Molecular species composition of rat liver phospholipids by ESI-MS/MS: the effect of chromatography.

Using electrospray ionization tandem mass spectrometry (ESI-MS/MS) this study shows that the loss of glycerophospholipid (GPL) after chromatography was unevenly distributed across the GPL molecular species. Both TLC and HPLC caused a preferential loss of GPL with 0 to 3 double bonds: 20% and 7.2% for choline glycerophosphates (PC) and 19.7% and 7.5% for ethanolamine glycerophosphates (PE), respectively. A consequence of these losses was that GPLs containing fatty acids with four or more double bonds had a greater contribution to the total after chromatography. ESI-MS/MS analysis also showed that PC molecular species with four or more double bonds migrated at the front of the TLC band of PCs. GPLs extracted from TLC plates occasionally contained PCs that were smaller than those in the original extract. These low molecular mass PCs were easily reduced to alcohols and formed derivatives with 2,4-dinitrophenylhydrazine, suggesting that aldehydes were generated by the oxidation of unsaturated fatty acids. Directly analyzing lipid extracts by ESI-MS/MS without preliminary chromatographic separation gives an accurate distribution of GPL molecular species in lipid mixtures. However, the ionization of the phospholipids in the electrospray jet maximized at relatively low concentrations of GPL. There was a linear response between phospholipid mass and ion intensity for concentrations around 1-2 nmol/ml for both PC and PE. The total ion intensity continued to increase with concentrations above 1-2 nmol/ml, but the response was non-linear.     

Analysis of phospholipid molecular species in brains from patients with infantile and juvenile neuronal-ceroid lipofuscinosis using liquid chromatography-electrospray ionization mass spectrometry

Phospholipids (PL) in cerebral cortex from patients with infantile (INCL or CLN1) and juvenile (JNCL or CLN3) forms of neuronal ceroid-lipofuscinosis (NCL) and controls were analysed by normal phase HPLC and on-line electrospray ionization ion-trap mass spectrometric detection (LC-ESI-MS). The method provided quantitative data on numerous molecular species of different PL classes, which are not achieved by using the conventional chromatographic methods. Compared with the controls, the INCL brains contained proportionally more phosphatidylcholine (PC), and less phosphatidylethanolamine (PE) and phosphatidylserine (PS). Different molecular species of PC, PE, PS, phosphatidylinositol and sphingomyelin were quantified using multiple internal PL standards that differed in fatty acyl chain length and thus allowed correction for chain length dependency of instrument response. In INCL cortex, which had lost 65% of the normal PL content, the proportions of polyunsaturated molecular species, especially the PS and PE that contained docosahexaenoic acid (22:6n-3), were dramatically decreased. The membranes may have adapted to this alteration by increasing the proportions of PL molecules substituted with monounsaturated and short-chain fatty acids. Lysobisphosphatidic acid was highly elevated in the INCL brain and consisted mostly of polyunsaturated species. It is possible that changes in the composition of PL membranes accelerate progression of INCL by altering signalling and membrane trafficking in neurons.     

Identification ofArcobacter species using phospholipid and total fatty acid profiles

High-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) were used to analyze the phospholipids and fatty acids of four Arcobacter species (becoming routinely isolated from a wide variety of food sources, especially of animal origin) to provide information for the identification within these species. Phospholipid differences were observed in the HPLC profiles. GC-MS analysis provided a complete fatty acid composition for each arcobacter that after pattern recognition analysis allows taxonomic classification of each species.     

Molecular Species of Diacylglycerols and Phosphoglycerides and the Postmortem Changes in the Molecular Species of Diacylglycerols in Rat Brains

