Alpha-hydroxy Fatty Acids Research Articles

Isolation and characterization of a novel phytosphingosine-containing GM2 ganglioside from mullet roe (Mugil cephalus).

The major ganglioside from the roe of striped mullet (Mugil cephalus) has been isolated and purified. Compositional analysis of this ganglioside revealed that it contained an equimolar ratio of the following residues: N-acetylneuraminic acid, N-acetylgalactosamine, galactose, glucose, and the long-chain base. Further structural studies by sequential enzymatic hydrolysis, permethylation analysis, and proton NMR spectroscopy indicated that the structure of the oligosaccharide moiety was identical to that of GM2 ganglioside from human brain: GalNAc beta 1----4Gal beta 1----4(3----2 alpha NeuAc)-Glc----ceramide. This ganglioside, however, differed from brain GM2 in its ceramide portion. The most striking differences are the presence of large amounts of C18 and C20 phytosphingosine (over 80% of the total long-chain bases) and the preponderance of monounsaturated alpha-hydroxy fatty acids (over 80%). Such a phytosphingosine-containing GM2 ganglioside has never been reported.

On the formation of alpha-hydroxy fatty acids. Evidence for a direct hydroxylation of nonhydroxy fatty acid-containing sphingolipids.

When Tetrahymena pyriformis, strain NT-1, was warmed from a growth temperature of 15 degrees C to 39 degrees C, there was a rapid conversion of its sphingolipids from the nonhydroxy fatty acid-containing ceramide aminoethylphosphonate (NCAEP) predominant at low temperature to the alpha-hydroxy fatty acid-containing analog (HCAEP). The fatty acid composition of the freshly formed HCAEP strongly resembled that of the original NCAEP pool. An extensive utilization of NCAEP long chain bases for HCAEP formation was shown by prelabeling the NCAEP bases with [14C]serine. The specific radioactivities of both fatty acids and long chain bases of the two sphingolipid classes of cells prelabeled with [3H]palmitic acid at 15 degrees C and then warmed to 39 degrees C were compatible with a direct hydroxylation of the intact ceramide aminoethylphosphonate or free ceramide formed from it. Exogenously added alpha-hydroxypalmitic acid was not incorporated into the sphingolipids intact, but there was an active alpha-oxidation of alpha-hydroxy fatty acids, yielding nonhydroxy fatty acids one carbon atom shorter in length. In vitro experiments failed to demonstrate a direct hydroxylation of NCAEP, and nonhydroxy fatty acid-containing free ceramides were hydroxylated only sparingly. This inefficient hydroxylation is attributed to the rapid enzymatic hydrolysis of these substrates in vitro and to a limited availability of the added ceramides to the hydroxylating enzymes. The weight of the evidence from this study strongly favors the alpha-hydroxylation of fatty acids only when they are bound as elements of sphingolipids.

Developmental profiles of glycolipids in mouse small intestine.

The major gangliosides were isolated from small intestine of 2-week-old mice of C3H/He strain and identified as GM3, GM1, and GD1a. These gangliosides characteristically contained a phytosphingosine moiety and alpha-hydroxy fatty acids. The gangliosides contained both N-glycolylneuraminic acid and N-acetylneuraminic acid. Gangliosides GM3, GM1, and GD1a contents increased in mouse small intestine during the suckling period, and decreased after weaning. In contrast, neutral glycosphingolipids appeared after weaning, except for monohexosyl ceramide, which was present at a fairly constant level throughout postnatal life. The same results were obtained with mice of other strains and germ-free ICR mice, although such changes could not be observed in rats. These observations indicate that the developmental change in the composition of glycosphingolipids occurs in a species-specific manner.

Presence of asialo GM1 and glucosylceramide in the intestinal mucosa of mice and induction of fucosyl asialo GM1 by conventionalization of germ-free mice.

