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The modified fatty acids, (Z,Z,Z)-(octadeca-6,9,12-trienyloxy)acetic acid, (Z,Z,Z)-(octadeca-9,12,15-trienyloxy)acetic acid, (all-Z)-(eicosa-5,8,11,14-tetraenyloxy)acetic acid, (all-Z)-(eicosa-5,8,11,14-tetraenylthio)acetic acid, 3-[(all-Z)-(eicosa-5,8,11,14-tetraenylthio)]propionic acid, (all-Z)-(eicosa-5,8,11,14-tetraenylthio)succinic acid, N-[(all-Z)-(eicosa-5,8,11,14-tetraenoyl)]glycine and N-[(all-Z)-(eicosa-5,8,11,14-tetraenoyl)]aspartic acid, all react with soybean 15-lipoxygenase. The products were treated with triphenylphosphine to give alcohols, which were isolated using HPLC. Analysis of the alcohols using negative ion tandem electrospray mass spectrometry, and by comparison with compounds obtained by autoxidation of arachidonic acid, shows that each enzyme catalysed oxidation occurs at the omega -6 position of the substrate. In a similar fashion, it has been found that (Z,Z,Z)-(octadeca-6,9,12-trienyloxy)acetic acid, (Z,Z,Z)-(octadeca-9,12,15-trienyloxy)acetic acid, (all-Z)-(eicosa-5,8,11,14-tetraenylthio)acetic acid and N-[(all-Z)-(eicosa-5,8, 11.14-tetraenylthio)]propionic acid each undergoes regioselective oxidation at the carboxyl end of the polyene moiety on treatment with potato 5-lipoxygenase. Neither (all-Z)-(eicosa-5,8,11,14-tetraenylthio)succinic acid nor N-[(all-Z)-(eicosa-5,8,11,14-tetraenoyl)]aspartic acid reacts in the presence of this enzyme, while N-[(all-Z)-(eicosa-5,8,11,14-tetraenoyl)]glycine affords the C11' oxidation product. The alcohol derived from (Z,Z,Z)-(octadeca-6,9, 12-trienyloxy)acetic acid using the 15-lipoxygenase reacts at the C6' position with the 5-lipoxygenase. (C) 2001 Elsevier Science Ltd. All rights reserved.

Hydroperoxide derivatives of beta-oxa-substituted polyunsaturated fatty acids were prepared by 15-lipoxygenase catalysed oxidation and perketal derivatives of fatty acid hydroperoxides were synthesized. The perketals are more stable than their parent fatty acid hydroperoxides, but less active as antimalarial agents in the in vitro growth inhibition of Plasmodium falciparum. (C) 1998 Elsevier Science Ltd. All rights reserved.

Our objective was to review the Australasian experience of X-linked adrenoleukodystrophy (ALD), to compare the spectrum of disease seen in Australasia with previously published data from elsewhere, and to assess the reliability of carrier testing. Study design was a retrospective review of records collected over a 15-year period, the setting was an international referral laboratory for the study of metabolic disease, and the subjects were all known cases of ALD diagnosed in Australia and New Zealand between 1981 and 1996 and their families. We estimate that the combined incidence of ALD and its variants in Australasia is at least 1.6 per 100,000. Of 95 affected males, 51 had cerebral adrenoleukodystrophy, 24 had adrenomyeloneuropathy, 15 had Addison's disease only, and 5 remained asymptomatic when last examined. However, the distribution of phenotypes among newly diagnosed patients has changed substantially over the last 15 years, with cerebral forms of the disease forming a decreasing proportion of new diagnoses. The measurement of plasma very long chain fatty acids (VLCFAs) alone detects 93% of women who can be proven to be carriers. The addition of genetic linkage studies or assay of VLCFAs in cultured fibroblasts improved this detection rate to the point that there were no obligate carriers who could not be detected using a combination of two or more techniques.

