and Fatty acid Composition of Mulberry (Morus Alba L) Seed oil of three Different Regions of Bangladesh

The physicochemical properties, glycerides, lipids and fatty acid compositions of mulberry (tut) seed oil have been studied. It was observed that tut, a shrub grown well under the soil and climatic conditions of Bangladesh contains 31.41% of orange yellow colored oil. The oil was fractionated into mono, di and triglycerides by silicic acid column chromatography. The triglyceride was varied from 90.08-91.05%, diglyceride from 3.25-4.10% and monoglyceride from 1.47-2.05%. The total lipids were fractionated into three major lipid groups, neutral lipids, glycolipids and phospho-lipids by silicic acid column chromatograpy. The neutral lipid ranges from 92.97-93.10%, glycolipids from 2.12-2.43% and phospholipid from 1.85-1.92% of the total oil of the lipid applied. Saturated and unsaturated fatty acids present in the oil were separated and the amount varied from 15.04-16.28% and 74.29-75.86%, respectively, depending on soil conditions of the areas in which the plant grows. The percentage of fatty acid compositions of the oil were analyzed by GLC and found to contain linoleic acid (74.29%), palmitic acid (10.60%), stearic acid (5.61%) and myristic acid (0.07%).

Studies on the Lipid Classes and Fatty Acid Compositions of Petuli (Trewia nudiflora Linn.) Seed oil

Studies were carried out on the lipid classes and fatty acid compositions of petuli seed oil. It was observed that petuli seed grown under the soil and climatic condition of Bangladesh contains about 22% pale yellow coloured oil. The total lipids were fractionated into three major lipid classes, neutral lipid glyco lipid and phospho lipid by silicic acid column chromatography. The neutral lipid was accounted to 92.5% of the total weight of the lipid applied. The oil was also fractionated into mono, di and triglyceride by silicic acid column chromatography. The triglyceride was counted for over 90% of the total weight of the oil. The fatty acid compositions of the oil were analyzed by Gas liquid Chromatography and found the major fatty acid ?-elaeostearic acid 38.50%, oleic acid 34.35%, linoleic acid 26.15% and small amount of arachidic acid 1%. DOI: http://dx.doi.org/10.3329/bjsir.v47i1.10716 Bangladesh J. Sci. Ind. Res. 47(1), 105-108, 2012

Deciphering sulfoglycolipids of Mycobacterium tuberculosis

For 4 decades, in vivo and in vitro studies have suggested that sulfoglycolipids (SGLs) play a role in the virulence or pathogenesis of the tubercle bacilli. However, the SGL structure and biosynthesis pathway remain only partially elucidated. Using the modern tools of structural analysis, including MALDI-time-of-flight MS, MS/MS, and two-dimensional NMR, we reevaluated the structure of the different SGL acyl (di-, tri-, and tetra-acylated) forms of the reference strain Mycobacterium tuberculosis H37Rv, as well as those produced by the mmpL8 knockout strains previously described to intracellularly accumulate di-acylated SGL. We report here the identification of new acyl forms: di-acylated SGL esterified by simple fatty acids only, as well as mono-acylated SGL bearing a hydroxyphthioceranoic acid, which were characterized in the wild-type strain. In a clinical strain, a complete family of mono-acylated SGLs was characterized in high abundance for the first time. For the mmpL8 mutant, SGLs were found to be esterified i) by an oxophthioceranoic acid, never observed so far, and ii) at nonconventional positions in the case of the unexpected tri-acylated forms. Our results further confirm the requirement of MmpL8 for the complete assembly of the tetra-acylated forms of SGL and also provide, by the discovery of new intermediates, insights in terms of the possible SGL biosynthetic pathways.

