Thin-layer chromatography-densitometry of minor acidic phospholipids: application to lipids from erythrocytes, liver, and kidney

A method for quantitative determination of acidic phospholipids by thin-layer chromatography (TLC) followed by densitometry is described. The total lipids were separated into neutral and acidic fractions by DEAE-Sephadex column chromatography. A clear-cut separation of acidic phospholipids was achieved by high-performance TLC with a solvent system of chloroform/acetone/acetic acid/formic acid/water (60/60/4/10/3). Each phospholipid band was quantitated by densitometry with the use of an internal standard. The lipid compositions of sheep and mouse erythrocytes and of rat liver and kidney were determined by the present method.     

Critical Assessment of Glyco- and Phospholipid Separation by Using Silica Chromatography

Phospholipid-derived fatty acids (PLFAs) are commonly used to characterize microbial communities in situ and the phylogenetic positions of newly isolated microorganisms. PLFAs are obtained through separation of phospholipids from glycolipids and neutral lipids using silica column chromatography. We evaluated the performance of this separation method for the first time using direct detection of intact polar lipids (IPLs) with high-performance liquid chromatography–mass spectrometry (HPLC-MS). We show that under either standard or modified conditions, the phospholipid fraction contains not only phospholipids but also other lipid classes such as glycolipids, betaine lipids, and sulfoquinovosyldiacylglycerols. Thus, commonly reported PLFA compositions likely are not derived purely from phospholipids and perhaps may not be representative of fatty acids present in living microbes.     

Lipid composition of isolated epiphyseal cartilage cells, membranes and matrix vesicles.

1. Intact cells, cell fragments (membranes) and matrix vesicles were isolated from the proliferating and calcifying layers of epiphyseal cartilage by sequential hyaluronidase and collagenase digestion and differential centrifugation. Lipids were extracted and analyzed for various lipid classes and their fatty acid composition by column, thin-layer, paper and gas-liquid chromatography. 2. On a protein basis the isolated matrix vesicles had more total lipid than either the membrane or cell fractions, the vesicles and membranes being richer in non-polar lipids and containing smaller quantities of phospholipids than whole cells. Expressed as a percentage of the total lipid, the cells were richer in triacylglycerols and lower in free fatty acids than in the membrane or vesicle fractions. The proportion of free cholesterol and the cholesterol/phospholipid ratio were nearly twice as high in the matrix vesicles as in the other tissue fractions. Choline and ethanolamine phosphoglycerides progressively declined in the membrane and matrix vesicle fractions, whereas serine phosphoglycerides and sphinogomyelin increased. Non-phosphorus-containing polar lipids were present in all fractions, the vesicles being richer in polyhexosyl ceramides, cerebrosides, glycosyldiacylglycerols and certain uncharacterized acidic polar lipids. 3. Fatty acid patterns of the matrix vesicles were distinctive from those of isolated cells, being generally richer in 18 : 0 and 18 : 2, and lower in 16 : 1 and 18 : 1 fatty acids. Monoacyl forms were similarly increased in 16 : 0 and/or 18 : 0, and reduced in 16 : 1, 18 : 1 or 20 : 2 fatty acids, depending on the lipid class. The fatty acid composition of diphosphatidylglycerol from cells and matrix vesicles was markedly different, providing evidence that the cardiolipin in the vesicles was not from mitochondrial components. 4. Based on the fact that the matrix vesicles were significantly enriched in free cholesterol, sphingomyelin, glycolipids and serine-phosphoglycerides, it is concluded that they are derived from the plasma membrane of the cell, supporting earlier conclusions based upon morphological and enzymological evidence.     