Two major neutral glycolipids of the intestinal mucosa were purified by a series of column chromatographies and the structures were determined to be glucosylceramide and asialo GM1 by gas liquid chromatography. The carbohydrate structure of asialo GM1 was also confirmed from the reactivity of the glycolipid to rabbit anti-asialo GM1 antibody by an enzyme linked-immunosorbent antibody. The ceramide portion of both glycolipids had an extremely hydrophilic nature and more than 90% of the ceramide was composed of phytosphingosine and alpha-hydroxy fatty acids. In the previous paper we reported that the induction of a fucolipid in the microvillus membrane of mouse intestinal mucosa by conventionalization was observed on monitoring the incorporation of radiolabeled fucose in vivo. A fucoglycolipid having the same mobility on an autoradiogram as the fucolipid labeled in vivo was produced by in vitro incubation of intestinal asialo GM1 and GDP-[14C]fucose with the mucosal homogenates. Moreover, asialo GM1 prepared from brain gangliosides exhibited a similar ability to accept fucose and it was converted to fucosyl asialo GM1 which moved faster than the product from intestinal asialo GM1. The difference is considered to be due to the ceramide composition. These results suggest that conventionalization can induce the fucosyl asialo GM1 in the microvillus membrane probably through the induction of fucosyltransferase. Understanding the molecular mechanism of interaction between the physiological flora and host is the matter of further study.

Immunochemical Study of Gangliosides at the Cell Surface of Sea Urchin Embryos

Two main gangliosides (G-1 and G-2) were isolated from eggs and embryos S. intermedius. They contain glucose, N-glucolylneuraminic acids, phytosphyngosine, fatty acids and alpha-hydroxy fatty acids. Molar ratios and sequence of these components are the same for both gangliosides, but G-2 contains sulphate residue which is attached to the terminal neuraminic acid. To obtain specific antisera rabbits were immunized by G-1 or G-2, which were mixed with bovine serum albumin and Freund's adjuvant. Both gangliosides possessed electrophoretic and antigenic heterogeneity. G-1 and G-2 gangliosides have common and individual antigenic determinants. Glucosylceramide of gangliosides is immunologically inactive. Individual antigenic specificity of the gangliosides depends on the presence of N-glycolylneuraminic acid (G-1) and SO3H-group (G-2). Egg gangliosides were demonstrated by immunofluorescence throughout the cell surface. After fertilization of immunofluorescent label was concentrated on one pole of the embryo only. During the development of specific fluorescence was again uniformly distributed at the blastomer surface. The most intense fluorescence was observed in the junction areas of the blastomers.

Triethyllead treatment of cultured brain cells. Effect on accumulation of radioactive precursors in galactolipids.

Cultured cells from chick embryo brains were studied for their sensitivity to triethyllead. Triethyllead chloride (3.16 microM) was added to the nutrient medium and incubated for 48 hr with the cells. Morphological changes in light microscope and radioactive labeling of galactolipids were assayed. Triethyllead treatment reduced the number of neuronal cells with processes. Morphological changes were not observed in glial cells. The [35S]sulfate labeling of sulfatides was reduced to 50%. The [3H]serine labeling of cerebrosides with alpha-hydroxy fatty acids was not influenced, while the [3H]serine labeling of cerebrosides with nonhydroxy fatty acids was inhibited 40% in one- and two- but not in three-week-old cultures. The results indicate that the nerve cell response to triethyllead in cultures is selective, since the neurons are more sensitive than the glia cells and the labeling of sulfatides is more sensitive than that of cerebrosides.

Changes in fatty acid composition of myelin cerebrosides after treatment of the developing rat with methylmercury chloride and diethylmercury.

Suckling rats were exposed to methylmercury chloride or diethylmercury in order to induce chronic sublethal intoxication during the period of active myelination. Doses of 5 mg Hg/kg body weight were injected every second day from 5-25 days of age. The rats were killed at 27-28 days of age, and the brains contained about 1 microgram Hg/g wet weight. No changes in brain weight, myelin content of proteins or phospholipids were found, whereas the cholesterol and galactolipid levels were slightly reduced. The most significant change observed was a decrease in the ratio between alpha-hydroxy fatty acid and the nonsubstituted fatty acid in the myelin cerebrosides. The biochemical changes were less pronounced in the animals given diethylmercury than in animals receiving methylmercury.

Conversion by rat brain preparation of lignoceric acid to ceramides and cerebrosides containing both alpha-hydroxy and nonhydroxy fatty acids. Effects of compounds which affect sphingolipid metabolism.