Effect of Stripping Methods on the Oxidative Stability of Three Unconventional Oils

AbstractStripped and non‐stripped oils from Sclerocarya birrea [marula oil (SCO)], Aspongopus viduatus [melon bug oil (MBO)] and Agonoscelis pubescens [sorghum bug oil (SBO)], traditionally used for nutritional applications in Sudan, were investigated for their fatty acid and tocopherol composition, and their oxidative stability. Three stripping methods were used, phenolic compounds extraction, silicic acid column, and aluminum oxide column. The stripping methods did not affect the fatty acid composition. Non‐stripped SCO, MBO and SBO contained oleic, palmitic, stearic and linoleic acids, which were not significantly (P < 0.05) different than stripped SCO, MBO and SBO. The stripping methods’ effect on the tocopherol composition of the studied oils, the total amount of tocopherol in non‐stripped oils decreased by extraction of phenolic compounds, mean that part of the tocopherols was extracted with the phenolic compounds. No traces of tocopherols were found in oils stripped using silicic and aluminum columns and the tocopherols were eliminated during the stripping processes. The stability of SCO, MBO and SBO oils was 43, 38 and 5.1 h, respectively, this stability decreased by 22.0, 37.6 and 23.5%, respectively after extraction of phenolic compounds. This stability decreased by 96.9, 98.2 and 90.2% respectively, when stripped using the aluminium column and decreased by 92.6, 96.1 and 86.3% when stripped by the silicic column. It is possible to assume that the tocopherols and phenolic compounds play a more active role in the oxidative stability of the oils than the fatty acid composition and phytosterols.

Partial Purification of Antimicrobial Compounds Isolated from Mycelia of Tropical Lentinus cladopus LC4

Lentinus cladopus LC4 produced at least eight antimicrobial compounds (ACs) which are active against plant and human pathogens. Three ACs in its crude mycelial were extracted with methanol and partial purification was carried out with silicic acid column chromatography and by thin layer chromatography (PTLC). The antimicrobial activity was tested by paper disc method and antibiographic method. The chromatography purification eluted with dichloromethane containing 5% methanol gave one active fraction (FII). This fraction which was active against X. campestris pv. glycines and showing two inhibition zones against Bacillus subtilis on bioautographic plates with the R f s 0.8 and 0.7. FI and FIII fractions eluted with dichloromethane containing 0 and 10% methanol performed one inhibition zone with R f s 0.8 and 0.7 respectively. However, their activities were lower than that of FII fraction. The PLTC purification gave one separate fraction with R f value of 0.73 and it was active against X. campestris pv. glycines. The compound of Rf 0.73 fraction should be further studied using TLC and HPLC to obtain the pure substance for molecule characterization.

Biochemical Characterization of the Oxygenation of Unsaturated Fatty Acids by the Dioxygenase and Hydroperoxide Isomerase of Pseudomonas aeruginosa 42A2

We have studied oxygenation of fatty acids by cell extract of Pseudomonas aeruginosa 42A2. Oleic acid ((9Z)-18:1) was transformed to (10S)-hydroperoxy-(8E)-octadecenoic acid ((10S)-HPOME) and to (7S,10S)-dihydroxy-(8E)-octadecenoic acid (7,10-DiHOME). Experiments under oxygen-18 showed that 7,10-DiHOME contained oxygen from air and was formed sequentially from (10S)-HPOME by isomerization. (10R)-HPOME was not isomerized. The (10S)-dioxygenase and hydroperoxide isomerase activities co-eluted on ion exchange chromatography and on gel filtration with an apparent molecular size of approximately 50 kDa. 16:1n-7, 18:2n-6, and 20:1n-11 were also oxygenated to 7,10-dihydroxy fatty acids, and (8Z)-18:1 was oxygenated to 6,9-dihydroxy-(7E)-octadecenoic acid. A series of fatty acids with the double bond positioned closer to ((6Z)-18:1, (5Z,9Z)-18:2) or more distant from the carboxyl group ((11Z)-, (13Z)-, and (15Z)-18:1) were poor substrates. The oxygenation mechanism was studied with [7S-(2)H]18:1n-9, [7R-(2)H]18:2n-6, and [8R-(2)H]18:2n-6 as substrates. The pro-R hydrogen at C-8 was lost in the biosynthesis of (10S)-HPODE, whereas the pro-S hydrogen was lost and the pro-R hydrogen was retained at C-7 during biosynthesis of the 7,10-dihydroxy metabolites. Analysis of the fatty acid composition of P. aeruginosa revealed relatively large amounts of (9E/Z)-16:1 and (11E/Z)-18:1 and only traces of 18:1n-9. We found that (11Z)-18:1 (vaccenic acid) was transformed to (11S,14S)-dihydroxy-(12E)-octadecenoic acid and to a mixture of 11- and 12-HPOME, possibly due to reverse orientation of (11Z)-18:1 at the active site compared with oleic acid. The reaction mechanism of the hydroperoxide isomerase suggests catalytic similarities to cytochrome P450.

