Elution factors of synthetic oxotriacylglycerols as an aid in identification of peroxidized natural triacylglycerols by reverse-phase high-performance liquid chromatography with electrospray mass spectrometry.

Selected elution factors were determined for model oxotriacylglycerols as an aid in identification of the peroxidation products of natural triacylglycerols by reverse-phase high-performance liquid chromatography (HPLC) with electrospray mass spectrometry (LC/ES/MS). For this purpose synthetic triacylglycerols of known structure were converted to hydroperoxides, hydroxides, epoxides, and core aldehydes and their dinitrophenylhydrazones by published procedures. The oxotriacylglycerols were resolved by normal-phase thin-layer chromatography and reverse-phase HPLC, and the identities of the oxotriacylglycerols confirmed by LC/ES/MS. Elution factors of oxotriacylglycerols were determined in relation to a homologous series of saturated triacylglycerols, ranging from 24 to 54 acyl carbons, and analyzed by reverse-phase HPLC, using a gradient of 20-80% isopropanol in methanol as eluting solvent and an evaporative light-scattering detector. It was shown that the elution times varied with the nature of the functional group and its regiolocation in the triacylglycerol molecule. A total of 31 incremental elution factors were calculated from chromatography of 33 oxygenated and nonoxygenated triacylglycerol species, ranging in carbon number from 36 to 54 and in double-bond number from 0 to 6.

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 nonenzymatic 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%.

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.

Preparation and characterization of glucosylated aminoglycerophospholipids.

Natural aminophospholipids were isolated from egg yolk and from human red blood cells. Glucosylated ethanolamine and serine phosphatides were prepared by exposing synthetic and natural aminophospholipids to glucose for 3-18 h at pH 7.4. The glucosylation products were resolved from parent phospholipids by normal-phase high-performance liquid chromatography and were identified by on-line mass spectrometry with an electrospray interface. The soft ionization method allowed us to detect the glucosylation products as molecular ions of the Schiff bases. The Schiff bases could be stabilized by sodium cyanoborohydride reduction. The molecular species of the ethanolamine and serine phosphatides reacted in proportion to their molar concentration in the mixtures. The yields of the glucosylation products varied with time of reaction and the concentration of glucose in the medium. At 50 mM glucose and 0.6 mg/mL phosphatidylethanolamine, 20% of the aminophospholipid was glycated in 18 h at 37 degrees C.

Lipid ester-bound aldehydes among copper-catalyzed peroxidation products of human plasma lipoproteins.

We have isolated the core aldehydes (aldehydes still bound to parent molecules) of phosphatidylcholine (PC) and cholesteryl esters (CE) from copper-catalyzed peroxidation of human plasma low (LDL) and high (HDL) density lipoproteins. The aldehydes were isolated by extraction with acidified chloroform-methanol containing 2,4-dinitrophenylhydrazine. The 2,4-dinitrophenylhydrazone (DNPH) derivatives formed were resolved by reversed phase high performance liquid chromatography (HPLC) and identified by on-line quadrupole mass spectrometry (LC/MS). The major PC core aldehydes from oxidized LDL and HDL were identified as 1-palmitoyl-(1-stearoyl) 2-(9-oxononanoyl)-, 1-palmitoyl-(1-stearoyl) 2-(8-oxooctanoyl)-, and 1-palmitoyl-(1-stearoyl) 2-(5-oxovaleroyl)-sn-glycerols after phospholipase C digestion of the DNPH derivatives of the phospholipids. The major aldehydes from peroxidation of cholesteryl esters were the 9-oxononanoyl, 8-oxooctanoyl, and 5-oxovaleroyl esters of cholesterol and 7-ketocholesterol. The core aldehydes were estimated to account for a minimum of 1-2% of the consumed linoleate and arachidonate esters. A relatively smaller yield of the PC core aldehydes from LDL compared to HDL was attributed to the presence of greater amounts of phospholipases in LDL than in HDL. More comparable yields of PC core aldehydes were obtained in the presence of phenylmethylsulfonylfluoride, which inhibits phospholipases. We conclude that peroxidation of LDL and HDL results in formation of detectable amounts of cholesteryl and glycerophospholipid esters containing aldehyde functions. The yield of PC aldehydes varies with the activity of the platelet activating factor (PAF) acetyl hydrolase.