The molecular species of 1,2-diacyl-sn-glycerol (DAG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2) from brains of adult rats (weighing 150 g) were determined. The DAG, isolated from brain lipid extracts by TLC, was benzoylated, and the molecular species of the purified benzoylated derivatives were separated from each other by reverse-phase HPLC. The total amount and the concentration of each species were quantified by using 1,2-distearoyl-sn-glycerol (18:0-18:0) as an internal standard. About 30 different molecular species containing different fatty acids at the sn-1 and sn-2 positions of DAG were identified in rat brains (1 min postmortem), and the predominant ones were 18:0-20:4 (35%), 16:0-18:1 (15%), 16:0-16:0 (9%), and 16:0-20:4 (8%). The molecular species of PC, PE, PS, and PI were determined by hydrolyzing the lipids with phospholipase C to DAG, which was then benzoylated and subjected to reverse-phase HPLC. PIP and PIP2 were first dephosphorylated to PI with alkaline phosphatase before hydrolysis by phospholipase C. The molecular species composition of phosphoinositides showed predominantly the 18:0-20:4 species (50% in PI and approximately 65% in PIP and PIP2). PS contained mainly the 18:0-22:6 (42%) and 18:0-18:1 (24%) species. PE was mainly composed of the 18:0-20:4 (22%), 18:0-22:6 (18%), 16:0-18:1 (15%), and 18:0-18:1 (15%) species. In PC the main molecular species were 16:0-18:1 (36%), 16:0-16:0 (19%), and 18:0-18:1 (14%). Studies on postmortem brains (30 s to 30 min) showed a rapid increase in the total amount (from 40-50 nmol/g in 0 min to 210-290 nmol/g in 30 min) and in all the molecular species of DAG. Comparatively larger increases (seven- to 10-fold) were found for the 18:0-20:4 and 16:0-20:4 species. Comparison of DAG species with the molecular species of different glycerolipids indicated that the rapid postmortem increase in content of DAG was mainly due to the breakdown of phosphoinositides. However, a slow but continuous breakdown of PC to DAG was also observed.     

Analysis of glycerophosphocholine molecular species as derivatives of 7-[(chlorocarbonyl)-methoxy]-4-methylcoumarin.

A method has been developed for the analysis of derivatized diradylglycerols obtained from glycerophosphocholine (GPC) of transformed murine bone marrow-derived mast cells that provided high performance liquid chromatography (HPLC) separation of GPC subclasses and molecular species separation with on-line quantitation using UV detection. In addition, the derivatized diradylglycerol species were unequivocably identified by continuous flow fast-atom bombardment mass spectrometry. GPC was initially isolated by thin-layer chromatography (TLC), the phosphocholine group was hydrolyzed, and the resultant diradylglycerol was derivatized with 7-[(chlorocarbonyl)-methoxy]-4-methylcoumarin (CMMC). After separation of the derivatized subclasses by normal phase HPLC, the individual molecular species of the alkylacyl and diacyl subclasses were quantitated and collected during a subsequent reverse phase HPLC step. With an extinction coefficient of 14,700 l mol-1 cm-1 at a wavelength detection of 320 nm, the CMMC derivatives afforded sensitive UV detection (100 pmol) and quantitation of the molecular species. Continuous flow fast-atom bombardment mass spectrometry of the alkylacyl CMMC derivatives yielded abundant [MH]+ ions and a single fragment ion formed by loss of alkylketene from the sn-2 acyl group, [MH-(R = C = O)]+. No fragmentation of the sn-1 alkyl chain was observed. Diacyl derivatives also produced abundant [MH]+ ions plus two fragment ions arising from loss of RCOOH from each of the acyl substituents and two fragment ions from the loss of alkyketene from each acyl group. Individual molecular species substituents were assigned from these ions.     

Biosynthesis of diacylglycerols by rat intestinal mucosa in vivo.

The biosynthesis of diacylglycerols was studied in rat intestinal mucosa during in vivo absorption of a low molecular weight fraction fraction of butter oil and of the corresponding medium and long chain fatty acids. The experimental fat solutions were given by stomach tube to the animals after a 24-h fast and mucosal scraping were collected 3 h later. The lipids were isolated and the acylclycerols determined by combined thin-layer chromatography gas-liquid chromatography techniques and stereospecific analyses. Free fatty acid feeding led mainly to sn-1,2-diacyl-glycerols, which contained exogenous and endogenous fatty acids. During triacylglycerol feeding, both sn-1,2-and sn-2,3-diacylglycerols were recovered in significant amounts from the intestinal mucosa. The composition of the sn-2,3-diacylglycerols corresponded to that with exogenous fatty acids but the sn-1,2-diacylglycerols clearly contained both exogenous and endogenous fatty acids. In all cases it was possible to isolate endogenous sn-1,2-diacylglycerols made up largely of species with linoleic and arachidonic acids in the 2 position and palmitic and stearic acids in the 1 position, which apparently were not converted to triacylglycerols. The in vivo reacylation of 2-monoacylglycerols via both sn-1,2- and sn-2,3-diacylglycerols is in agreement with similar findings in vitro with everted sacs of rat intestinal mucosa.     

