High-performance liquid chromatographic analysis of acylated lipids containing pyrene fatty acids

A high-performance liquid chromatographic method for the separation and quantification of acylated lipids containing pyrene fatty acids is described. The method is adapted from a procedure originally developed for the analysis of tissue lipids (Christie, W. W. (1985) J. Lipid Res. 26, 507-512). Pyrenyl lipid analogs ranging in polarity from cholesteryl ester to lysophosphatidylcholine are completely resolved on a silica column in 50 min by gradient elution with a ternary solvent system. Furthermore, pyrene-labeled triglycerides are resolved according to the number of pyrene fatty acid residues incorporated. Pyrenyl lipids are detected at levels of 10(-13) mol by high-sensitivity fluorescence detection. Accurate quantification of pyrenyl lipids is obtained by correcting peak areas for mobile-phase quenching effects. The close correspondence between chromatograms obtained for the separation of labeled lipids extracted from Hep-G2 cells incubated with either 12-(1-pyrenyl)dodecanoic acid (fluorescence detection) or [1-14C]oleic acid (radioactivity detection) indicates that this HPLC method is equally suitable for analysis of native lipids.     

Rapid extraction of oxygenated metabolites of arachidonic acid from biological samples using octadecylsilyl silica

A rapid procedure for the efficient extraction of prostaglandins, thromboxanes and hydroxy fatty acids from urine, plasma and tissue homogenates has been developed. Fractions containing these substances are acidified and passed through a column of octadecylsilyl silica, which retains oxygenated metabolites of arachidonic acid. Phospholipids, proteins and very polar materials either are not retained or can be eluted with dilute aqueous ethanol. Nonpolar lipids and monohydroxy fatty acids are then eluted with petroleum ether or benzene. Subsequent elution of the column with methyl formate gives a fraction containing prostaglandins and thromboxanes which is much less contaminated with extraneous material than that obtained by conventional extraction of aqueous media with organic solvents. The methyl formate can be removed rapidly under a stream of nitrogen and the components of the sample purified directly by high pressure liquid chromatography (HPLC). An improved method for the purification of prostaglandins and TXB2 by HPLC on silica columns is reported.     

Rapid extraction of oxygenated metabolites of arachidonic acid from biological samples using octadecylsilyl silica

A rapid procedure for the efficient extraction of prostaglandins, thromboxanes and hydroxy fatty acids from urine, plasma and tissue homogenates has been developed. Fractions containing these substances are acidified and passed through a column of octadecylsilyl silica, which retains oxygenated metabolites of arachidonic acid. Phospholipids, proteins and very polar materials either are not retained or can be eluted with dilute aqueous ethanol. Nonpolar lipids and monohydroxy fatty acids are then eluted with petroleum ether or benzene. Subsequent elution of the column with methyl formate gives a fraction containing prostaglandins and thromboxanes which is much less contaminated with extraneous material than that obtained by conventional extraction of aqueous media with organic solvents. The methyl formate can be removed rapidly under a stream of nitrogen and the components of the sample purified directly by high pressure liquid chromatography (HPLC). An improved method for the purification of prostaglandins and TXB₂ by HPLC on silica columns is reported.     

Improved high-performance liquid chromatographic method in the analysis of adenovirus particles

We developed a HPLC method on a novel continuous bed matrix (UNO Q, Bio-Rad) for the direct quantification of adenoviral type 5 (Ad5) particles produced in 293S Human Embryonic Kidney cells and compared this with an existing HPLC method on a conventional ion-exchange resin (Resource Q, Pharmacia). The 293S cell extract contained large amounts of DNA. This contaminated the viral peak on the Resource Q column and only after Benzonase treatment was it possible to quantify the viral particles in the cell extract. In contrast, the virus peak on the UNO Q column was resolved from the DNA which eliminates the need for pretreatment of the sample with Benzonase. Cross-analysis of the Ad5 fraction from the UNO Q column using a size-exclusion HPLC column revealed no additional contaminating peaks. We conclude that the purity of the Ad5 virus peak on the continuous bed matrix UNO Q column was superior to the purity of the virus on the conventional Resource Q column, which is essential for reliable quantification.     

