Resolution and chromatographic configuration analysis of 2-hydroxy fatty acids

2-Hydroxyoctadecanoic acid was resolved into D and L isomers as salts of 1-phenylethylamine enantiomers The diastereomers of phenylethylamides of 2-hydroxy fatty acids and the corresponding derivatives with protected hydroxy group (acetyl, methyl, trifluoro-acetyl, trimethylsilyl) are well separated by thin-layer or gas-liquid chromatography. This allows a simple microanalysis of configuration and optical purity of 2-hydroxy fatty acids. With this method 2-hydroxy fatty acids from sphingomyelin of the honey-bee were shown to belong exclusively to the D series.     

Stereospecificity of fatty acid 2-hydroxylase and differential functions of 2-hydroxy fatty acid enantiomers

FA 2-hydroxylase (FA2H) is an NAD(P)H-dependent enzyme that initiates FA α oxidation and is also responsible for the biosynthesis of 2-hydroxy FA (2-OH FA)-containing sphingolipids in mammalian cells. The 2-OH FA is chiral due to the asymmetric carbon bearing the hydroxyl group. Our current study performed stereochemistry investigation and showed that FA2H is stereospecific for the production of (R)-enantiomers. FA2H knockdown in adipocytes increases diffusional mobility of raft-associated lipids, leading to reduced GLUT4 protein level, glucose uptake, and lipogenesis. The effects caused by FA2H knockdown were reversed by treatment with exogenous (R)-2-hydroxy palmitic acid, but not with the (S)-enantiomer. Further analysis of sphingolipids demonstrated that the (R)-enantiomer is enriched in hexosylceramide whereas the (S)-enantiomer is preferentially incorporated into ceramide, suggesting that the observed differential effects are in part due to synthesis of sphingolipids containing different 2-OH FA enantiomers. These results may help clarify the mechanisms underlying the recently identified diseases associated with FA2H mutations in humans and may lead to potential pharmaceutical and dietary treatments. This study also provides critical information to help study functions of 2-OH FA enantiomers in FA α oxidation and possibly other sphingolipid-independent pathways.     

Occurrence of 2-hydroxy acids in microalgae

2-Hydroxy acids were believed to be absent in algae until this study, in which the analysis of microalgae belonging to Chlorophyta (Chlamydomonas reinhardtii and Chlorella pyrenoidosa), Rhodophyta (Cyanidium caldarium M-8 and Cyanidium caldarium RK-1) and Cyanophyta (Anbaena variabilis, Anacystis nidulans, Oscillatoria species and Phormidium foveolarum) is reported. 2-Hydroxy adds with carbon chain lengths of C₁₆-C₂₆, were found in all the algal samples studied, ranging in concentrations from 4.0 to 320μg/g dry alga. The dominant constituents are 2-hydroxyhexadecanoic, 2-hydroxynonadecanoic, 2-hydroxyhexacosanoic and a branched 2-hydroxy-C₁₉ acid. The distribution patterns of the acids differed significantly among the algal samples. Hence 2-hydroxy acids may be useful for the classification of algal species as well as being an important source of 2-hydroxy acids in the natural environment.     

Bioconversion of 2-amino acids to 2-hydroxy acids by Clostridium butyricum

Analysis by gas chromatography-mass spectrometry (GC-MS) of 24-h cultures of Clostridium butyricum type strain in synthetic BMG medium supplemented with various 2-amino acids (10 mM) revealed the presence of the corresponding 2-hydroxy acids. C. butyricum was able to bioconvert l-valine, dl-norvaline, l-leucine, dl-norleucine, l-methionine and l-phenylalanine as well as unusual 2-amino acids, i.e., l-2-aminobutyric acid, l-2-amino-4-pentenoic acid, dl-2-aminooctanoic acid, and dl-2-amino-4-phenylbutanoic acid. l-Isoleucine and cycloleucine were not converted into their corresponding 2-hydroxy acids. The bioconversion rate was maximal with dl-norvaline (6.2%). Chiral GC analysis demonstrated that only d-2-hydroxy-4-methylpentanoic acid is formed from l-leucine, indicating that the bioconversion is stereospecific, with inversion of configuration. d-Leucine and d-methionine were also converted to the corresponding 2-hydroxy acids. This observation opens new aspects in the study of C. butyricum and raises questions about the amino acid metabolism by this species.     

