1-O-acylceramides are natural components of human and mouse epidermis

Lysosomal phospholipase A2 (LPLA2) is characterized by increased activity toward zwitterionic phospholipid liposomes containing negatively charged lipids under acidic conditions. The effect of anionic lipids on LPLA2 activity was investigated. Mouse LPLA2 activity was assayed as C2-ceramide transacylation. Sulfatide incorporated into liposomes enhanced LPLA2 activity under acidic conditions and was weakened by NaCl or increased pH. Amiodarone, a cationic amphiphilic drug, reduced LPLA2 activity. LPLA2 exhibited esterase activity when p-nitro-phenylbutyrate (pNPB) was used as a substrate. Unlike the phospholipase A2 activity, the esterase activity was detected over wide pH range and not inhibited by NaCl or amiodarone. Presteady-state kinetics using pNPB were consistent with the formation of an acyl-enzyme intermediate. C2-ceramide was an acceptor for the acyl group of the acyl-enzyme but was not available as the acyl group acceptor when dispersed in liposomes containing amiodarone. Cosedimentation of LPLA2 with liposomes was enhanced in the presence of sulfatide and was reduced by raising NaCl, amiodarone, or pH in the reaction mixture. LPLA2 adsorption to negatively charged lipid membrane surfaces through an electrostatic attraction, therefore, enhances LPLA2 enzyme activity toward insoluble substrates. Thus, anionic lipids present within lipid membranes enhance the rate of phospholipid hydrolysis by LPLA2 at lipid-water interfaces.

Sphingolipid Metabolism in Organotypic Mouse Keratinocyte Cultures

Retronasal discrimination between vapor-phase fatty acids was tested in 40 participants. Linoleic, oleic, or stearic fatty acids were used in stimulus delivery containers (SDC) at concentrations previously determined to be detectable when presented vapor phase retronasally (Bolton and Halpern in Chem Senses 35:229–238, 2010). For each of these 18-carbon fatty acids, participants received, retronasally, 5 SDC on 2 trials, with 4 of the containers holding, in random order, 1 fatty acid, and the 5th container, a different fatty acid. In addition, for a “negative control”, on two trials, participants received very dilute linoleic acid (0.005%) versus mineral oil. For all trials, the task was to select the one different SDC. It was found that the one different SDC was selected on 83% of the stearic versus linoleic acid trials, 75% of stearic versus oleic acid trials, 58% of linoleic versus oleic acid trials, and 12% of very dilute linoleic acid versus mineral oil trials. An ANOVA found a difference between these distributions, p < 0.0001. Seventy percent of participants discriminated, i.e., selected the 1 different SDC on both trials (chance p = 0.04) between stearic and linoleic acids, 65% discriminated between stearic and oleic acids, and 38% discriminated between linoleic and oleic acids. No participants discriminated on “negative control” trials. These data suggest that retronasal vapor-phase stearic fatty acid is discriminated from vapor-phase linoleic or oleic fatty acids and that oleic and linoleic fatty acids, at concentrations known to be detectable, are discriminated from each other by a substantial minority of participants.

