Skin lipids from Saudi Arabian birds

Skin lipids play an important role in the regulation of cutaneous water loss (CWL). Earlier studies have shown that Saudi desert birds exhibit a tendency of reduced CWL than birds from temperate environment due to adaptive changes in composition of their skin lipids. In this study, we used thin-layer chromatography (TLC) for separation and detection of non-polar and polar lipids from the skin of six bird species including sooty gull, brown booby, house sparrow, Arabian waxbill, sand partridge, and laughing dove. The lipids were separated and detected on Silica gel G coated TLC plates and quantified by using densitometric image analysis. Rf values of the non-polar lipids were as follows: cholesterol (0.29), free fatty acids (0.58), triacylglycerol (0.69), fatty acids methyl esters (0.84) and cholesterol ester (0.97). Rf values for the polar lipids were: cerebroside (0.42), ceramide (0.55) and cholesterol (0.73). The results showed the abundance of fatty acids methyl esters (47.75–60.46%) followed by triacylglycerol (12.69–24.14%). The remaining lipid compositions were as follows: cholesterol (4.09–13.18%), ceramide (2.18–13.27%), and cerebroside (2.53–12.81%). In conclusion, our findings showed that TLC is a simple and sensitive method for the separation and quantification of skin lipids. We also reported a new protocol for lipid extraction using the zirconia beads for efficient disruption of skin tissues. This study will help us better understand the role of skin lipids in adaptive physiology towards adverse climatic conditions.     

Use of nile red for the rapid in situ quantitation of lipids on thin-layer chromatograms

We describe the use of the fluorescent dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, as a general-purpose reagent for the rapid detection and quantitation of a wide variety of lipids and other hydrophobic compounds separated by thin-layer chromatography. After samples are applied to silica gel plates and chromatographed, the plate is briefly dipped into a nile red solution (8 micrograms/ml of methanol-water 80:20, v/v). Background fluorescence of nile red dye adsorbed to the silica gel is then preferentially destroyed by dipping the plate in a dilute aqueous solution of bleach. After drying, lipid bands are visualized under ultraviolet light. Reflectance fluorometry (Ex: 580 nm; Em: 640 nm) is utilized for in situ quantitative analysis of the fluorescence of the lipids on the nile red-stained plate. Neutral lipids, phospholipids, sphingolipids, and fatty acids can be examined, although the nile red fluorescence intensity varies significantly among the lipid classes. Also, staining is stronger for unsaturated lipids than for saturated lipids. The lower detection limit of the assay is 25-100 ng for cholesterol, cholesteryl esters, triacylglycerols, and phospholipids.     

Complete separation of lipid classes on a single thin-layer plate

A double-development procedure employing first a polar and then a nonpolar solvent system is described for the complete separation by thin-layer chromatography of the main lipid classes encountered in natural lipids. For better quantification, long plates (34 cm) are employed. Diglycerides were separated from cholesterol, 1,2- from 1,3-diglycerides, and monoglycerides from phospholipids.     

Estimation of lipid concentrations on thin-layer plates by densitometry of transparent copies

A relatively simple procedure for the quantitative estimation of phospholipids resolved on thin-layer plates has been developed. After resolution in an appropriate solvent, the lipids are visualized by staining with iodine, ninhydrin, or molybdate and then photocopied onto transparent sheets with a standard office copy machine. The density of each spot on the photocopy, measured with a simple silicon cell area densitometer, is a direct function of each lipid applied to the plate over at least a six- to eightfold range in concentration. Under controlled conditions the staining and photocopying steps are quite reproducible. Known concentrations of the choline, ethanolamine, serine, and inositol derivatives of L-alpha-phosphatidic acid applied either separately or as mixtures can be determined essentially quantitatively (100 +/- 5%) by this procedure following their resolution on the thin-layer plates.     

Direct analysis of glycolipids on thin-layer plates by matrix-assisted secondary ion mass spectrometry: application for glycolipid storage disorders

The lipids accumulated in organs of patients with Gaucher's, Tay-Sachs, and Fabry's disease were identified by means of the combination of thin-layer chromatography and matrix-assisted secondary ion mass spectrometry. The total lipid extract of each lipidosis tissue was chromatographed on a TLC plate and then analyzed directly by mass spectrometry without elution of the sample from the TLC plate. The amount of material needed to obtain an adequate spectrum is in the order of a few micrograms of lipids per band for both positive and negative ion detection. By scanning the plates, mass spectral and chromatographic information can be obtained simultaneously, which was shown to be useful for the qualitative identification of the components on the plates.     

