Biosynthesis of very long chain monounsaturated fatty acids by subcellular fractions of developing seeds

Subcellular fractions from developing seeds of mustard (Sinapis alba), honesty (Lunaria annua) and nasturtium (Tropaeolum majus) synthesize very long chain cis (n−9) monounsaturated fatty acids, e.g. gadoleic (20:1), erucic (22:1) and nervonic (24:1) acid, from oleoyl-CoA and malonyl-CoA by condensation reactions. The particulate 2000 × g and 15 000 × g fractions exhibit considerably higher elongase activities compared to the microsomal or oil body fractions, whereas the soluble (150 000 × g supernatant) fraction is devoid of such activities.     

Biosynthesis of long chain fatty acids by oligodendroglia isolated from bovine white matter.

Abstract— Radioactively labelled fatty acids were incubated with interfascicular oligodendroglial preparations isolated from 9 month fetal and adult bovine CNS white matter to study their metabolism by these cells. Of the various acids studied, the uptake was greatest for palmitic acid and decreased with decreasing chain length. Laurie acid was converted to the greatest extent to other fatty acids. The incorporation of oleic and linoleic acids in the oligodendroglia from both the fetal and adult brains was higher than that of linolenic acid. Fatty acids underwent chain elongation, desaturation and oxidation. Oleic acid was elongated to nervonic acid. Fatty acids were incorporated into both cerebrosides and phospholipids, with preferential incorporation into ethanolamine phosphoglyceride.     

Very long chain fatty acids: Occurrence and biosynthesis in membrane fractions from etiolated maize coleoptiles

The endoplasmic reticulum from maize coleoptiles elongates stearoyl-CoA more effectively than the plasmalemma-enriched fraction. The alkane and very lo     

Synthesis of saturated long chain fatty acids from sodium acetate-1-C14 by Mycoplasma

Three strains of Mycoplasma, M. laidlawii A and B, and Mycoplasma sp. A60549, were grown in broth containing sodium acetate-1-C(14). The methyl esters of the phospholipid fatty acids of harvested radioactive cells were prepared and identified by comparison of their mobilities to known radioactive fatty acid methyl esters by use of a modified reversed-phase partition-thin layer chromatographic technique. No radioactive methyl oleate or methyl linoleate was detected. Compounds migrating as radioactive methyl myristate, stearate, palmitate, and, with less certainty, laurate and octanoate were detected. The qualitative findings for all three organisms appeared similar. M. laidlawii B synthesized a radioactive substance, presumably a saturated fatty acid detected as the methyl ester derivative, which migrated in a position intermediate to methyl myristate-1-C(14) and methyl palmitate-1-C(14). This work indicates that M. laidlawii A and B and Mycoplasma sp. A60549 are capable, in a complex medium containing fatty acids, of synthesizing saturated but not unsaturated fatty acids entirely or in part from acetate.     

Nutritional significance and metabolism of very long chain fatty alcohols and acids from dietary waxes

Very long chain fatty alcohols obtained from plant waxes and beeswax have been reported to lower plasma cholesterol in humans. This review discusses nutritional or regulatory effects produced by wax esters or aliphatic acids and alcohols found in unrefined cereal grains, beeswax, and many plant-derived foods. Reports suggest that 5-20 mg per day of mixed C24-C34 alcohols, including octacosanol and triacontanol, lower low-density lipoprotein (LDL) cholesterol by 21%-29% and raise high-density lipoprotein cholesterol by 8%-15%. Wax esters are hydrolyzed by a bile salt-dependent pancreatic carboxyl esterase, releasing long chain alcohols and fatty acids that are absorbed in the gastrointestinal tract. Studies of fatty alcohol metabolism in fibroblasts suggest that very long chain fatty alcohols, fatty aldehydes, and fatty acids are reversibly inter-converted in a fatty alcohol cycle. The metabolism of these compounds is impaired in several inherited human peroxisomal disorders, including adrenoleukodystrophy and Sj?gren-Larsson syndrome. Reports on dietary management of these diseases confirm that very long chain fatty acids (VLCFA) are normal constituents of the human diet and are synthesized endogenously. Concentrations of VLCFA in blood plasma increase during fasting and when children are placed on ketogenic diets to suppress seizures. Existing data support the hypothesis that VLCFA exert regulatory roles in cholesterol metabolism in the peroxisome and also alter LDL uptake and metabolism.     

