Monohydroxy Fatty Acids Research Articles

Adsorption and reversed-phase high-performance liquid chromatography of p-nitrobenzyl esters of monohydroxy fatty acids

To enhance the UV detectability of hydroxy fatty acids, p-nitrobenzyl (PNB) esters of twenty-two different monohydroxy fatty acids of various chain-lengths (C 16C 22) and differing positional isomers were formed using O-( p-nitrobenzyl)-N,N-(diisopropyl)-isourea (PNBDI) as alkylating agent. Reversed-phase and adsorption high-performance liquid chromatography (HPLC) of the twenty-two monohydroxy fatty acid PNB esters were studied. The PNB group did not dominate the chromatographic properties of monohydroxy fatty acids and it did not interfere with the HPLC separation of positional isomers. PNBDI was, however, found to be less than ideal for formation of PNB derivatives of monohydroxy fatty acids because UV absorbing contaminants of PNBDI interfered with the HPLC analyses.

Metabolism of Arachidonic Acid in Acetic Acid Colitis in Rats: Similarity to Human Inflammatory Bowel Disease

We recently reported that human inflammatory bowel disease mucosa contains large amounts of leukotriene B4, a potent chemotactic agent formed from arachidonic acid through the lipoxygenase pathway. To more fully evaluate the role of arachidonic acid metabolites in the mediation of intestinal inflammation, we studied arachidonate metabolism in an animal model: acetic acid colitis in the rat. Incubation of acetic acid colitis mucosa with arachidonic acid resulted in the production of leukotriene B4 and a series of monohydroxy fatty acids, all products of the lipoxygenase pathway, plus much smaller amounts of cyclooxygenase products including prostaglandin E2. All of these metabolities were made in significantly greater quantities by mucosa from acetic acid-treated rats than by controls. The pattern of arachidonate metabolism in acetic acid colitis was strikingly similar to that in human inflammatory bowel disease. Moreover, the concentration of leukotriene B4 in acetic acid-treated mucosa was almost identical to that in human inflammatory bowel disease mucosa and was 50 times greater than that in normal rat colonic mucosa. These data indicate that lipoxygenase products, including leukotriene B4, may be important mediators of intestinal inflammation in a wide variety of inflammatory conditions. Moreover, the similarities in the metabolism of arachidonate by human inflammatory bowel disease and by acetic acid colitis may allow the use of this model, and perhaps other animal models of intestinal inflammation, in the screening of potential therapeutic agents for inflammatory bowel disease.

Characterization of lipoxygenase metabolites of arachidonic acid in cultured human skin fibroblasts

Cultured human skin fibroblasts were incubated in the presence of [ 14Clarachidonic acid (50 μM; 1 mCi/mmol) and the divalent cation ionophore A23187 at 37°C for 60 min. The metabolites formed were extracted from the cell-free medium in diethyl ether, separated by thin layer chromatography and identified unequivocally by GC-MS. The distribution of the arachidonic acid metabolites as estimated from the recovered radioactivity showed as major product prostaglandin E 2 (26%). Minor amounts of other prostaglandins, i.e., 6-oxo-prostaglandin F 1α (1%), prostaglandin F 2α (1%), prostaglandin D 2 (0.5%) and prostaglandin A 2 (1%) were also present. In addition to the prostaglandins, monohydroxy fatty acids (4.5%) were also detected. This fraction contained 33% 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), 22% 11-hydroxy-5,8,11,14-eicosatetraenoic acid (11-HETE) and 31% 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). Lipid extracts of the cells did not show any detectable amount of the monohydroxy fatty acids, indicating that they are not incorporated metabolically in the cellular lipids. The monohydroxy fatty acids originate mainly from the exogenously added arachidonic acid as evidenced by the 2H/H ratio (30:1) from experiments with octadeuterated arachidonic acid ([ 2H 8]arachidonic acid). Indomethacin inhibited the formation of all prostaglandins, HHT and 11-HETE; moreover, eicosatetraynoic acid (ETYA) also blocked the formation of 15-HETE. From these results, it can be concluded that in human skin fibroblasts prostaglandin E 2 is the major product of the cycloxygenase pathway, while 15-HETE is the main lipoxygenase product.

