Unique molecular species of phosphatidylcholine containing very-long-chain (C24-C38) polyenoic fatty acids in rat brain.

Rat brain has been shown to contain polyenoic very-long-chain fatty acids (VLCFA) belonging to the n-3 and n-6 series with four, five and six double bonds and even-carbon chain lengths from 24 to 38. These fatty acids are almost exclusively located in unusual molecular species of phosphatidylcholine at the sn-1 position of the glycerol backbone, whereas saturated, monoenoic and polyenoic fatty acids with less than 24 carbon atoms are present at the sn-2 position. Polyenoic VLCFA phosphatidylcholine in neonatal rat brain is enriched with n-6 pentaenoic and n-3 hexaenoic VLCFA with up to 36 carbon atoms, whereas the corresponding phospholipid in adult rat brain mainly contains n-6 tetraenoic and n-3 pentaenoic VLCFA with up to 38 carbon atoms. The total amount of polyenoic VLCFA associated with phosphatidylcholine is highest in the brain of immature animals. Polyenoic VLCFA phosphatidylcholine appears to be predominantly confined to nervous tissue in rats, and it is envisaged that this phospholipid is of physiological significance.     

Structure and lipid distribution of polyenoic very-long-chain fatty acids in the brain of peroxisome-deficient patients (Zellweger syndrome).

The polyenoic fatty acids with carbon chain lengths from 26 to 38 (very-long-chain fatty acids, VLCFA) previously detected in abnormal amounts in Zellweger syndrome brain have been shown to be n-6 derivatives and therefore probably derived by chain elongation of shorter-chain n-6 fatty acids such as linoleic acid and arachidonic acid. Polyenoic VLCFA are also present in Zellweger syndrome liver, but this tissue differs significantly from brain in that the saturated and mono-unsaturated derivatives are the major VLCFA. Zellweger syndrome brain polyenoic VLCFA are present in the neutral lipids predominantly in cholesterol esters, with smaller amounts in the non-esterified fatty acid and triacylglycerol fractions. These fatty acids are barely detectable in any of the major phospholipids, but are present in significant amounts in an unidentified minor phospholipid. The polyenoic VLCFA composition of this lipid differs markedly from that observed for all other lipids, as it contains high proportions of pentaenoic and hexaenoic fatty acids with 34, 36 and 38 carbon atoms. A polar lipid with the chromatographic properties in normal brain contains similar fatty acids. It is postulated that the polyenoic VLCFA may play an important role in normal brain and accumulate in Zellweger syndrome brain because of a deficiency in the peroxisomal beta-oxidation pathway, although a possible peroxisomal role in the control of carbon-chain elongation cannot be discounted.     

The occurrence of polyenoic very long chain fatty acids with greater than 32 carbon atoms in molecular species of phosphatidylcholine in normal and peroxisome-deficient (Zellweger''s syndrome) brain.

The n-6 tetra- and pentaenoic fatty acids with carbon chain lengths greater than 32 found in normal brain are located predominantly in a separable species of phosphatidylcholine. A similar phospholipid is found in increased amounts in the brain of peroxisome-deficient (Zellweger's syndrome) patients, but the fatty acid composition differs in that penta- and hexaenoic derivatives predominate. Our data strongly suggest that the polyenoic very long chain fatty acids are confined to the sn-1 position of the glycerol moiety, while the sn-2 position is enriched in saturated, monounsaturated and polyunsaturated fatty acids with less than 24 carbon atoms. It is postulated that these unusual molecular species of phosphatidylcholine may play some, as yet undefined, role in brain physiology.     

Very long chain (C24 to C36) polyenoic fatty acids of the n-3 and n-6 series in dipolyunsaturated phosphatidylcholines from bovine retina

A complete series of even-carbon chain polyenoic fatty acids having 20-36 carbons occur in dipolyunsaturated molecular species of phosphatidylcholine from bovine retina. Using oxidative ozonolysis, it is shown that very long chain tetraenes belong to the n-6 series, hexaenes to the n-3 series, and major pentaenes to the n-3 series of fatty acids (very long chain n-6 pentaenes also occur). Molecular ions are obtained by electron impact mass spectrometry of methyl ester derivatives which conclusively identify the major components of this novel group of fatty acids. Mass spectral patterns are similar for the major very long chain tetraenes, for the pentaenes, and for the hexaenes, but different for each group of unsaturation. Very long chain (C24 to C36) polyenes account for about half the weight (40 mol %) of the acyl chains of major dodecaenoic, undecaenoic, and decaenoic molecular species of bovine retina phosphatidylcholine, the other half being made up by docosahexaenoate (22:6 n-3).     