Determination and pharmacokinetics of orientin in rabbit plasma by liquid chromatography after intravenous administration of orientin and Trollius chinensis Bunge extract

A high-performance liquid chromatography (HPLC) method was developed and validated for the determination of orientin in rabbit plasma using ultraviolet (UV) absorbance detection. Orientin is the active constituent of purified herbal extract (TRO PE) from the flower of Trollius chinensis Bunge. Protein precipitation was used as the sample preparation technique. A Diamonsil C18 column (150 mmx4.6 mm, 5 microm) was equilibrated with a mobile phase composed of 0.1% acetic acid/methanol/acetonitrile (80/5/15, v/v/v). The calibration curve of orientin in rabbit plasma was linear in the concentration range of 0.530-53.0 microg/mL. This validated method was successfully applied to a pharmacokinetic study in rabbits after the intravenous administrations of orientin and TRO PE at three different doses.     

Reversed phase LC/MS/MS method for targeted quantification of glycerophospholipid molecular species in plasma

The relationship between lipid status and metabolism, infant development and health has widely been studied, but the importance of individual glycerophospholipid species for biological functions in infants has hardly been considered. We developed a method for quantitative analyses of plasma glycerophospholipids from small sample volume. Proteins were precipitated with methanol, which eliminated further sample preparation. The supernatant was analysed by reversed-phase HPLC using a gradient of water, methanol and isopropanol as mobile phase. Electrospray ionisation in negative mode in combination with tandem mass spectrometry enabled detection of specific fatty acids as fragments of glycerophospholipid species. With this combination of chromatography and mass spectrometry, PC, lyso-PC, PE and lyso-PE species and their relevant isobaric compounds were quantified. Method validation showed a linear working range between 0.05 μmol/L and 10 μmol/L in diluted plasma samples. The intra-assay coefficients of variation (n=6) ranged from 1.1% to 13.9%. Results were comparable with data of the human metabolome database and gas chromatographic fatty acid analyses. All quantitatively important PE and PC species are covered. The method can be applied for investigating dietary effects on plasma GP composition from small plasma volumes.     

Phospholipids of the differentiating species of Tetrahymena

The whole-cell phospholipid composition of the six known polymorphic species of Tetrahymena has been examined by [(3)H]acetate and [(3)H]myristic acid radiolabeling, and by gas-liquid chromatography of total phospholipid-bound fatty acids. Five of the polymorphic species contained similar phospholipid profiles following radiolabeling in that phosphatidylethanolamine (PE) was the predominant phospholipid; however, in cells of Tetrahymena patula LFF, aminoethylphosphonolipid was present in amounts nearly equal to PE. Tetrahymena patula LFF contained an unusually large percentage of sphingolipid (16.2% by [(3)H]acetate radiolabeling). Substantial differences were found in the fatty acid profiles of the polymorphic species, which included the degree of fatty acid unsaturation and relative weight percentages of odd-chain fatty acids. Tetrahymena vorax contained a low ratio of unsaturated C(18) fatty acids to saturated C(18) fatty acids as compared with all other species examined. The differentiating species generally contained a lesser percentage of monoenoic fatty acids and a lower ratio of unsaturated C(16) fatty acids to saturated C(16) fatty acids as compared with the two monomorphic species examined.     

Aminophospholipid molecular species asymmetry in the human erythrocyte plasma membrane

The transbilayer distribution of the molecular species of aminophospholipids in human red blood cell plasma membrane has been investigated using a covalent labelling technique. Separation and quantitative analysis of the molecular species of phosphatidylethanolamine (PE) and phosphatidylserine (PS) was performed using high-performance liquid chromatography with UV detection of the trinitrophenyl derivatives obtained after reaction with trinitrobenzenesulfonic acid (TNBS). When the molecular species distribution obtained with intact cells was compared to that of the whole membrane, a molecular species asymmetry was evident. This phenomenon was most clearly evident when the reaction was performed at low temperatures (0 degrees C) and was obscured by the excessive labelling or probe permeation associated with higher temperatures or longer incubation times. The monoene species were enriched in the outer leaflet, they comprised about 30% of the PE species in this leaflet. The polyunsaturates were preferentially localized in the inner leaflet and this was true of the arachidonyl species in particular as they represented up to 35% of this pool. The w-3 polyunsaturated fatty acids displayed a preferential localization in the plasmalogen subclass in comparison to the diacyl fraction, i.e., they comprised about 58 of the former and 42% of the latter subclass of cellular PE w-3 species. Data concerning the separation, identification and quantification of PS molecular species in human erythrocytes is also presented. The internal localization of the polyunsaturated species as well as the compartmentalization of the w-3 and w-6 pools will have metabolic, structural and physical implications for membrane function.     

