J. J. Myher

Molecular species of glycerophospholipids and sphingomyelins of human erythrocytes: Improved method of analysis

This study reports the application of modern methods of molecular species analysis in determination of the structure of both major and minor glycerophospholipids and sphingomyelins of human erythrocytes. Individual phospholipid classes were resolved from total lipid extracts by thin-layer chromatography. Diradylglycerols were released by phospholipase C and converted into trimethylsilyl ethers, which were resolved into the alkenylacyl, alkylacyl and diacylglycerol subclasses by normal phase high performance liquid chromatography. Molecular species of diradylglycerols and ceramides were quantitated according to carbon and double bond number by gas liquid chromatography using a fused silica capillary column wall-coated with bonded RTx-2330. The molecular species of ceramides were determined by GC/MS. The diradyl glycerophosphocholines contained 93.0% diacyl, 4.6% alkylacyl and 2.5% alkenylacyl, white the diradyl glycerophosphoethanolamines were made up of 48.8% diacyl, 47.8% alkenylacyl and 3.4% alkylacyl subclasses. Analysis of the molecular species showed that the long chain polyunsaturated acids were mainly combined with C16 in all diradyl GPC subclasses and in diacyl GPE, while in the alkylacyl and alkenylacyl GPE and in diacyl glycerophosphoinositol and diacyl glycerophosphoserine they were combined mainly with C18 saturated fatty chains. In addition to the C16 and C18 alkyl and alkenyl, the ether fractions also contained significant proportions of C20, C22 and C24 chains. The molecular species of the ceramide moieties of the SPH were made up largely of mono- and diunsaturated species. Over 200 molecular species were identified and quantitated in a representative sample of human red blood cells.

Molecular Species of Glycerophospholipids and Sphingomyelins of Human Plasma: Comparison to Red Blood Cells

In addition to diacyl glycerophosphocholine and sphingomyelin, human plasma also contains small amounts of other glycerophospholipids, which may have special metabolic function. The structure and origin of these minor plasma lipids has not been determined. Knowledge of the detailed composition of the phospholipids of red blood cells (Myheret al., Lipids 24, 1989) permits evaluation of one of the possible sources. This study reports the detailed analyses of plasma glycerophospholipids made in parallel to those of the erythrocyte lipids obtained from the same blood using HPLC and GLC methods. The proportions of the major phospholipid classes in the plasma and erythrocytes were similar to published values, including the essential absence of diradyl glycerophosphoserine from plasma. Plasma diradyl glycerophosphocholine contained 93.0% diacyl, 3.4% alkylkacyl and 3.6% alkenylacyl, whereas the diradyl glycerophosphoethanolamine consisted of 71.8% alkenylacyl, 19.9% diacyl and 8.3% alkylacyl subclasses. The diradyl glycerophosphoinositol was 100% diacyl. The content of the minor subclasses of plasma diradyl glycerophosphocholine is similar to that of the red cells, but the ether content of the diradyl glycerophosphoethanolamine is higher in plasma than in cells. The lipid ether subclasses of plasma glycerophospholipids also contained a higher proportion of the C20, C22 and C24 alkyl and alkenyl chains than those of the cells. Furthermore, the C16 and C18-containing species in diradyl glycerophosphoethanolamine subclasses varied with the nature of the polyunsaturated acid, whereas in diradyl glycerophosphocholine subclasses the polyunsaturated acids were combined with the C16 and C18 acids in equal proportions. The significant differences in the molecular species of glycerophospholipids and sphingomyelin between plasma and red cells would appear to limit any direct transfer or equilibration of their lipid components.

Triglyceride structure of milk fats.

The enantiomeric nature of the triglycerides of bovine milk fat was reinvestigated by determining the stereospecific distribution of fatty acids in rearranged butterfat, following partial hydrolysis with pancreatic lipase, and in certain molecular distillates of native butterfat, following Grignard degradation. The results with rearranged butterfat confirmed the validity of pancreatic lipase hydrolysis as a means of generating representative diglycerides from milk fat triglycerides. The Grignard degradation and lipolysis gave identical distributions for fatty acids when included as part of the assay system in the stereospecific analysis. Characteristically, butyric acid and the other short chain acids occupied the 3 position in the native butterfat, while in the rearranged oil they were distributed more or less randomly. Gas chromatographic analysis of the short chain glycerides on polyester columns allowed an effective resolution of butyryl, caproyl and caprylyl glycerides of identical numbers of total acyl carbons and double bonds. The method was especially well suited for resolution of the 2,3-diglycerides, which were recovered either as the more polar fraction from thin layer chromatography of the X-1,2-diacylglycerols, or by acetolysis of the residual phenolphosphatides resulting from phospholipase A digestion. It was shown that butyric acid in the 3 position was preferentially paired with myristic, palmitic and oleic acid in the 2 position, and palmitic and oleic acid in the 1 position, which was also characteristic of the other short chain acids.

General strategies in chromatographic analysis of lipids

Lipid extracts of natural sources contain a large number of lipid classes and molecular species. Completely reproducible samples are obtained only with great care and skill. Analytical methods other than chromatography and/or mass spectrometry are of little use for resolution and identification of lipid molecules even in simple mixtures. The analytical information desired governs the selection of the chromatographic and mass spectrometric method, which determine the sample preparation and derivative needed. Usually a combination of chromatographic methods is necessary to identify specific species of lipids. The recent development of soft ionization techniques, that are readily interfaced with mass spectrometers, have greatly simplified the sample preparation and have largely eliminated the need for derivatization. Because these techniques require expensive equipment and dedicated operators, the methods selected must be consistent with the true analytical needs and the available resources. Although personal preference cannot be eliminated entirely, the general strategies outlined below should help to reduce the number of possibilities facing a lipid analyst to a few practical choices.