Three synthetic compounds which affect sphingolipid metabolism in vitro were examined for their effects on the synthesis in a rat brain system of nonhydroxyceramide, hydroxyceramide, nonhydroxycerebroside, and hydroxycerebroside from [1-14C]ignoceric acid. These compounds are N-octanoyl-D-threo-p-nitrophenylaminopropanediol (Compound I), N-(2,3-epoxydecanoyl)-norephedrine (Compound II), and N-decyl-N'-glucosylthiourea (Compound III). In the presence of up to 0.5 mM Compound I, only the hydroxyceramide fromation increased, while the synthesis of the other three lipids decreased. Compound II strongly inhibited the formation of all four lipids at all concentrations tested (0.1 to 1 mM). Compound III increased the synthesis of both ceramides approximately 2-fold at 1 mM, but the conversion of lignoceric acid to both cerebrosides remained relatively unchanged. Several significant conclusions from these observations are discussed.

Isolation and characterization of human liver hematoside.

The monosialoganglioside hematoside (GM3) is an important precursor in the synthesis of the more complex gangliosides. To obtain large quantities of GM3 for use in ganglioside biosynthetic experiments, we have devised a column chromatographic procedure for the isolation and purification of GM3 from human liver. A total ganglioside mixture was obtained from a 550 g sample of normal human liver. Quantitative analysis by gas-liquid chromatography indicated about 66 microgram of lipid-bound N-acetylneuraminic acid per gram of fresh tissue. Hematoside appeared as double bands on thin-layer plates and represented 86.4% of the total sialic acid content. Additional ganglioside species, notably GD3, were also seen. Hematoside was separated from the other gangliosides by GM3 obtained was 120 mg, which represented a 90% recovery. Although the long-chain base and sugar compositions of the upper and lower GM3 fractions were similar, striking fatty acid differences were detected. The upper fraction contained predominantly unsubstituted fatty acids, while the lower fraction showed a preponderance of alpha-hydroxy fatty acids. The unsubstituted fatty acid and hydroxy fatty acid composition of the total GM3 fraction was calculated to be 56.9% and 43.1%, respectively.

Alpha-Hydroxylation of lignoceric and nervonic acids in the brain. Effects of altered thyroid function on postnatal development of the hydroxylase activity.

Rat brain postnuclear preparations catalyzed the alpha-hydroxylation of nervonic acid with an apparent Km of 3 muM. Evidence has been presented which suggests that nervonic acid in the brain is hydroxylated by the same enzyme system which hydroxylates lignoceric acid. The hydroxylase activity in brains of normal (euthyroid) rats increased rapidly from a low in the period immediately following birth to a maximum at the 23rd day and then declined to a low level characteristic of the mature brain. Neonatal hypothyroidism retarded the development of the activity and shifted its peak to the 39th day after birth. Conversely, neonatal hyperthyroidism accelerated the entire developmental pattern and shifted the peak to the 16th day after birth. The hydroxylase activity in mouse brain was also increased by thyroid hormone administration from the 13th through the 18th day after birth. Unlike normal mice, the low activity in jimpy mice was not affected by this treatment. It is concluded that thyroid hormones play an important role in the control of brain fatty acid alpha-hydroxylation. The stimulation of alpha-hydroxy fatty acid synthesis in response to hyperthyroidism during the early postnatal period may be one of the major effects of thyroid hormones in accelerating myelination of the central nervous system.

Isolation and identification of cerebrosides from the marine sponge Chondrilla nucula

A cerebroside mixture has been isolated from the marine sponge Chondrilla nucula. Acid-catalyzed methanolysis of this cerebroside mixture afforded methyl glucosides, three long-chain bases, and a mixture of alpha-hydroxy fatty acid methyl esters. The bases were identified as saturated C(17), C(18), and C(19) trihydroxy bases (1,3,4-trihydroxy-2-aminoalkanes) by gas-liquid chromatographic-mass spectrometric analysis of their corresponding trimethylsilyl derivatives. The methyl ester fraction consisted of a mixture of homologous C(16) to C(26) saturated straight-chain alpha-hydroxy esters plus a trace of saturated C(25) iso alpha-hydroxy ester.

Stereochemistry of alpha-hydroxy fatty acids isolated from a strain of Streptomyces

A strain ofStreptomyces contains a mixture (ca. 3:1) of 2d-hydroxy-12l-methyltetradecanoic and 2d-hydroxy-13-methyltetradecanoic acids; it has the same properties as a similar mixture prepared by synthesis.

Identification of growth-promoting alpha-hydroxy fatty acids produced by Lactobacillus casei.

Identification of growth-promoting alpha-hydroxy fatty acids produced by Lactobacillus casei.