Cytotoxic and inhibitory activity of ceramide on cancer cell lines

The inhibitory activity of ceramide on cancer cells was evaluated and its cytotoxicity on different cancer cell lines was measured. Ceramide was separated from bovine brain and spinal cord, and extracted by organic solvents. The crude extract was purified by using silicic acid column. Detection and identification of purified extract were carried out by using three assays: visualization, spectrophotometry and infrared. Cytotoxic effect of different concentrations (7, 15, 30 and 60 μM) of ceramide on HEp-2, RD, AMGM5, REFAM3 and AMN3 cancer cell lines was studied. Results showed that ceramide at 30 μM imposed cytotoxic activity on all cancer cell lines especially on AMGM5. Effect of ceramide at 30 μM on cell division of human lymphocyte was also examined. Significant reductions in mitotic and blast indices were observed. In addition, No genotoxic effects or chromosomal aberration were detected in lymphocyte chromosomes when ceramide was tested in vitro.

Triacylglycerols from viper bugloss (Echium vulgare L.) seed bio‐oil

AbstractTriacylglycerols (TAG) in viper bugloss oil were isolated from raw pressed oil by silicic acid column chromatography. The obtained blend of TAG was separated by silver ion thin‐layer chromatography (TLC Ag+) into nine fractions, varying in terms of unsaturation level and molecular polarity. The composition of TAG in viper bugloss oil was determined by HPLC coupled with a diode‐array detector and an evaporative light‐scattering detector. The results showed that the first three fractions were combinations of TAG containing palmitic, oleic and linoleic acids. Fractions 4 and 6 contained TAG of a similar acid composition as above, but with the addition of γ‐linolenic acid. The remaining fractions (7–9) were the most varied in acid composition. They were found to contain 26–39% palmitic acid, 12–15% oleic acid, 13–41% linoleic acid 8–24% γ‐linolenic acid, 1.5–5.5% α‐linolenic acid and 1–5% stearidonic acid. The analysis of fatty acid allocation in TAG of viper bugloss lipids revealed that linoleic acid (ranging from 2 to 100%) was the only acid found in all isolated fractions. In the investigated oil, the predominant TAG included: LnLnG (11.38%), LnLnSt (11.17%), LnGSt (7.71%), LnStSt (6.19%) and LnLnLn (5.44%). Almost 86% of the TAG contained α‐linolenic acid, while γ‐linolenic and stearidonic acids amounted to 49 and 38%, respectively.

Extraction and classification of lipids from seeds of persea Americana miller and chrysophyllum albidum g. Don.

Seed lipids of Persea americana and Chrysophyllum albidum were studied. Lipids were extracted with chloroform-methanol, analysed with silicic column chromatography and thin layer chromatography. The total lipid content of Persea americana was 10.8% while that of Chrysophyllum albidum was 7.7%. Fractionation of the total lipid fraction of Persea americana using silicic acid column chromatography showed the occurrence of neutral lipids 6.5%, glycolipids 2.1% and phospholipids 2.2% while those of Chrysophyllum albidum were 4.2%, 1.7% and 1.8% for neutral lipids, gycolipids and phospholipids, respectively. Thin layer chromatography separation showed three classes of lipids in Persea americana and Chrysophyllum albidum which were oleic acid, palmitic acid and cholesterol. The colour of these lipids was yellow and solid at room temperature. These studies suggest that oil extracted from seeds of Persea americana and Chrysophyllum albidum could be used for the manufacture of industrial products.