A comparison of extraction methods for the isolation of phospholipids from biological sources

Four classical methods, as well as a method presented in this paper, were compared as to their efficiency in extracting phospholipids from animal tissue. After the extractions, total lipids were separated quantitatively by DEAE-Sephadex chromatography into their acidic and nonacidic fractions. The two fractions were then further analyzed by gradient saturation high-performance thin-layer chromatography (HPTLC) combined with scanning photodensitometry after coloration with copper acetate. Of the five methods compared, the present and Christiansen's methods based upon single-phase solvent systems proved to be more efficient than biphasic extraction procedures. The undesirable discriminatory effect exhibited by biphasic solvent systems toward acidic phospholipids which were partly retained in the aqueous phase was confirmed by statistical evaluation of the HPTLC results. Total chromogenic response of acidic phospholipids extracted using biphasic solvent systems was shown to be lower by 10-35% in comparison to the single-phase method of Christiansen. The suitability of the present method for studies involving phospholipid synthesis was confirmed by monitoring the elimination of water-soluble compounds from the single-phase extracts using a classical phospholipid precursor, 2-[3H]glycerol-3-phosphate. The labeled compound was eliminated (99.3-100%) from the single-phase postcentrifugation supernatant, followed by DEAE-Sephadex chromatography.     

Separation of lipid classes by solid phase extraction

A rapid and reliable method for the separation of lipid classes is described using aminopropyl disposable columns. This method is a modification to an existing procedure that allows the separation of both neutral and acidic phospholipid fractions and a high recovery of the latter. Acidic phospholipids were eluted with a mixture of hexane-2-propanol-ethanol-0.1 M ammonium acetate-formic acid 420:350:100:50:0.5 containing 5% phosphoric acid after neutral phospholipids had been eluted with methanol. It was verified that extremely high recoveries of cholesterol (CH), triglycerides (TG), free fatty acids (FFA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA), sphingomyelin (SM), and cerebrosides were obtained with this method. In addition, there appeared to be no preferential losses or degradation of any particular molecular species as the fatty acid distribution of bovine brain PS and the molecular species profile of plant PI were unaltered by the procedure. Depending on the tissue, this method may yield fractions containing pure lipid classes and/or simple mixtures of lipid classes of similar polarity. These fractions may then be more easily separated by thin-layer chromatography or high performance liquid chromatography for a complete lipid class analysis.     

Isolation and comparative analysis of glycolipid fractions in bifidobacteria

A comparative TLC analysis of lipid extracts from Bifidobacterium longum B 379 M, B. bifidum 791, and B. adolescentis 94 BIM has been performed. It is demonstrated that carbohydrate-containing lipid components were present in the bacteria, which differed in their chromatographic mobility (Rf) from similar compounds isolated from actinomycetes Stomatococcus mucilaginosus PCM 2415T, Nocardiopsis dassonvillei PCM 2492, Propionibacterium propionicum PCM 2431, Saccharopolyspora hirsuta PCM 2279 (= ATCC 27875T), Rhodococcus equi PCMT 559 (= ATCC 3969), and Gordonia bronchialis PCM 2167. Polar lipids of bifidobacteria exhibited the closest similarity to their counterparts from propionic acid bacteria. Preparative chromatography (silica gel column I; elution with chloroform, acetone, and methanol) of the lipid extract of B. adolescentis 94 BIM made it possible to isolate fractions containing nonpolar lipids, glycolipids, and phospholipids. Further purification of the glycolipid fraction (column II; eluant, methanol gradient in chloroform) produced preparations of glycolipids and phospholipids. The preparations were studied by two-dimensional TLC using solvent systems chloroform-methanol-H2O MiLi Q (65 : 25 : 4, v/v/v) and n-butanol-acetic acid-H2O MiLi Q (60 : 20 : 20, v/v/v) for directions I and II, respectively. Two major glycolipids were revealed (G1 and G2), in addition to compounds characteristic of the polar lipid group and minor glycolipids (g), the latter being present in considerably lesser amounts.     

Rapid quantitative measurement of lung tissue phospholipids

A rapid procedure for the separation of phospholipids of lung tissue into acidic and nonacidic fractions by means of diethylaminoethyl cellulose acetate microcolumns is described. The fractions are then resolved into individual phospholipids by thin-layer chromatography and quantified by transmission densitometry.     

