Search

TLC has traditionally been used to analyze lipids isolated from membrane complexes. Here, we describe a method based on the combination of TLC and SDS-PAGE to qualitatively analyze the protein/lipid profile of membrane complexes such as those of lung surfactant. For this purpose, native lung surfactant was applied onto a silica TLC plate in the form of an aqueous suspension, preserving not only hydrophilic proteins associated with lipids but also native protein-lipid interactions. Using native membrane complexes in TLC allows the differential migration of lipids and their separation from the protein components. As a result, (partly) delipidated protein-enriched bands can be visualized and analyzed by SDS-PAGE to identify proteins originally associated with lipids. Interestingly, the hydrophobic surfactant protein C, which interacts tightly with lipids in native membrane complexes, migrates through the TLC plate, configuring specific bands that differ from those corresponding to lipids or proteins. This method therefore allows the detection and analysis of strong native-like protein-lipid interactions.

Gangliosides and sulfatides (STs) are acidic glycosphingolipids (GSLs) that have one or more sialic acids or sulfate substituents, in addition to neutral sugars, attached to the C-1 hydroxyl group of the ceramide long chain base. TLC is a widely employed and convenient technique for separation and characterization of GSLs. When TLC is directly coupled to MS, it provides both the molecular mass and structural information without further purification. Here, after development of the TLC plates, the structural analyses of acidic GSLs, including gangliosides and STs, were investigated using the liquid extraction surface analysis (LESA™) and CAMAG TLC-MS interfaces coupled to an ESI QSTAR Pulsar i quadrupole orthogonal TOF mass spectrometer. Coupling TLC with ESI-MS allowed the acquisition of high resolution mass spectra of the acidic GSLs with high sensitivity and mass accuracy, without the loss of sialic acid residues that frequently occurs during low-pressure MALDI MS. These systems were then applied to the analysis of total lipid extracts from bovine brain. This allowed profiling of many different lipid classes, not only gangliosides and STs, but also SMs, neutral GSLs, and phospholipids.

Five mutants of apolipoprotein A-I (apoA-I), apoA-I(Δ63–73), apoA-I(Δ140–150), apoA-I(63–73@140–150), apoA-I(R149V), and apoA-I(P143A) were compared with human plasma apoA-I for their ability to promote cholesterol and phospholipid efflux from HepG2 cells. A significantly lower capacity to promote cholesterol and phospholipid efflux was observed with lipid-free apoA-I(Δ63–73), while mutations apoA-I(Δ140–150) and apoA-I(P143A) affected phospholipid efflux only. When added as apoA-I/palmitoyloleoyl phosphatidylcholine (POPC) complex, mutations apoA-I(63–73@140–150) and apoA-I(Δ140–150) affected cholesterol efflux. None of the mutations affected α-helicity of the lipid-free mutants or their self-association. Five natural mutations of apoA-I, apoA-I(A95D), apoA-I (Y100H), apoA-I(E110K), apoA-I(V156E), and apoA-I (H162Q) were studied for their ability to bind lipids and promote cholesterol efflux. None of the mutations affected lipid-binding properties, cholesterol efflux, or α-helicity of lipid-free mutants. Two mutations affected self-association of apoA-I: apoA-I(A95D) was more prone to self-association, while apoA-I(E100H) did not self-associate.The following conclusions could be made from the combined data: i) regions 210–243 and 63–100 are the lipid-binding sites of apoA-I and are also required for the efflux of lipids to lipid-free apoA-I, suggesting that initial lipidation of apoA-I is rate limiting in efflux; ii) in addition to the lipid-binding regions, the central region is important for cholesterol efflux to lipidated apoA-I, suggesting its possible involvement in interaction with cells.