Bidimensional reversed-phase high-pressure liquid chromatography analysis of cultured cell neuropeptides: application to atrial natriuretic factor

We report here a one-step procedure for extraction and analysis of neuropeptides in chromaffin cell culture media and acid extracts using reversed-phase HPLC. The bidimensional HPLC system consists of a precolumn connected to a six-port switching valve which is on-line with an analytical column. The direct injection of the biological samples onto the precolumn previously equilibrated with 15% acetonitrile allows the elimination of interfering substances. The samples purified on the precolumn can then be eluted onto the analytical column via the switching valve for neuropeptide separation. This trace-enrichment system allows a minimum of sample handling, both saving time and reducing possibilities of loss and contamination. This method has been applied to monitor the precursor and mature forms of atrial natriuretic factor from chromaffin cell secretion media and cell content extracts. The recovery of atrial natriuretic factor is in the range of 80-100%. This procedure could be applied to the study of the precursor-product relationship of any neuropeptide, e.g., from radiolabeled extracts of pulse-chase experiments performed on cultured chromaffin cells.     

High-performance liquid chromatography analysis of molecular species of sphingolipid-related long chain bases and long chain base phosphates in Saccharomyces cerevisiae after derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.

The molecular species of dihydrosphingosines and phytosphingosines and their 1-phosphates with carbon chain lengths from 16 to 20 have been tagged with the fluorescent amino group reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. All these derivatives could be resolved by reversed phase HPLC on a C18 column. A convenient one-pot method is described whereby lipid extracts from strains of Saccharomyces cerevisiae containing carbon chain length homologs of sphingolipid long chain bases and their phosphorylated derivatives were directly reacted with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, ester lipids were deacylated, and the reaction mixtures were subjected to liquid chromatography. Five molecular species of both sphingolipid long chain bases and their phosphorylated derivatives are for the first time separated and analyzed. The procedure is quite sensitive, requiring only approximately 10(8) wild-type cells.     

A method for determination of iododeoxyuridine substitution of thymidine using reversed-phase high-performance liquid chromatography

An assay for thymidine substitution by iododeoxyuridine (IdUrd) using reversed-phase high-performance liquid chromatography (HPLC) has been developed. Three principal steps in this procedure are: extraction of DNA from cell or tissues, hydrolysis of DNA into deoxynucleosides and separation using HPLC. Approximately 1 microgram of DNA was recovered from 10(5) cells by phenol extraction, and subjected to hydrolysis into deoxynucleosides which required a three-stage DNA digestion using enzymes DNAse I. phosphodiesterase I and alkaline phosphatase. The deoxynucleosides were separated on the Microsorb C18 column with isocratic elution; 90-100% of the DNA was recovered as deoxynucleosides on the column. The method was used to determine quantitatively the percent IdUrd substitution of thymidine in Chinese hamster lung cells in vitro and BA1112 rhabdomyosarcoma in WAG/Rij rats perfused with IdUrd. It was possible to determine the thymidine substitution by IdUrd as small as 1% using a few micrograms of DNA. The close correspondence between the percent substitutions determined by HPLC and those determined by radioactive assay using [125I]-labelled IdUrd, confirmed the accuracy of our HPLC method. The HPLC analysis is especially suitable for the determination of percent IdUrd substitution of thymidine in tissue biopsies from animals used in in vivo experiments or humans undergoing radiation treatment.     

A simplified and sensitive fluorescent method for disaccharide analysis of both heparan sulfate and chondroitin/dermatan sulfates from biological samples

Sulfated glycosaminoglycans regulate the biological functions of a wide variety of proteins, primarily through high affinity interactions mediated by specific sugar sequences or patterns/densities of sulfation. Disaccharide analysis of such glycosaminoglycans yields important diagnostic and comparative structural information on sulfate patterning. When applied to specific oligosaccharides it can also make a vital contribution to sequence elucidation. Standard UV detection of lyase-generated disaccharides resolved by HPLC can lack sufficient sensitivity and be compromised by contaminating UV signals, when dealing with scarce tissue- or cell culture-derived material. Various methods exist for improved detection, but usually involve additional HPLC hardware and often necessitate different procedures for analyzing different glycosaminoglycans. We describe a simple procedure, requiring only standard HPLC instrumentation, involving prederivatization of disaccharides with 2-aminoacridone with no cleanup of samples, followed by a separation by reverse-phase HPLC that is sensitive to as little as approximately 100 pg (approximately 10(-13) mol) of an individual disaccharide, thereby allowing analyses of >10 ng of total glycosaminoglycan. Importantly, separate analysis of both HS/heparin and CS/DS species within a mixed glycosaminoglycan pool can be performed using the same procedure on a single column. We demonstrate its applicability in dealing with small quantities of material derived from rat liver (where we demonstrate a high abundance of the unusual CS-E species within the CS/DS pool) and MDCK cells (which revealed a HS species of relatively low N-sulfation, but high O-sulfation). This simplified method should find a widespread utility for analyzing glycosaminoglycans from limited animal and cell culture samples.     

