Ernesto E. Aeberhard

Fatty Acid Synthesis in the Developing Brain

Extract: Fatty acid biosynthesis in the developing brain was studied. During development, total fatty acid synthesis was maximal at 15–16 days of age—a time of rapid myelination for this species. Results were expressed as mμmoles of 14C-labeled acetyl-CoA incroported into fatty acids per milligram of brain mitochondrial of microsomal protein, and as 14C-labeled malony-CoA incorporated per milligram of microsmal protein.Elongation of saturated fatty acids by microsoms followed a similar development pattern. The peak incorporation of malaony-CoA into saturated fatty acids occurred at 15 days of age, while synthesis of polyunsaturated fatty acid did not change significantly with maturation. In the mitochondrial-acetyl-CoA system, elongation of potyunsaturated fatty acids was unchanged significantly with maturation. These result support the concept that chain elongation of fatty acids is directly ralated to myelination. The marked increase in the rate of elongation of saturated fatty acid by the micosmol fraction, occurring at this time, suggests that this system is involved in synthesis of myelin fatty acids.Speculation: At the time myelination there is an increase in the activity of microsomes with resect to incroporating malony-CoA into saturated fatty asids, and into polyunsaturated fatty acids (20:2 and 20:4). This is unexpected because of the well established finding that in mature liver the microsomal fraction preferentially synthesizes polyunsaturated fatty acids, while the mitochondrial fraction forms saturated fatty acids. Polyunsaturated fatty acids constitute a relatively minor proportion of myelin fatty acids, and it is unlikely that the striking increase i microsomal activity observed at 15–16 days is necessary for their formation. It is possible that maturation changes the selectivity of the fatty acid synthesizing system, or that there are specificity differenced between the hepatic and cerebral microsomal chain elongation systems.

Stimulation of phosphatidylcholine synthesis by fatty acids in fetal rabbit type II pneumocytes

After 24 h exposure to 0.1 mM oleate or 0.1 mM palmitate there was a 2- and 1.7-fold increase, respectively, in the incorporation of choline into the lipids of type II pneumocytes. Palmitate increased the labeling of disaturated phosphatidylcholine (PC) from 23.0% of total labeled PC in control cultures to 56.6% and oleate decreased labeling of disaturated PC to 9.4%. The percentage of total cellular radioactivity found in the lipid fraction was also markedly higher in the fatty acid-treated cells (83.3% for oleate and 78.7% for palmitate) than in control cultures (64.0%). Radioactivity in water-soluble choline metabolites was correspondingly lower, with phosphocholine representing more than 95% of the label in both control and experimental cultures. After a 3 h pulse-chase period, oleate and palmitate significantly increased the percentage of total cellular radioactivity in PC and decreased the percentage in phosphocholine. Similar results were obtained by adding melittin (1-2 micrograms/ml) or phospholipase C (0.05 U/ml) to the culture medium. The stimulation of PC synthesis by fatty acids was demonstrated as early as 1 h after exposure to oleate or palmitate and at all concentrations from 0.025 to 0.25 mM. Cytidylyltransferase activity in total cell homogenates was also enhanced by long-term exposure to fatty acids and short-term addition of fatty acids or phospholipase C and melittin to the culture medium. A similar increase in cytidylyltransferase activity was found in the 100 000 X g particulate fraction of type II cells exposed to fatty acids, whereas no differences were found between the cytosolic fractions of control and treated cells. These results support the concept that an increase in intracellular level of fatty acids either from an exogenous source or following the activation of endogenous phospholipases regulates PC synthesis in fetal type II pneumocytes.

Effects of cyclic AMP analogues and phosphodiesterase inhibitors on phospholipid biosynthesis in fetal type II pneumocytes