Assessment of free fatty acid (FFA) concentration and isotopic enrichment is useful for studies of FFA kinetics in vivo. A new procedure to recover the major FFA from plasma for concentration and isotopic enrichment measurements is described and validated. The procedure involves extraction of plasma lipids with hexane, methylation with iodomethane (CH3I) to form fatty acid methyl esters (FAME), and subsequent purification of FAME by solid phase extraction (SPE) chromatography. The new method was compared with a traditional method using thin-layer chromatography (TLC) to recover plasma FFA, with subsequent methylation by BF3/methanol. The TLC method was found to be less reliable than the new CH3I method because of contamination with extraneous fatty acids, chemical fractionation of FFA species, and incomplete recovery of FFA associated with TLC. In contrast, the CH3I/SPE method was free of contamination, did not exhibit chemical fractionation, and had higher recovery. The iodomethane reaction was specific for free fatty acids; no FAME were formed when esterified fatty acids (triglycerides, cholesteryl esters, phospholipids) were subjected to the methylation reaction. We conclude that the CH3I/SPE method provides rapid and convenient recovery of plasma fatty acids for quantification or GC/MS analysis as methyl esters, and is not subject to the problems of contamination, reduced recovery, and chemical fractionation associated with recovery of FFA by TLC.—Patterson, B. W., G. Zhao, N. Elias, D. L. Hachey, and S. Klein. Validation of a new procedure to determine plasma fatty acid concentration and isotopic enrichment. J. Lipid Res. 1999. 40: 2118–2124.

Seven radioactive methyl branched isomers of eicosa-8, 11, 14-trienoic acid, in which a methyl branch was located at carbon 2, 5, 10, 13, 17, 18, or 19, were used as substrates to study their enzymatic conversion into homologues of prostaglandin E1 by microsomes from bovine vesicular glands. The products of this reaction were partially characterized. When the methyl branch was moved towards position 13, the site of stereospecific hydrogen removal by prostaglandin synthetase, the rate of prostaglandin formation declined rapidly. When a methyl branch was at positions 10, 13, 17, 18, or 19, the rates of prostaglandin formation were less than 25% of that from eicosa-8, 11, 14-trienoic acid. However, when a methyl branch was at positions 2 or 5, these isomers were converted to prostaglandins at least 50% as rapidly as eicosa-8, 11, 14-trienoic was converted to prostaglandins. The apparent Km and Vmax values for eicosa-8, 11, 14-trienoic acid were respectively 224 μM and 4.62 nmoles/min/mg protein. Kinetic data suggest that the enzyme converted eicosa-8, 11, 14-trienoic acid to prostaglandins 3.6 times faster than the 5-methyl branched isomer, although the enzyme bound the branched isomer twice as firmly as eicosa-8, 11, 14-trienoic acid. The concentration-dependent inhibition of prostaglandin formation from eicosa-8, 11, 14-trienoic acid by the 13-methyl branched isomer was also demonstrated.

Glucosylsphingosine has been isolated for the first time as a natural constituent from Gaucher's spleen. On thinlayer chromatography, it migrates with authentic glucosylsphingosine, yielding a positive color reaction with ninhydrin for the amino group and with α-naphthol–sulfuric acid for the carbohydrate residue. N-Acylation with palmitic acid gave rise to glucosylceramide, which was cleaved by purified glucosylceramide: β-glucosidase to ceramide. Gas–liquid chromatography of the trimethylsilyl derivative showed a retention time similar to authentic glucosylsphingosine. Gas–liquid chromatographic analysis of the trimethylsilyl derivatives after methanolysis revealed the presence of only glucose and C18-sphingosine. Mass spectral data further supported the structural identity with glucosylsphingosine.

Phospholipid compositions were determined in samples of gastrocnemius muscles of individuals from fetal stage to the age of 55 yr. The lipids were separated by thin-layer chromatography. In order to enable characterization of the individual phospholipids, a lipid extract was prepared from pooled samples of gastrocnemius muscles from adult males and separated by ion-exchange chromatography on TEAE-cellulose. The individual phosphoglycerides were purified by thin-layer chromatography and then characterized according to their content of fatty acids, aldehydes, phosphorus, and the identity of the bases. The relative amounts of the major phospholipids, choline and ethanolamine phosphoglycerides, changed little with age, and in adult males they constituted 47% and 24%, respectively. Cardiolipin increased from 3% in the fetal stage to 9% by the end of the first year of life. Sphingomyelin and serine phosphoglyceride decreased with increasing age, while inositol phosphoglyceride increased. In adult males, cardiolipin constituted 10% of total lipid phosphorus, inositol phosphoglyceride constituted 9%, serine phosphoglyceride 3%, and sphingomyelin 7%.