Manipulation of fatty acid composition of membrane phospholipid and its effects on cell growth in mouse LM cells

Fatty acid composition of the phospholipids of mouse LM cells grown in suspension culture in serum-free chemically defined medium was modified by supplementing the medium with various fatty acids bound to bovine serum albumin.Following supplementation with saturated fatty acids of longer than 15 carbons (100 μM) profound inhibition of cell growth occurred; this inhibitory effect was completely abolished when unsaturated fatty acids were added at the same concentration. Supplementing with unsaturated fatty acids such as linoleic acid, linolenic acid or arachidonic acid had no effect on the cell growth.Fatty acid composition of membrane phospholipids could be manipulated by addition of different fatty acids. The normal percentage of unsaturated fatty acids in LM cell membrane phospholipids (63%) was reduced to 35–41% following incorporation of saturated fatty acids longer than 15 carbon atoms and increased to 72–82% after addition of unsaturated fatty acids.A good correlation was found between the unsaturated fatty acid content of membrane phospholipids and cell growth. When incorporated saturated fatty acids reduced the percentage of unsaturated fatty acids in membrane phospholipids to less than 50%, severe inhibition of the cell growth was found. Simultaneous addition of an unsaturated fatty acid completely abolished this effect of saturated fatty acids.     

Lipids of higher fungi. III. The fatty acids and 2-hydroxy fatty acids in some species of Basidiomycetes

Total fatty acids derived from 12 species of mushrooms were separated into fatty acid and 2-hydroxy fatty acid fractions (FA and HFA), and analyzed quantitatively. The HFA content varied from 0 to 22% of total fatty acids. The major fatty acids were 16:0, 18:0, 18:1, 18:2, and the major 2-hydroxy fatty acids were h16:0, h18:0, h22:0, h24:0. The predominant HFA in the mushrooms investigated had chain-lengths greater than 20 C-atoms, and were derived from sphingolipids — ceramides and cerebrosides.     

Analysis and quantitation of free ceramide containing nonhydroxy and 2-hydroxy fatty acids, and phytosphingosine by high-performance liquid chromatography.

Reaction of ceramides containing nonhydroxy fatty acids with benzoyl chloride in pyridine at 70 degrees C for 1 hr resulted in N-benzoylation to form N,N-acyl,benzoyl derivatives; O-benzoylation also occurred. However with ceramides containing 2-hydroxy fatty acids and phytosphingosine only O-benzoylation occurred even on prolonged treatment. Only O-benzoylation occurred on reaction with benzoic an hydride. However, the benzoylation of ceramides with phytosphingosine could not be achieved with benzoic anhydride and this benzoylation was performed by reaction with benzoyl chloride at 70 degrees C for 4 hr. Because N,N-acyl,benzoyl derivatives of ceramides containing nonhydroxy fatty acids produced by treatment with benzoyl chloride overlap methyl benzoate on high-performance liquid chromatography, benzoic anhydride was preferable for benzoylation of ceramides with nonhydroxy and 2-hydroxy fatty acids. On the other hand, the reaction with benzoyl chloride at 70 degrees C for 4 hr was used for quantitation of benzoylated ceramides containing 2-hydroxy fatty acids and phytosphingosine. 3-(p-Phenylbenzoyl)estrone was used as an internal standard for both reactions and values for ceramides containing 2-hydroxy fatty acids obtained by the two reactions were in good agreement. This procedure was applied to measurement of the ceramide levels in the brain, liver, and kidney of rats during development. The levels of ceramides containing nonhydroxy and 2-hydroxy fatty acids in the brain, liver, and kidney increased to the adult levels and then remained unchanged. Ceramide with phytosphingosine was detected in the liver and kidney, where its concentration gradually increased with age, but it was not found in the brain. The composition of nonhydroxy fatty acids were also analyzed.     