Consecutive analysis of sphingoglycolipids on the basis of sugar and ceramide moieties by high performance liquid chromatography

A quantitative consecutive method was developed for analysis of sphingoglycolipids in biological materials by high performance liquid chromatography (HPLC). Crude lipid extracts were separated into neutral and acidic fractions on a DEAE-Sephadex column. Glycolipid fractions were obtained by acetylation and Florisil column chromatography, and the acetylated glycolipids were N-p-nitrobenzoylated by treatment with p-nitrobenzoyl chloride in pyridine at 60 degrees C for 6 h. Excess reagent and by-products were removed by solvent partition and gel filtration. The glycolipid derivatives were analyzed by their absorption at 254 nm on Zorbax SIL, a silica gel column, with a gradient of 0.5--7% isopropanol in hexane-chloroform (2 : 1, v/v) at a flow rate of 0.5 ml/min. The detector response was linear with up to 60 nmol of injected glycolipids. The practical lower limit of detection was about 50 pmol. The derivatives were separated on the basis of their sugar chains. Effluents corresponding to each peak were collected and analyzed further on the basis of their lipid portion on mu-Bondapak C18, a reversed phase column. This combined procedure was applied to the analysis of erythrocyte glycolipids. Samples containing as little as 20 micrograms of glycolipids could be analyzed by this method.     

Separation of chloroplast polar lipids and measurement of galactolipid metabolism by high-performance liquid chromatography

Procedures are described for the separation of polar lipids from plant chloroplasts by high-performance liquid chromatography, using a polar-modified silica column. Glycolipids and phospholipids were eluted with a gradient of 2-propanol/n-hexane (80:55, v/v) and 2-propanol/n-hexane/water/methanol (80:55:15:10, v/v). The lipids were detected by uv absorbance at 202 nm. Diacylglycerol and mono-, di-, and trigalactosyldiacylglycerol and phosphatidylcholine were separated on a LiChrosorb NH2 column (7-microns particles, Merck, FRG), but acidic lipids were retained. These lipids could be quantified from their 202-nm absorbance recording. The absorption coefficients obtained depended on the mean number of double bonds in the different lipid classes. The separation was applied for a rapid monitoring of the lipid composition in thylakoids and in fractionated inner and outer envelopes. The activities of galactosyltransferases involved in galactolipid metabolism, UDPGal:diacylglycerol galactosyltransferase and galactolipid:galactolipid galactosyltransferase, could be measured quantitatively in specific assays for both enzymes.     

Characterization of the seed and leaf lipids of high and low linolenic acid flax genotypes

The total seed lipids of four flax (Linum usitatissimum) genotypes, differing markedly in their acyl composition, were extracted and fractionated using column, preparative, and thin-layer chromatography. In the total lipid extract of seeds, the lower linolenate content of the cultivar Glenelg (39.1% compared to that of cv. Croxton (50.5%) was associated with a higher oleate content. Further reductions in linolenate content in the induced mutants of cv. Glenelg, M1722 (17.2%) and "Zero" (1.9%) were accompanied by equivalent increases in linoleate but only minor increases in oleate. Similar changes were observed in the major triacylglycerol fraction of the simple lipids (fatty acid esters of glycerol and sterols), but there was considerable heterogeneity for acyl composition in the minor simple lipid components, including both diacylglycerols and sterol esters, and the complex lipids (glycolipids and phospholipids). The induced mutations substantially reduced linolenate content of all lipid fractions but in no case was it eliminated. Maturation of "Zero" seed at 15/10 degrees C (compared to 24/19 degrees C) increased linoleate and decreased stearate and oleate contents in all lipid fractions. In contrast to seed lipids, the acyl composition of the leaf lipids of the mutant genotypes was the same as those of their parent.     