The fatty acids in cardiolipin, choline phosphoglyceride, ethanolamine phosphoglyceride, inositol phosphoglyceride, and serine phosphoglyceride in the human gastrocnemius muscle were studied for changes between early fetal age to middle age. In cardiolipin, choline phosphoglyceride, and ethanolamine phosphoglyceride, the relative total amount of the linoleic acid series increased considerably during the prenatal period and reached maximum values during the first year of life. In inositol phosphoglyceride and serine phosphoglyceride, only slight bechanges in the sum of fatty acids of the linoleic acid series were found. An increase in the concentration of fatty acids of the linolenic acid series was found in ethanolamine phosphoglyceride and serine phosphoglyceride, while only minor amounts of these fatty acids were present in choline phosphoglyceride and inositol phosphoglyceride and not at all in cardiolipin. During the period studied, the amount of plasmalogen in ethanolamine phosphoglyceride decreased slightly during the first year of life and then remained at 40%. The amount of plasmalogen increased in choline phosphoglyceride from 8% at the middle of the gestational period to 15% 1 yr after birth. The individual phospholipids were found to have characteristic fatty acid patterns.

The extent of the side chain hydroxylation of cholesterol, campesterol (24 α-methylcholesterol), and β-sitosterol (24 α-ethylcholesterol) in rat liver mitochondria has been compared. Two β-sitosterol metabolites, tentatively identified by liquid chromatography, thin-layer chromatography, gas-liquid chromatography, combined with radioactivity detection, and gas-liquid chromatography-mass spectrometry as the 26- and 29-hydroxy derivatives, were formed in the proportion 1:1. The sum of 26-hydroxy- and 29-hydroxy-β-sitosterol obtained amounted only to about one-fourth of the yield of 26-hydroxycholesterol. Campersterol appeared to give rise only to 26-hydroxycampesterol (tentatively identified), which was formed in similar yields as 26-hydroxycholesterol (0.2-0.4%). The formation of 29-hydroxy-β-sitosterol but not of 28-hydroxycampesterol indicates that the omega-hydroxylation of the steroid side chain is dependent on the length of the side chain. Cholesterol gave rise to identifiable amounts of a 25-hydroxy derivative but the formation of 25-hydroxy derivatives of β-sitosterol and campesterol could not be established with certainty. 24-hydroxycholesterol was also found to be formed in the mitochondrial system. The ratio between the yields of 26- and 25-hydroxychoelsterol ranged between 2 and 3, and that between 26- and 24-hydroxycholesterol was about 10.

A sensitive radioenzymatic assay for glycerol and acylglycerols is described. The assay depends on the quantitative phosphorylation of glycerol to glycerophosphate by glycerol kinase using [γ-32P]ATP as a substrate. The 32P content of the formed glycerophosphate is determined and gives a measure of the original glycerol content. Acylglycerols can be determined by prior hydrolysis to glycerol. The assay is sensitive to about 0.1 nmol of glycerol and can be extended to 100 nmol. The assay can be applied to the determination of acylglycerols separated by thin-layer chromatography in amounts as low as 0.5 nmol. The assay is particularly useful in the determination of the specific activity of 14C- or 3H-labeled glycerol moeities.

An assay using radioactive substrates is described that permits rapid determinations of glycerophospholipid-hydrolyzing activity in postheparin plasma or its fractions. Optimal conditions are described for hydrolysis of phosphatidylcholine and phosphatidylethanolamine. A minimum of only 2 µl of normal postheparin plasma is used and no extraction of the reaction products is required before their separation by thin-layer chromatography. We found that after an optimal heparin dose of 60 units/kg body weight the rate of hydrolysis for diacyl glycerophosphocholine and diacyl glycerophosphoethanolamine is 1.16 µmoles/ml per hr and 22.4 µmoles/ml per hr, respectively.

The four fractions obtained by argentation thin-layer chromatography of intact rat liver lecithins can be further subdivided by reversed-phase partition thin-layer chromatography on hydrophobic kieselguhr. The resultant eight fractions contain virtually only one saturated and one unsaturated acid each.

Several crystalline N-hydroxysuccinimide esters of short- and long-chain fatty acids have been synthesized. These compounds react with free amino acids to form preferentially N-acylamino acids. The reaction of the N-hydroxysuccinimide esters with hydroxylamine and the behavior of the N-acylamino acids on thin-layer chromatography are described.

Procedures are described for the isolation of free fatty aldehydes from total lipid extracts of rat, dog, and bovine heart muscle. These aldehydes did not arise from hydrolysis of the naturally occurring alkenyl ethers in either the extraction or isolation procedures.The free fatty aldehydes were present in heart muscle in amounts between 0.27 and 0.56 μmole per 100 mg of lipid.Hexadecanal and octadecanal are the principal free fatty aldehydes present, except in dog heart muscle where an unidentified, unusual aldehyde constitutes 20% of the mixture.

