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Acyl-CoA-dependent O-acyltransferases catalyze reactions in which fatty acyl-CoAs are joined to acyl acceptors containing free hydroxyl groups to produce neutral lipids. In this report, we characterize a human multifunctional O-acyltransferase (designated MFAT) that belongs to the acyl-CoA:diacylglycerol acyltransferase 2/acyl-CoA:monoacylglycerol acyltransferase (MGAT) gene family and is highly expressed in the skin. Membranes of insect cells and homogenates of mammalian cells overexpressing MFAT exhibited significantly increased MGAT, acyl-CoA:fatty acyl alcohol acyltransferase (wax synthase), and acyl-CoA:retinol acyltransferase (ARAT) activities, which catalyze the synthesis of diacylglycerols, wax monoesters, and retinyl esters, respectively. Furthermore, when provided with the appropriate substrates, intact mammalian cells overexpressing MFAT accumulated more waxes and retinyl esters than control cells. We conclude that MFAT is a multifunctional acyltransferase that likely plays an important role in lipid metabolism in human skin.

Sjogren-Larsson syndrome (SLS) is an inherited disorder associated with deficient oxidation of long-chain aliphatic alcohols. Previous studies have reported modest elevations in total (free 1 esterified) fatty alcohols in SLS, but free fatty alcohols have not been selectively measured, in part because of their low concentrations in most tissues and the presence of trace fatty alcohol contaminants in some solvents used for their analysis. We adapted methods to measure free fatty alcohols in cultured cells and plasma that minimize exogenous alcohol contamination. Fatty alco- hols were analyzed as acetate derivatives, using capillary column gas chromatography. By this method, cultured skin fibroblasts from SLS patients were found to have 7- and 8-fold elevations in the mean content of hexadecanol (16:0- OH) and octadecanol (18:0-OH), respectively. The mean plasma 16:0-OH and 18:0-OH concentrations in SLS pa- tients (n 5 11) were 9- and 22-fold higher than in normal controls, respectively. In SLS fibroblasts, most of the fatty alcohol (59%) that accumulated was free rather than esteri- fied alcohol, whereas free alcohol accounted for 23% of the total alcohol in normal cells. These results indicate that elevations in free fatty alcohols provide a sensitive marker for the enzymatic defect in SLS. The ability to measure free fatty alcohols in cultured cells and plasma should prove use- ful for investigations of normal fatty alcohol metabolism and the deranged metabolism in SLS. —Rizzo, W. B., and D. A. Craft. Sjorgren-Larsson syndrome: accumulation of free fatty alcohols in cultured fibroblasts and plasma . J. Lipid Res . 2000. 41: 1077-1081.

Pneumocystis carinii carinii and rat lung phospholipids contained 3–6% 1-alkyl-2-acyl glycerols composed of the glyceryl ether species, 1-O-octadecyl glycerol (batyl alcohol), 1-O-octadec-9-enyl glycerol (selachyl alcohol), 1-O-hexadecyl glycerol (chimyl alcohol), and 1-O-hexadec-9-enyl glycerol. Of the major phospholipid classes, phosphatidylinositol (PI) and phosphatidylserine contained the highest percentage of alkyl acyl glycerols. Methylprednisolone treatment caused an increase in alkyl acyl PI of rat lung lipids from 12% to 45%. As the PI concentration in lung phospholipids increases in rats treated with methylprednisolone, the increase in alkyl acyl PI was substantial; the proportions of alkyl acyl phosphatidylethanolamine and alkyl acyl lyso phosphatidylcholine (PC) also increased. Pneumocystis phospholipids contained higher proportions of alkyl acyl PC than the phospholipids of the lungs from normal and immunosuppressed uninfected rats. The glyceryl ether compositions of P. carinii carinii PC and lyso PC were similar, which suggests that lyso PC in the organism is derived by phospholipase A2 action on PC. This was not the case for PC and lyso PC of the lung controls. Analysis of the free fatty alcohols, precursors of glyceryl ethers identified only saturated species in P. carinii carinii and rat lung controls. Thus, the introduction of a double bond in the alcohol moiety of glyceryl ethers occurs after formation of the ether linkage between fatty alcohol and the glyceryl backbone.—Kaneshiro, E. S., Z. Guo, D. Sul, K. A. Kallam, K. Jayasimhulu, and D. H. Beach. Characterization of Pneumocystis carinii and rat lung lipids: glyceryl ethers and fatty alcohols. J. Lipid. Res. 1998. 39: 1907–1917.