Group XV phospholipase A2, a lysosomal phospholipase A2

The photosynthetic apparatus of higher plants consists of discrete cytoplasmic organelles, the chloroplasts, which contain proteins, lipids, pigments, water, and other substances. Relatively little is known, apparently, concerning the composition of the lipid fraction of the chloroplast. Menke (8), studying chloroplasts of spinach, Spinacia oleracea, found that essentially all the lipids of the leaf were located in the chloroplasts. The chloroplasts had an average lipid content of 30.9 % of the dry weight, while the cytoplasm had a lipid content of only 0.5 %. For this reason, a number of earlier studies of the lipid content and composition of green leaves as a whole are pertinent to the present problem. Chibnall and Channon (3), studying the fatty acids of leaves of cabbage, Brassica oleracea, found that approximately 10.7 % of the fatty acid fraction consisted of saturated acids, and 89.3 % of unsaturated acids. The saturated fraction was composed of 70 % palmitic acid and 30 % stearic acid. The unsaturated fraction included large quantities of linoleic and linolenic acids. Speer et al. (13) examined the lipids of spinach leaves, finding that 53 % of the fatty acidls occurred in the free form, with 47 % as glycerides. The saturated acids consisted chiefly of palmitic and stearic acids. Oleic acid made up 26.3 %, linoleic acid 34.7 %, and linolenic acid 12.7 % of the unsaturated lipid fraction. The fatty acids of the forage grasses Dactylis glomierata and Lolium perenne were examined by Smith and Chibnall (12). Saturated acids composed 11 % of the total in Dactylis and 12 % in Lolium; the corresponding figures for oleic acid were 16 and 22 %, for linoleic acid 31 and 26 %, and for linolenic acid 42 and 40 %, respectively. According to Chibnall (2), cabbage leaves contain 17.5 % glycerides, 12.3 % waxes, 4.5 % sterols, and 18.4 % phospholipids. Comparable figures for cocksfoot grass, Dactylis glomnerata, were 38.0 % glycerides, 23.5 % waxes, 3.0 % sterols, and 1.5 % phospholipids. Menke (9) isolated chloroplasts from spinach leaves by centrifugation; he found that their lipid content was approximately 37 %. The ether-soluble fraction of the chloroplasts is a complex mixture of pigments, fatty acids, glycerides, phosphatides, and sterols. Bot (1) isolated grana from chloroplasts of Lathyrus odorattis and Spinacia oleracea. These were shown to have an ether-soluble fraction, excluding chlorophyll, of 18 to 30 % of the dry weight in sweet pea and 26 to 32 % in spinach. Comar (4) determined the lipid content of spinach chloroplasts as 34%. Menke and Jacob (10) extracted isolated chloroplasts of spinach with ether or ether-alcohol mixtures. The extracts were fractionated into a larger acetonesoluble fraction, making up about 80 % of the total, and containing the pigments, triglycerides and sterols, and an acetone-insoluble fraction including phosphatides and waxes. The pigment-free portion of the acetone-soluble fraction consisted of 5.1 % glycerol, 42.2 % fatty acids, 49.0 % triglycerides, and 2.1 % sterols. Sisakyan and Smirnov (11) found that fatty acids composed 57.8 % of the dry weight of the chloroplasts of sugar beet, 48.8 % in sunflower, and 60.0 % in red clover. Isolated chloroplasts of sunflower incorporated acetate-C'4 into higher fatty acids in either light or darkness, but bean chloroplasts did not. In manometric experiments, chloroplasts of bean oxidized palmitic, oleic, linoleic, and linolenic acids. Therefore, the chloroplasts are metabolically active in both fatty acid synthesis and oxidation. Zill and Harmon (14) investigated the chloroplast lipids of spinach by the technique of chromatography on silicic acid columns. Of the crude whole spinach extract 24 % was represented by two monoglycerides, which were not specifically identified. Further studies of chloroplast lipids by these workers are in progress (personal communication). Debuch (5) has recently analyzed the fatty acid composition of chloroplasts of Antirrhinum majus, a plastid mutant of this species, and Alliumn porrum by means of gas chromatography. C1l6 fatty acids composed 10 to 20 % and C18 fatty acids composed 80 to 90 % of the total. Linolenic acid was the component present in greatest amount (44.1-71.3 % of the total), linoleic acid (15.0-29.2 %) and palmitic acid (9.519.7 %) were the other principal constituents. The studies reported in this paper were designed to determine the fatty acid composition of chloroplasts obtained from spinach leaves.

Phospholipids are required for epidermal lamellar body formation. Glycerol 3-phosphate acyltransferases (GPATs) catalyze the initial step in the biosynthesis of glycerolipids. Little is known about the expression and regulation of GPATs in epidermis/keratinocytes. Here, we demonstrate that GPAT 1, 3, and 4 are expressed in epidermis/keratinocytes, whereas GPAT2 is not detected. In mouse epidermis, GPAT 3 and 4 are mainly localized to the upper layers whereas GPAT1 is found in both the upper and lower layers. GPAT1 and 3 mRNA increase during fetal rat epidermal development. No change in GPAT expression was observed in adult mice following acute permeability barrier disruption. Calcium-induced human keratinocyte differentiation increased GPAT3 mRNA whereas both GPAT1 and 4 mRNA levels decreased. In parallel, total GPAT activity increased 2-fold in differentiated keratinocytes attributable to an increase in N-ethylmaleimide (NEM) sensitive GPAT activity localized to microsomes with little change in NEM resistant activity, consistent with an increase in GPAT3. Furthermore, PPARγ or PPARδ activators increased GPAT3 mRNA, microsomal GPAT activity, and glycerol lipid synthesis without affecting the expression of GPAT1 or 4. Finally, both PPARγ and PPARδ activators increased GPAT3 mRNA via increasing its transcription. Thus, multiple isoforms of GPAT are expressed and differentially regulated in epidermis/keratinocytes.