The molar ratios of N-acetyl neuraminic acid, hexose, hexosamine, and sphingosine have been determined for the abnormal ganglioside in Tay-Sachs disease that was previously detected as a fast-moving band in thin-layer chromatography, and in two abnormal fast-moving bands of gangliosides from the cortex and white matter of the brain in cases of gargoylism and Niemann-Pick's disease. The fastest-moving ganglioside band in these two conditions contains neither hexosamine nor glucose.

The chemical synthesis and characterization of three intermediates in the Β oxidation of palmitic acid-1-14C by rat liver mitochondria, namely, 3-ketohexadecanoic acid-1-14C, dl-3-hydroxyhexadecanoic acid-1-14C, and trans-2-hexadecenoic acid-1-14C, are described.

Gluco- and galactocerebrosides can be separated by thin-layer chromatography on Silica Gel G prepared with sodium borate solution instead of water. The most successful developing system was chloroform-methanol-water-15 m NH4OH 280:70:6:1.

The mono- and disubstituted cholanoic acids present in human feces have been investigated. Extracts of feces were fractionated on silicic acid column and individual bile acids were isolated by preparative thin-layer chromatography. The isolated compounds were studied by gas-liquid chromatography of the methyl esters, partial trimethylsilyl ethers, oxidation products, and trifluoroacetates. The probable structures deduced were confirmed by gas chromatography-mass spectrometry and by comparisons with authentic compounds.The following derivatives of 5Β-cholanoic acid not previously isolated from human feces were identified: 3,12-diketo, 3-keto-12α-hydroxy, 3α,l2Β-dihydroxy, 3Β,12Β-dihydroxy, 3-keto-7α-hydroxy, 3α-hydroxy-7-keto, 3Β,7α-dihydroxy, 3α,7α-dihydroxy, and 3α,7Β-dihydroxy.The presence of 3-keto-, 3Β-hydroxy-, 3α-hydroxy-, 3Β-hydroxy-12-keto-, 3α-hydroxy-12-keto-, 3Β,12α-dihydroxy-, and 3α,12α-dihydroxy-5Β-cholanoic acids was confirmed.Evidence was obtained for the presence of two bile acids having at least one hydroxyl group at a carbon atom other than C3, C7, or C12.

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.

A double-development procedure employing first a polar and then a nonpolar solvent system is described for the complete separation by thin-layer chromatography of the main lipid classes encountered in natural lipids. For better quantification, long plates (34 cm) are employed. Diglycerides were separated from cholesterol, 1,2- from 1,3-diglycerides, and monoglycerides from phospholipids.

A method for isolation and quantification of fecal neutral steroids is described which allows studies to be made of sterol balance in man or in small laboratory animals without requiring the use of radioisotopes in vivo. The critical separations of cholesterol plant sterols and their conversion products depend upon preliminary separations into three subfractions by thin-layer chromatography. Individual components in the three subfractions thus obtained are then quantitatively measured by gas–liquid chromatography of the unsubstituted 3-ketosteroids and of the trimethylsilyl ethers of the sterols.Extractions of cholesterol-7-α-H3 added in vitro and of C14-labeled neutral steroids synthesized in vivo were quantitative and highly reproducible. Several lines of evidence validate the determination of individual fecal neutral steroids by GLC.Examples are given of the application of this technique: a sterol balance study of 27 days' duration is described in a patient whose diet included plant sterols as well as cholesterol. Representative results in man and in rats are compared to others obtained by previously described methods.The sensitivity of the method is such that 1-g fecal aliquots containing as little as 25 μg of mixed neutral steroids can be analyzed accurately, but the procedure lends itself well to preparative scale work for more definitive study of individual neutral steroids.

A method for isolation and quantification of fecal bile acids is described which allows sterol balance studies to be made in man or in small laboratory animals without requiring the use of radioisotopes in vivo. Bile acids are purified by column and thin-layer chromatography, converted to the trimethylsilyl ethers of their methyl esters, and quantified by GLC with detection by hydrogen flame ionization. Recoveries are complete when internal standard corrections are applied, with an error

A two-step thin-layer chromatographic procedure is described for separation of cholesterol from its naturally occurring companions. Impregnation of Silica Gel G with silver nitrate retards cholesterol relative to cholestan-3Β-ol and Δ7-cholestenol, which can be separated from each other on a second plate by reversed-phase thin-layer chromatography at low temperature.