Methods for the rapid separation and estimation of the major lipids of arteries and other tissues by thin-layer chromatography on small plates followed by microchemical assays

Methods are described for the rapid separation of the major individual phospholipids and neutral lipids of tissues by thin-layer chromatography on small glass plates (75 × 75 mm), and for the specific microchemical estimation of separated lipids and for determination of fatty acid composition and radioactivity. The overall method, involving tissues extraction, thin-layer chromatographic separation and assay has been evaluated using pure standards and biological samples and gives good reproducibility and almost complete recovery of lipids.     

Visualization method for sulfolipids and its application to the determination of nanomole quantities of lipid sulfur

A simple and sensitive visualization method for sulfolipids on a thin-layer chromatogram is described. By spraying with an acidic solution of azure A, a complex was formed between an anionic sulfolipid and a blue cationic compound. After the unbound dye was washed out by brief soaking in methanol, sulfolipids were visualized as clear dark-blue bands on a light-blue background. As little as 0.5 nmol could be detected. Sulfolipid-dye complex was estimated by densitometry or colorimetric measurement after extraction with chloroform/methanol. For the quantitative determination of sulfolipids having long sugar chains, it is necessary to treat thin-layer chromatography plates with acetic anhydride before color development. Of the other tissue lipids not containing sulfuric acid ester that were tested none were stained significantly. A linearity of quantitative determination was observed over the range of 1-8 nmol.     

Modification of the Dittmer-Lester reagent for the detection of phospholipid derivatives on thin-layer chromatograms.

A simple modification of the Dittmer-Lester reagent is described that allow the detection of phospholipid derivatives at very low concentrations on silica gel and reversed-phase thin-layer plates. This modification, which involves the addition of acetic acid to the mixture, permits the observation of sharp blue spots on a white background. The specificity and sensitivity of the spray are discussed.     

A silver stain for the rapid quantitative detection of proteins or nucleic acids on membranes or thin layer plates

A relatively simple silver stain which takes less than 15 min to perform has been developed for the detection of nanogram quantities of proteins and DNA on cellulose membranes and thin layer plates. This stain demonstrates a reproducible curvilinear relationship between silver density and the amount of protein or DNA, over an averaged concentration range from 1 to 300 ng for proteins and 10 to 710 ng for DNA. The ease of staining proteins and DNA on membranes, combined with the stain's sensitivity and reproducibility, permits the use of this procedure for the quantitative determination of nanogram amounts of proteins and DNA. The simplicity of this silver stain has also permitted a survey of the staining properties of individual amino acids, purine and pyrimidine bases, nucleosides, nucleotides, homopolymers, and small peptides of known sequence. This survey demonstrated the importance of the basic amino acids, particularly lysine and histidine, and the sulfur-containing amino acids in the detection of proteins. It also indicated that the purine bases may play an important role in the detection of DNA.     

A simple and sensitive method for lipoprotein and lipids profiles analysis of individual micro-liter scale serum samples

A simple and sensitive method to determine lipoprotein and lipids profiles in micro-liter scale individual serum sample is not presently available. Traditional lipoprotein separation techniques either by ultra-centrifugation or by liquid chromatography methods have their disadvantages in both lipoprotein separation and lipids component quantification. In this study we used small volume needing size-exclusion fast protein liquid chromatography to separate different lipoprotein subclasses in 50μL serum. And lipids contents, such as cholesterol, cholesterol ester and triacylglycerol, were measured by using two different fluorescence-based lipid detection methods. With this method, very low density lipoprotein, low density lipoprotein and high density lipoprotein could be easily separated, and follow-up lipid detection was completed by simple kinds of reactions. Serum lipoprotein and lipids profiling from C57BL/6 mice (n=5) and human (n=5) were analyzed. The elution profiles of five individuals were highly reproducible, and there were lipoprotein and lipids distribution variations between C57BL/6 mice and human beings. In conclusion, this method which combined small volume needing size-exclusion fast protein liquid chromatography and fluorescence-based lipids measurement, provided a simple, efficient, integrity and reproducible procedure for determining serum lipoprotein and lipids profiles in micro-liter scale levels. It becomes possible that determination of lipoprotein profiles and gaining information of lipids in different lipoproteins can be accomplished simultaneously.     