Monohydroxy and Dihydroxy Fatty Acid Composition of Legionella Species

A total of 28 strains, representing 10 species of Legionella and including strains belonging to most of the known serogroups of each species, were examined for location, content, and composition of cellular nonhydroxy, monohydroxy, and dihydroxy fatty acids. The nonhydroxy fatty acid profiles were in agreement with previously published values. All species contained 5 to 15 mol% (depending on species) monohydroxy fatty acid, the profile of each species being readily distinguishable from the profiles of the other species. Only two species, Legionella pneumophila and Legionella micdadei, contained detectable levels of dihydroxy fatty acids, which comprised 1 to 5 mol% of the total fatty acids. The dihydroxy fatty acid profiles of these two species were significantly different. In all species, the nonhydroxy fatty acids were readily extractable by lipid solvents and were labile when subjected to mild alkaline methanolysis (lipid associated, ester linked), whereas the hydroxylated components were not extractable, were stable when subjected to alkaline methanolysis, and were labile when subjected to acid hydrolysis (bound, amide linked). The monohydroxy fatty acids were shown to be acids of the 3-hydroxy family. The dihydroxy fatty acids were shown to be members of the 2,3-dihydroxy family. The profiles of the hydroxylated fatty acids provide a powerful method for differentiating the Legionella species, particularly those species whose nonhydroxy fatty acid profiles are quite similar.

Lipoxygenase products : A novel gas chromatographic—mass spectrometric assay for monohydroxy fatty acids

We present a novel mass spectrometric method for the profiling of monohydroxy fatty acids and illustrate its use for the analysis of lesional skin of patients with psoriasis. By combination of vapour phase hydrogenation and gas chromatography—mass spectrometry with selected ion monitoring the position of the substituted hydroxyl group of each fatty acid was determined in their methyl ester trimethylsilyl ether derivatives. Reduction was instantaneous and quantitative allowing detection of less than 100 picogram of each compound. Biological extracts, derivatised as methyl ester tert.-butyldimethylsilyl ethers, were purified by reversed-phase high-performance liquid chromatography. Separation was dependent on carbon chain length and degree of unsaturation, but not on the position of the silyl ether group. Subsequent conversion of the tert.-butyldimethylsilyl ethers to trimethylsilyl ethers facilitated detection of each of the positional hydroxyl isomers as described. Distinction between double bond isomers was possible when they were separated on gas chromatography prior to reduction.

Arachidonic acid metabolism by rabbit fetal membranes of various gestational ages

The ability of rabbit fetal membranes to convert arachidonic acid to both lipoxygenase and cyclooxygenase products was studied by separation and identification of products derived from incubations of 1- 14 C-arachidonate with subcellular fractions obtained by differential centrifugation of tissue homogenates. Both amnion and splanchnopleure (the chorion-equivalent of the rabbit) produced a mixture of 11-, 12-, and 15-hydroxyeicosatetraenoic acids when stimulated by calcium ions; these products were produced in greater quantity in middle pregnancy (20–22d) than later (28–30d). Cyclooxygenase products included PGD 2, PGE 2, TxB 2 and PGF 2α, all of which were made more actively in late pregnancy than the middle in both amnion (which was more active) and chorion-equivalent. These data suggest that arachidonate metabolism by rabbit fetal membranes in middle pregnancy is directed primarily toward production of monohydroxy fatty acids, but that as pregnancy nears term, the PG-producing enzymes are induced, preparing the uterine smooth muscle for parturition.