Solvent systems are described which permit class separations of various peroxidized lipids by thin-layer chromatography. This procedure has proved useful in biological studies and for testing the deterioration of lipids.Proof is given that the least polar peroxides of methyl esters, glycerides, and fatty acids are monoperoxides; it is then assumed that the least polar peroxides in the other tested groups (cholesterol, cholesterol esters, and phospholipids) are also monoperoxides. The more polar peroxides probably represent highly peroxidized and polymeric forms.

A micro spectrophotometric procedure for the quantitative determination of plasmalogen aldehydes is described which utilizes simultaneous methanolysis and formation of 2,4-dinitrophenylhydrazones. After isolation of the hydrazones by thin-layer chromatography, the aldehydes can be regenerated, reduced, acetylated, and then analyzed by gas–liquid chromatography. Identification of the plasmalogen aldehydes obtained from rumen holotrich protozoa is described.

By column chromatography two lipid fractions, x1 and x2 of which there are only traces in the lipids of other organs, have been isolated from the pancreatic lipids. The lipid fraction x2, which forms a very considerable proportion of the total lipids of the pancreas, has been found to be composed of the methyl esters of lauric, myristic, tetradecenoic, palmitic, palmitoleic, stearic, oleic, linoleic, and arachidic acids. Fraction x1 was not analyzed.

An improved procedure for the quantitative assay of phospholipids separated by TLC is described, in which a specially washed Silica Gel H and a newly designed chromatography unit are employed.Analysis of phospholipid phosphorus and phospholipid fatty acids was accomplished in the presence of silica gel scraped directly from the chromatoplates. Recovery of phosphorus ranged from 96 to 100%.The washing of the Silica Gel H with chloroform–methanol–formic acid resulted in extremely low phosphorus blanks on the silica gel, and also removed impurities which were found to cause various peaks on GLC.

Cholesterol preparations from 14 commercial sources were purified by the dibromide method. The cholesterol contents of the original and purified preparations were determined by gas–liquid chromatography. Calculation of the results of the gas chromatographic analyses from the areas of the peaks rather than from their heights gave more precise results. Purified samples had higher contents of cholesterol, higher melting points, and narrower melting point ranges, and exhibited fewer contaminants on thin-layer chromatoplates than did the original preparations.

Intact, naturally occurring lecithins were fractionated according to degree of unsaturation by thin-layer chromatography on silica gel plates impregnated with silver nitrate and heated at 175–180° for several hours.

Purified sphingosine was resolved into dihydrosphingosine, sphingosine, and a fast-moving component thin-layer chromatography on Silica Gel G. The fast-moving component was generated by the adsorbent, and consisted of a mixture of the cationic forms of dihydrosphingosine and sphingosine. Other sphingosine derivatives gave rise to similar artifacts.Sphingosine formed two degradation products when stored in solution or on thin-layer plates; related long-chain bases with some of their reactive sites blocked did not deteriorate when stored in solution.

Homologs of methyl esters of very polar fatty acids were obtained by methanolysis of cerebrin phosphate isolated from baker's yeast. The major ester component was isolated by preparative gas–liquid chromatography and was found to be 2,3-dihydroxyhexacosanoic acid as deduced from the mass spectra of its trimethylsilyl ether and isopropylidene derivative, reaction with periodate, and comparison of its chromatographic behavior with that of synthetic erythro- and threo-dihydroxy acids. Its infrared spectrum supported the above conclusions. From their retention times by gas–liquid chromatography, the homologs were found to be saturated, unbranched 2,3-dihydroxy fatty acids with 24–27 carbon atoms. The synthesis of the new fatty acids, erythro- and threo-2,3-dihydroxyhexacosanoic acids, is also reported. A method for separating trans-2-hexacosenoic acid, a key intermediate of the above synthesis, and its isomer, trans-3-hexacosenoic acid, both formed by dehydrobromination of 2-bromohexacosanoic acid, is also described.

A simple and relatively rapid technique has been developed for the separation of several 4,4-dimethyl steryl acetates, some of which contain sterically hindered nuclear double bonds. The method involves thin-layer chromatography on silver nitrate-impregnated silica gel and silver nitrate-impregnated alumina. The separated steryl acetates may then be analyzed quantitatively by gas–liquid chromatography.