Yeast sphingolipids do not need to contain very long chain fatty acids

Synthesis of VLCFAs (very long chain fatty acids) and biosynthesis of DHS (dihydrosphingosine) both are of vital importance for Saccharomyces cerevisiae. The bulk of VLCFAs and DHS are used for ceramide synthesis by the Lag1p (longevity-assurance gene 1)/Lac1p (longevity-assurance gene cognate 1)/Lip1p (Lag1p/Lac1p interacting protein) ceramide synthase. LAG1 and LAC1 are redundant but LIP1 is essential. Here we show that 4Delta (lag1Deltalac1Deltaypc1Deltaydc1Delta) cells devoid of all known endogenous ceramide synthesis pathways are unviable but can be rescued by the expression of Lass5, a mouse LAG1 homologue. Ceramide synthase activity of 4Delta.Lass5 cells only utilizes C16 and C18 fatty acids and does not require the help of Lip1p, an essential cofactor of Lag1p/Lac1p. HPLC-electrospray ionization-MS/MS analysis demonstrated that in IPCs (inositolphosphorylceramides) of 4Delta.Lass5, the very long chain fatty acids (C26 and C24) account for <1% instead of the normal >97%. Notwithstanding, IPCs incorporated into glycosylphosphatidylinositol anchors of 4Delta.Lass5 show normal mobility on TLC and the ceramide- and raft-dependent traffic of Gas1p (glycophospholipid-anchored surface protein) from endoplasmic reticulum to Golgi remains almost normal. Moreover, the biosynthesis of C24:0 fatty acids remains essential. Thus, C(24:0) and dihydrosphingosine are both necessary for survival of yeast cells even if they utilize C16 and C18 fatty acids for sphingolipid biosynthesis.     

Direct demonstration that the deficient oxidation of very long chain fatty acids in X-linked adrenoleukodystrophy is due to an impaired ability of peroxisomes to activate very long chain fatty acids

A method was developed to prepare peroxisome-enriched fractions depleted of microsomes and mitochondria from cultured skin fibroblasts. The method consists of differential centrifugation of a postnuclear supernatant followed by density gradient centrifugation on a discontinuous Metrizamide gradient. The activity of hexacosanoyl-CoA synthetase was subsequently measured in postnuclear supernatants and peroxisome-enriched fractions prepared from cultured skin fibroblasts from control subjects and patients with X-linked adrenoleukodystrophy. Whereas the hexacosanoyl-CoA synthetase activity in postnuclear supernatants of X-linked adrenoleukodystrophy fibroblasts was only slightly decreased (77.8 +/- 4.4% of control (n = 15], enzyme activity was found to be much more markedly reduced in peroxisomal fractions isolated from the mutant fibroblasts (19.6 +/- 6.7% of control (n = 5]. This is a direct demonstration that the defect in X-linked adrenoleukodystrophy is at the level of a deficient ability of peroxisomes to activate very long chain fatty acids, as first suggested by Hashmi et al. [Hashmi, M., Stanley, W. and Singh, I. (1986) FEBS Lett. 86, 247-250].     

Peroxisomal disorders: complementation analysis using beta-oxidation of very long chain fatty acids

Complementation studies, using fused cell lines from patients with peroxisomal disorders, have shown correction of defective plasmalogen synthesis and phytanic acid oxidation as well as an increase in the number of peroxisomes. At least six complementation groups have been reported. We demonstrate here that complementing cell lines also acquire the ability to oxidize very long chain fatty acids (VLCFA), and that complementation groups defined with this technique are identical to those reported previously when plasmalogen synthesis was used as the criterion for complementation. This VLCFA complementation technique is of particular value in the study of patients in whom defective VLCFA is the only or major enzymatic defect, and we show complementation between cell lines from two patients each with an isolated defect in one of the peroxisomal fatty acid beta-oxidation enzymes.     