The present study was conducted to determine lysosomal phospholipase A2 (LPLA2) activity in the aqueous humor (AH) and to identify the possible sources of the LPLA2 found in the AH. To detect LPLA2 activity in pig AH and ocular tissues, liposomes consisting of 1,2-dioleoylphosphatidylglycerol/N-acetylsphingosine were used as substrates in an activity assay under acidic conditions. The reaction products were separated by thin-layer chromatography. To identify the LPLA2 in pig AH, the AH was analyzed by Western immunoblot analysis with an anti-LPLA2 antibody. Distribution of the LPLA2 in pig ocular tissues was studied by determining its activity in individual tissue extracts. LPLA2 activity was detected in the AH obtained from pig eyes. Consistent with the known properties of LPLA2, the activity was heat-labile and undetectable at neutral pH. The immunoblot of pig AH showed the anti-LPLA2 antibody-reactive protein band. In addition, the specific activity of the enzyme, when normalized to volume, was higher in pig AH than in pig serum. Individual tissue extracts obtained from pig ocular tissues showed different specific activity of LPLA2. In particular, the extract prepared from the trabecular meshwork provided the highest specific activity. The present findings suggest that the phospholipase A2 activity found in pig AH under acidic conditions is due to LPLA2 and that it originates from ocular tissues surrounding the anterior chamber as well as plasma.

Calcium salts of polyunsaturated fatty acids deliver more essential fatty acids to the lactating dairy cow

The metabolism of the linolenic acid family (n-3) of fatty acids, e.g., linolenic, eicosapentaenoic, and docosahexaenoic acids, in cultured smooth muscle cells from rabbit aorta was compared to the metabolism of linoleic and arachidonic acids. There was a time-dependent uptake of these fatty acids into cells for 16 hr (arachidonic greater than docosahexaenoic, linoleic, eicosapentaenoic greater than linolenic), and the acids were incorporated mainly into phospholipids and triglycerides. Eicosapentaenoic and arachidonic acids were incorporated more into phosphatidylethanolamine and phosphatidylinositol plus phosphatidylserine and less into phosphatidylcholine than linolenic and linoleic acids. Docosahexaenoic acid was incorporated into phosphatidylethanolamine more than linolenic and linoleic acids and into phosphatidylinositol plus phosphatidylserine less than eicosapentaenoic and arachidonic acids. Added linolenic acid accumulated mainly in phosphatidylcholine and did not decrease the arachidonic acid content of any phospholipid subfraction. Elongation-desaturation metabolites of linoleic acid did not accumulate. Cells treated with eicosapentaenoic acid accumulated both eicosapentaenoic and docosapentaenoic acids mainly in phosphatidylethanolamine and the arachidonic acid content was decreased. Added docosahexaenoic acid accumulated mainly in phosphatidylethanolamine and decreased the content of both arachidonic and oleic acids. The following conclusions are drawn from these results. The three n-3 fatty acids are utilized differently in phospholipids. The arachidonic acid content of phospholipids is reduced by eicosapentaenoic and docosahexaenoic acids, but not by linolenic acid. Smooth muscle cells have little or no desaturase activity, but have significant elongation activity for polyunsaturated fatty acids.

The subcellular distribution of the phospholipase activities and metabolism of lysophosphatidylcholine in cultured human cell line (FL cells) during "fusion-from-within" induced by measles virus were studied. During cell fusion, fairly high activities of phospholipase A1 and A2, the optimal pHs of which were acidic, appeared in the cytosol. These phospholipases were confirmed to be of lysosomal origin by the following facts: (1) the properties of these phospholipases were the same as those of lysosomal phospholipase A1 and A2; (2) a decrease in the total activity of phospholipases A1, A2 and acid phosphatase in the lysosomal fraction of the infected cells coincided with an increase in their activity in the cytosol. The release of lysosomal phospholipase A1 and A2 into the cytosol of the infected cells appeared to be related to the extent of cell fusion-from-within. Phospholipase A1 and A2 released to the cytosol hydrolyzed the cell membrane-bound phospholipids to form lysophospholipids. 1-[1-14C]Palmitoyl-GPC, incorporated into the cells from the culture medium, was rapidly converted into phosphatidylcholine, triacylglycerol and phosphatidylethanolamine in the cells during fusion-from-within, but the radioactive lysophosphatidylcholine and fatty acids were hardly detectable, indicating a rapid turnover of cellular lysophosphatidylcholine during cell fusion. The subcellular lysophospholipid acyl-hydrolase activities of the infected cells were higher than those of normal cells. Possible relation between the membrane fusion induced by measles virus and the phospholipid metabolism in the infected cells were discussed.