High performance thin-layer chromatography and densitometry of synaptic plasma membrane lipids

Previously, it has been shown that phospholipids, cholesterol, and glycolipids could be quantitated using the same high performance thin-layer chromatography (HPTLC) method. Here we examined that method in terms of linearity of standards in the nanogram range, recovery of nonacidic and acidic lipids after Sephadex column chromatography, and quantitation of lipids in mouse synaptic plasma membranes (SPM) where lipid content is low. Nonacidic and acidic fractions were separated by Sephadex column chromatography, applied to plates using contact spotting, chromatographed, visualized with cupric acetate, and quantitated using in situ densitometry. Recovery of nonacidic and acidic fractions off the columns was determined with radiolabeled phospholipids. Standards for each lipid class were linear in the nanogram range. Quantitation of SPM lipid classes could be made with as little as 1.5 micrograms of total lipid. Recovery of the nonacidic fraction after Sephadex column chromatography was approximately 100% whereas the acidic fraction was approximately 91%. Phospholipids, cholesterol, and glycolipids could be determined in nanogram amounts using the same method. This method is an efficient method for examining different lipid classes and in samples where lipid content is low.     

Uptake and Metabolism of L-Serine by Pneumocystis carinii carinii

ABSTRACT. Serine is an important amino acid that is utilized in the biosyntheses of proteins and lipids. It is directly incorporated into the head group of phosphatidylserine, which in turn can be converted to other phospholipids. Also, it is required for the formation of long chain bases, precursors of sphingolipids. Uptake and incorporation of radiolabeled serine into both lipids and acid-precipitable material were demonstrated in Pneumocystis carinii carinii organism preparations freshly isolated from infected rat lungs. Radioactivity in proteins was about double that observed in lipids. Liquid scintillation spectrometry of metabolically radiolabeled lipids separated by thin-layer chromatography showed 53% of the total radioactivity were in phosphatidylserine, 12% in phosphatidylethanolamine, 24% in ceramides, and 11% in long chain bases and other compounds. Four long chain bases were detected by thin-layer chromatography in hydrolyzed P. carinii ceramides metabolically labeled with radioactive serine. Phytosphingosine and dihydrosphingosine were tentatively identified by their migrations on thin-layer plates. Radiolabeled ethanolamine was incorporated into P. carinii phosphatidylethanolamine, but relatively low incorporation of radiolabeled choline into phosphatidylcholine occurred. The observations made in this study indicated that P. carinii has the biosynthetic capacity to metabolize phospholipid head groups and to de novo synthesize sphingolipids. L-Cycloserine and β-CI-D-alanine, inhibitors of long chain base synthesis, reduced the incorporation of serine into P. carinii long chain bases and ceramides, which supported the conclusion that the pathogen synthesizes sphingolipids.     

Perhexiline maleate-induced lipidosis in cultured human fibroblasts: cell kinetics, ultrastructural and biochemical studies

Perhexiline maleate reduced the growth of human skin fibroblasts in cell culture at a concentration range of 0.3-3 micrograms/ml. At the highest concentration, the cells survived only four days. Pleomorphic inclusions characteristic of drug-induced phospholipidoses appeared in cultured cells. Analysis of the major lipid classes was performed on cells exposed to 3 micrograms/ml at four days. Gangliosides, phospholipids and cholesterol levels four to six times above controls were found. No major qualitative abnormalities were detected in phospholipids. On the contrary, an abnormal pattern of gangliosides was seen by densitometry of silica gel thin-layer plates with increases of GD3 and of an unknown ganglioside. Drug induced lipidosis may involve other lipids than phospholipids, particularly gangliosides.     

Characterisation of sphingolipids in the human lens by thin layer chromatography–desorption electrospray ionisation mass spectrometry

The lipidome of the human lens is unique in that cholesterol and dihydrosphingomyelin are the dominant classes. Moreover, the lens lipidome is not static with dramatic changes in several sphingolipid classes associated with both aging and cataract. Accordingly, there is a clear need to expand knowledge of the molecular species that constitute the human lens sphingolipidome. In this study, human lens lipids have been extracted and separated by thin-layer chromatography (TLC). Direct analysis of the TLC plates by desorption electrospray ionisation–mass spectrometry (DESI–MS) allowed the detection over 30 species from 11 classes of sphingolipids. Significantly, novel classes of lens lipids including sulfatides, dihydrosulfatides, lactosylceramide sulfates and dihydrolactosylceramide sulfates were identified.     