Plasma and urine enrichments following infusion of L-[1-13C]phenylalanine and L-[ring-2H5]phenylalanine in humans: evidence for an isotope effect in renal tubular reabsorption.

Amino acids labeled with 13C or deuterium are commonly used in studies of amino acid metabolism. Traditionally, amino acid flux has been estimated by measurement of isotopic enrichment in the plasma pool; however, urine sampling as a noninvasive means of determining isotope enrichment has been increasing. The isotope enrichments and fluxes estimated from plasma and urine sampling were compared when two phenylalanine tracers (L-[1-13C]phenylalanine and L-[ring-2H5]phenylalanine) were intravenously infused for 4 hours in seven healthy men. This is the first evaluation of these isotopes as urinary tracers for assessing amino acid metabolism in adult humans. Before infusion, the mean ratio of plasma to urine (P:U) isotope enrichment was 0.99 +/- 0.03 (SD) and 0.99 +/- 0.02 for [13C]phenylalanine and [13C]tyrosine, respectively (isotope enrichment of [2H5]phenylalanine is zero at baseline). At isotopic steady state, the ratio was 1.06 +/- 0.05, 0.98 +/- 0.03, and 0.60 +/- 0.10 for [13C]phenylalanine, [13C]tyrosine, and [2H5]phenylalanine, respectively. The [13C]phenylalanine isotope showed a high correlation (R2 = .96) between enrichment in plasma and urine. However, use of [2H5]phenylalanine resulted in a significantly higher enrichment in urine than in plasma. Since amino acid flux is inversely related to enrichment, urine sampling would result in an underestimation of flux. The plasma to urine difference is probably due to discrimination of the [2H5]phenylalanine isotope in renal transport; therefore, this isotope may not be suitable for in vivo use where cellular transport mechanisms are involved.

Identification of core aldehydes among in vitro peroxidation products of cholesteryl esters.

Synthetic cholesteryl 5-oxovalerate and 9-oxononanoate were used as reference standards for the isolation and identification of cholesteryl ester core aldehydes from tert-butyl hydroperoxide/Fe++ oxidation of synthetic and natural cholesteryl esters. The core aldehydes were recovered from the peroxidation products by thin-layer chromatography as the free aldehydes or the 2,4-dinitrophenylhydrazones and were identified, respectively, by gas-liquid chromatography (GLC) and by GLC combined with mass spectrometry (GC/MS) or by reverse-phase high-performance liquid chromatography (HPLC) and by HPLC with MS (LC/MS). The core aldehydes produced by peroxidation of cholesteryl linoleate were identified as mainly 9-oxononanoates of cholesterol and oxycholesterols, with smaller amounts of the 8-oxooctenoates, 10-oxodecenoates, 11-oxoundecenoates and 12-oxododecenoates. Peroxidation of cholesteryl arachidonate yielded 5-oxovalerates of cholesterol and the oxycholesterols as the main products with smaller amounts of the 4-oxobutyrates, 6-oxohexenoates, 7-oxoheptenoates, 8-oxooctenoates, 9-oxononenoates, 9-oxononadienoates and 10-oxodecadienotes. The oxycholesterols resulting from the peroxidation of the steroid ring were identified as mainly 7-keto-, 7 alpha-hydroxy- and 7 beta-hydroxy-cholesterols and 5 alpha,6 alpha- and 5 beta,6 beta-epoxy-cholestanols. Cholesteryl palmitate and oleate did not yield core aldehydes in the present peroxidation system. In these esters, the sterol and linoleic acid moieties appeared to be oxygenated at about the same rate, while the arachidonic acid moiety reacted more rapidly than did the sterol moiety.

Identification of cholesterol-bound aldehydes in copper-oxidized low density lipoprotein.