A simple modification of a silicic acid lipid fractionation protocol to eliminate free fatty acids from glycolipid and phospholipid fractions

When fractionating natural and standard lipid mixtures according to the most widely used method for the analysis of soil and other environmental materials, free fatty acids (FFAs) were not recovered quantitatively in the fraction expected to contain all simple lipids. Rather than being eluted from activated silicic acid adsorption chromatographic columns with chloroform, FFAs in standard lipid mixtures appeared only in the two subsequent fractions eluted with acetone and methanol, respectively. Substantial quantities of FFAs from cow dung lipid samples were eluted using chloroform as expected but appreciable amounts also eluted in the acetone (glycolipid) fraction. The fatty acid distribution of the methanol (phospholipid) fraction from dung displayed slightly more FFA compositional character than when FFAs were excluded but this influence was not significant ( p = 0.08). A simple modification to the silicic acid column technique ensures that FFAs are reproducibly eluted in the chloroform/acetic acid (100:1 v/ v; simple lipid) fraction. The modification had no deleterious effect upon the elution characteristics of any phospholipid from a variety of headgroup classes tested. The carry-over of FFAs into glycolipid fractions was confirmed by nanospray ionisation mass spectrometry of the intact polar lipid fractions. As far as could be inferred from the distinctive molecular weight distributions of the glycolipid and phospholipid fractions, none of the complex lipids in dung eluted in both fractions.

The Journal of Biological Chemistry, Volume 245, 1970: Evidence for the biologically active form of cholecalciferol in the intestine.

Abstract Fifteen hours after the intracardiac administration of a physiological dose (0.65 nmole) of radioactive cholecalciferol (vitamin D3) to rachitic chicks, the bulk of the radioactivity in the plasma, liver, and intestine exists as three polar metabolites. These are designated Metabolites 4A, 4B, and 4C on the basis of their chromatographic mobility on silicic acid columns. After doses of 0.65 to 6.5 nmoles of cholecalciferol, the predominant metabolite found in the plasma is 4A with smaller amounts of 4B and 4C. In the intestine, a primary target tissue of cholecalciferol, the predominant metabolite is 4B, with much smaller amounts of 4A and 4C. Metabolite 4A from both the plasma and intestine is tentatively identified as 25-hydroxycholecalciferol on the basis of its migration with 25-hydroxycholecalciferol during silicic acid column chromatography. The biological activity of Metabolites 4A and 4B was examined with an assay which measures intestinal calcium transport, in vivo. Both 4A and 4B have biological activity significantly greater than equivalent amounts of cholecalciferol. The kinetics of the appearance of Metabolite 4B in the intestine and its binding to intestinal chromatin are consistent with the lag in the physiological response to cholecalciferol. When radioactive Metabolite 4A is administered to rachitic chicks, the bulk of the plasma radioactivity remains as 4A, but the predominant metabolite found in the intestine is 4B. These data support the concept that Metabolite 4B may be the biologically active form of cholecalciferol in the intestine and that 25-hydroxycholecalciferol is an intermediate in its formation.

Isolierung und Analytik von Glykolipiden aus Humansperma

Glycolipids from human semen of two different diagnoses were separated from a total lipid extract by silicic acid column chromatography. Fatty acids and carbohydrates were determined by gas-liquid chromatography. Quantitative differences in the fatty acid and carbohydrate contents were found between the two diagnose groups.

Sphingomyelins in Four Ascidians, Ciona intestinalis, Halocynthia roretzi, Halocynthia aurantium, and Styela clava

Sphingomyelin is rarely found in lower animals, while sphingophospholipid is a characteristic of higher animals. In this study, sphingomyelin was first isolated and characterized from ascidian Ciona intestinalis. Ascidian sphingomyelin was prepared using ion exchange (QAE-Sphadex-A25) and silicic acid (Florisil and Iatrobeads) column chromatographies. The chemical structure was characterized by fatty acid analysis, sphingoid analysis, hydrogen fluoride degradation, acid hydrolysis, enzymatic hydrolysis, infrared analysis, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The ceramide moieties of C. intestinalis sphingomyelin consisted primarily of C16:0, C18:0, and C18:1 fatty acids and d18:2 sphingadiene. Furthermore, sphingomyelins were isolated and characterized from 3 other ascidians, Halocynthia roretzi, Halocynthia aurantium, and Styela clava using the same methods. Comparative analysis of the sphingomyelin structures in 4 ascidian species-C. intestinalis (Enterogona) and H. roretzi, H. aurantium, and S. clava (Pleurogona)-revealed that the major fatty acid composition of the ceramides was similar, and that they differed in minor components.