Purified type II pneumocytes grown in monolayer cultures after isolation from fetal rabbit lung organotypic cultures were employed to investigate effects of cAMP analogues and phosphodiesterase inhibitors on [methyl-14C]choline and [9-10(n)3H]palmitate incorporation into cell lipids. After 24 h exposure to 0.5 mM N6,O2-dibutyryl-cAMP or 8-bromo-cAMP, a significant increase was found in the rate of incorporation of choline into phospholipids. Addition of 1 mM 1-methyl-3-isobutylxanthine or aminophylline also increased incorporation of choline into phospholipids but did not significantly change the incorporation of choline into sphingomyelin. These effects were not due to increased uptake of choline or changes in the pool size of the precursor. Cyclic AMP analogues also stimulated the rate of incorporation of palmitate into most lipid fractions but did not alter the relative percentages of incorporation of either precursor into any of the phospholipids. Phosphodiesterase inhibitors did not significantly change the rate of incorporation of palmitate into neutral lipids and most phospholipids, except for a decrease into sphingomyelin, phosphatidylinositol and phosphatidylethanolamine. However, they increased the percentage of incorporation of palmitate into phosphatidylcholine and decreased the percentage of incorporation into most other phospholipids. These data clearly indicate that cAMP can stimulate the synthesis of phospholipids within the type II pneumocytes. This effect is probably a general stimulation effect for the cAMP analogues but methylxanthines may selectively increase the synthesis of surfactant lipids such as phosphatidylcholine while decreasing that of other membrane-associated phospholipids.

Biosynthesis of fatty acids byTrypanosoma cruzi

The incorporation of [1-14C] acetate into fatty acids by cultured epimastigotes ofTrypanosoma cruzi was studied. After 8, 24, and 48 hr incubation with labeled precursor, up to 2.8% of the initial radioactivity added to the medium was found in theT. cruzi long chain fatty acids. Saturated (16∶0 and 18∶0), monounsaturated (18∶1ω9), and diunsaturated (18∶2ω6) fatty acids were synthesized. Both the pattern of incorporation of labeled acetate into the fatty acids and the decarboxylation ratios found suggest that de novo synthesis of fatty acids has taken place.

Maternal phenylketonuria: The composition of cerebral lipids in an affected offspring

The chemical composition of cerebral lipids and the fatty acid pattern of the major white matter and myelin glycolipids were examined in a severely retarded heterozygote offspring of a phenylketonuric mother. The lipid abnormalitis seen were comparable with those previously found in patients with phenylketonuria. They included a slight reduction in cholesterol, cerebrosides, and sulfatides. As was observed in 3 phenylketonuric patients, the relative amounts of nonhydroxylated sulfatides were reduced. A loss in sulfatide cerebronic acid (24h:0), usually accompanied by an increase in alpha-hydroxy nervonic acid (24h:1), was seen in all 3 specimens analyzed in this patient and was also present in all 3 phenylketonuric brains previously studied by us. The presence of large amounts of esterified kerasin cerebrosides was documented. While they may represent an artifact, it is more likely that they were formed in vivo as a consequence of esterification of long-chain extra-myelin cerebrosides. The similarity of the biochemical abnormalities to those seen in the brain of the homozygous phenylketonuric patient provides evidence that exposure of the fetus to a deranged amino acid environment produces irreversible neurochemical alterations.

Desaturation of fatty acids inTrypanosoma cruzi

Uptake and metabolism of saturated (16∶0, 18∶0) and unsaturated [18∶1(n−9), 18∶2(n−6), 18∶3(n−3)] fatty acids by cultured epimastigotes ofTrypanosoma cruzi were studied. Between 17.5 and 33.5% of the total radioactivity of [1-14C]labeled fatty acids initially added to the culture medium was incorporated into the lipids ofT. cruzi and mostly choline and ethanolamine phospholipids. As demonstrated by argentation thin layer chromatography, gas liquid chromatography and ozonolysis of the fatty acids synthesized, exogenous palmitic acid was elongated to stearic acid, and the latter was desaturated to oleic acid and 18∶2 fatty acid. The 18∶2 fatty acid was tentatively identified as linoleic acid with the first bond in the Δ9 position and the second bond toward the terminal methyl end. Exogenous stearic acid was also desaturated to oleic and 18∶2 fatty acid, while oleic acid was only converted into 18∶2. All of the saturated and unsaturated fatty acids investigated were also converted to a small extent (2–4%) into polyunsaturated fatty acids. No radioactive aldehyde methyl ester fragments of less than nine carbon atoms were detected after ozonolysis of any of the fatty acids studied. These results demonstrate the existence of Δ9 and either Δ12 or Δ15 desaturases, or both, inT. cruzi and suggest that Δ6 desaturase or other desaturases of the animal type are likely absent in cultured forms of this organism.

On the mechanisms of fatty acid transformations in membranes

Concentrations of albumin in excess of 1% in the incubation mixture inhibited the elongation of added fatty acids and their incorporation into microsomal lipids whereas these reactions were not inhibited with endogenous microsomal membrane fatty acids. The results of these and other studies support the idea that such reactions of membrane lipid fatty acids with membrane-bound enzymes normally occur entirely within the membrane without release of free fatty acids to equilibrate with the fatty acid pool during the process.