Lipid-soluble cholesteryl ester core aldehydes (aldehydes still bound to the cholesterol ring) were identified among the products of copper-catalyzed peroxidation of human low density lipoprotein (LDL). The LDL was exposed to oxygenated buffer and 5 microM CuSO4 for 24 h. The core aldehydes were isolated as the dinitrophenylhydrazones, and were identified by reverse-phase HPLC with mass spectrometry. The major components were the C4-C10 oxoalkanoyl esters of cholesterol and 7-ketocholesterol, and accounted for 1-2% of the cholesteryl linoleate and arachidonate consumed.

Plasma lipid profiling by liquid chromatography with chloride-attachment mass spectrometry.

A sensitive high-performance liquid chromatographic assay was developed using chloride attachment negative chemical ionization mass spectrometry for detection of glyceryl esters and ceramides, and positive chemical ionization mass spectrometry for detection of free cholesterol and cholesteryl esters in minimal quantities of plasma. The novel technique was validated by high temperature gas-liquid chromatography with flame ionization detection. Sample preparation was achieved by phospholipase C digestion of whole plasma, total lipid extraction and derivatization of any free carboxyl and hydroxyl groups by trimethyl- or tert-butyldimethyl-chlorosilane. The lipids were separated by reverse phase HPLC with 20-90% propionitrile in acetonitrile containing 1% dichloromethane, which served as the reagent and the source of chloride. Negative chemical ionization with chloride attachment is estimated to provide about 100 times higher response for the triacylglycerols and the trimethylsilyl or tert-butyldimethylsilyl ethers of diacylglycerols, and about 500 times higher response for the trimethylsilyl or tert-butyldimethylsilyl ethers of ceramides than positive chemical ionization mass spectrometry. Determination of the full negative chemical ionization mass spectra showed that each glycerolipid and ceramide species yielded a single ionic species corresponding to the chloride-attachment product of the parent ion. The cholesteryl esters and ethers failed to attach chloride and remained undetected by negative chemical ionization. However, the cholesteryl esters and ethers gave a high response for the steroid nucleus in positive chemical ionization mass spectrometry. Chloride attachment negative chemical ionization mass spectrometry is suitable for the unequivocal identification of plasma glycerolipids and ceramides in high-performance liquid chromatography and for the quantitation of molecular species in any unresolved peaks following appropriate calibration of the instrument response.

HPLC resolution of diacylglycerol moieties of natural triacylglycerols on a chiral phase consisting of bonded (R)-(+)-1-(1-naphthyl)ethylamine.

Chiral phase high performance liquid chromatographic resolution of sn-1,2(2,3)- and X-1,3-diacylglycerols generated by partial Grignard degradation from natural triacylglycerols was carried out using a chiral column (25 cm x 4.6 mm i.d.) containing (R)-(+)-1-(1-napthyl)ethylamine polymer chemically bonded to 300A wide pore spherical silica (5 microns particles). The diacylglycerols were chromatographed as 3,5-dinitrophenyl-urethanes and detected at 226 or 254 nm UV. By an isocratic elution with n-hexane- 1,2-dichloroethane-ethanol 40:10:1 (v/v/v) as the mobile phase, the sn-1,2(2,3)-diacylglycerols from corn, linseed, and menhaden oils were resolved into two clearly distinguishable enantiomer groups, although some peak overlappings between the enantiomers were observed in the linseed and menhaden oil diacylglycerols. In addition to the excellent enantiomer resolution, each enantiomer and the X-1,3-isomers were partially resolved into several peaks, which could be tentatively identified on the basis of equivalent carbon number. It is concluded that chiral phase high performance liquid chromatography can be utilized for effective resolution, identification, and quantitation of enantiomeric diacylglycerols from complex natural mixtures.

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.

Stereochemical course of intestinal absorption and transport of mustard-seed oil triacylglycerols in the rat.