The biosynthesis of prostaglandin E2 (PGE2) from [1-14C]arachidonic acid has been demonstrated in homogenates and subcellular fractions of human epidermis. This biosynthetic capacity is localized in the microsomal fraction, indicating the presence of an active prostaglandin synthetase system associated with membranes of the skin. The incorporation of 14C from [1-14C]arachidonic acid into PGE2 by the microsomal fraction was enhanced by EDTA. This apparent increase in 14C incorporation into PGE2 in the presence of EDTA could be due at least in part to its chelating properties of removing the divalent cations in the homogenate that enhance the selective formation of PGF2α and the suppression of the activity of epidermal phospholipase A, which causes the release of nonradioactive fatty acid precursors from endogenous phospholipids. This study has also demonstrated that the formation of PGE2 from arachidonic acid by the microsomal fraction from human skin could be inhibited by polyunsaturated fatty acids, suggesting a possible regulatory role of fatty acids released from endogenous phospholipids on prostaglandin synthesis in this tissue. The inhibitory effects of some anti-inflammatory drugs on skin microsomal prostaglandin synthetase were also demonstrated in these studies. Results from these studies indicate that the skin is therefore a useful tissue for the study of mechanisms of prostaglandin biosynthesis and the mode of action of various anti-inflammatory drugs.

A sulfur-containing glycolipid, accounting for ca. 25% of the total polar lipids, has been isolated from the extreme halophile Halobacterium cutirubrum. The ammonium salt of the lipid was found to have the molecular formula C61H117O21S·NH4, and on strong acid hydrolysis it yielded 2,3-di-O-phytanyl-sn-glycerol, glucose, mannose, galactose, and sulfate in equimolar proportions. Infrared and NMR spectra indicated the presence of a secondary sulfate group. Solvolysis of the lipid in 0.004 m HCl in tetrahydrofuran resulted in rapid release of inorganic sulfate and formation of galactosyl-mannosyl-glucosyl diphytanyl glycerol ether. With higher acid concentration (0.25 m methanolic HCl), stepwise hydrolysis of monosaccharide units occurred, giving mannosyl-glucosyl glycerol diphytanyl ether and monoglucosyl glycerol diphytanyl ether. The position of attachment of the sugars and of the sulfate group was determined by methylation of the free acid form of the glycolipid sulfate, followed by acid hydrolysis and gas–liquid chromatographic analysis of the partially methylated sugars as the alditol acetates. The configuration of the glycosidic linkages was established both by optical rotation measurements and by specific enzymatic hydrolysis. The results obtained established the structure as 2,3-di-O-phytanyl-1-O-[β-d-galactopyranosyl-3′-sulfate-(1′→6′)-O-α-d-mannopyranosyl-(1′→2′)-O-α-d-glucopyranosyl]-sn-glycerol.

Free fatty acids, aldehydes, alcohols, and 1-O-alkyl and alk-1-enyl glycerols were identified and quantified in lipid extracts from bovine cardiac muscle.Although a number of components present in the free fatty aldehydes were also noted in the fatty chains in the 1-O-alk-1-enyl glycerols, a direct qualitative similarity did not exist as would be expected if the free fatty aldehydes were artifactual in origin. Also, a qualitative similarity did not exist between the fatty chains of the 1-O-alkyl and alk-1-enyl glycerols. This latter observation would suggest a mechanism other than biodehydrogenation of the alkyl ethers for the origin of the alk-1-enyl glycerols.Free fatty aldehydes were distributed evenly between the 105,000 g supernatant and particulate fractions of cardiac muscle, while the 1-O-alk-1-enyl glycerols were associated primarily with the particulate fraction. Free fatty alcohols were noted only in the supernatant fraction, while the 1-O-alkyl glycerols were present in both fractions.

Saturated and unsaturated fatty aldehydes were synthesized 99+% pure with yields of up to 80% by the reduction of 1-acylaziridines with lithium aluminum hydride, and in yields of up to 87% by oxidation of the corresponding alcohol with 1-chlorobenzotriazole. It was found for the reduction that optimum aldehyde yield was obtained with a mole ratio of reactants, consisting of acid chloride–ethylenimine–triethylamine–LiAlH4, equal to 1:2:2:2. Optimum conditions for alcohol oxidation were found to be a mole ratio of oxidant to alcohol of 1:1.3 with refluxing for 45 min in methylene chloride containing 25% pyridine. Methods for the purification of the final product are also described. Purity criteria were thin-layer and gas–liquid chromatography and infrared and nuclear magnetic resonance spectroscopy.