Although tumor necrosis factor-alpha (TNF-alpha) has been shown to induce marked changes in the physiology/pathophysiology of cells, little is known about the effects of this cytokine on cellular lipid metabolism. In this study we examined the effects of TNF-alpha on the metabolism of eicosatetraenoic acid (arachidonic acid, (20:4(n-6)) in human neutrophils. Pretreatment of neutrophils with TNF-alpha caused a rapid increase in the incorporation of [1-14C]20:4(n-6) substrate into cellular phosphatidylinositol and phosphatidic acid and a slower rise in the incorporation into phosphatidylcholine and phosphatidylethanolamine. Radioactivity was exclusively associated with the sn-2 position of each molecule. The labeling pattern of other phospholipids, neutral lipids, and eicosanoids was unchanged. TNF-alpha had no effect on the distribution of radioactivity in 1-acyl, 1-alkyl, and 1-alk-1-enyl subclasses of phosphatidylcholine, phosphatidylethanolamine, and triglyceride. Chain elongation, beta-oxidation and desaturation of [1-14C]20:4(n-6) were not modulated by the cytokine. TNF-alpha stimulated the release of [3H]20:4(n-6) from prelabeled neutrophils and also induced the production of endogenous unesterified 20:4(n-6). Concomitantly, treatment with the cytokine caused a decrease in the mass of cellular phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine and an increase in the levels of corresponding lysophospholipids, but had no significant effect on sphingomyelin, phosphatidic acid, diglyceride, and other lipids. TNF-alpha did not evoke neutrophils prelabeled with [3H]lyso platelet activating factor to produce [3H]phosphatidylethanol, [3H]phosphatidic acid, or [3H]diglyceride in the presence of ethanol, indicating that phospholipases D and C were not activated. Treatment of the leukocytes with the cytokine had no effect on the activity of neutral and acidic sphingomyelinase. These data collectively provide evidence that TNF-alpha specifically induces the turnover of neutrophil phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine, which are enriched with 20:4(n-6) by the activation of phospholipase A2.

The two branched chain fatty acids pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) and phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) were converted to co-enzyme A thioesters by rat liver mitochondrial outer membranes. However, these branched chain fatty acids could not be converted to pristanoyl and phytanoyl carnitines, respectively, by mitochondrial outer membranes. As expected, the unbranched long chain fatty acids, stearic acid and palmitic acid, were rapidly converted to stearoyl and palmitoyl carnitines, respectively, by mitochondrial outer membranes. These observations indicate that the branched chain fatty acids could not be transported into mitochondria. The data presented strongly suggest that in rat liver, α-oxidation of phytanic acid occurs in organelles other than mitochondria.

Hydroperoxide derivatives of beta-oxa-substituted polyunsaturated fatty acids were prepared by 15-lipoxygenase catalysed oxidation and perketal derivatives of fatty acid hydroperoxides were synthesized. The perketals are more stable than their parent fatty acid hydroperoxides, but less active as antimalarial agents in the in vitro growth inhibition of Plasmodium falciparum. (C) 1998 Elsevier Science Ltd. All rights reserved.

Saturated very long chain fatty acids (fatty acids with greater than 22 carbon atoms; VLCFA) accumulate in peroxisomal disorders, but there is little information on their turnover in patients. To determine the suitability of using stable isotope-labeled VLCFA in patients with these disorders, the metabolism of 22-methyl[23,23,23-2H3]tricosanoic (iso-lignoceric) acid was studied in rats in vivo and in human skin fibroblasts in culture. The deuterated iso-VLCFA was degraded to the corresponding 16- and 18-carbon iso-fatty acids by rats in vivo and by normal human skin fibroblasts in culture, but there was little or no degradation in peroxisome-deficient (Zellweger's syndrome) fibroblasts, indicating that its oxidation was peroxisomal. Neither the 14-, 20-, and 22-carbon iso-fatty acids nor the corresponding odd-chain metabolites could be detected. In the rat, the organ containing most of the iso-lignoceric acid, and its breakdown products, was the liver, whereas negligible amounts were detected in the brain, suggesting that little of the fatty acid crossed the blood-brain barrier. Our data indicate that VLCFA labeled with deuterium at the omega-position of the carbon chain are suitable derivatives for the in vivo investigation of patients with defects in peroxisomal beta-oxidation because they are metabolized by the same pathways as the corresponding n-VLCFA. Moreover, as iso-VLCFA and their beta-oxidation products are readily separated from the corresponding n-fatty acids by normal chromatographic procedures, the turnover of VLCFA can be more precisely measured.

Lipid mediators such as arachidonic acid (AA) generated during inflammation play an important role in stimulating phagocytic cell responses. Since cord blood neutrophils show reduced responses to agonists such as the bacterial tripeptide f-Met-Leu-Phe (fMLP), it would be of interest to know whether cord blood neutrophils show normal or reduced responses to AA and other fatty acids. The data showed that the polyunsaturated n-6 fatty acid (PUFA) AA stimulated cord blood neutrophils to produce a respiratory response (measured by chemiluminescence) and degranulation. Other PUFAs, eicosapentanoic acid and docosahexaenoic acid, elicited similar responses in cord blood neutrophils. Specific granule release and chemiluminescence response in cord blood neutrophils were evident at 0.1–0.5 μg/ml of PUFA, concentrations normally found in vivo during inflammation or following diets enriched with n-3 fatty acids. Neutrophil responses to PUFA were significantly better than those to either fMLP or phorbol myristate acetate. Cord blood neutrophils primed with PUFA showed enhanced responses to fMLP. These results suggest that cord blood neutrophils respond to a similar degree to adult neutrophils to the AA which is generated during the inflammatory response and to the n-3 fatty acids eicosapentaenoic acid/docosahexaenoic acid, both of which may be used in diet manipulation of neurological function and immunological reactions.