Sterol transformation by Escherichia]

The use of the Liebermann-Burhard reaction and the thin-layer chromatography of nonsaponifiable lipids of culture medium (donor blood serum) permitted the isolation of three biological variants of Escherichia in the process of their 48-hour cultivation in this medium. The cholesterol-destroying variant of Escherichia is characterized by a decrease in the content of total, free, esterified cholesterol and a decrease in the occurrence of fractions corresponding to cholesterol, delta 4-cholestenone-3, delta 5-cholestenone-3), as well as nonsaponifiable lipid, where Rf was equal to 0.36; two fractions of labeled nonsaponifiable lipids, not corresponding to cholesterol, appeared on plasma with sodium acetate-1-14C. Cholesterol-transforming biovars produced insignificant changes in the content of chemically determined cholesterol in the medium, but in plasma nonsaponifiable lipid with Rf = 0.26 and other less polar lipids were found. Escherichia strains increasing the amount of chemically determined cholesterol in the process of their growth more frequently transformed or used nonsaponifiable lipids with Rf = 0.26 and 0.42. As a rule, the occurrence of cholesterol and less polar lipids increased. The sodium acetate-1-14C was incorporated into 3-4 fractions of nonsaponifiable lipids, one of them being identified as cholesterol.     

A non-destructive sensitive reagent for lipid detection on TLC plates

The aqueous solutions of 3 acidic and 14 basic dyes used as reagents for detection of lipids without their destruction are tested on plates for highly effective TLCh with the silicagel layer fixed by the silicic acid sol. It is shown that 0.002% solution of basic fuchsin in 1% boric acid reveals neutral, phospho- and glycolipids with the sensitivity compared with such in detection of sulphuric acid. It is established that detection of lipids by this reagent with the use of TLCh does not change the composition of their fatty acids.     

Lipid content of human platelets quantitated by thin-layer chromatography in combination with flame ionization detection

The lipid contents of human platelets from twenty-one healthy adults were analysed using thin-layer chromatography in combination with flame ionization detection.The weight per cent of neutral lipids in human platelets was 14.6%, which consisted mainly of free cholesterol, that of phosphatidylethanolamine 24.9%, phosphatidylserine plus phosphatidylinositol 6.8%, phosphatidylcholine 35.2% and sphingomyelin 18.6%. Free cholesterol in 10⁸ platelets was estimated as 7 μg and phospholipids as 46 μg from calibration standards. The reproducibility was satisfactory and the procedure could be performed quickly and simply.     

Aflatoxin biosynthesis: detection of transient, acetate-dependent intermediates in Aspergillus by kinetic pulse-labeling.

A simple technique was developed for the detection of intermediary metabolites of Aspergillus versicolor that are putative precursors of aflatoxin. Minicolony populations were allowed to metabolize [1,2-14C]acetate over various time intervals. The biosynthetic reactions were quenched by quick-freezing the minicolonies, the cells were disrupted, and the metabolites were extracted into acetone. Small silica thin-layer chromatographic plates were then used to separate any radioactive metabolites present. Elution in two or three different directions was often necessary. Radioautography of the thin-layer chromatography plates provided a sensitive assay for the appearance of the various intermediates in a timing pattern which implicated the sequence of formation. Transient intermediates were distinguished from dead-end metabolites by the rapid formation and disappearance of the former. At least five unknown precursors of versicolorin A, a dead-end metabolite, were recognized. The kinetic pulse-labeling technique should be generally applicable to other fungal species whenever the entrapment of intermediary metabolites in the mycelium poses and technical problem.     

Permanent sensitive stain for choline-containing phospholipids on thin-layer chromatograms

The phosphomolybdic blue stain for choline of Levine and Chargaff can be used for detection of small amounts of choline-containing phospholipids on thin-layer chromatograms if they are first fixed with polyvinyl propionate to permit a washing step.     

Application of improved iodine-azide procedure for the detection of thiouracils in blood serum and urine with planar chromatography

The application of iodine-azide reaction for the determination of thiouracils in thin-layer chromatography and high-performance thin-layer chromatography is described. The developed plates were sprayed with a freshly prepared mixture of sodium azide, adjusted to a proper pH, and starch solution, and exposed to iodine vapour for 5 s. The detection limits were established at pmol level. The factors depending on the detection limits were described. A comparison of iodine-azide tests reaction with other procedures is presented. The developed method was applied to detection of thiouracils in blood serum and urine. The possibility of detection of a thiouracils mixture was demonstrated.