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Sphingosine 1-phosphate (S1P), a bioactive lipid involved in various physiological processes such as cell proliferation and apoptosis, can be irreversibly cleaved by S1P lyase, yielding phosphoethanolamine and (2E)-hexadecenal (2EHD). The latter metabolite, an α,β-unsaturated fatty aldehyde, may be susceptible to nucleophilic attack by cellular biomolecules. Hence, we studied whether 2EHD forms reaction products with GSH and proteins in vitro. Using LC-MS/MS and stable isotopically labeled reference material, we identified a total of nine novel reaction products of 2EHD in a cell-free approach: two GSH conjugates and seven l-amino acid adducts. Both GSH conjugates were also found in HepG2 cell lysates incubated with 2EHD. Likewise, we detected four out of seven amino acid adducts released from the model protein, BSA, and proteins extracted from HepG2 cells. On this occasion, the 2EHD Michael adduct with l-histidine proved to be the most prominent adduct. Most interestingly, inhibition of the enzymatically driven oxidative degradation of 2EHD resulted in increased levels of both GSH conjugates and protein adducts in HepG2 cell lysates. Hence, our data provide new insights into sphingolipid metabolism and will be useful to investigate certain disorders linked to an impaired fatty aldehyde metabolism in more detail.

Fatty aldehyde dehydrogenase (EC 1.2.1.48) converts long-chain fatty aldehydes to the corresponding acids. Deficiency in this enzyme causes the Sjogren Larsson Syndrome, a rare inherited disorder characterized by ichthyosis, spasticity, and mental retardation. Using a fluorescent aldehyde, pyrenedecanal, and HPLC with fluorescence detection, we developed a novel method to monitor fatty aldehyde dehydrogenase activity by quantification of the product pyrenedecanoic acid together with the substrate pyrenedecanal and possible side products, such as aldehyde adducts. As shown with recombinant enzymes, pyrenedecanal showed a high preference for fatty aldehyde dehydrogenase compared with other aldehyde dehydrogenases. The method allowed detection of fatty aldehyde dehydrogenase activity in nanogram amounts of microsomal or tissue protein and microgram amounts of Sjogren Larsson syndrome patients' skin fibroblast protein. It could successfully be adapted for the analysis of fatty aldehyde dehydrogenase activity in gel slices derived from low-temperature SDS-PAGE, showing that fatty aldehyde dehydrogenase activity from solubilized rat liver microsomes migrates as a dimer. Thus, monitoring of pyrenedecanoic acid formation from pyrenedecanal by HPLC with fluorescence detection provides a robust and sensitive method for determination of fatty aldehyde dehydrogenase activity.-Keller, M. A., K. Watschinger, G. Golderer, M. Maglione, B. Sarg, H. H. Lindner, G. Werner-Felmayer, A. Terrinoni, R. J. A. Wanders, and E. R. Werner. Monitoring of fatty aldehyde dehydrogenase by formation of pyrenedecanoic acid from pyrenedecanal. J. Lipid Res. 2010. 51: 1554-1559

Modified galactosylceramide with a long-chain cyclic acetal at the sugar moiety, plasmalogalactosylceramide, was isolated from equine brain. To identify the isomeric stereostructure of the natural product, the plasmalo derivative was chemically synthesized from galactosylceramide through acetalization. The presence of cyclic acetal linkage, the linked position and length of the acetal chain of the synthesized and natural products were determined by proton nuclear magnetic resonance spectroscopy and fast-atom bombardment–mass spectrometry, as well as gas chromatography–mass spectrometry and gas–liquid chromatography. The orientation of the acetal chain linked to galactoside was characterized by connectivity between the cyclic acetal proton and ring proton(s) on the sugar moiety using the homonuclear Overhauser effect. This revealed that, of the two positional isomers of the acetal linkage with 4,6-O-acetal and 3,4-O-acetal derivatives obtained from the acetalization reaction, the former positional isomer, separated into two spots, was identified to 'endo'- and 'exo'-type acetal chains. In comparison to the NMR data of the synthesized derivative, equine brain acetalized lipid was found to be an 'endo'-type 4,6-O-acetal derivative.—Yachida, Y., M. Kashiwagi, T. Mikami, K. Tsuchihashi, T. Daino, T. Akino, and S. Gasa. Stereochemical structures of synthesized and natural plasmalogalactosylceramides from equine brain. J. Lipid Res. 1998. 39: 1039–1045.