Pre-packed reverse phase columns for isolation of complex lipids synthesized from radioactive precursors

Pre-packed reverse phase columns (Bond Elut) were used for the separation of complex lipids, such as phosphatidylcholine, cerebrosides, sulfatides, and gangliosides, from their respective water-soluble radioactive precursors after their in vitro biosynthesis. After an incubation in vitro, the entire reaction mixture is passed through the column, where complex lipids are retained and the hydrophilic radioactive precursors are washed away from the column. The retained lipids are then eluted with a more nonpolar organic solvent. The procedure is shown to be simpler and more efficient than the normally used Folch partitioning method or other procedures.     

It’s a Lipid’s World: Bioactive Lipid Metabolism and Signaling in Neural Stem Cell Differentiation

Lipids are often considered membrane components whose function is to embed proteins into cell membranes. In the last two decades, studies on brain lipids have unequivocally demonstrated that many lipids have critical cell signaling functions; they are called “bioactive lipids”. Pioneering work in Dr. Robert Ledeen’s laboratory has shown that two bioactive brain sphingolipids, sphingomyelin and the ganglioside GM1 are major signaling lipids in the nuclear envelope. In addition to derivatives of the sphingolipid ceramide, the bioactive lipids discussed here belong to the classes of terpenoids and steroids, eicosanoids, and lysophospholipids. These lipids act mainly through two mechanisms: (1) direct interaction between the bioactive lipid and a specific protein binding partner such as a lipid receptor, protein kinase or phosphatase, ion exchanger, or other cell signaling protein; and (2) formation of lipid microdomains or rafts that regulate the activity of a group of raft-associated cell signaling proteins. In recent years, a third mechanism has emerged, which invokes lipid second messengers as a regulator for the energy and redox balance of differentiating neural stem cells (NSCs). Interestingly, developmental niches such as the stem cell niche for adult NSC differentiation may also be metabolic compartments that respond to a distinct combination of bioactive lipids. The biological function of these lipids as regulators of NSC differentiation will be reviewed and their application in stem cell therapy discussed.     

Comparison of GC and HPLC for the quantification of organic acids in coffee

A GC and an HPLC method for the quantification of organic acids OAs in coffee have been compared. The GC procedure, employing trimethylsilyl derivatives, was found to be very tedious. The HPLC method, which employed an ion exchange column using a flow gradient of water containing 1% phosphoric acid and UV detection (210 nm), was found to be much simpler for the quantification of eight organic acids (oxalic, succinic, fumaric, malic, tartaric, citric, quinic and fumaric acids) in four representative coffee samples. The HPLC procedure was more convenient than that described in the literature since no pre-purification was required for quantification of the OAs.     

Liquid chromatographic analysis of sebum lipids and other lipids of medical interest

A technique is described for the high-pressure liquid chromatographic (HPLC) analysis of sebum lipid classes. The lipid classes present in sebum are separated by gradient elution HPLC from a microparticulate silica column and detected using a moving-wire detector. The system described can be linked to a computer. Quantitation can be carried out by comparing peak areas obtained with those of an internal standard. Peak trapping for further investigations of the separated components, for example by gas chromatography—mass spectrometry, is very easy.Sebum lipids are separated into the following lipid classes: hydrocarbons and squalene, cholesterol esters and wax esters, fatty acids as their methyl esters, triglycerides, 1,3-diglycerides, 1,2-diglycerides, free cholesterol, monoglycerides and other polar materials. Besides to sebum, the method has been successfully applied to other lipid mixtures, such as serum lipids. Examples of other applications are shown.     

