Alfred Poulos

Activation of neutral sphingomyelinase in human neutrophils by polyunsaturated fatty acids.

Although unesterified polyunsaturated fatty acids (PUFA) have been shown to elicit marked changes in neutrophil function, the associated signal transduction processes require clarification. In this study we examined the effect of PUFA on the sphingomyelin (SM)‐signalling cycle in human neutrophils. Treatment of neutrophils with eicosatetraenoic acid [ arachidonic acid, 20:4(n‐6)] caused a decrease in the mass of cellular SM and an increase in the level of ceramide. 20:4(n‐6)‐stimulated neutral sphingomyelinase (SMase) activity of the leucocytes in a time‐ and concentration‐dependent manner. Other unsaturated fatty acids, docosahexaenoic [22:6(n‐3)], eicosapentaenoic [20:5(n‐3)], octadecenoic [oleic, 18:1(n‐9)] and octadecadienoic [linoleic, 18:2(n‐6)] acids also had the capacity to activate neutral SMase; however, certain 20:4(n‐6) derivatives {20:4(n‐6) methyl ester [20:4(n‐6)ME], 15‐hydroperoxyeicosatetraenoic (15‐HPETE) and 15‐hydroxyeicosatetraenoic (15‐HETE) acids}, very‐long‐chain PUFA {tetracosatetraenoic [24:4(n‐6)] and octacosatetraenoic [28:4(n‐6)] acids} and saturated fatty acids [octadecanoic (stearic, 18:0) and eicosanoic (arachidic, 20:0) acids] had no significant effect. Activation of neutral SMase by 20:4(n‐6) appeared to involve metabolism via 20:4(n‐6)CoA (arachidonoyl CoA) and was not dependent on prostaglandin and leukotriene synthesis. All of the fatty acids and derivatives tested failed to activate acidic SMase of neutrophils. Ceramide was found to inhibit 20:4(n‐6)‐induced superoxide generation by the cells. It is envisaged that the PUFA‐induced ceramide production in neutrophils plays a role in the regulation of biological responses.

Antimalarial properties of n-3 and n-6 polyunsaturated fatty acids: in vitro effects on Plasmodium falciparum and in vivo effects on P. berghei.

The polyunsaturated fatty acids docosahexaenoic acid (C22:6,n-3), eicosapentaenoic acid, arachidonic acid, and linoleic acid caused marked in vitro growth inhibition of Plasmodium falciparum, assessed by a radiometric assay. In contrast, negligible parasite killing was seen with oleic acid or docosanoic acid. Parasite killing was significantly increased when oxidized forms of polyunsaturated fatty acids were used. Antioxidants greatly reduced the fatty acid-induced killing. Mice infected with P. berghei and treated for 4 d with C22:6,n-3 showed marked reduction in parasitemia. The anemia associated with the infection was also alleviated by treatment with C22:6,n-3. The data provide new information that could be explored in order to develop new strategies in malaria treatment.

Neutrophil oxygen radical generation. Synergistic responses to tumor necrosis factor and mono/polyunsaturated fatty acids.

In inflammatory reactions there are complex interactions of protein mediators (cytokines) and mediators derived from lipids. An important event in inflammation is superoxide production, in relation to microbicidal activity as well as tissue damage. We have studied interactions of lipid mediators with a cytokine mediator tumor necrosis factor alpha (TNF) in stimulating superoxide production by human neutrophils for this reason and because it throws light on intracellular signals activating this response. Pretreatment of neutrophils with TNF markedly augmented the amount of superoxide produced in response to AA but not to either a 20 carbon saturated fatty acid, or the hydroxy- or hydroperoxy-derivatives of AA. Not only were other polyunsaturated fatty acids (eicosapentanoic, docosahexaenoic, linolenic, linoleic acid) as effective as AA but so was the monounsaturated fatty acid, oleic acid. Indeed TNF primed the neutrophils for an increased response to a major mediator of inflammation, leukotriene B4, which is a product of AA metabolism via the lipoxygenase pathway. The data demonstrate that two major types of mediators generated during an inflammatory response have synergistic action on neutrophils in the generation of reactive oxygen species. In contrast, neutrophils primed with TNF and challenged with PGE2, a product of AA metabolism via the cyclooxygenase pathway, showed a reduced chemiluminescence response. This identifies an important interaction between unsaturated lipids and cytokines which is likely to play a critical role in disease processes and nutrient modulation of the immune responses.

Neutrophil migration inhibitory properties of polyunsaturated fatty acids. The role of fatty acid structure, metabolism, and possible second messenger systems.