The identification of hydroxy fatty acids in psoriatic skin

Psoriasis is a common chronic inflammatory and proliferative skin disease characterised by epidermal neutrophil infiltration which may be induced by chemotactic substances in the involved epidermis. Superficial psoriatic scale was shown to contain biologically active amounts of leukotriene B 4 and monohydroxy-eicosatetraenoic acid (HETE)- like material as determined by assay for chemokinetic activity in high performance liquid chromatography (HPLC) fractions of scale extracts. Extracts of scale and chamber fluid from abraded lesional and uninvolved psoriatic skin were purified by HPLC and appropriate fractions were analysed by gas chromatography - mass spectrometry (GC-MS). The following monohydroxy metabolites of arachidonic, linoleic and 11,14-eicosadienoic acids were identified : 15-HETE, 12-HETE, 11-HETE, 9-HETE, 8-HETE, 5-HETE, 13-hydroxy-octadecadienoic acid (13-HODD), 9-HODD and 15-hydroxy-eicosadienoic acid (15-HEDE). The results suggested that 12-HETE, 13-HODD and 9-HODD are the most abundant monohydroxy fatty acids in the psoriatic skin extracts described above. Assays of 13-HODD, 9-HODD and 15-HEDE for chemokinetic activity were negative with concentrations up to 10 −4M. The biological significance of these three compounds in not known, but some of the hydroxylated metabolites of arachidonic acid may, by virtue of their chemotactic properties, be relevant to the pathogenesis of the psoriatic neutrophil infiltrate.

Esterification of monohydroxyfatty acids into the lipids of a macrophage cell line

Cells of a mouse macrophage-like tumor cell line, J774.2, were incubated with 0.6μM radiolabeled mono- and di-hydroxyfatty acids. Monohydroxyfatty acid products of the neutrophil and platelet lipoxygenase pathways (5-HETE, 15-HETE, and 12-HETE) were rapidly taken up (42–64% of the counts cell associated at 1 min) and esterified into triglycerides and phospholipids. 5-HETE and 12-HETE were found in triglycerides and distributed among phospholipid classes while 50% of added 15-HETE was esterified into phosphatidyl inositol. Treatment of phospholipids from cells incubated with 5-HETE, 12-HETE, and 15-HETE with phospholipase A 2 resulted in release of the respective monohydroxyfatty acid. HHT, a monohydroxyfatty acid product of the cyclooxygenase pathway, was taken up and esterified more slowly than the lipoxygenase products. In addition, HHT was not released when the phospholipids from cells incubated with HHT were treated with phospholipase A 2. LTB 4, a dihydroxyfatty acid product of neutrophil lipoxyegnase, was not taken up by J774.2 cells. The unique patterns of uptake and intracellular distribution of the different monohydroxyfatty acids suggests that the enzymes involved in the esterification of these compounds have substrate specificity and may also relate to the specific biologic effects of the compounds.

Simple method for the separation of monohydroxy fatty acid metabolites of arachidonate metabolism

Simple method for the separation of monohydroxy fatty acid metabolites of arachidonate metabolism

Formation of trihydroxyheptadecenoic acids, monohydroxyicosatrienoic acids, and prostaglandins from 8,11,14-icosatrienoic acid by adult and fetal aorta.

trienoic acid (12h-20:3), a lipoxygenase product, was also detected. The major metabolites of 20:3 formed by a homogenate of rat aorta were 12h-17:2, llh-20:3, and 15h-20:3 along with a small amount of 12h-20:3. Methemoglobin increased the formation of all prod- ucts except PGEl and PGF1, by about 50-100% above the control. Adrenaline stimulated the formation of the monohydroxy fatty acids by 5-6-fold and that of

Prostaglandins, hydroxy fatty acids, leukotrienes and inflammation of the skin.

There is good evidence that prostaglandins E2, I2 and D2 are mediators of the vascular events of experimentally induced acute inflammation in human skin, but their role in inflammatory skin diseases such as atopic eczema remains to be determined. The relevance of arachidonic acid metabolites in inflammation has been expanded by the recent discovery of many biologically active arachidonate lipoxygenase products. These include monohydroxy fatty acids and a dihydroxy acid (leukotriene B4), which, especially in the latter case, are potent chemotactic agents. Other products include leukotriene C4 and D4, which are vasoactive and appear to be the functionally critical components of slow reacting substance of anaphylaxis. Much work is needed to determine the role of these compounds in inflammatory skin diseases.