The excretion of sulfatides in human urine was studied. 24-hr urine collections were filtered. Urinary glycolipids were extracted from the filter paper and fractionated on diethylaminoethyl cellulose and silicic acid columns, and by thin-layer chromatography. Fatty acids and long-chain bases were analyzed by gas–liquid chromatography of the corresponding esters and aldehydes. Glycosyl ceramide concentration was determined by gas–liquid chromatography of the trimethylsilyl ethers of the methyl glycosides.Normal females were found to excrete larger amounts of dihexosyl ceramides than males. Sulfatides were detected in all urine specimens. In sulfatidosis, a hereditary sulfatide storage disorder known as metachromatic leukodystrophy, a large increase in sulfatide was readily apparent on a thin-layer chromatogram of the crude lipid extract. On comparing samples from normal individuals and patients with sulfatidosis, urinary sulfatide composition was remarkably similar to that previously reported in the kidney, including differences in fatty acid pattern. The determination of urinary sulfatides was a valuable confirmation of the deficiency in arylsulfatase A activity characteristic of sulfatidosis.

Two improvements are described for the assay of HMG CoA reductase. These are a simple synthesis of the substrate precursor HMG-3-14C anhydride and a double-label (14C and 3H) method for determining the amount of mevalonate-3-14C that is formed from the substrate.

Dialkyl, alkyl acyl, and diacyl glycerols were resolved as trimethylsilyl ethers and as acetates by gas-liquid chromatography on a nonpolar stationary phase (OV-1). The two types of derivatives proved suitable for quantitative gas chromatographic analysis.

3α-Hydroxy-5β-cholanoic acid (lithocholic acid) and some unidentified acids (one of them perhaps 3α-hydroxycholest- 5-en-26-oic acid) have been detected in the brains of guinea pigs afflicted with experimental “allergic” encephalomyelitis. None of the acids could be identified in comparable amounts of brain tissue from normal guinea pigs. Thin-layer chromatography of the free acids, and thin-layer and gasliquid chromatography combined with mass spectrometry of the methyl esters were used for identification.The occurrence of these acids may possibly be the result of cholesterol oxidation in the brain. The acids, or compounds related to them, may play a role in development of demyelination in experimental allergic encephalomyelitis.

The ethanolamine phosphoglycerides were prepared from lipid extracts of ox and mouse brains by preparative thin-layer chromatography. The cyclic acetal derivatives of the alk-1-enyl groups were made by treating the ethanolamine phosphoglycerides with 1,3-propanediol. The resulting monoacyl glycerophosphoryl ethanolamines were separated from the unchanged ethanolamine phosphoglycerides by preparative thin-layer chromatography. Methyl ester derivatives of the acyl groups from both of these fractions were prepared by alkaline methanolysis. The cyclic acetal and methyl ester derivatives were analyzed by gas-liquid chromatography.Substantial differences were found in the composition of the side chains when the combined alk-1-enyl and acyl side chains of the alk-1 ‘-enyl acyl glycerophosphoryl ethanolamines were compared with the side chains of the diacyl glycerophosphoryl ethanolamines. The side chains from the 1-position of these two ethanolamine phosphoglycerides are different in chain length and unsaturation as well as in chemical bonding. The acyl groups from the 2-position of the alk-1 ‘-enyl acyl glycerophosphoryl ethanolamines were predominantly unsaturated. Therefore, acyl group compositions of the total ethanolamine phosphoglyceride from brain are of limited value and individual types should be analyzed.