Involvement of ω-O-acylceramides and protein-bound ceramides in oral permeability barrier formation

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Optical resolution of racemic 2-hydroxy octanoic acid by lipase-catalyzed hydrolysis in a biphasic membrane reactor

Racemic 2-hydroxy octanoic acid methyl ester was optically resolved by lipase-catalyzed hydrolysis in a biphasic membrane reactor using hydrophilic/hydrophobic capillary membranes. In a buffer/hexane biphasic membrane reactor using hydrophilic ultrafiltration membranes, (S)-2-hydroxy octanoic acid was recovered from the aqueous phase at 59–67% yield and 0.9–0.92 enantiomeric excess (ee), and the ester of (R)-isomer was recovered from the organic phase at 73–75% yield and 0.92–0.99 ee.     

Chemotaxonomy of planktonic cyanobacteria based on non-polar and 3-hydroxy fatty acid composition

Twenty-eight axenio planktonic cyanobacterial strains (10 Microcystis, three Oscillatoria, one Spirulina, one Aphanizomenon, 13 Anabaena) were investigated for their fatty acid composition by measurement of non-polar and hydroxy fatty acids. No 2-hydroxy fatty acids were detected in any strain, but 3-hydroxy fatty acids were detected in minor quantities in 24 strains. The highest portion of total fatty acids were non-polar fatty acids. Qualitative and quantitative analyses of 3-hydroxy fatty acids showed no taxonomic value in these strains, while the type of non-polar fatty acid composition was shown to be consistent within Microcystis and Anabaena strains, distinguishing them as type 4, characterized by the presence of 18:4, and type 2, characterized by 18:3 (α) of the Kenyon-Murata system. Two Oscillatoria agardhii Gomont strains were also included in the type 2 group due to the presence of 18: 3 (α), but the difference in characteristics of 16:2 and 16:3 between O. agardhii and Anabaena further divided type 2 into two subgroups: type 2A for Anabaena and type 2B for O. agardhii. A simplified unweighted pair group method with arithmetic averages (UPGMA) dendrogram demonstrated that the classification of 28 strains (Microcystis spp., Anabaena spp., Aphanizomenon flos-aquae (Lemmermann) Ralfs f. gracile (Lemmermann) Elenkin, O. agardhii and Spirullnasubsalsa Oersted ex Gomont based on numerical analysis of non-polar fatty acids corresponded to morphological species criteria, suggesting that non-polar fatty acid composition is a valuable chemical marker in the taxonomy of planktonic cyanobacteria. However, the fatty acid composition in Oscillatoria raciborskii is similar to that of Microcystis and very different from that of O. agardhii, suggesting its special position in Oscillatoria and the chemical diversity in the genus Oscillatoria.     

A novel method for the measurement of in vitro fatty acid 2-hydroxylase activity by gas chromatography-mass spectrometry

Fatty acid 2-hydroxylase (FA2H), encoded by the FA2H gene, is an enzyme responsible for the de novo synthesis of sphingolipids containing 2-hydroxy fatty acids. 2-Hydroxy sphingolipids are highly abundant in the brain, as major myelin galactolipids (galactosylceramide and sulfatide) contain a uniquely high proportion ( approximately 50%) of 2-hydroxy fatty acids. Other tissues, such as epidermis, epithelia of the digestive tract, and certain cancers, also contain 2-hydroxy sphingolipids. The physiological significance of the 2-hydroxylation on N-acyl chains of subsets of sphingolipids is poorly understood. To study the roles of FA2H and 2-hydroxy sphingolipids in various tissues, we developed a highly sensitive in vitro FA2H assay. FA2H-dependent fatty acid 2-hydroxylation requires an electron transfer system, which was reconstituted in vitro with an NADPH regeneration system and purified NADPH:cytochrome P-450 reductase. A substrate [3,3,5,5-D(4)]tetracosanoic acid was solubilized in alpha-cyclodextrin solution, and the 2-hydroxylated product was quantified by gas chromatography-mass spectrometry after conversion to a trimethylsilyl ether derivative. When the microsomes of FA2H-transfected COS7 cells were incubated with the electron transfer system and deuterated tetracosanoic acid, deuterated 2-hydroxy tetracosanoic acid was formed in a time- and protein-dependent manner. With this method, FA2H activities were reproducibly measured in murine brains and tissue culture cell lines.     