Separation of neutral lipids and free fatty acids by high-performance liquid chromatography using low wavelength ultraviolet detection

Normal phase, isocratic high-performance liquid chromatography methods are described for the separation of neutral lipid and fatty acid classes using low wavelength detection. Prior to high-performance liquid chromatography, methods were developed and are described for the separation of phospholipids from neutral lipids and fatty acids using small (600 mg) silica Sep-PaksTM. Recoveries of cholesteryl esters, triglycerides, fatty acids, and phospholipids from the silica columns were greater than 95%. Two mobile phases are described for lipid class separation by high-performance liquid chromatography. The first mobile phase, hexane-2-propanol-acetic acid 100:0.5:01, resulted in incomplete separation of cholesteryl ester and triglyceride but excellent separations of fatty acids and cholesterol. The second mobile phase, hexane-n-butyl chloride-acetonitrile-acetic acid 90:10:1.5:0.01, resulted in complete separation of the four lipid classes. This mobile phase also separated individual triglycerides and fatty acids based on the number of double bonds. Recoveries of radiolabeled lipids for the four lipid classes from high-performance liquid chromatography was greater than 95% with both mobile phases.     

In vivo covalent binding of 14CCl4 metabolites in liver microsomal lipids

Covalently bound ¹⁴C from ¹⁴CCl₄ is preferentially localized in the lipids of hepatic microsomes of rats within 15 min. Label was recovered in all classes of lipids isolated from the microsomal lipid extract by diethylaminoethyl column chromatography. Among phospholipids, specific activity was the highest in the fraction containing phosphatidyl serine and lowest in phosphatidyl choline. Cholesterol esters had more than ten times the specific activity of cholesterol.     

Identification and characterization of lipids from endosomes purified by electromagnetic chromatography

Traditional separation techniques do not yield endolysosomes of sufficient purity to permit detailed biochemical characterization of this important class of intracellular vesicles. Here, we have used a magnetic chromatography technique to isolate the endosomes from rat peritoneal macrophages and studied their lipid composition. Electromagnetic isolation works by retention of colloidal iron containing vesicles on magnetic column. The data suggested that both early and late endosomes were rich in cholesterol, whereas sphingomyelin (SM) and specific phospholipids like phosphatidylcholine. phosphatidylethanolamine, phosphatidylglycerol and phosphatidylserine are enriched in the late compartments. Our results also indicated that the purified fractions are enriched in raft lipids like SM, but not in cholesterol. The endosomal purification method described here yields pure endosomes with little or no contamination from mitochondria and hence could be used for further biochemical and marker analysis, giving insight into mechanisms of endocytic traffic.     

Formation and properties of retinylphosphate galactose.

Crude cell membrane fractions from a number of tissues can form acidic glycolipids. The formation of acidic galactose lipid and mannose lipid was greatly reduced in vitamin A deficiency, primarily in tissues known to be mucus-producing. Mouse mastocytoma tissue was active in forming acidic galactose lipids with UDP-galactose as substrate. One of the products was identified as retinylphosphate galactose. The synthetase reaction producing this compound exhibited an apparent pH optimum at 6.3. The presence of detergent and retinol stimulated the synthetase reaction, which exhibited an absolute requirement for Mn2+ or Mg2+. The synthetase reaction was readily reversible. Incubation of particulate enzyme with retinylphosphate galactose and UDP yielded UDP-galactose and a compound tentatively identified as retinylphosphate. The galactose lipid was isolated by column chromatography on DEAE-cellulose and silica gel. The retinylphosphate galactose was homogeneous when examined by thin layer chromatography. Mild acid hydrolysis of labeled retinylphosphate galactose yields [14C]galactose, whereas alkaline hydrolysis and hydrogenolysis produced [14C]galactose 1-phosphate. Retinylphosphate galactose bound to vitamin A-depleted, retinol-binding protein.     