Long-chain alcohols are synthesized in the mouse preputial gland tumor (ESR-586) by NADPH:acyl-CoA oxidoreductase. In this study, a series of labeled acids was tested as substrates for the oxidoreductase in a cell-free system from the tumor, and the distribution of label into alcohols, waxes, and other products was determined. The system contained the labeled acid, an acyl-CoA-generating system, an NADPH-generating system, and tumor homogenate. The highest rates of alcohol synthesis were obtained with palmitic (16:0), heptadecanoic (17:0), stearic (18:0), myristic (14:0), elaidic (18:1 trans), and linoleic (18:2) acids, which yielded, respectively, 151, 124, 102, 76, 65, and 35 pmol alcohol/min per mg protein. Decanoic (10:0), lauric (12:0), oleic (18:1 cis), linolenic (18:3), arachidonic (20:4), and behenic (22:0) acids all gave lower activities. Acyl-CoA formation did not appear to be rate limiting with any of the substrates tested except behenic acid. In addition to the fatty alcohol product, a small amount of fatty aldehyde was formed in the system. Incorporation of the labeled fatty acids into wax esters was examined and the distribution of label between the alcohol and acid components of the waxes was determined. Incubation of [1-(14)C]palmitic acid yielded 3.4% free alcohol, 8.3% alcohol esterified in waxes, and 7.7% palmitoyl groups esterified into waxes, whereas, at the other extreme, [1-(14)C]linolenic acid yielded 0.8%, 0.6%, and 38%, respectively, into the homologous components.-Wykle, R. L., B. Malone, and F. Snyder. Acyl-CoA reductase specificity and synthesis of wax esters in mouse preputial gland tumors.

The seasonal variations in the total lipid content, lipid class composition, fatty acid composition, and fatty alcohol composition of Meganyctiphanes norvegica (M. Sars), Thysanoessa inermis (Krøyer), and T. raschii (M. Sars) have been examined. The total lipid content was highest in the autumn and early winter months and lowest in the spring. In M. norvegica, triacylglycerols served as the only depot lipids, whereas in T. inermis and T. raschii triacylglycerols, wax esters, and glycerophospholipids varied in proportion to the total lipid content. This suggests that glycerophospholipids, as well as wax esters and triacylglycerols, constitute depot lipids in these species. Wax esters and glycerophospholipids were the dominating depot lipids in T. inermis, whereas triacylglycerols and glycerophospholipids were most important in T. raschii. Results suggest that non-depot glycerophospholipids may constitute 3.5-4.5% of the dry weight of the three species of krill examined. T. inermis and T. raschii, from the same catches, had very similar fatty acid compositions for each of the major lipid classes, with the exception of a few minor fatty acids. The major lipid classes in all three species showed complex seasonal variations in the content of the fatty acids that typically reflect the diet, particularly in the case of the triacylglycerols. The results suggest that all the species examined are more herbivorous during the summer than during the autumn and winter. M. norvegica seemed to be significantly more carnivorous than the two Thysanoessa species. The degree of incorporation of individual fatty acids from the diet is probably specific for each lipid class in each krill species. The proportion of polyenoic fatty acids in the glycerophospholipids and the proportion of monoenoic fatty acids in the wax esters may be of importance for the temperature adaptation of T. inermis and T. raschii.

Biosynthesis of wax esters, one of the two major products of the meibomian gland, was found to be catalyzed mainly by the microsomes of the bovine meibomian gland. The microsomal preparation catalyzed hexadecanoyl-CoA reduction to hexadecanol without any accumulation of the aldehyde intermediate. Maximal rates of reduction occurred at pH 6.5 and required both NADH and NADPH; the latter alone gave considerable rates whereas NADH alone was ineffective. Exogenous hexadecanal reduction catalyzed by the same preparation showed a preference for NADH. The hexadecanoyl-CoA saturation pattern was slightly sigmoidal and concentrations higher than 125 microM inhibited reduction. The fatty alcohol generated from hexadecanoyl-CoA was found as free alcohol and as wax esters. Esterification of hexadecanol to wax esters catalyzed by the meibomian gland microsomal preparation required exogenous acyl-CoA or ATP and CoA and was not affected by exogenous cholesterol. Maximal rates of esterification were observed at neutral pH. Hexadecanoyl-CoA concentrations higher than 125 microM inhibited esterification. Hexadecanol showed a typical substrate saturation pattern with an apparent Km of 125 microM. Radio gas-liquid chromatography showed that, in the presence of exogenous hexadecanoyl-CoA, hexadecanol gave hexadecyl hexadecanoate whereas in the presence of ATP and CoA both C16 and C18 endogenous acids were used to esterify the alcohol. Consistent with the composition of the meibomian gland secretion, exogenous acyl-CoA longer than C14 and shorter than C20 gave maximal rates of esterification of hexadecanol.