Nutritional intervention with omega-3 fatty acids enhances tumor response to anti-neoplastic agents

Nutritional intervention with specific fatty acids depresses tumor growth and enhances tumor responsiveness to chemotherapy. Supplementation of tumors with long chained omega-3 polyunsaturated fatty acids results in enrichment of tumor phospholipid fractions with omega-3 fatty acids resulting in an altered membrane composition and function. Tumors enriched with long chained omega-3 polyunsaturated fatty acids possess membranes with increased fluidity, an elevated unsaturation index, enhanced transport capabilities that results in accumulation of selective anti-cancer agents, increased activity of selected drug activating enzymes, and alteration of signaling pathways important for cancer progression. These nutritionally induced changes in tumor fatty acid composition result in increased sensitivity to chemotherapy, especially in tumor lines that are resistant to chemotherapy and cause specific enhancement of cytotoxicity to tumor cells and protection of normal cells. Pre-disposing tumors to increased chemo-sensitivity through nutritional intervention with specific fatty acids has the potential to improve patient response to chemotherapy with fewer untoward side effects if these pre-clinical findings carry over into a clinical setting.     

Genome analysis of sphingolipid metabolism-related genes in Tetrahymena thermophila and identification of a fatty acid 2-hydroxylase involved in the sexual stage of conjugation

Sphingolipids are bioactive lipids present in all eukaryotes. Tetrahymena thermophila is a ciliate that displays remarkable sphingolipid moieties, that is, the unusual phosphonate-linked headgroup ceramides, present in membranes. To date, no identification has been made in this organism of the functions or related genes implicated in sphingolipid metabolism. By gathering information from the T. thermophila genome database together with sphingolipid moieties and enzymatic activities reported in other Tetrahymena species, we were able to reconstruct the putative de novo sphingolipid metabolic pathway in T. thermophila. Orthologous genes of 11 enzymatic steps involved in the biosynthesis and degradation pathways were retrieved. No genes related to glycosphingolipid or phosphonosphingolipid headgroup transfer were found, suggesting that both conserved and innovative mechanisms are used in ciliate. The knockout of gene TTHERM_00463850 allowed to identify the gene encoding a putative fatty acid 2-hydroxylase, which is involved in the biosynthesis pathway. Knockout cells have shown several impairments in the sexual stage of conjugation since different mating types of knockout strains failed to form cell pairs and complete the conjugation process. This fatty acid 2-hydroxylase gene is the first gene of a sphingolipid metabolic pathway to be identified in ciliates and have a critical role in their sexual stage.     

Unique thermal behavior of sphingomyelin species with nonhydroxy and 2-hydroxy very-long-chain (C28-C32) PUFAs

In rat germ cells and spermatozoa, sphingomyelin (SM) contains molecular species with nonhydroxy (n) and 2-hydroxy (h) very-long-chain polyunsaturated fatty acids (V), the most abundant being SMs with (n- and h-) 28:4n-6, 30:5n-6, and 32:5n-6 as acyl chains. The aim of this study was to gain information about their thermotropic behavior and interactions with other lipids. After isolation from rat testis, multilamellar and giant unilamellar vesicles from these SMs were examined using fluorescent probes. Only n-32:5 SM and h-32:5 SM displayed a gel-liquid transition temperature (Tt ∼ 21–22°C), the rest remaining in the liquid state in the 5°C–45°C range. The degree of order was larger in bilayers of any of the h-V SMs than in those of their chain-matched n-V SMs. Both, but n-V SM relatively more than h-V SM, decreased the Tt of dimyristoylphosphatidylcholine as their proportion increased in binary phosphatidylcholine:SM liposomes. In contrast to the established ability of 16:0 SM to form lateral cholesterol/SM-rich ordered domains in ternary dioleoylphosphatidylcholine:cholesterol:SM bilayers, neither n-V SM nor h-V SM showed a tendency to do so. Thus, these SMs are in the fluid state and are not involved in this type of domains in spermatozoa at physiological temperatures. However, this state could be altered at the very low temperatures at which these gametes are usually preserved.     