Determination of trace levels of fatty acid metal salts by high-performance liquid chromatography with fluorescence prelabeling

A simple method is described for picomole determinations of fatty acid metal salts. Fatty acid salts are directly labeled with 4-bromomethyl-7-methoxycoumarin in the presence of excess ethylenediaminetetraacetic acid tripotassium salt without any solvent extractions. The fluorescence derivatives of fatty acids are separated by reverse-phase high-performance liquid chromatography followed by fluorometric detection. The response of each fatty acid (C8-C18) calcium salt is linear from 1 to 50 micrograms/ml of samples. The detection limit is about 7 pmol. Good recoveries are obtained for the calcium salts of myrystic acid and soap (C8-C18, C18:1,2). The new method is successfully applied to the study on biodegradation of fatty acids in river water.     

Molecular species of diacylphosphatidylethanolamine in rat and mouse heart given the same diet

Diacylphosphatidylethanolamine (PE) was isolated from mouse and rat heart given the same standard diet. The molecular species of PE were determined after conversion of PE into diglycerides by means of hydrolysis with phospholipase C, subsequent hydrolysis with pancreatic lipase and separation of the products by argentation TLC and capillary gaschromatography. Docosahexaenoic acid (22:6n3) containing molecular species and arachidonic acid (20:4n6) containing molecular species represented the major fractions. A preference for stearic acid to combine with poly-unsaturated fatty acids was found. Despite an abundant presence of linoleic acid (18:2n6) in the diet, molecular species containing this fatty acid represented only a minor fraction. The possible physico-chemical and physiological meaning of the presence of molecular species containing many double bonds is discussed.     

Diaglycerol biosynthesis in everted sacs of rat intestinal mucosa.

The molecular specificity in the biosynthesis of diacylglycerols by rat intestinal mucosa was examined by means of radioactive markers, thin-layer chromatography with silver nitrate and gas-liquid chromatography with radioactivity monitoring. Bile salt micelles of alternately labeled monoacyglycerols and free fatty acids were incubated with everted sacs of intestinal mucosa for various periods of time and the diacylglycerols were isolated by solvent extraction and thin-layer chromatography. Sterospecific analyses of the X-1,2-diacylglycerols labeled from 2-monoacylglycerols showed that the sn-1,2-isomers (45-55%) were slightly in excess of the sn-2,3-isomers (34-45%) with the X-1,3-diacylglycerols accounting for the rest of the radioactivity (5-10%). This suggests that racemic diacylglycerols may be intermediates in the resynthesis of dietary fat in rat intestinal mucosa. Detailed analyses of the molecular species of the sn-1,2-diacylglycerols labeled from free fatty acids revealed that 10-45% of the total did not contain the acid present in the 2-monoacylglycerol supplied, and therefore had originated from the phosphatidic acid pathway. These findings are at variance with those obtained in isolated microsomes, which have suggested an inhibition of the phosphatidic acid pathway by monoacylglycerols as well as have given evidence of an exclusive synthesis of sn-1,2-diacylglycerols from 2-monoacylglycerols.     

Analysis of molecular species of plant polar lipids by high-performance and gas liquid chromatography

Total lipid extracts from potato tubers and tobacco leaves are separated into lipid classes by two step HPLC using a silicic column. Elution is first performed for 20 min with a programmed linear gradient of two mixed solvents running from 100% of solution A (isopropanol-hexane, 4:3) to 100% of solution B (isopropanol-hexane-water, 8:6:1.5); the column is then eluted with pure solution B in an isocratic mode for 20 min more. The main polar lipids (MGDG, DGDG, PC, PE, PG) from both plant tissues can be collected and further separated into component molecular species on a simplified HPLC system with a C₁₈ column eluted in an isocratic mode with a polar solvent. Molecular species separations are achieved within 35 min; quantifications are made through GLC analysis of attached fatty acids. Three to five main molecular species are thus clearly identified in each lipid class. In potato tuber, phospholipids (PC, PE) 18:2/18:2 species are predominant. In tobacco leaf, six double bond species (18:3/18:3 and 16:3/18:3) are predominant in galactolipids, whereas PC contains a greater number of molecular species varying by their degree of unsaturation (from 18:3/18:3 to 16:0/18:2). Only certain molecular species of PG contain Δ³-trans-hexadecenoic acid.     