Occurrence of conjugated polyenoic fatty acids in seaweeds from the Indian Ocean

Three species of red marine macro algae (Rhodophyta) from the Indian Ocean were analysed for the occurrence of conjugated polyenes. The composition of different lipid classes in these seaweeds along with their fatty acid composition has also been reported. Analysis of lipid classes of these seaweeds revealed that both Acanthophora spicifera (Ceramiales, Rhodophyta) and two species of Gracilaria, viz. G. edulis and G. folifera (Gracilariales, Rhodophyta) were rich in glycolipids followed by neutral- and phospholipids. The fatty acid composition of these seaweeds revealed C16:0 as the predominant fatty acid in all three species. However, A. spicifera had significantly higher amounts of eicosapentaenoic acid (EPA) and arachidonic acid (AA) as compared to negligible amount of these fatty acids in both species of Gracilaria. The red seaweed Acanthophora spicifera contained conjugated eicosapentaenoic acid (CEPA) and conjugated arachidonic acid (CAA) in all lipid classes except glycolipids.     

Occurrence of novel nonmethylene-interrupted C24 polyenoic fatty acids in female gonad lipids of the limpet Cellana grata

The occurrence of positional isomers of minor C(24) unsaturated fatty acids in female gonad lipids of the limpet Cellana grata was clarified by gas chromatography-mass spectrometry of the combination of their 4,4-dimethyloxazoline and picolinyl ester derivatives. In this study, in addition to 5,9-24:2, 9,15-24:2, 5,9,15-24:3, and 5,9,17-24:3, previously identified, 24:4n-6, 24:5n-3, and four novel nonmethylene-interrupted fatty acids, 9,17-24:2, 9,15,18-24:3, 5,9,15,18-24:4, and 5,9,15,18,21-24:5, were newly recognized. All C(24) unsaturated fatty acids detected were present only in triacylglycerols.     

Oxidation of very-long-chain fatty acids in rat brain: cerotic acid is beta-oxidized exclusively in rat brain peroxisomes

We studied the effect of sodium 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA), a potent inhibitor of mitochondrial carnitine palmitoyltransferase I, on fatty acid oxidation by rat brain cells. In cultured glial cells as well as in dissociated brain cells from adult rats palmitic acid (16:0) oxidation was inhibited by about 85% of control values when 25 microM POCA was added to the medium, whereas no inhibition of cerotic acid (26:0) oxidation was observed. Furthermore, omission of carnitine from the culture medium resulted in a 57.7% decrease in palmitic acid oxidation in cultured glial cells, whereas cerotic acid oxidation was not influenced. These results indicate that rat brain peroxisomes contribute only little (about 15%) to palmitic acid oxidation and provide conclusive evidence that cerotic acid is oxidized exclusively in rat brain peroxisomes.     

Beta-oxidation of medium chain (C8-C14) fatty acids studied in isolated liver cells

The beta-oxidation and esterification of medium-chain fatty acids were studied in hepatocytes from fasted, fed and fructose-refed rats. The beta-oxidation of lauric acid (12:0) was less inhibited by fructose refeeding and by (+)-decanoyl-carnitine than the oxidation of oleic acid was, suggesting a peroxisomal beta-oxidation of lauric acid. Little lauric acid was esterified in triacylglycerol fraction, except at high substrate concentrations or in the fructose-refed state. With [1-14C]myristic acid (14:0), [1-14C]lauric acid (12:0), [1-14C]octanoic acid (8:0) and [2-14C]adrenic acid (22:4(n - 6] as substrate for hepatocytes from carbohydrate-refed rats, a large fraction of the 14C-labelled esterified fatty acids consisted of newly synthesized palmitic acid (16:0), stearic acid (18:0) and oleic acid (18:1) while intact [1-14C]oleic acid substrate was esterified directly. With [9,10-3H]myristic acid as the substrate, small amounts of shortened 3H-labelled beta-oxidation intermediates were found. With [U-14C]palmitic acid, no shortened fatty acids were detected. It was concluded that when the mitochondrial fatty acid oxidation is down-regulated such as in the carbohydrate-refed state, medium-chain fatty acids can partly be retailored to long-chain fatty acids by peroxisomal beta-oxidation followed by synthesis of C16 and C16 fatty acids which can then stored as triacylglycerol.     