Simultaneous extraction and preparation for high-performance liquid chromatography of prostaglandins and phospholipids

A method for the maximum recovery of prostaglandins from brain tissue with simultaneous recovery of neutral lipids and phospholipids was developed. Hexane:2-propanol was used to extract lipids from bovine brain. This method, which does not require a washing step to remove nonlipid contaminants, was compared to extraction according to Folch et al. [(1957) J. Biol. Chem. 226, 497-509] for efficiency of lipid extraction. Recoveries of prostaglandins were 12-37% greater with hexane:2-propanol than with the Folch extraction procedure with washing. The ratios of cholesterol to lipid phosphorus and absolute phospholipid recoveries were comparable for the two methods. A new elution sequence was devised for separation of lipid classes on silicic acid columns. The elution sequence was chloroform (neutral lipids and free fatty acids), methyl formate (prostaglandins and cerebrosides), acetone (remaining glycolipids), and methanol (phospholipids). Reverse-phase HPLC of the methyl formate fraction was used to separate the prostaglandins. The method permits simultaneous quantitative recovery of prostaglandins and phospholipids (which contain the 20:4(n-6) precursor for prostaglandin synthesis), and therefore allows changes in phospholipid composition and prostaglandin synthesis to be studied in the same tissue sample.     

High-performance liquid chromatography of lipids for the identification of human metabolic disease.

We describe a system for quantitative lipid analysis employing ternary gradient high-performance liquid chromatography with evaporative light scattering detection. This technique was applied to extracts of cultured fibroblasts, cultured lymphocytes, and leukocytes and to liver and spleen biopsy specimens. Separation of nonpolar lipids, glycolipids, phospholipids, and sphingolipids was achieved in a single run. Detection did not depend on the presence of any specific chemical reactions, uv absorption, or fluorescence. The sensitivity of the technique is well below 200 ng for individual lipids, and many individual lipid classes were detected in samples as small as 1 mg of total protein, the yield of a single flask of cultured skin fibroblasts. The characteristic stored lipids cholesterol ester and sphingomyelin were seen in excess in human fibroblast cultures from patients with Wolman's disease and Niemann-Pick disease, respectively. A biopsy spleen sample from a patient with Gaucher's disease showed a large glucosylceramide peak. This system provides a tool for detecting lipids that accumulate in tissues of patients with currently unidentified metabolic storage disorders.     

Human epidermal lipids: characterization and modulations during differentiation

Using thin-layer chromatography and glass capillary gas-liquid chromatography, we have quantitated the lipids in the germinative, differentiating, and fully cornified layers in human epidermis. As previously noted in nonhuman species, we found progressive depletion of phospholipids coupled with repletion of sterols and sphingolipids during differentiation. The sphingolipids, present only in small quantities in the lower epidermis, accounted for about 20% of the lipid in the stratum corneum, and were the major repository for the long-chain fatty acids that predominate in the outer epidermis. Although the absolute quantities of sphingolipids increased in the outer epidermis, the glycolipid:ceramide ratio diminished in the stratum corneum, and glycolipids virtually disappeared in the outer stratum corneum. Squalene and n-alkanes were distributed evenly in all epidermal layers, suggesting that these hydrocarbons are not simply of environmental or pilosebaceous origin. Cholesterol sulfate, previously considered only a trace metabolite in epidermis, was found in significant quantities, with peak levels immediately beneath the stratum corneum in the stratum granulosum. These studies: 1) provide new quantitative data about human epidermal lipids; 2) implicate certain classes of lipids for specific functions of the stratum corneum; and, 3) shed light on possible product-precursor relationships of these lipids.     

Lipid composition of the electron transport membrane of Haemophilus parainfluenzae