Phospholipids are a major class of lipids in epidermis, where they serve as a source of free fatty acids that are important for the maintenance of epidermal permeability barrier function. The phospholipid biosynthetic enzyme, 1-acyl-sn-glycerol-3-phosphate acyltransferase (AGPAT), catalyzes the acylation of lysophosphatidic acid to form phosphatidic acid, the major precursor of all glycerolipids. We identified an expression pattern of AGPAT isoforms that is unique to epidermis, with relatively high constitutive expression of mouse AGPAT (mAGPAT) 3, 4, and 5 but low constitutive expression of mAGPAT 1 and 2. Localization studies indicate that all five isoforms of AGPAT were expressed in all nucleated layers of epidermis. Furthermore, rat AGPAT 2 and 5 mRNAs increased in parallel with both an increase in enzyme activity and permeability barrier formation late in rat epidermal development. Moreover, after two methods of acute permeability barrier disruption, mAGPAT 1, 2, and 3 mRNA levels increased rapidly and were sustained for at least 24 h. In parallel with the increase in mRNA levels, an increase in AGPAT activity also occurred. Because upregulation of mAGPAT mRNAs after tape-stripping could be partially reversed by artificial barrier restoration by occlusion, these studies suggest that an increase in the expression of AGPATs is linked to barrier requirements.

Human epidermis contains endogenous retinoids (retinol and retinyl esters) and carotenoids (mostly beta-carotene). Previous studies have shown that the enzymes involved in retinoid metabolism are present in human epidermis. There is still a controversy about the presence in the skin of the enzymes able to convert beta-carotene into vitamin A (retinol), although a recent study demonstrated the conversion of beta-carotene into retinol in human cultured epidermal cells. In this study, we addressed the question of the possible bioconversion of topical beta-carotene into vitamin A or derivatives by human and mouse skin. Surgically excised human abdominal skin was mounted on Franz perfusion chambers to assess the cutaneous penetration of topical beta-carotene as well as its metabolism, after a 24-h incubation period, whereas hairless mice received topical beta-carotene 24 h before assaying epidermal beta-carotene and retinoid concentrations. Epidermal retinoid and beta-carotene concentrations were determined by high-pressure liquid chromatography. Topical beta-carotene penetrated well into human and mouse epidermis and induced a 10-fold (human) and a threefold (mouse) increase of epidermal retinyl esters, which demonstrates that topical beta-carotene is converted into retinyl esters by human and mouse epidermis and thus appears as a precursor of epidermal vitamin A.

Ten matings among adapted oat (Avena sativa L.) cultivars were employed to study the inheritance of fatty‐acid composition of oat groat oil. Heritability estimates in six matings ranged from 0.33 to 0.56 using unreplicated hill plots for palmitic, oleic, linoleic, and linolenic acids. Heritabilities based on progeny means of the same six matings over two replicates at each of three locations were 0.68, 0.72, and 0.64 for palmitic, oleic, and linoleic acids, respectively. Effects of matings and matings ✕ locations interaction and variation among lines within the six matings were significant (P &lt; 0.01) for palmitic, oleic, and linoleic acids. Correlations between contents of fatty acids in groat oil were: palmitic and oleic acid, −0.47; palmitic and linoleic, −0.30; palmitic and linolenic, −0.10; oleic and linoleic, −0.55; oleic and linolenic, −0.43; and linoleic and linolenic, −0.04. Additive gene action was the most important genetic component of variation among generation means of four additional matings for palmitic, oleic, and linoleic acids. Additive ✕ additive effects were significant for oleic acid in one mating and for linoleic acid in a second, but dominance and epistatic effects involving dominance were of no importance in fatty‐acid inheritance. It should be possible to breed oat genotypes with high or low levels of either saturated or unsaturated fatty acids in the groat oil. The importance of additive gene action implies that desired allelic combinations from these matings could be obtained in pureline cultivars.

Studies on thermal adaptation in Tetrahymena membrane lipids Positional distribution of fatty acid in diacyl- and alkyl-acyl-phosphatidylcholinesand -(2-aminoethyl)phosphonolipids from cells grown at different temperatures

In corporation of fatty acids into rat heart lipids. In Vivo and in vitro studies