Screening for ω−1-hydroxy fatty acid over-producing mutants for bioconversion of oleic acid by combining general mutagenesis and specific selection

Chemical mutation of a strain producing hydroxy-fatty acid from oleic acid (OA) using NTG (N-methyl-N’-nitro-N-nitrosoguanidine) resulted in a high percentage of improved mutants. A positive screening procedure yielded several high producers, specifically the strain Bacillus pumilus M-F641 (BP M-F641). This strain produced predominantly ω_?1-hydroxy fatty acid and could utilize higher concentrations of OA than the parent strain. In shake flask culture, the best ω_?1-hydroxy fatty acid concentration and yield (the ratio of ω_?1-hydroxy fatty acid accumulation to OA consumption) reached 570 mg L^?1 and 11.5%, respectively. Repeated tests showed that the mutant BP M-F641 was genetically stable.     

Limitations in the use of 3-hydroxy fatty acid analysis to determine endotoxin in mammalian samples

3-Hydroxy fatty acids (3-OH FAs) of 10–18-carbon chain lengths are constituents of the lipopolysaccharide of Gram-negative bacteria. These acids are used as chemical markers for determining endotoxin in environmental samples. The present communication addresses the question whether this type of analysis also would be applicable to mammalian samples. Low levels (6.1±1.6–94.0±23.2 pmol/ml) of the studied 3-OH FAs were detected in blood from both conventional and germ-free rats. The levels were considerably higher (0.0–1.06±0.17 nmol/mg) in livers. The amounts of the 3-OH FAs did not differ between the two groups of rats. All analyses were made by gas chromatography–tandem mass spectrometry (GC–MSMS) for unequivocal identification. The results illustrate a limitation in using 3-OH FA analysis to determine endotoxin in mammalian samples since these acids may represent not only endotoxin but also products from mammalian mitochondrial fatty acid β-oxidation.     

Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion

We examined the effect of Niemann-Pick disease type 2 (NPC2) protein and some late endosomal lipids [sphingomyelin, ceramide and bis(monoacylglycero)phosphate (BMP)] on cholesterol transfer and membrane fusion. Of all lipid-binding proteins tested, only NPC2 transferred cholesterol at a substantial rate, with no transfer of ceramide, GM3, galactosylceramide, sulfatide, phosphatidylethanolamine, or phosphatidylserine. Cholesterol transfer was greatly stimulated by BMP, little by ceramide, and strongly inhibited by sphingomyelin. Cholesterol and ceramide were also significantly transferred in the absence of protein. This spontaneous transfer of cholesterol was greatly enhanced by ceramide, slightly by BMP, and strongly inhibited by sphingomyelin. In our transfer assay, biotinylated donor liposomes were separated from fluorescent acceptor liposomes by streptavidin-coated magnetic beads. Thus, the loss of fluorescence indicated membrane fusion. Ceramide induced spontaneous fusion of lipid vesicles even at very low concentrations, while BMP and sphingomyelin did so at about 20 mol% and 10 mol% concentrations, respectively. In addition to transfer of cholesterol, NPC2 induced membrane fusion, although less than saposin-C. In this process, BMP and ceramide had a strong and mild stimulating effect, and sphingomyelin an inhibiting effect, respectively. Note that the effects of the lipids on cholesterol transfer mediated by NPC2 were similar to their effect on membrane fusion induced by NPC2 and saposin-C.     

Application of metabolic flux analysis to identify the mechanisms of free fatty acid toxicity to human hepatoma cell line

Chronic exposure to elevated levels of free fatty acids (FFAs) has been shown to cause cell death (lipotoxicity), but the underlying mechanisms of lipotoxicity in hepatocytes remain unclear. We have previously shown that the saturated FFAs cause much greater toxicity to human hepatoma cells (HepG2) than the unsaturated ones (Srivastava and Chan, 2007). In this study, metabolic flux analysis (MFA) was applied to identify the metabolic changes associated with the cytotoxicity of saturated FFA. Measurements of the fluxes revealed that the saturated FFA, palmitate, was oxidized to a greater extent than the non-toxic oleate and had comparatively less triglyceride synthesis and reduced cystine uptake. Although fatty acid oxidation had a high positive correlation to the cytotoxicity, inhibitor experiments indicated that the cytotoxicity was not due to the higher fatty acid oxidation. Application of MFA revealed that cells exposed to palmitate also had a consistently reduced flux of glutathione (GSH) synthesis but greater de novo ceramide synthesis. These predictions were experimentally confirmed. In silico sensitivity analyses identified that the GSH synthesis was limited by the uptake of cysteine. Western blot analyses revealed that the levels of the cystine transporter xCT, but not that of the GSH-synthesis enzyme glutamyl-cysteine synthase (GCS), were reduced in the palmitate cultures, suggesting the limitation of cysteine import as the cause of the reduced GSH synthesis. Finally, supplementing with N-acetyl L-cysteine (NAC), a cysteine-provider whose uptake does not depend on xCT levels, reduced the FFA-toxicity significantly. Thus, the metabolic alterations that contributed to the toxicity and suggested treatments to reduce the toxicity were identified, which were experimentally validated.     