The n-3 polyunsaturated fatty acids (PUFA) appear to have antiinflammatory properties that can be partly explained by their biological activity on leukocytes. Since leukocyte emigration is an essential component of the inflammatory response, we have examined the effects of the n-3 PUFA (eicosapentaenoic and docosahexaenoic acids) on neutrophil random and chemotactic movement. Preexposure of neutrophils for 15-30 min to 1-10 micrograms/ml PUFA reduced the random and chemotactic migration to both FMLP- and fungi-activated complement. The inhibitory effect diminished with increasing saturation and carbon chain length, and methylation abolished this activity. Arachidonic and docosahexaenoic acids were the most active fatty acids. The PUFA concentration required to inhibit migration was dependent on cell number, suggesting that the fatty acid effects on leukocyte migration in vivo may be governed by the stage of the inflammatory response. It was concluded that the PUFA rather than their metabolites were responsible for the inhibition since: (a) antioxidants did not prevent the PUFA-induced migration inhibition and the hydroxylated intermediates were less active, and (b) inhibitors of the cyclooxygenase and lipoxygenase pathways were without effect. Inhibitors of protein kinases and calmodulin-dependent enzyme system did not prevent the PUFA-induced migration inhibition, which was also independent of phospholipase D-catalyzed hydrolysis of phospholipids. It is also shown that PUFA decrease the FMLP-induced Ca2+ mobilization.

Peroxisomal assembly defects: Clinical, pathologic, and biochemical findings in two patients in a newly identified complementation group

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 patients phenotype was atypical.

Altered responses of human macrophages to lipopolysaccharide by hydroperoxy eicosatetraenoic acid, hydroxy eicosatetraenoic acid, and arachidonic acid. Inhibition of tumor necrosis factor production.

The regulation of allergic and autoimmune inflammatory reactions by polyunsaturated fatty acids and their metabolic products (eicosanoids) continues to be of major interest. Our data demonstrate that arachidonic acid 5,8,11,14-eicosatetraenoic acid (20:4n-6) and its hydroxylated derivatives 15(s)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) and 15(s)-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) regulate agonist-induced tumor necrosis factor alpha (TNF) production, a cytokine that plays a role in inflammatory diseases. Although 20:4n-6 and 15-HETE caused a reduction in production of TNF in mononuclear leukocytes stimulated with phytohaemagglutinin, pokeweed mitogen, concanavalin A, and Staphylococcus aureus, 15-HPETE was far more active. 15-HPETE was also found to dramatically depress the ability of bacterial lipopolysaccharide to induce TNF production in monocytes and the monocytic cell line Mono Mac 6. These fatty acids depressed the expression of TNF mRNA in Mono Mac 6 cells stimulated with LPS; 15-HPETE was fivefold more active than 20:4n-6 and 15-HETE. While 15-HPETE treatment neither affected LPS binding to Mono Mac 6 cells nor caused a decrease in CD14 expression, the fatty acid significantly reduced the LPS-induced translocation of PKC (translocation of alpha, betaI, betaII, and epsilon isozymes), suggesting that 15-HPETE acts by abrogating the early signal transduction events. The findings identify another molecule that could form the basis for development of antiinflammatory pharmaceuticals.

Oxidation of pristanic acid in fibroblasts and its application to the diagnosis of peroxisomal beta-oxidation defects.

Pristanic acid oxidation measurements proved a reliable tool for assessing complementation in fused heterokaryons from patients with peroxisomal biogenesis defects. We, therefore, used this method to determine the complementation groups of patients with isolated defects in peroxisomal beta-oxidation. The rate of oxidation of pristanic acid was reduced in affected cell lines from all of the families with inherited defects in peroxisomal beta-oxidation, thus excluding the possibility of a defective acyl CoA oxidase. Complementation analyses indicated that all of the patients belonged to the same complementation group, which corresponded to cell lines with bifunctional protein defects. Phytanic acid oxidation was reduced in fibroblasts from some, but not all, of the patients. Plasma samples were still available from six of the patients. The ratio of pristanic acid to phytanic acid was elevated in all of these samples, as were the levels of saturated very long chain fatty acids (VLCFA). However, the levels of bile acid intermediates, polyenoic VLCFA, and docosahexaenoic acid were abnormal in only some of the samples. Pristanic acid oxidation measurements were helpful in a prenatal assessment for one of the families where previous experience had shown that cellular VLCFA levels were not consistently elevated in affected individuals.

X‐linked adrenoleukodystrophy: The Australasian experience

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 Addisons 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.

Cord Blood Neutrophil Responses to Polyunsaturated Fatty Acids: Effects on Degranulation and Oxidative Respiratory Burst

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 microgram/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.

Substrate specificity of rat liver mitochondrial carnitine palmitoyl transferase I: evidence against α‐oxidation of phytanic acid in rat liver mitochondria

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.