Separation of icosenoic acids, monohydroxyicosenoic acids, and prostaglandins by high-pressure liquid chromatography on a silver ion-loaded cation-exchange column

Prostaglandins and monohydroxy fatty acids derived from 8,11,14-icosatrienoic acid and arachidonic acid have been separated by high-pressure liquid chromatography using a cation-exchange column loaded with silver ions. The retention times in a variety of solvent systems have been determined for prostaglandin E 1(PGE 1), PGF 1α, PGD 2, PGE 2, PGF 2α, 6-oxoPGF 1α, 15-hydroxy-8,11,13-icosatrienoic acid, 5-hydroxy-6,8,11,14-icosatetraenoic acid, 8-hydroxy-5,9,11,14,-icosatetraenoic acid, 9-hydroxy-5,7,11,14-icosatetraenoic acid, 11-hydroxy-5,8,12,14-icosatetraenoic acid, 12-hydroxy-5,8,10,14-icosatetraenoic acid, 15-hydroxy-5,8,11,13-icosatetraenoic acid, 8,11,14,-icosatrienoic acid, and arachidonic acid. The mechanisms involved in the interaction of solutes with the stationary phase have been investigated. Retention times on silver ion columns appear to be determined by a combination of interactions between (a) the silver ions of the stationary phase and double bonds of the solute and (b) polar groups of the stationary phase and polar groups of the solute. The relative contributions of these two types of interactions to the retention of solutes can be varied over a wide range by altering the composition of the solvent. In this way the selectivity of the stationary phase can be controlled in order to optimize the separation of any given group of solutes. The maximum separation of solutes on the basis of the number of double bonds they possess is obtained by using polar solvents containing low concentrations of acetonitrile. As the polarity of the mobile phase is reduced or the concentration of acetonitrile increased, the selectivity of the stationary phase tends to resemble that of normal-phase chromatography on silicic acid.

Rapid extraction of oxygenated metabolites of arachidonic acid from biological samples using octadecylsilyl silica

A rapid procedure for the efficient extraction of prostaglandins, thromboxanes and hydroxy fatty acids from urine, plasma and tissue homogenates has been developed. Fractions containing these substances are acidified and passed through a column of octadecylsilyl silica, which retains oxygenated metabolites of arachidonic acid. Phospholipids, proteins and very polar materials either are not retained or can be eluted with dilute aqueous ethanol. Nonpolar lipids and monohydroxy fatty acids are then eluted with petroleum ether or benzene. Subsequent elution of the column with methyl formate gives a fraction containing prostaglandins and thromboxanes which is much less contaminated with extraneous material than that obtained by conventional extraction of aqueous media with organic solvents. The methyl formate can be removed rapidly under a stream of nitrogen and the components of the sample purified directly by high pressure liquid chromatography (HPLC). An improved method for the purification of prostaglandins and TXB 2 by HPLC on silica columns is reported.

Bitterness of Lipids

AbstractWe have been able to demonstrate, that the hydrophobicity of the amino acid side chains is responsible for the bitterness of a peptide. Now we investigated lipids (and carbohydrates) in the following classes: Monoglycerides, diglycerides, phosphatides, lysophosphatides, monohydroxy fatty acids, dihydroxy fatty acids, trihydroxy fatty acids, monosaccharides, disaccharides. It was found that for lipids and carbohydrates the ratio from the number of the C‐atoms of a molecule to the number of the hydroxyl groups called the R‐values, gave a good indication as to whether the lipid or carbohydrate was sweet, bitter or non‐bitter. For sweet compounds we found R = 1.00 ‐ 1.99, for bitter compounds we found R =2.00 ‐ 6.99, and for non‐bitter compounds we found a value of R higher than 7.00. These results confirm our earlier assumption, that like in the case of peptides, the bitterness of lipids and carbohydrates is caused by a certain range of the hydrophobicity.