Analysis of aminophospholipid molecular species by high performance liquid chromatography

A new method is described for the separation of individual molecular species of the aminophospholipids, phosphatidylethanolamine and phosphatidylserine. Trinitrobenzene-sulfonic acid was used to derivatize both aminophospholipids and the derivatives were purified by thin-layer chromatography. A reversed-phase high performance liquid chromatography technique was developed to separate and quantify individual molecular species based upon ultraviolet detection of the attached chromophore. The retention times of the molecular species on the C18 reversed-phase column were longer with increasing carbon chain length and decreasing degree of unsaturation of fatty acyl chain. The overall procedure allowed a quantitative recovery of the aminophospholipid species. The lower limit of detection was about 10 pmol and a linear response was observed in the range of 0.1-10 nmol of phospholipid. Using this method, we were able to separate and quantify trinitrophenyl-phosphatidylethanolamine molecular species of both subclasses (diacyl and alkenyl) from human red blood cells and rat brains. Separation of species was confirmed by gas-liquid chromatographic analysis of the fatty acid content of each peak and by thermospray liquid chromatography-mass spectrometry. This new method provides a convenient and sensitive technique for studies of aminophospholipid molecular species composition. Furthermore, it appears to be a useful tool for the analysis of asymmetric distribution of these species in biological membranes.     

Homeoviscous adaptation under pressure. III. The fatty acid composition of liver mitochondrial phospholipids of deep-sea fish

Homeoviscous adaptation of biological membranes to high hydrostatic pressure has been investigated by determining the differences in lipid composition of membranes from fish obtained from depths between 200 and 4000 m. The fatty acid composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine/inositol and cardiolipin from a liver mitochondrial fraction was analysed by capillary gas-liquid chromatography. The ratio of saturated to unsaturated fatty acids significantly and negatively related to depth in PC and PE as predicted by homeoviscous adaptation to pressure. Thus, deep sea species possess greater proportions of unsaturated fatty acids than shallow species. Cardiolipin showed the opposite trend. An unsaturation index was not significantly related to depth in any phospholipid fraction.     

Structural analysis of phosphatidylcholine of plant tissue

Pure preparations of phosphatidylcholine were isolated from spinach leaf chloroplasts, spinach leaf microsomes, and cauliflower inflorescence. The isolated phosphatidylcholine was treated with snake venom phospholipase A, and the fatty acid distribution and composition of the fatty acid methyl esters prepared from the lysophosphatidylcholine and the freed fatty acid were determined by gas-liquid chromatography. The results showed that saturated fatty acids were preferentially esterified at position 1 and unsaturated fatty acids at position 2. The phosphatidylcholine from cauliflower was also treated with phospholipase C. The resulting diglycerides were fractionated on AgNO(3)-impregnated thin-layer plates. The diglyceride fractions were transesterified and the fatty acid composition of each was determined by gas-liquid chromatography. The predominant species contained linolenic acid only (22% of the total), linolenic and oleic acids (19%), and linolenic and palmitic acids (37%). These molecular species could not be accounted for by random distribution of the fatty acids.     

Determination of molecular species of enantiomeric diacylglycerols by chiral phase high performance liquid chromatography and polar capillary gas-liquid chromatography

A simple method is described for the determination of molecular species of enantiomeric sn-1,2- and sn-2,3-diacylglycerols derived from natural triacylglycerols by Grignard degradation. The method is based on a preparative separation of the enantiomeric diacylglycerols as 3,5-dinitrophenylurethane (DNPU) derivatives by high performance liquid chromatography (HPLC) on a chiral column (25 cm x 4.6 mm ID) containing R-(+)-1-(1-naphthyl)ethylamine as a stationary phase. This is followed by polar capillary gas-liquid chromatography (GLC) of the trimethylsilyl (TMS) ether derivatives of the enantiomeric diacylglycerols derived from the DNPU derivatives using trichlorosilane, which does not cause acyl migration and racemization during the reaction. The cleavage is better than 94% complete. The method was standardized with synthetic sn-1,2- and sn-2,3-dipalmitoyl- and rac-1,2-dioleoylglycerols and was applied to the identification and quantitation of individual molecular species of enantiomeric diacylglycerols generated by Grignard degradation of the triacylglycerols from corn oil, cocoa butter, and lard.     

Molecular analysis of the phospholipids of Escherichia coli k12

Phospholipids from Escherichia coli K12 were converted to 1,2-diacylglycerols with phospholipase C from Bacillus cereus. High-pressure liquid chromatography of 1,2-diacylglycerol p-methoxybenzoates on LiChrosorb RP-18 using 2-propanol/acetonitrile (35:65) as eluant permitted separation of 14 molecular species. The main combinations of fatty acids were 1-16:0-2-16:1, 1-16:0-2-cyclo-17:0 and 1-16:0-2-18:1. Positional isomers were not present. The 1,2-di-16:0 compound was present at a significant level (7-10 mol%). Proportions of molecular species varied between phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Phospholipid from the outer membrane of E. coli K12 contained a lower level of molecules with two unsaturated chains than was present in the cytoplasmic membrane. The method is sensitive, has good resolving power and employs readily available equipment.