Invited review: nutrient-sensing receptors for free fatty acids and hydroxycarboxylic acids in farm animals

Data on nutrient sensing by free fatty acid receptors (FFAR1, FFAR2, FFAR3, FFAR4) and hydroxycarboxylic acid receptors (HCAR1, HCAR2) are increasing for human or rodent models. Both receptor families link intestinal fermentation by the microbiota and energy metabolism with cellular responses. Therefore, this finding provides a link that is independent of the only function of the fermentation products as energy substrates. For example, these reactions are associated with insulin secretion, regulation of lipolysis, adipose tissue differentiation and innate immune responses. In farm animals, the available data on both receptor families from the intestine and other tissues increase. However, currently, the data are primarily linked with the distribution of receptor messenger RNAs (mRNAs) and more rarely with proteins. Functional data on the importance of these receptors in farm animal species is not abundant and is often associated with the immune system. In certain farm animal species, the receptors were cloned and ligand binding was characterised. In chicken, only one FFAR2 was recently identified using genome analysis, which is contradictory to a study using an FFAR1 small interfering RNA. The chicken FFAR2 is composed of more than 20 paralogs. No data on HCAR1 or HCAR2 exist in this species. Currently, in pigs, most available data are on the mRNA distribution within intestine. However, no FFAR1 expression has been shown in this organ to date. In addition to FFAR2, an orthologue (FFAR2-like) with the highest abundance in intestine has been reported. The data on HCAR1 and HCAR2 in pigs is scarce. In ruminants, most of the currently available information on receptor distribution is linked to mRNA data and shows the expression, for example, in mammary gland and adipose tissue. However, some protein data on FFAR2 and FFAR1 protein has been reported and functional data availability is slowly increasing. The receptor mRNAs of HCAR1 and HCAR2 are expressed in bovine. The HCAR2 protein has been demonstrated in certain tissues, such as liver and fat. Because of the physiological importance of both receptor families in human life science, more studies that analyse the physiological significance of both receptor families in animal science may be performed within the next several years.     

Loss-of-function mutations and inducible RNAi suppression of Arabidopsis LCB2 genes reveal the critical role of sphingolipids in gametophytic and sporophytic cell viability

Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis, and downregulation of this enzyme provides a means for exploring sphingolipid function in cells. We have previously demonstrated that Arabidopsis SPT requires LCB1 and LCB2 subunits for activity, as is the case in other eukaryotes. In this study, we show that Arabidopsis has two genes ( AtLCB2a and AtLCB2b ) that encode functional isoforms of the LCB2 subunit. No alterations in sphingolipid content or growth were observed in T-DNA mutants for either gene, but homozygous double mutants were not recoverable, suggesting that these genes are functionally redundant. Reciprocal crosses conducted with Atlcb2a and Atlcb2b mutants indicated that lethality is associated primarily with the inability to transmit the lcb2 null genotype through the haploid pollen. Consistent with this, approximately 50% of the pollen obtained from plants homozygous for a mutation in one gene and heterozygous for a mutation in the second gene arrested during transition from uni-nucleate microspore to bicellular pollen. Ultrastructural analyses revealed that these pollen grains contained aberrant endomembranes and lacked an intine layer. To examine sphingolipid function in sporophytic cells, Arabidopsis lines were generated that allowed inducible RNAi silencing of AtLCB2b in an Atlcb2a mutant background. Studies conducted with these lines demonstrated that sphingolipids are essential throughout plant development, and that lethality resulting from LCB2 silencing in seedlings could be partially rescued by supplying exogenous long-chain bases. Overall, these studies provide insights into the genetic and biochemical properties of SPT and sphingolipid function in Arabidopsis.