Male rats with thoracic duct cannulae were intubated with mustard-seed oil or the corresponding fatty acid methyl esters and the lymph was collected over 0-24 h. The chylomicron and very low density lipoprotein fractions were obtained by conventional ultracentrifugation. The triacylglycerols and glycerophospholipids were isolated and the positional distribution and molecular association of fatty acids were determined by stereospecific and chromatographic methods. The oleic, linoleic, and linolenic acids were recovered in the lymph in the proportion in which they occurred in the fat fed, while eicosenoic, erucic, and lignoceric acids were rejected to about the same extent by the two pathways of intestinal triacylglycerol biosynthesis. It is shown that the lymph triacylglycerols arising via the monoacylglycerol or the phosphatidic acid pathway possess structures that are closely similar to each other and to that of the original mustard-seed oil. It is proposed that this is a result of comparable fatty acid and positional specificity of the acyltransferases associated with the acylglycerol synthesis in the animal and plant tissues and the wide range of fatty acid chain lengths in the mustard-seed oil.

Usefulness of gas chromatographic profiles of plasma total lipids in diagnosis of phytosterolemia.

A Cambodian male (aged 5 years and 9 months) presented with subcutaneous and tendon xanthomas in association with hypercholesterolemia. He was erroneously diagnosed as having familial hypercholesterolemia and treated with a low cholesterol diet (+/- cholestyramine) to which he did not respond. A determination of plasma total lipid profile by high-temperature gas chromatography revealed elevated plasma levels of free and esterified plant sterols along with the hypercholesterolemia. Introduction and maintenance of a diet low in cholesterol and plant sterols resulted in significant reduction in the blood concentration of these sterols, which returned to pretreatment level upon discontinuation of the low sterol regimen. The rapid identification and quantitation of the plant sterols by high-temperature gas chromatography provides a sensitive means of distinguishing phytosterolemia, which might be more common than previously suspected, from other forms of dyslipidemia, and for following the course of any treatment.

Fatty acid composition of individual plasma steryl esters in phytosterolemia and xanthomatosis.

The bulk of the plasma plant sterol in phytosterolemia occurs in the esterified form and is carried mostly in the low and high density lipoproteins. We have determined the fatty acid composition of the individual plasma steryl esters from a newly discovered subject with phytosterolemia and xanthomatosis. For this purpose the intact steryl esters were subject to high temperature gas liquid chromatography (GLC) on a polar capillary column, which separated the major esters on the basis of molecular weight and degree of unsaturation of the fatty acids. The saturated and unsaturated sterols esterified to saturated, monoenic, dienoic and tetraenoic fatty acids were identified by GLC analysis of the sterol moieties of the corresponding AgNO3-TLC fractions of the steryl esters. The GLC results were confirmed by reversed phase high performance liquid chromatography combined with mass spectrometry via direct liquid inlet interface. It was found that, in general, each fatty acid was esterified to the same complement of sterols, and that the esterified sterols possessed a composition comparable to that of the free plasma sterols, which was comprised of about 75% cholesterol, 6% campesterol, 4% 22,23-dihydrobrassicasterol and 15% beta-sitosterol. The fatty acid composition of the steryl esters differed from that of the 2-position of the plasma phosphatidylcholines, which contained significantly less palmitic and oleic and more linoleic acid. On the basis of these results and a review of the literature it is suggested that the plasma cholesteryl and plant steryl esters in phytosterolemia originate from both synthesis in plasma via the lecithin-cholesterol acyltransferase and synthesis in tissues via the acylCoA-cholesterol acyltransferase.

Stereospecific analysis of fatty acid esters of chloropropanediol isolated from fresh goat milk.