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.

Saturated and unsaturated fatty aldehydes were prepared in good yield by the reduction of acid chlorides with lithium aluminum tri-t-butoxy hydride. Saturated odd and even numbered aldehydes were prepared by the ozonolysis- reduction of 1-alkenes. Ozonides were hydrogenated with a Lindlar catalyst or reduced with dimethyl sulfide. Several diols, including 1,3-propanediol and ethylene gly- col, were used to synthesize cyclic acetals from aldehydes and plasmalogens in quantitative yield. Cyclic acetals were synthe- sized from 2,4-dinitrophenyl hydrazones when an exchanger such as acetone or acetylacetone was included in the reaction mixture. A number of physical and chemical properties indicate that cyclic acetals are stable compounds which do not decompose during storage or gas-liquid chromatographic (GLC) analysis. The cyclic acetals have unusually long retention volumes which are probably related to the large dipole moments found with cyclic compounds. These derivatives are, therefore, readily separated from their aldehyde and methyl ester analogues on polar and nonpolar stationary phases. GLC analysis of the aldehydogenic moieties in plasmologens may be conveniently carried out by direct conversion to cyclic acetals without pre- liminary isolation of aldehydes or removal of methyl esters by saponification.

Improved methods for lipid analysis that have been developed recently were employed to reevaluate the phospholipid composition, the fatty acid and fatty aldehyde composition of the total phospholipid, and the fatty acid composition of the individual phospholipids of normal human red cells. Thirty-three fatty acids and five fatty aldehydes were estimated and tentatively identified in the total phospholipid of normal human red cells. Additional minor components were evident. The major individual phospholipids were isolated by silicic acid thin-layer chromatography and quantified. The fatty acid compositions of phosphatidyl ethanolamine, phosphatidyl serine, lecithin, and sphingomyelin were determined. Each of these phospholipids showed a distinctive and characteristic fatty acid pattern.

Gray matter, white matter, and myelin were isolated from the frontal lobes of the brains of humans aged 10 months, 6 yr, 9 yr, and 55 yr. The major lipids, including ethanolamine glycerophosphatides (EGP), serine glycerophosphatides (SGP), choline glycerophosphatides (CGP), sphingomyelin, cerebroside, cerebroside sulfate, and ceramide were isolated by column chromatography and their fatty acid and fatty aldehyde compositions were determined by gas–liquid chromatography. EGP and SGP from myelin had a fatty aldehyde composition which differed from that of EGP and SGP from gray matter; octadecenaldehydes were present in much higher proportions in these lipids from myelin than in those from gray matter. EGP and SGP also contained high proportions of 20- and 22-carbon polyunsaturated fatty acids, whereas CGP contained small proportions of these acids. Each glycerophosphatide from gray matter contained approximately 3- to 6-fold higher proportions of polyunsaturated fatty acids than did the same glycerophosphatide from myelin. Sphingomyelin, cerebroside, cerebroside sulfate, and ceramide also differed in their fatty acid compositions depending upon their tissue source; each sphingolipid from myelin in the younger subjects contained 5- to 9-fold higher proportions of long-chain fatty acids (C19-C26) than did the same sphingolipid from gray matter. The lipids from myelin in the baby (10 months) were very similar to those from myelin in the adult, both with respect to their content of polyunsaturated fatty acids and to their content of long-chain fatty acids. These findings suggest that myelin in the baby is “chemically mature” in its lipid composition at an early age.