Diether glycerophosphocholines containing one long and one short chain were synthesized in moderate yield. The synthesis of a diether glycerophosphocholine containing a branched chain and a straight chain is also described. Alkylation of fatty alcohol with allyl bromide and dimethyl sulfoxide anion gave fatty alkyl allyl ether, which was hydroxylated and tritylated. By procedures described previously, 1-O-alkyl-3-O-triphenyl-methylglycerol was alkylated, detritylated, and converted into the phosphocholine product. This method is of general utility for the preparation of a variety of mixed-chain racemic glycerophosphocholines whose physical properties are of interest in studies of biological membranes.

Lipids secreted from the bovine meibomian glands were assigned to the following classes: cholesteryl esters (A) (fatty acyl chain lengths from 15 to 27 carbon atoms), 41%; wax esters, 29%; triacylglycerols, 10%; and cholesteryl esters (B) (fatty acyl chain lengths from 14 to 18 carbons), 15%. The remaining 5% consisted of choles- terol, fatty acids, and highly polar material. Analysis of the lipids showed that only the cholesteryl esters (A) con- tained fatty acyl chains 2 Cz0:,. One-third of the fatty acyl chains of the wax esters and the cholesteryl esters (B) was iC15:o. The fatty alcohol moieties of the wax esters were found to be branched chain, Cz3:o-Cz7:o. A computer- based programmable gas-liquid chromatographic pro- cedure using 10% apolar 10C on Gas Chrom Q could be used to distinguish both is0 and anteis0 fatty alcohols and esters of fatty acids. The oily secretions of the meibomian glands are a major constituent of the precorneal tear film. These glands, located at the upper and lower margins of the eyelid, secrete materials that are thought to be responsible for the lubrication between the lid mar- gin and the cornea and that act as a hydrophobic barrier preventing evaporation of water from the tear film (1). The oils from the human meibomian glands are composed mainly of nonpolar lipids, principally cholesteryl esters and wax esters (2, 3), but they also appear to contain a surface active component (4). Analysis of the composition of the principal ester fraction from human meibomian secretions showed the major fatty acyl moieties to be nC18,1, UC~~:~, ic26:o and nC,,:,, while the major alcohols are cholesterol, ic26:0, nC2,:,, iC24:0, and nCZk0 (2). In this paper, we shall describe the characteriza- tion of bovine meibomian secretions. The principal nonpolar lipids, in particular the wax esters and cholesteryl esters, were separated by TLC and then the resulting fatty acyl and alcohol moieties were analyzed by GLC. MATERIALS AND METHODS

Procedures are described for the preparation of alkyl iodides from fatty alcohols and glyceryl ethers. Alkyl iodides were separated directly by gas-liquid chromatography (GLC) on ethylene glycol succinate columns and do not require calibration factors in their analysis with a thermal conductivity detector. Iodide derivatives have absolute retention times on GLC similar to the corresponding fatty alcohol acetates. Alkyl iodides were converted to alcohol acetates with silver acetate or to hydrocarbons with lithium aluminum hydride and these derivatives were separated by GLC. The elimination of hydriodic acid from secondary iodides during GLC is described. The resulting monoene iodides are readily separated from saturated iodides by GLC. Alkoxy structures have been assigned by comparison of the retention time data of iodide derivatives with standard alcohol derivatives. Unsaturation was confirmed by catalytic hydrogenation. Branched components were identified as the alkanes. Glyceryl ethers were prepared from diacyl glyceryl ethers by hydrogenolysis with lithium aluminum hydride. The glyceryl ether-fatty alcohol mixture was separated quantitatively by thin-layer chromatography (TLC) and sufficient material obtained from one TLC plate for the preparation of alkyl iodide derivatives.