The function of very long chain polyunsaturated fatty acids in the pineal gland

The mammalian pineal gland is a prominent secretory organ with a high metabolic activity. Melatonin (N-acetyl-5-methoxytryptamine), the main secretory product of the pineal gland, efficiently scavenges both the hydroxyl and peroxyl radicals counteracting lipid peroxidation in biological membranes. Approximately 25% of the total fatty acids present in the rat pineal lipids are represented by arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3). These very long chain polyunsaturated fatty acids play important roles in the pineal gland. In addition to the production of melatonin, the mammalian pineal gland is able of convert these polyunsaturated fatty acids into bioactive lipid mediators. Lipoxygenation is the principal lipoxygenase (LOX) activity observed in the rat pineal gland. Lipoxygenation in the pineal gland is exceptional because no other brain regions express significant LOX activities under normal physiological conditions. The rat pineal gland expresses both 12- and 15-lipoxygenase (LOX) activities, producing 12- and 15-hydroperoxyeicosatetraenoic acid (12- and 15-HpETE) from arachidonic acid and 14- and 17-hydroxydocosahexaenoic acid (14- and 17-HdoHE) from docosahexaenoic acid, respectively. The rat pineal also produces hepoxilins via LOX pathways. The hepoxilins are bioactive epoxy-hydroxy products of the arachidonic acid metabolism via the 12S-lipoxygenase (12S-LOX) pathway. The two key pineal biochemical functions, lipoxygenation and melatonin synthesis, may be synergistically regulated by the status of n-3 essential fatty acids.     

A novel group of very long chain polyenoic fatty acids in dipolyunsaturated phosphatidylcholines from vertebrate retina

Dipolyunsaturated phosphatidylcholines from bovine retina contain a whole series of unusual fatty acids. Methyl esters from these acids are very strongly retained on polar and nonpolar gas-liquid chromatography stationary phases. On thin layers of silica-AgNO3, they separate as tetra-, penta-, and hexaenoic fatty acid methyl esters. After hydrogenation, the three polyunsaturated fractions give the same series of saturated methyl esters, having 20 (or 22)-36 carbon atoms. High pressure liquid chromatography, as well as gas-liquid chromatography, indicates that the new components of the three fractions are even-carbon homologs of well known polyenoic fatty acids of the n-6 and n-3 families, since they behave as series of 20-36-carbon tetraenoic (n-6), pentaenoic (n-3 and n-6), and hexaenoic (n-3) fatty acids. Their occurrence in phospholipid molecules also having docosahexaenoate (22:6) explains the separation of major dipolyunsaturated phosphatidylcholines from retina into dodecaenoic, undecaenoic, and decaenoic fractions after argentation thin layer chromatography. Using high pressure liquid chromatography, the latter are resolved into individual species having 10-12 double bonds and 42-58 carbon atoms. The unusual PCs are thus endowed not only with the highest degree of unsaturation, but with the longest hydrocarbon chains yet reported for vertebrate glycerophospholipids. It is shown that phosphatidylcholines containing the novel fatty acids are highly concentrated in photoreceptor membranes and that they occur in the retina of vertebrates so distant in evolution as fish, birds, and various mammals.     

Identification of very long chain fatty acids from sugar cane wax by atmospheric pressure chemical ionization liquid chromatography-mass spectroscopy

A method is described for the enrichment of very long chain fatty acids (VLCFAs) from total fatty acids of sugar cane wax and their identification as picolinyl esters by means of liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-MS/APCI). The method is based on the use of preparative reversed phase HPLC of 100 mg amounts and their subsequent identification by microbore APCI LC-MS. The combination of these two techniques was used to identify unusual saturated VLCFAs up to C(50).     