We describe the clinical, pathologic, and biochemical findings for two peroxisome-deficient patients in a newly identified complementation group. Both patients had biochemical findings typical of patients with peroxisome biogenesis disorders. However, whereas one patient had the typical clinicopathologic features of Zellweger syndrome, the other patient's phenotype was atypical.

The fatty acid composition of postmortem brain and liver from an adrenoleukodystrophy patient whose diet was supplemented with Lorenzo's oil (glycerol trioleate and glycerol trierucate) for 9 months was determined. The diet depressed plasma and liver saturated very long chain fatty acids (24:0 and 26:0) and increased plasma and liver erucic (22:1) and nervonic (24:1) acids. The levels of plasma linoleic (18:2 n-6), eicosopentaenoic (20:5 n-3), and docosahexaenoic (22:6 n-3) acids were also reduced, while the biochemical marker for essential fatty acid deficiency (20:3 n-9) was markedly increased in liver. However, we were unable to detect any corresponding changes in brain indicating that little erucic acid crossed the blood-brain barrier. Our findings suggest that dietary supplementation with Lorenzo's oil is of limited value in correcting the accumulation of saturated very long chain fatty acids in the brain of patients with adrenoleukodystrophy.

The metabolism of [1-14C]eicosatetraenoic (arachidonic, 20:4(n - 6)), [1-14C]tetracosatetraenoic (24:4(n - 6)) and [1-14C]triacontatetraenoic (30:4(n - 6)) acids was studied in intact human neutrophils. [1-14C]20:4(n - 6) and [1-14C]24:4(n - 6) were efficiently taken up by the neutrophils, esterified into neutral lipids and phospholipids, and elongated by up to four carbon units. In contrast, [1-14C]30:4(n - 6) was poorly incorporated into the cells and remained predominantly in the original unesterified form. The [1-14C]tetraenoic fatty acids were mainly esterified into triacylglycerol, suggesting that this lipid class is important in the intracellular trafficking of polyunsaturated fatty acids. The leukocytes demonstrated a low capacity to beta-oxidize and desaturate the fatty acid substrates. In the presence of calcium ionophore A23187 the neutrophils converted [1-14C]20:4(n - 6) to a variety of radiolabelled oxygenated fatty acid derivatives including prostaglandins, thromboxanes, mono- and dihydroxylated fatty acids and leukotrienes. The major eicosanoid products were 5-monohydroxy-20:4(n - 6) and leukotriene B4. In contrast, [1-14C]24:4(n - 6) was metabolized to radiolabelled monohydroxylated fatty acids (predominantly the 9-hydroxy positional isomer) but not to other lipoxygenase or cyclooxygenase products by the calcium ionophore-stimulated cells. Negligible oxygenated fatty acid compounds were formed from [1-14C]30:4(n - 6), indicating that it is a poor substrate for the neutrophil cyclooxygenase and lipoxygenase enzymes.

Krabbe's globoid cell leukodystrophy is a rare hereditary progressive neurological disease of infants, in which there is deficient activity of galactosylceramide beta-galactosidase. The pathological hallmark is the presence of multinucleated globoid cells in the white matter associated with severe myelin depletion and gliosis. We report a second case where galactosylceramide beta-galactosidase deficiency was proven but no globoid cells were found in the brain. Symptoms began within the first 10 months of life and a deficiency of galactosylceramide beta-galactosidase activity was demonstrated in peripheral blood leukocytes and skin fibroblasts. The child survived till 8 yrs 7 mths. The reason for the absence of globoid cells is not clear but may be related to different effects of the gene mutation on the four substrates or possibly the interaction of sphingolipid activator protein-2.

Lysosomal acid hydrolase activities have been measured in extracts of peripheral blood leucocytes of approximately 1600 patients referred from throughout Australia, each of whom was suspected of having a neurolipidosis. Assays for 12 different lysosomal enzymes were performed on each patient as a routine; ten assay systems were based on commercially available substrates, and four used radiolabelled glycosphingolipids prepared in our own laboratory. Of the 85 patients with positive results, 81 were diagnosed as being homozygous-deficient for a particular lysosomal enzyme. These patients comprised nine with GM1-gangliosidosis, 12 with GM2-gangliosidosis (11 of Tay-Sachs' disease and one of Sandhoff's disease), 18 with trihexosylceramide lipidosis (Fabry's disease), eight with beta-galactosylceramide lipidosis (Krabbe's disease), 14 with beta-glucosylceramide lipidosis (Gaucher's disease), two with sphingomyelin lipidosis (Niemann-Pick disease), 13 with metachromatic leucodystrophy and five with alpha-mannosidosis. In addition, four patients were diagnosed as being affected with mucolipidosis Type II (I-cell disease), based on elevated plasma lysosomal enzyme activities, making a total of 85 homozygous-affected patients. Clinically the patients showed wide phenotypic variability within each of the enzyme-deficient states, which did not appear to correlate with the level of "residual" enzyme activity in their leucocyte extracts.