The principal lipids associated with the electron transport membrane of Haemophilus parainfluenzae are phosphatidylethanolamine (78%), phosphatidylmonomethylethanolamine (0.4%), phosphatidylglycerol (18%), phosphatidylcholine (0.4%), phosphatidylserine (0.4%), phosphatidic acid (0.2%), and cardiolipin (3.0%). Phospholipids account for 98.4% of the extractible fatty acids. There are no glycolipids, plasmalogens, alkyl ethers, or lipo amino acid esters in the membrane lipids. Glycerol phosphate esters derived from the phospholipids by mild alkaline methanolysis were identified by their staining reactions, mobility on paper and ion-exchange column chromatography, and by the molar glycerol to phosphate ratios. Eleven diacyl phospholipids can be separated by two-dimensional thin-layer chromatography. Each lipid served as a substrate for phospholipase D, and had a fatty acid to phosphate ratio of 2:1. Each separated diacyl phospholipid was deacylated and the glycerol phosphate ester was identified by paper chromatography in four solvent systems. Of the 11 separated phospholipids, 3 were phosphatidylethanolamines, 2 were phosphatidylserines, and 2 were phosphatidylglycerols. Phosphatidylcholine, cardiolipin, and phosphatidic acid were found at a single location. Phosphatidylmonomethylethanolamine was found with the major phosphatidylethanolamine. Three distinct classes of phospholipids are separable according to their relative fatty acid compositions. (i) The trace lipids consist of two phosphatidylethanolamines, two phosphatidylserines, phosphatidylcholine, phosphatidic acid, and a phosphatidylglycerol. Each lipid represents less than 0.3% of the total lipid phosphate. These lipids are characterized by high proportions of the short (C(10) to C(14)) and long (C(19) to C(22)) fatty acids with practically no palmitoleic acid. (ii) The major phospholipids (93% of the lipid phosphate) are phosphatidylethanolamine, phosphatidylmonomethylethanolamine, and phosphatidylglycerol. These lipids contain a low proportion of the short (C(19)) fatty acids. Palmitic and palmitoleic acids represent over 80% of the total fatty acids. (iii) The fatty acid composition of the cardiolipin is intermediate between the other two classes. Both palmitoleic and the longer fatty acids represent a significant proportion of the total fatty acid.     

A novel fluorescent fatty acid, 5-methyl-BDY-3-dodecanoic acid, is a potential probe in lipid transport studies by incorporating selectively to lipid classes of BHK cells.

The 5-methyl-BDY-3-dodecanoic acid (B12FA) labelling of BHK cell lipids was analyzed by thin layer and reverse phase column chromatography. Incorporation to phospholipids was selective: over 90% of B12FA label was enriched in phosphatidylcholine. The major molecular species of PC was that containing palmitate as the unlabelled fatty acid. Small amounts of label was also found in other phosphoglycerides, but not in sphingomyelin. Triglycerides and diglycerides constituted the main B12FA-labelled neutral lipid classes; however, no label was found in cholesterol esters. B12FA was degraded to shorter homologues, which had significantly slower lipid incorporation rates. B12FA-labelled cells displayed in a microscope initially green reticular type fluorescence, but later red spherical structures, representing neutral lipid droplets, could also be seen. It is concluded that B12FA does not incorporate indiscriminately to all lipid classes of BHK cells, but is enriched to PC, diglycerides and triglycerides, which could be utilized in studies on lipid transport as well as metabolism.     

Comparison of acetate and glucose incorporation into rat and horse skin lipids

The relative efficiency of acetate and glucose as substrates for the biosynthesis of lipids in the skin of the rat and horse was examined using in vivo pulse labelling of skin with [1-14C]acetate and [U-14C]glucose by intradermal injections. The resulting radiolabelled lipids were recovered in the rat by punch biopsy as well as by daily, long-term skin surface lipid collections and in the horse by punch biopsy of the injection sites. The lipids were examined by liquid scintillation and by a combination of thin-layer chromatography and autoradiography. In both species the recovery of radiolabel in the non-polar lipids was much higher after a pulse of [1-14C]acetate than after a pulse of [U-14C]glucose. In the rat, the skin surface lipids labelled through acetate contained sufficient radiolabel to allow observation of the time course of excretion of 14C in the major non-polar lipid classes. The results suggest that the biosynthesis of these lipid classes in the sebaceous glands of the rat are not entirely synchronous. In the skin lipid extracts of the horse, all of the major lipid classes, including phospholipids and glycolipids, were labelled through acetate. In contrast, none of the non-polar lipids and very little of the polar lipids were labelled through glucose.