Identification of very long chain unsaturated fatty acids from Ximenia oil by atmospheric pressure chemical ionization liquid chromatography-mass spectroscopy

A method is described for the enrichment of very long chain unsaturated fatty acids from total fatty acids of Ximenia oil and their identification as picolinyl esters by means of liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-MS/APCI). The method is based on the use of preparative reversed phase HPLC and their subsequent identification by microbore LC-MS/APCI. The combination of these two techniques was used to identify unusual unsaturated VLCFAs up to tetracontenoic acid. All four positional isomers of tetratriacontenoic acid were also synthesized to unambiguously confirm their structure.     

Inhibition of chymase activity by long chain fatty acids

The activity of chymase was markedly inhibited by fatty acids with carbon chain lengths of 14-22 at doses greater than 0.02 microM, irrespective of the number of double bonds. Cis acids with a carbon chain length of 18, such as stearic acid, oleic acid, linoleic acid, and linolenic acid were potent inhibitors, whereas the trans isomer of oleic acid, elaidic acid, showed less inhibitory activity. The extent of inhibition by oleyl alcohol was almost the same as that by oleic acid, suggesting that the acid moiety itself was not necessary for the inhibition; but a fatty acid with a terminal functional amide, oleamide, showed little inhibitory activity. The inhibition was noncompetitive and was reversible, and the Ki value of oleic acid was 2.7 microM. Stearic acid and oleic acid inhibited all chymotrypsin-type serine endopeptidases tested. The ID50 values of these fatty acids for atypical mast cell protease were higher than those for the other chymotrypsin-type serine endopeptidases tested. Other proteases, such as papain, trypsin, collagenase, and carboxypeptidase A, except cathespin D, were not affected by stearic or oleic acid.     

Monensin blocks the transfer of very long chain fatty acid containing lipids to the plasma membrane of leek seedlings. Evidence for lipid sorting based on fatty acyl chain length.

Delivery of newly synthesized fatty acids and lipids to the plasma membrane in leek seedlings via the endoplasmic reticulum (ER)-Golgi apparatus pathway is primarily by bulk transport (without sorting). However, pulse-chase experiments revealed kinetics of transport of lipids with VLCFA (very long chain fatty acids having more than 18 carbon atoms) in favor of a preferential transfer of these molecules to the plasma membrane. Use of monensin showed the accumulation of lipids in the Golgi apparatus and a related decrease of the amount of lipids transported to the plasma membrane. Lipid and fatty acid analyses revealed that transport of VLCFA-containing phospholipids was most strongly inhibited by the monensin block. These results taken together with an inability of the plasma membrane to synthesize VLCFA support a role for the Golgi apparatus in VLCFA delivery to the plasma membrane and leads to the hypothesis of a sorting function as well, based on fatty acyl chain length.     

Effect of ciprofibrate on the activation and oxidation of very long chain fatty acids

The effect of ciprofibrate, a hypolipidemic drug, was examined in the metabolism of palmitic (C_16:0) and lignoceric (C_24:0) acids in rat liver. Ciprofibrate is a peroxisomal proliferating drug which increases the number of peroxisomes. The palmitoyl-CoA ligase activity in peroxisomes, mitochondria and microsomes from ciprofibrate treated liver was 3.2, 1.9 and 1.5-fold higher respectively and the activity for oxidation of palmitic acid in peroxisomes and mitochondria was 8.5 and 2.3-fold higher respectively. Similarly, ciprofibrate had a higher effect on the metabolism of lignoceric acid. Treatment with ciprofibrate increased lignoceroyl-CoA ligase activity in peroxisomes, mitochondria and microsomes by 5.3, 3.3 and 2.3-fold respectively and that of oxidation of lignoceric acid was increased in peroxisomes and mitochondria by 13.4 and 2.3-fold respectively. The peroxisomal rates of oxidation of palmitic acid (8.5-fold) and lignoceric acid (13.4-fold) were increased to a different degree by ciprofibrate treatment. This differential effect of ciprofibrate suggests that different enzymes may be responsible for the oxidation of fatty acids of different chain length, at least at one or more step(s) of the peroxisomal fatty acid -oxidation pathway.