The quantities of ethanolamine glycerophosphatides (EGP), choline glycerophosphatides (CGP), and serine glycerophosphatides (SGP) were determined in the grey and white matter from three apparently normal adult human brains. In each locale the quantities decreased from EGP through CGP to SGP. The quantities of aldehydes in these lipids were also determined. In grey matter the aldehyde content (expressed as per cent of fatty acids plus aldehydes) of EGP, CGP, and SGP was 22, 0.3, and 0.2% respectively; while in white matter the proportions were 49, 0.8, and 13%, respectively. Palmitaldehyde, stearaldehyde, and olealdehyde made up 90% of the aldehydes found. White matter glycerophosphatides contained much more olealdehyde than those from grey matter. EGP and SGP contained much larger proportions of C20 and C22 polyenoic acids from grey matter than from white matter. CGP, on the other hand, had a similar fatty acid composition, comprised mainly of 16:0, 18:0, and 18:1 acids, in each locale. The differences in fatty acid composition of these three glycerophosphatides may be related to the higher myelin content of white matter.

A method is described for the identification and determination of sphingolipid long-chain bases in various animal and plant lipids. Sphingosine and related bases, isolated as a mixture from acid hydrolysates of sphingolipids, are oxidized by sodium metaperiodate and the fatty aldehyde reaction products are isolated and analyzed by gas-liquid partition chromatography. The records thus obtained reflect the composition of the sphingolipid base fraction. A preliminary survey of various tissues is reported and the types of long-chain base found for each tissue are given. Evidence is presented for the presence of a new long-chain base associatcd with the sphingomyelin fraction of human plasma lipids.

Myofibrillar, mitochondrial, and microsomal fractions were prepared from normal and dystrophic mouse limb muscle by differential centrifugation and analyzed for phospholipids and cholesterol. Fatty acids and aldehydes of neutral lipids and of phospholipids from whole muscle and particulate fractions were also determined. Normal microsomes contained more lecithin and less total ethanolamine phospholipids and cardiolipin than mitochondria. The myofibrils had an intermediate phospholipid composition, but their cholesterol–phospholipid ratio was smaller than that of the other two fractions. Except for an increased percentage of phosphatidalethanolamine in the dystrophic mitochondria, only the composition of the dystrophic microsomes differed from normal by containing less lecithin but more total ethanolamine phospholipid, phosphatidalethanolamine, sphingomyelin, and cholesterol. No significant differences were found in the fatty acid composition of neutral lipid extracts from normal and dystrophic preparations, but there was a significant decrease in the percentage of 22:6 in phospholipids from both dystrophic whole muscle and microsomes (–25% and –37%, respectively), whereas the 20:4 content was unaltered. By contrast, the percentages of 18:0 and total fatty aldehyde increased significantly. Phospholipid extracts from all dystrophic samples showed a significant decrease in 16:0 and an increase in 18:1 as compared with the normal.

The behavior of palmitaldehyde and linolealdehyde and of their dimethyl acetals during gas-liquid chromatography on beta-cyclodextrin acetate (beta-CDX acetate) and ethylene glycol succinate polyester-phosphoric acid (EGSP) columns was studied. The aldehydes were well separated from their dimethyl acetals on the beta-CDX acetate column. However, on the EGSP column the retention times of palmitaldehyde and its dimethyl acetal were identical; a mixture of linolealdehyde and its dimethyl acetal gave a split peak. The aldehydes were recovered unchanged in 80-85% yield by preparative GLC from both columns, but the dimethyl acetals were quantitatively converted to the corresponding alk-1-enyl methyl ethers. The structure of these compounds was elucidated by infrared spectroscopy, mass spectrometry, and chemical means. Upon hydrolysis at low temperatures with 100% H(2)SO(4) they yielded the corresponding aldehydes, which were identified as 2,4-dinitrophenylhydrazones.