We have studied the effect of long-term hyperlipemia and atherosclerosis in squirrel monkeys on the metabolism of lysolecithin-14C (1-palmitoyl-1′-14C sn-glycerol 3-phosphorylcholine) in order to explain elevated plasma and arterial concentrations of lysolecithin. The die-away curves of lysolecithin-14C from plasma and the timing of appearances of other 14C-labeled moieties in plasma and other tissues demonstrated a complex pattern of metabolic reactions. There was a rapid equilibration of specific activities of lysolecithin of plasma, liver, and aortic intima plus inner media. The specific activities of lecithin peaked first in liver, then in plasma, and rose slowly in aortic intima plus inner media. The appearance of lecithin-14C in heart and skeletal muscle was also slower than in the liver and some other tissues. Triglycerides, and to a lesser extent, cholesteryl esters contained radioactivity. The concentrations of aortic lysolecithin in the atherosclerotic aortas were several times greater than comparable values for control aortas, and the time of equilibration of plasma and aorta lysolecithin-14C was much greater for the atherosclerotic group. The quantities of lysolecithin in plasma and in the pool of which the plasma was a part, were increased with hyperlipemia and atherosclerosis, as was the rate of lysolecithin production in the fast pool. Hyperlipemia was also associated with an early increase in plasma lecithin:cholesterol acyltransferase (LCAT) activity in vitro. Furthermore, nutritional hyperlipemia influenced the distribution of lysolecithin-14C and lecithin-14C between different plasma lipoproteins. The increase in concentrations of lysolecithin in the aorta occurred more slowly than that in plasma after we had induced hyperlipemia in the monkeys.

The fractionation of lipoproteins of normal and hyperlipidemic subjects on a column of 2% agarose was compared with ultracentrifugation and paper electrophoresis procedures. The following results were obtained. (a) Plasma lipoproteins were eluted successively from the column in the four overlapping peaks of chylomicrons, very low density lipoproteins, low density lipoproteins, and high density lipoproteins. (b) Very low density lipoproteins and high density lipoproteins (d > 1.063, containing nonlipoprotein proteins) showed continuous progressive changes in lipid composition as these fractions emerged, while low density lipoproteins showed a relatively constant lipid composition. (c) A discontinuous transition of lipid composition was observed when consecutive ultracentrifugal fractions were placed on the column. (d) The “trail” of pre-beta lipoprotein seen on paper electrophoresis was shown to consist of particles whose molecular sizes range between chylomicrons and pre-beta lipoproteins. A reverse relationship was observed between electrophoretic mobilities of “trail” components and their particle size. (e) Gel with an agarose content of 2% seemed to fractionate chylomicrons and very low density lipoproteins more effectively than other lipoprotein classes.

The dienic long-chain base (sphingadienine) of human plasma sphingomyelins has been identified as d-erythro-1,3-dihydroxy-2-amino-4-trans-14-cis-octadecadiene. A similar sphingosine was also detected in plasma sphingomyelins of rat, rabbit, and cat. The key reaction in the structural studies was partial reduction of sphingadienine with hydrazine to cis-14-sphingenine and 4-sphingenine.

A method has been developed and validated for the determination of the six major conjugated bile acids, cholesterol, and total phospholipids in bile of human subjects previously injected with 4-14C-cholesterol.The procedure is designed for use with 5-10 ml of duodenal or T-tube bile and eliminates difficulties associated with existing methods for bile acid determination, in particular the requirement for preliminary saponification under pressure or the use of paper chromatography. Saponification under pressure is employed only in steps where partial destruction of the steroid moiety of conjugated bile acids is not a crucial matter.A preliminary Folch extraction and washing step separated free cholesterol and phospholipids (bottom layer) from the six major conjugated bile acids (top layer). The conjugated bile acids were then fractionated cleanly by thin-layer chromatography to give four groups, the 14C content of each of which was determined. A second aliquot of the top layer was used to determine (after deconjugation) the radioactivity ratio of deoxycholic acid to chenodeoxycholic acid for the two unresolved groups (dihydroxycholanoic acid conjugates with glycine and taurine, respectively). A third aliquot was used for determination of specific activities of the methyl esters of cholic, chenodeoxycholic, and deoxycholic acids derived from the total bile salts. Appropriate calculations yielded the concentration in bile of all six major bile acid conjugates.

A homologous series (C10, C12, C14, C16, C18) of synthetic O-alkyl and S-alkyl ethers of glycerol was analyzed by gas-liquid chromatography (GLC) and thin-layer chromatography (TLC), and examined by IR and n.m.r. spectroscopy; the physical properties of the O-alkyl and S-alkyl ethers were compared. Isopropylidene derivatives of the glycerol ethers and thioethers were quantitatively analyzed by GLC on polar and nonpolar liquid phases. On a medium polar liquid phase (ethylene glycol succinate), mixtures of the O-alkyl and S-alkyl ethers were completely resolved. Isopropylidene derivatives of glycerol ethers and of thioethers could be separated as classes (though not into individual homologues) by TLC. O-hexadecyl and S-hexadecyl ethers of glycerol are easily distinguished by IR and n.m.r. spectroscopy.