Effects of pentoxifylline on in vivo leukocyte function and clearance of group B streptococci from preterm rabbit lungs.

ObjectivesPentoxifylline was evaluated for its ability to enhance inactivation of group B streptococci in lungs of prematurely born rabbits. Mechanisms associated with intra-pulmonary streptococcal clearance and the phar-macodynamics of pentoxifylline were also investigated. DesignRandomized, controlled animal trial. SettingUniversity research laboratory. SubjectsA total of 123 New Zealand rabbits were delivered prematurely by cesarian section and were used for clearance studies. Twenty-three preterm pups were additionally utilized to study the pharmacodynamics of pentoxifylline. InterventionsPreterm rabbits were infected with group B streptococcal aerosols and given intraperitoneal injections of either pentoxifylline (25,12.5, and 12.5 mg/kg) or placebo at 0,6, and 12 hrs after infection. Measurements and Main ResultsAt 0, 4, and 24 hrs, the numbers of streptococci were determined in the left lung, while the right lung underwent bronchoalveolar lavage to quantify intra-alveolar leukocytes, phagocytosis of inhaled bacteria, and concentrations of lysozyme and tumor necrosis factor. In a separate experiment, blood and bronchoalveolar fluid from infected animals were analyzed for pentoxifylline content.Streptococcal proliferation was less in pentoxifylline-treated animals than in controls at 24 hrs (p < .01). Pulmonary macrophages and poly-morphonuclear leukocytes recovered from bronchoalveolar lavage fluid did not differ in numbers or phagocytic activity. Pentoxifylline-treated animals had lower levels of lysozyme (p < .02) and tumor necrosis factor (p < .005) in bronchoalveolar lavage fluid compared with placebo-treated pups. Therapeutic levels of pentoxifylline were achieved in blood and bronchoalveolar lavage fluid. ConclusionsDespite lowering lysozyme and tumor necrosis factor content in epithelial lining fluid, pentoxifylline improves the inactivation of group B streptococci in preterm rabbit lungs. These findings suggest that increased group B streptococcal clearance was coincident with an anti-inflammatory effect due to pentoxifylline. We conclude pentoxifylline may be clinically useful as an adjunctive therapy for group B streptococcal pneumonia in newborns. (Crit Care Med 1993; 21:712–720)

Regulation of phospholipid synthesis by intracellular phospholipases in fetal rabbit type II pneumocytes.

Exposure of fetal type II pneumocytes to phospholipase A2 inhibitors led to significantly reduced choline uptake and decreased synthesis of total and disaturated phosphatidylcholines from both [methyl-14C]choline and [9,10(n)-3H]palmitate precursors. The percentage of the total synthesized phosphatidylcholine recovered as disaturated phosphatidylcholine was increased when compared to that in control cultures, suggesting that unsaturated phosphatidylcholine synthesis was reduced to a greater extent than that of the disaturated species. Synthesis of sphingomyelin and phosphatidylethanolamine from labeled palmitate was also reduced, whereas that of phosphatidylinositol and phosphatidylglycerol was significantly increased. Addition of phospholipase C resulted in increased synthesis of phosphatidylcholine from both labeled precursors; no significant changes were found in synthesis of most of the other 3H-labeled lipids. Added phospholipase A2 did not lead to any changes in either choline or palmitate incorporation. However, when melittin (a phospholipase A2 activator) was added to the cultures, greater incorporation of both palmitate and choline was observed, along with a significant increase in the percentage of total cellular radioactivity in 14C-labeled lipids, indicating also stimulation of phosphatidylcholine synthesis. A marked increase in CTP: phosphorylcholine cytidylyltransferase activity was found after treatment of the cultures with phospholipase C. Exposure to quinacrine also increased the activity of this enzyme. Addition of phospholipase C and melittin to prelabeled pneumocyte cultures accelerated degradation of cell phospholipids and the release of free fatty acids as the main degradation products. These findings suggest that intracellular phospholipases are regulators of synthesis of surfactant phospholipids in fetal type II pneumocytes, and that activation or inhibition of these phospholipases could represent a mechanism through which hormones and pharmacological agents modify surfactant and other phospholipid synthesis.