The fatty acid esters of chloropropanediol isolated from goat milk fat in small quantities were subjected to a stereospecific analysis via phospholipase C and phosphocholine esters as intermediates. Synthetic rac-1-chloro-2,3-dioleoyl-propanediol was prepared by standard methods and was used as a control. The stereospecific analyses were performed following a release of the fatty acids from the primary positions of each chloropropanediol diester with pancreatic lipase. The resulting X-1-chloro-2-acylpropanediols were then converted into the corresponding phosphocholine derivatives by a stepwise reaction with phosphorus oxychloride and choline chloride. The X-1-chloro-2-acyl-3-phosphocholinepropanediols were subjected to hydrolysis with phospholipase C (C. perfringens), which hydrolyzed 50% of the phosphatide within two min and the rest of it in two hr. From previous experience with glycerol esters, it was assumed that the more rapidly hydrolyzed molecules were the sn-1-chloro-2-acyl-propanediol derivatives and the more slowly hydrolyzed ones the sn-2-acyl-3-chloropropanediol derivatives. A hydrolysis with phospholipase A2 (Crotalus adamanteus) released 50% of the total fatty acid along with the corresponding lyso compound within 10 min, after which there was no further reaction. The hydrolysis products were assayed directly by gas liquid chromatography (GLC) or were isolated by thin layer chromatography (TLC) prior to quantitation by GLC. Both naturally occurring and synthetic chloropropanediol diesters behaved similarly on stereospecific analysis and were therefore concluded to be racemic.

Comparative study of the molecular species of chloropropanediol diesters and triacylglycerols in milk fat.

In an effort to establish the origin of the fatty acid esters of 3-chloropropanediol, which recently have been isolated in small amounts from goat milk, we compared the molecular species composition of the chlorohydrin diesters and of goat milk triacylglycerols. The chloropropanediol diesters were found to be composed of molecular species containing C10-C18 fatty acids and corresponded closely in carbon number to those calculated for the long chain sn-1,2-diacylglycerol moieties of goat milk triacylglycerols. The molecular species of goat milk total triacylglycerols contained C4-C18 fatty acids. It is suggested that triacylglycerols and chloropropanediol diesters are derived from the same pool of long chain fatty acids. A molecular distillate of bovine milk fat did not contain chloropropanediol diesters, while the available samples of human milk fat were shown to contain alkyldiacylglycerols as the major components of a neutral lipid fraction corresponding in polarity to the chloropropanediol diesters.

Simultaneous analysis of low plasma levels of deuterium-labeled saturated and unsaturated fatty acids as t-butyldimethylsilyl esters.

A sensitive and accurate method for detection and quantitation of deuterated fatty acids in the presence of large amounts of unlabeled fatty acids is described using mass fragmentography in combination with the preparation of tertiarybutyldimethylsilyl esters (t-BDMS). The method has been applied to determination of deuterated stearic, oleic, elaidic and linoleic acids in human plasma lipoproteins following duodenal perfusion with a micellar mixture of acids. Over a concentration range of 10-1000 ng/ml, the average coefficient of variation for the linoleate was 3% and for the oleate (elaidate) ester was 2%.

Deacylation of endogenously deuterated rat liver microsomal phospholipids by endogenous phospholipases.

The relative deacylation of microsomal phospholipid molecular species was reexamined. Microsomal membranes were prepared from the livers of rats injected, over a period of 20 h, with perdeuterated ethanol. The phosphatidylcholine and phosphatidylethanolamine were isolated by thin-layer chromatography of the total lipid extracts and the distribution of deuterium among the molecular species of the diacylglycerol moieties was determined by reversed-phase high pressure liquid chromatography in combination with chemical ionization mass spectrometry. Deuterium was found to be incorporated into newly formed glycerol and newly synthesized palmitic and stearic acids (4-22% replacement) which were distributed throughout the molecular species in proportion to their relative rates of turnover (10-45% replacement). Within each unsaturation class, the palmitoyl species were labelled more extensively than the corresponding stearoyl species. Following in vitro incubation of the membranes with 10 mM Ca2+ at pH 8.5, there occurred a rapid degradation of the phosphatidylethanolamines (to 60% of control values after 90 min), while the phosphatidylcholines remained essentially unaffected. The various molecular species of the phosphatidylethanolamines were degraded linearly and in proportion to their masses. The deuterium content of the phosphatidylethanolamine and phosphatidylcholine remained constant throughout the incubation. It was concluded that under the present experimental conditions all molecular species of phosphatidylethanolamine, both old and newly synthesized, are equally accessible to the endogenous phospholipase.

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.