ω-1, ω-2 and ω-3 Hydroxylation of long-chain fatty acids, amides and alcohols by a soluble enzyme system from Bacillus megatyerium

A soluble enzyme preparation from Bacillus megaterium, previously shown to hydroxylate free fatty acids to isomeric mixtures of ω-1, ω-2 and ω-3 monohydroxy fatty acids in the presence of NADPH and O 2, has now been shown to act also on fatty alcohols and fatty amides but not on hydrocarbons or fatty acid methyl esters. Using 14C-labelled substrates, both the chainlength specificity and the positional specificity of hydroxylation was determined for fatty acids, alcohols and amides. The most active saturated fatty acid (pentadecanoic) was hydroxylated at a rate 10 times greater than the most active amide (myristamide) and 14 times faster than the most active alcohol (1-tetradecanol). Among the saturated fatty acids, the order of activity as hydroxylation substrates was C 15 > C 16 > C 14 > C 17 > C 13 > C 18 = C 12. For amides the order was C 14 > C 12 > C 15 > C 16 while for alcohols it was C 14 > C 13 = C 15 > C 12 > C 16. Four cis-monounsaturated fatty acids were also tested. Oleic, palmitoleic and cis-12-octadecenoic acids were more active than their saturated analogs but cis-5-tetradecenoate was less active than myristate. For all of the substrates mentioned above, with the possible exception of several unsaturated acids, the alkyi chains were monohydroxylated to give isomeric mixtures of the ω-1, ω-2 and ω-3 derivatives. The distribution of these three isomers varied with chain-length and type of substrate but generally, the ω-2 position was favored. The terminal methyl (ω) group of these substrates was never hydroxylated and there did not appear to be significant hydroxylation of methylene carbons beyond the ω-3 position. Based on the data presented here and in a previous paper, a model is proposed for the enzyme-substrate complex which involves hydrophobic binding and sequestering of the terminal methyl group of the substrate and electrostatic binding of the substrate's polar functional group.

1,3-diglycerides from eggs of Culex pipiens quinquefasciatus and Culex pipiens pipiens

1. 1. Ether washings of egg-rafts of the mosquitoes Culex pipiens quinquefasciatus and C. p. pipiens which attracted ovipositing females of these species were prepared. 2. 2. The largest fraction from each of these extracts consisted of a mixture of estrolide 1,3-diglycerides and could not be distinguished by spectrometric or thin-layer chromatographic methods from the analogous fraction from C. tarsalis. However, on further investigation, differences were found in the monohydroxy fatty acid composition of the 1,3-diglycerides.

Characterization of the phospholipids and the normal and hydroxy acid containing glycosphingolipids (globosides) of pig erythrocytes

The chemical characterization of certain phospholipids and glycolipids of the pig erythrocyte is reported. The main phospholipids under investigation were phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine and sphingomyelin, with lesser amounts of phosphatidyl inositol. Perhaps the most unique characteristics of the phosphoglycerides were the lack of any vinyl ether or glyceryl ether groups and the occurrence of significant concentrations of phosphatidyl serine (containing only C 18 fatty acids). The major glycolipids present in these erythrocytes were of the globoside (“tetraglycosyl” ceramide) type, with smaller amounts of other glycosyl ceramides and traces of gangliosides. Two major forms of the globosides, labeled glycolipid C and glycolipid D, were isolated and identified. Both C and D were of the same basic structure, i.e. N-acetyl-galactosaminyl(→)galactosyl(→)galactosyl(→)glucosyl(→)ceramide, with the major difference being that C contained normal straight chain fatty acids whereas D contained only monohydroxy fatty acids. The composition of these fatty acids from these two glycolipids, as related to chain length and unsaturation, were comparable and contained mainly 22:0, 24:0 and 24:1 types. On the basis of periodate-permanganate cleavage results, it was concluded that the double bond in the 24h:1 acid was located at the 8:9 position (from carboxyl end). Unequivocal proof is offered that the hydroxy function was located at the 2 position of the fatty acids. Other glycolipids containing both hydroxy and normal fatty acids were detected in these erythrocyte lipids.