The in vivo incorporation of 1-(14)C-palmitic acid and 1-0-[8,9-(3)H] hexadecyl glycerol (chimyl alcohol) by plasmodia of the slime mold,Physarum polycephalum has been studied.(14)C-palmitate rapidly enters ester and alkyl ether side chains of phospholipids, but alk-1-enyl side chains are labeled more slowly.(3)H-chimyl alcohol is incorporated into the alkyl ether phospholipids, which appear to undergo enzymatic desaturation, producing plasmalogens. The feasibility ofPhysarum as the source of a cell-free enzyme system for plasmalogen synthesis is discussed.

Rapidly growing plasmodia of the acellular slime mold,Physarum polycephalum, contain large amounts of ether-linked lipids. The ether bonds occur principally in the phospholipids, where plasmalogens account for 21–24 mole per cent of the total and alkyl ether phospholipids comprise 12 mole per cent of the total. Plasmalogens account for over half of the ethanolamine phosphatides, while the alkyl ether derivatives are more uniformly distributed among the various phospholipid fractions. The 16 carbon side chain is by far the major component of both the saturated and the α,β-unsaturated ether derivatives. The nature and amounts of ether lipids present inPhysarum add to the growing evidence that Myxomycetes are more closely related to protozoa than to fungi.

Animal complex lipids contain both hydrophobic and hydrophilic moieties and may be broadly sub-divided into phospholipid and sphingolipid classes and together with protein are localized in membranes. Phospholipids are derivatives of diacylglycerophosphate: the acyl groups have 14–22 carbon atoms and 0–6 double bonds. The phosphate group is almost invariably linked to a hydrophilic residue, e.g. choline, serine. Sphingolipids are derivatives of ceramide—an acylated long chain β-aminoalcohol. Similar to the phospholipids, the acyl groups may have 14–26 carbon atoms but usually no more than one double bond. The alcohol grouping may be linked to phosphorylchohne to form the ubiquitous sphingomyelin, or linked either to one or a chain of carbohydrate moieties to form neutral glycosphingolipids. Further addition of one or more polar sialic acid residues form gangliosides. The sialo- and asialogly-cosphingolipids display haptenic properties and may be involved in cellular interactions.

Sphingomyelin, galactosylceremide, glucosylceremide and GM 2 (or Tay-Sachs) ganglioside were isolated and purified. The lipids were then dissolved in methanol and hydrogenated with tritium gas in the presence of platinum oxide catalyst. After hydrogenation and removal of excess tritium the lipids were re-purified by preparative thin-layer chromatography, or by column chromatography. The radiochemical and chemical purities of the various compounds were evaluated by thin-layer and gas-liquid chromatography of products formed after hydrolysis in 0.75 M methanolic hydrogen chloride. All lipids incorporated tritium almost exclusively into the hydrophobic part of the molecule. The ratio of the specific activities of fatty acid to long-chain base in the various sphingolipids was of the order of 4, thus indicating that the bulk of the radioactivity had been incorporated into the fatty acid moieties. Initial experiments with two of the radioactive lipids prepared in this manner, sphingomyelin and galactosyl-ceramide, have confirmed their value in the diagnosis of Niemann-Pick and Krabbe's diseases respectively.

We examined the degradation of a labeled phosphatidylglycerol (PG) by fibroblasts from a normal control and a patient with Niemann-Pick (NP) disease. The control homogenate had both phospholipase A and phospholipase C activities toward PG, but NP cells had only phospholipase A. The PG phospholipase C of control fibroblasts was solubilized by sonication and freezing and thawing, was most active at pH 5.0, and was inhibited by Ca2-, detergents, sphingomyelin, and 5' AMP. Assay of PG phospholipase C in fibroblast cultures from NP patients with sphingomyelinase deficiency (three designated type A and four type B) confirmed absence of activity, whereas cultures from NP patients without sphingomyelinase deficiency (three designated type C and one with neurovisceral lipidoses and vertical supranuclear ophthalmoplegia) had activities close to those of normal controls. These findings substantiate previous observations of low phosphodiesterase activities in NP disease and suggest that the enzymatic function affected by the NP genes includes specificity toward PG and sphingomyelin. Deficiency of PG phospholipase C may explain the accumulation of bis(monoacylglycero)phosphate in NP disease.