ANTIBACTERIAL ACTIVITY OF ASPONGOPUS VIDUATUS (MELON BUG) OIL

ABSTRACTAspongopus viduatus (melon bug) oil is insect oil used as famine food in western parts of Sudan and has traditional medicinal uses. The antibacterial activities of melon bug crude oil, silicic acid column purified oil and phenolic compounds‐free oil were determined by agar diffusion assay against seven bacterial isolates, four of them are food‐related bacterial species: Staphylococcus aureus, Salmonella enterica serovar Paratyphi, Escherichia coli and Bacillus cereus, and the three other isolates are: B. subtilis, Enterococcus faecalis and Pseudomonas aeruginosa. The main constituents of this oil were examined. The crude oil and the phenolic compounds‐free oil showed high antibacterial activities against some test species while the silicic acid column purified oil showed no antibacterial activity. The study highlights the possibility of using this oil in food preservation.PRACTICAL APPLICATIONSIn this study, the antimicrobial properties and main components of insect oil (Aspongopus viduatus) were identified. The oil has traditional medicinal uses in human and animal skin diseases and is also used for meat dressing before drying in western regions of Sudan. The oil has long stability against rancidity because of its high oleic acid content. This oil in its crude form or phenolic compounds‐free form could be used in meat and meat products preservation processes to control gram‐positive bacteria.

Quantification of Pentafluorobenzyl Oxime Derivatives of Long Chain Aldehydes by GC–MS Analysis

Negative ion mass spectrometric techniques, for compounds having good ionization properties, such as pentafluorobenzyl derivatives, are believed to be more sensitive than positive ion methods. Preparation of PFB oximes of fatty aldehydes from crude lipid extracts is problematic due to the release of free aldehydes from plasmalogens during derivatization. Accordingly, in these studies plasmalogens were removed by silicic acid column chromatography prior to pentafluorobenzyl derivatization. This simple purification step to remove plasmalogens is shown to facilitate the quantification of long-chain aldehydes by analysis of their pentafluorobenzyl oxime derivatives utilizing gas chromatography-mass spectrometry in the negative ion chemical ionization mode. The limit of detection for long chain fatty aldehydes using this method is 0.5 pmol and it is linear over two orders of magnitude. Silicic acid column chromatography followed by electrospray ionization mass spectrometry demonstrated that plasmalogens were removed (the detection limit for this analyses was <or=0.3 pmol). Furthermore, we have exploited the utility and sensitivity of this method to identify increases in hexadecanal and octadecanal in 3-amino-1,2,4-triazole treated human neutrophils.

Stereospecific Analysis of Fatty Acid Composition of Chufa (Cyperus esculentus L.) Tuber Oil

We determined the positional fatty acid distribution of chufa tuber at stereospecific positions; the sn-1, sn-2, and sn-3 positions in the triacylglycerols of chufa tuber oil. Chufa tubers were washed with water, and oven dried to a moisture content of 20%. The total lipid (TL) was extracted in a Soxhlet apparatus using a mixture of chloroform and methanol at 40 � C. The total lipid extracted was dissolved in chloroform and separated into neutral lipids (NL), glycolipids (GL) and phospholipids (PL) using silicic acid column chromatography [7]. Stereospecific analysis of chufa oil was carried out using pancreatic lipase, phospholipase A2, TLC and GC [8]. The fatty acid composition was determined using gas chromatography [9]. The fatty composition at the sn-3 position was calculated based on the equation, 3 · [TG]‐[sn-1 + sn-2 position].