Growth regulation of the interstitial cell population in hydra : I. Evidence for global control by nerve cells in the head

The interstitial cells of hydra form a multipotent stem cell system, producing terminally differentiated nerve cells and nematocytes during asexual growth. Under well-fed conditions the interstitial cell population doubles in size every 4 days. We have investigated the possible role of nerve cells in regulating this behavior. Nerve cells are normally found in highest concentrations in the head region of hydra, while interstitial cells are primarily located in the body column. Our experimental approach was to construct, by grafting, animals in which the density of nerve cells varied in (1) the head region, or (2) the body column. The growth of the interstitial cell population was then measured in these hydra. The results indicate that differences in head nerve cell density are closely correlated with how fast the interstitial cell population increases in size. Variations in the level of either nerve cells or interstitial cells in the body column showed no such correlation. These findings suggest the existence of a signaling mechanism in the head region. This signal, which is a function of the density of head nerve cells, emanates from the head tissue and exerts global control on the growth of the interstitial cell population in the body column.     

Large-scale purification of synthetic oligonucleotides and carcinogen-modified oligodeoxynucleotides on a reverse-phase polystyrene (PRP-1) column

A procedure is described for the large-scale purification of synthetic oligonucleotides using a polystyrene (PRP-1, Hamilton Co.) high-performance liquid chromatography (HPLC) column with a phosphate/methanol/acetonitrile solvent system. Pure oligonucleotides are obtained with a three-step procedure that involves only one column purification step. The dimethoxytrityl group is left on the oligomer for the HPLC purification. The use of the PRP-1 polystyrene column with a phosphate/methanol/acetonitrile solvent system provides excellent separation of the desired dimethoxytrityl-bearing oligonucleotide from failure sequences. The dimethoxytrityl group is removed by treatment with acetic acid and the oligonucleotide is desalted on a C-18 Sep-Pak cartridge. The oligodeoxynucleotides obtained are shown to be essentially pure by HPLC, polyacrylamide gel electrophoresis, and 500-MHzNMR spectroscopy. This procedure is especially useful for the large-scale purification of oligonucleotides required for NMR studies. The PRP-1 column and the phosphate/methanol/acetonitrile solvent system is useful for purifying modified oligonucleotides containing lipophilic groups such as the carcinogen 2-(acetylamino)fluorene.     

Preparation of lipid-free tissue extracts for chromatographic determination of thyroid hormones and metabolites

We have developed a new method of preparing tissues for analysis of thyroid hormones and metabolites which eliminates troublesome lipids and proteins. Frozen tissue is homogenized by grinding with dry ice in a Waring blender, and the moist powder obtained is extracted with chloroform:methanol (2:1). In a modification of the Folch procedure for the total separation of lipids, the filtered extracts then are partitioned into polar and nonpolar layers by the addition of 0.05% aqueous calcium chloride. The upper phase contains the iodocompounds of interest as well as all salts and small polar molecules. The lipids remain dissolved in the lower phase after it is back-extracted with pure upper phase. The combined upper or aqueous methanol layers are lyophilized and the residue is taken up in methanol to yield a concentrated solution ready for analysis of iodocompounds. The greater clarity of the extract permits application of larger samples for two-dimensional paper chromatography than has been customary. For gradient analysis by reverse-phase ion-pair high-performance liquid chromatography (HPLC), the nitrogen-evaporated methanol extract is dissolved in the initial mobile phase, 0.1% H₃PO₄, for injection onto the column. Using these methods we have achieved the first reported separation of ¹²⁵I-labeled tissue iodothyronine metabolites by HPLC. The new method of extraction is of general applicability to any biological sample which might be analyzed in thyroid hormone metabolism research.     

Structural organization of mammalian lipid phosphate phosphatases: implications for signal transduction.

This article describes the regulation of cell signaling by lipid phosphate phosphatases (LPPs) that control the conversion of bioactive lipid phosphates to their dephosphorylated counterparts. A structural model of the LPPs, that were previously called Type 2 phosphatidate phosphatases, is described. LPPs are characterized by having no Mg(2+) requirement and their insensitivity to inhibition by N-ethylmaleimide. The LPPs have six putative transmembrane domains and three highly conserved domains that define a phosphatase superfamily. The conserved domains are juxtaposed to the proposed membrane spanning domains such that they probably form the active sites of the phosphatases. It is predicted that the active sites of the LPPs are exposed at the cell surface or on the luminal surface of intracellular organelles, such as Golgi or the endoplasmic reticulum, depending where various LPPs are expressed. LPPs could attenuate cell activation by dephosphorylating bioactive lipid phosphate esters such as phosphatidate, lysophosphatidate, sphingosine 1-phosphate and ceramide 1-phosphate. In so doing, the LPPs could generate alternative signals from diacylglycerol, sphingosine and ceramide. The LPPs might help to modulate cell signaling by the phospholipase D pathway. For example, phosphatidate generated within the cell by phospholipase D could be converted by an LPP to diacylglycerol. This should change the relative balance of signaling by these two lipids. Another possible function of the LPPs relates to the secretion of lysophosphatidate and sphingosine 1-phosphate by activated platelets and other cells. These exogenous lipids activate phospholipid growth factor receptors on the surface of cells. LPP activities could attenuate cell activation by lysophosphatidate and sphingosine 1-phosphate through their respective receptors.     