Supply of polyenoic fatty acids to the mammalian brain: The ease of conversion of the short-chain essential fatty acids to their longer chain polyunsaturated metabolites in liver,brain, placenta and blood

The metabolism of linoleic and linolenic acids to the longer polyunsaturated fatty acids of mammalian brain is discussed. Differences in metabolic activity are considered between tissues, between species, and during different stages of development. Available evidence suggests that:

• 1.1. The sites of metabolism are confined mainly to liver and the brain itself.
• 2.2. The similar metabolic pathways are subject to a complex control which is most effective at the first step in the sequence.
• 3.3. During early development there are reciprocal changes in the metabolic capacity of brain and liver.
• 4.4. Metabolic activity varies between species and may be absent in obligate carnivores.

     

Determination of C20-C30 fatty acids by reversed-phase chromatographic techniques: An efficient method to quantitate minor fatty acids in serum of patients with adrenoleukodystrophy

An analytical method for the determination of saturated very long chain (VLC) fatty acids in the serum has been devised. Free fatty acids obtained after hydrolysis of total lipid extracts were converted intop-bromophenacyl esters. The derivatives were purified in two sequential steps by clean-up on C₁₈ reversed-phase cartridge and fractionation by reversed-phase thin-layer chromatography (TLC), and then quantitated by high performance liquid chromatography (HPLC) analysis. This technique provides a reliable and alternative method for the biochemical identification of patients and carriers of an inherited metabolic disease characterized by the accumulation of saturated VLC fatty acids (C₂₄–C₂₆) such as Adrenoleukodystrophy (ALD). In four cases of diagnosed ALD the fatty acid composition of serum total lipids was dramatically enriched in saturated VLC fatty acids compared to controls. The ratio of hexacosanoic acid (C₂₆₀) to docosanoic acid (C₂₂₀) in ALD patients was approximately six-fold higher than that of healthy controls or patients affected by metabolic or neurological disorders other than ALD.     

Urinary excretion of dicarboxylic acids from patients with the Zellweger syndrome. Importance of peroxisomes in beta-oxidation of dicarboxylic acids

The urinary excretion of adipic acid, suberic acid and sebacic acid from two patients with the cerebrohepato-renal syndrome of Zellweger was studied. The patients had a complete lack of peroxisomes in the liver as judged by electron microscopy. In the non-ketotic state, the total excretion of free and conjugated adipic acid, suberic acid and sebacic acid was increased by about 100%, 200% and 350%, respectively, as compared to the corresponding excretion from six healthy infants of the same age. The excretion of free dicarboxylic acid was increased to a considerably lesser extent than the free + conjugated dicarboxylic acid. In view of the presence of adipic acid in urine of the Zellweger patients, it is concluded that peroxisomes are not obligatory for beta-oxidation of medium-chain dicarboxylic acids in vivo. The relative accumulation of suberic acid and sebacic acid as compared to adipic acid is, however, consistent with a relative block in the conversion of suberic acid and sebacic acid into adipic acid in patients with the Zellweger syndrome.     

Oxidation of fatty alcohols to acids in the caecum of a gourami (Trichogaster cosby).

Oxidation of fatty alcohols to acids in gourami caeca was investigated by measuring the reduction of NAD+ and the formation of labeled hexadecanoic acid from [1(-14)C]hexadecanol. Virtually all dehydrogenase activity is in the microsomal fraction. Maximal activity is obtained with NAD+ as cofactor whereas with NADP+ 60% of that activity is obtained. The enzyme is rather specific for long chain alcohols and 2 NADH are formed for each molecule of hexadecanol oxidized to acid. It is stabilized by mercaptoethanol, and completely inhibited by p-chloromercuribenzoate. The activity is optimal at pH 9.5. At higher pH, small amounts of aldehyde are found. The first reaction in the sequence, fatty alcohol leads to aldehyde leads to acid seems to occur under the more physiological condition at a much slower rate than the second reaction so that free aldehyde is not detected. Addition of palmitic acid indicated an uncompetitive product inhibition. The oxidation of alcohol to acid is reversible only to a very minor extent even in the presence of NADPH, CoA, ATP and Mg2+. Location, activity and properties of the enzyme are in agreement with the earlier observation from dietary experiments that in the gourami fatty alcohols of wax esters are oxidized to acids in the course of absorption.