Isolation and identification of glycated aminophospholipids from red cells and plasma of diabetic blood

Glycosylation is a major pathway for posttranslational modification of tissue protein and begins with non‐enzymatic addition of carbohydrate to the primary amino groups. Excessive glycation of tissue protein has been implicated in the pathogenesis of diabetes and ageing. While glycation of aminophospholipids has also been postulated, glycated aminophospholipids have not been isolated. Using normal phase HPLC with on‐line electrospray mass spectrometry we found glycated ethanolamine phospholipids to make up 10–16% of the total phosphatidylethanolamine (PE) of the red blood cells and plasma of the diabetic subjects. The corresponding values for glycated PE of control subjects were 1–2%.

Identification of the more complex triacylglycerols in bovine milk fat by gas chromatography—mass spectrometry using polar capillary columns

The fourth most volatile 2.5% molecular distillate of butteroil obtained by redistillation of the most volatile 10% cut was examined by gas chromatography on a polar capillary column (RSL-300) with electron impact and chemical ionization mass spectrometry. For this purpose the distillate was first freed from the acetyldiacylglycerols by thin-layer chromatography on plain silica gel and the remainder resolved into long and short chain length saturates, cis- and trans-monoenes, dienes and trienes by thin-layer chromatography on silver nitrate-silica gel. The order of gas chromatographic elution was established for more than 100 major and minor species making up the bulk of the molecular distillate. The results were used to derive the quantitative composition of the triacylglycerol species making up the various peaks obtained by polar capillary column gas chromatography of the total molecular distillate, which closely resembles the lower half of the molecular mass distribution of whole bovine milk fat.

Determination of lipid ester ozonides and core aldehydes by high-performance liquid chromatography with on-line mass spectrometry

Unsaturated triacylglycerols (TG) and choline (PC) and ethanolamine (PE) phosphatides of known structure were subjected to ozonization and reduction with triphenylphosphine to yield the corresponding lipid ester core aldehydes. Mono- and di-C9 aldehyde palmitoylglycerols were prepared from oleoyldipalmitoyl and oleoyllinoleoylpalmitoyl glycerols, respectively, while egg yolk PC and PE provided the mono-C5 and mono-C9 aldehydes of palmitoyl-and stearoyl glycerophospholipids. The aldehydes were isolated in the free form and as the dinitrophenylhydrazone (DNPH) derivatives by thin-layer chromatography (TLC). The intermediate ozonides, free aldehydes and hydrazones were identified by reversed phase high performance liquid chromatography (HPLC) with on-line negative ion thermospray and normal phase HPLC with on-line positive ion electrospray mass spectrometry (LC-MS). The synthetic aldehydes were used as carriers during isolation from natural sources and as reference compounds in quantitative analyses.

Acylglycerol structure of peanut oils of different atherogenic potential.

Detailed investigation was made of the triacylglycerol structure of native, simulated, and interesterified peanut oils, which had previously been shown to differ markedly in their atherogenic potential. By means of chromatographic and stereospecific analyses, it was shown that the more atherogenic native oil contains a significantly greater proportion of triacylglycerols with linoleic insn-2-position and arachidic, behenic, and lignoceric acids insn-3-position than the synthetic oils. It is suggested that the atherogenicity may arise from a relative metabolic unavailability of the linoleic acid from the native oil, which may be due in part to the presence of long chain saturated acids in the outer position. This might render the oil metabolically more saturated than the interesterified oils of the same total fatty acid composition, which contain a much greater proportion of the linoleic acid in the primary positions of the triacylglycerol molecule. The identification of specific triacylglycerols may allow the experimental testing of this hypothesis by feeding synthetic triacylglycerols incorporating the potentially atherogenic features.

Determination of plasma total lipid profiles by capillary gas-liquid chromatography

We have obtained improved qualitative and quantitative information about plasma lipid composition by adopting capillary gas-liquid chromatography to the determination of plasma total lipid profiles. The new procedure takes advantage of the good recoveries of high molecular weight solutes provided by short capillary columns as well as of the absence of sample fractionation during on-column injection. The major improvements in the gas-liquid chromatographic determination of plasma lipid profiles are the greatly increased carbon number resolution of the solutes and the minimal sample size requirements on the capillary columns. In many instances there is a partial resolution of the saturated and unsaturated molecules within a carbon number, which provides clues to the fatty acid composition of the molecular species. In other respects the new plasma total lipid profiles are similar to those obtained on packed columns. The plasma lipids are dephosphorylated by phospholipase C and trimethylsilylated before analysis.

Quantitation of natural triacylglycerols by reversed-phase liquid chromatography with direct liquid inlet mass spectrometry

Using acetonitrile and propionitrile as eluting solvents and reagent gases the yields of both quasi-molecular and fragment ions were found to vary with the molecular weight, degree of unsaturation and positional distribution of the fatty acids in the triacylglycerol molecule, and appropriate calibration factors were necessary for accurate quantitation. In the absence of pure structural isomers and mixed acid standards, preliminary calibration factors have been determined for total ion and specific ion current responses by comparing the peak area ratios obtained by liquid chromatography-mass spectrometry with the proportions of the molecular species known to be present in randomized oils and in natural oils of known chemical composition. Although the derived factors include both chromatographic and mass spectrometric effects and are obtained with a gradient of reagent gases, they appear to be generally applicable. It was shown that positional isomers affected the yield of the (MH-RCOOH)+ ions over a 1-3-fold range of intensities, while the nature of the fatty acid affected it over a range of 1.25-fold. After suitable calibration of the relative ion responses it was possible to determine the identities and amounts of the individual molecular species in natural fats and oils.