Purification of a hydrophobic surfactant peptide using high-performance liquid chromatography

A 4- to 6-kDa hydrophobic peptide (SP4-6) was purified from human pulmonary surfactant. Sep Pak Florisil cartridges removed most of the lipids and the 18-kDa peptide. Analytical wide-pore reversed-phase HPLC column separated a single peptide that contained no detectable lipids (less than 1 nmol/2.5 micrograms protein). N-terminal analysis indicated that this peptide was pure, but the N-terminal amino acid was blocked. The peptide was capable of restoring the in vitro surface properties of synthetic phospholipids, which is characteristic of native lung surfactant.     

Structural organization of mammalian lipid phosphate phosphatases: implications for signal transduction

This article describes the regulation of cell signaling by lipid phosphate phosphatases (LPPs) that control the conversion of bioactive lipid phosphates to their dephosphorylated counterparts. A structural model of the LPPs, that were previously called Type 2 phosphatidate phosphatases, is described. LPPs are characterized by having no Mg²⁺ requirement and their insensitivity to inhibition by N-ethylmaleimide. The LPPs have six putative transmembrane domains and three highly conserved domains that define a phosphatase superfamily. The conserved domains are juxtaposed to the proposed membrane spanning domains such that they probably form the active sites of the phosphatases. It is predicted that the active sites of the LPPs are exposed at the cell surface or on the luminal surface of intracellular organelles, such as Golgi or the endoplasmic reticulum, depending where various LPPs are expressed. LPPs could attenuate cell activation by dephosphorylating bioactive lipid phosphate esters such as phosphatidate, lysophosphatidate, sphingosine 1-phosphate and ceramide 1-phosphate. In so doing, the LPPs could generate alternative signals from diacylglycerol, sphingosine and ceramide. The LPPs might help to modulate cell signaling by the phospholipase D pathway. For example, phosphatidate generated within the cell by phospholipase D could be converted by an LPP to diacylglycerol. This should change the relative balance of signaling by these two lipids. Another possible function of the LPPs relates to the secretion of lysophosphatidate and sphingosine 1-phosphate by activated platelets and other cells. These exogenous lipids activate phospholipid growth factor receptors on the surface of cells. LPP activities could attenuate cell activation by lysophosphatidate and sphingosine 1-phosphate through their respective receptors.     

Elastic membrane heterogeneity of living cells revealed by stiff nanoscale membrane domains

Many approaches have been developed to characterize the heterogeneity of membranes in living cells. In this study, the elastic properties of specific membrane domains in living cells are characterized by atomic force microscopy. Our data reveal the existence of heterogeneous nanometric scale domains with specific biophysical properties. We focused on glycosylphosphatidylinositol (GPI)-anchored proteins, which play an important role in membrane trafficking and cell signaling under both physiological and pathological conditions and which are known to partition preferentially into cholesterol-rich microdomains. We demonstrate that these GPI-anchored proteins reside within domains that are stiffer than the surrounding membrane. In contrast, membrane domains containing the transferrin receptor, which does not associate with cholesterol-rich regions, manifest no such feature. The heightened stiffness of GPI domains is consistent with existing data relating to the specific condensation of lipids and the slow diffusion rates of lipids and proteins therein. Our quantitative data may forge the way to unveiling the links that exist between membrane stiffness, molecular diffusion, and signaling activation.     

Isolation of plasma membrane exovesicles from BHK cells using merocyanine 540.

Treatment of cultured BHK cells with merocyanine 540 caused the non-lytic release of vesicular material having the phospholipid composition characteristic of plasma membrane. The protein composition of the vesicles closely resembled that of the soluble fraction of the cell, as expected for exovesicles budding from the cell surface. Vesicles prepared from cells surface-iodinated with 125I contained no obvious iodinated membrane polypeptides, suggesting that no major proteins in the plasma membrane of the BHK cell are free to diffuse with lipids. The procedure described should represent a general method, applicable to a wide range of cell types, for isolating plasma membrane vesicles.