Effects of fatty acids on superoxide radical generation in leukocytes

Acetylated ferricytochrome c was employed for the detection of superoxide radicals (O₂^?) generated both in intact cells and in subcellular fractions of leukocytes. Certain saturated fatty acids, myristate in particular, induced the production of O₂^? in leukocytes, suggesting a correlation between the formation of O₂^? and the hydrophobic interaction of fatty acids with the leukocyte plasma membrane. As compared with O₂^? radical generation from phagocytizing leukocytes, a greater stimulation of O_2? formation was observed in cells in which myristate was added. The enhanced activity which generated O₂^? in the cell-free system was located in a particulate fraction but not in the cytosol. The rate of O₂^? generation in the particulate fraction was higher in the presence of NADPH than in the presence of NADH. The effects of reagents such as KCN, 2,4-dichlorophenol and aminotriazole on the O₂^? generation in this fraction are examined and the nature of the O₂^? generating system is discussed.     

Studies on the biochemical basis of oxygen toxicity

The toxic effects of high pressure oxygen on the isolated toad urinary bladder have been studied. Sodium transport in this system is reversibly inhibited by high pressure O₂. This inhibition is potentiated by adrenal steroid hormones, and occurs despite both increased glycolytic and Kreb's cycle flux and tissue ATP content. High pressure O₂ leads to increased pyruvate/lactate and pyruvate/malate redox couples, as well as to a decrease in the weight percentage of phospholipid long-chain unsaturated fatty acids and [2-¹⁴C]pyruvate incorporation into tissue lipid. During recovery from high pressure O₂ treatment, [2-¹⁴C]Pyruvate incorporation into lipid is increased and the weight percentage of long-chain unsaturated fatty acids increases. These data indicate that high pressure O₂ poisoning in this tissue does not result from an inhibition of carbohydrate metabolism, but may result from the formation of toxic lipid peroxides.     

On the chemical structure of the apical droplet from eggs of Culex pipiens

The chemical nature of the apical droplet from eggs of Culex pipiens was investigated by chromatographic techniques. Results indicated that the hydrolysate of the apical drop contains C-12, C-14, C-16, and C-18 straightchain aliphatic fatty acids. A C-12β-hydroxy fatty acid was also found, but the largest component of the fatty acid mixture of the apical drop was shown to be a C-14β-OH fatty acid. Two other fractions appear to be unsaturated fatty acids, probably C-12 and C-14. Quantitative estimation of the percentage of each fatty acid in the mixture showed that about 85 per cent of the fatty acid content of the apical drop consisted of hydroxy fatty acids. By thin-layer chromatography, the largest component coincided with β-OH myristic acid.Glycerol was confirmed to be present in the hydrolysate. Feeding studies with radioactive ³²PO₄^?3 and ³⁵SO₄^?2 showed no significant incorporation of phosphorus, but a sulphur-containing anionic compound could be detected in the apical drop. Infrared analysis showed the presence of an ester group, double bond, primary and secondary alcohol groups, suggesting the presence of hydroxy-, unsaturated-, saturated straight-chain fatty acids, as well as mono-and diglycerides. The structural evidence explains in part the surfactant properties of the apical drop.     

Formation of superoxide anion and carbon-centered radicals by photosystem II under high light and heat stress—EPR spin-trapping study

In this study, electron paramagnetic resonance spin-trapping spectroscopy was used to study the light-induced production of superoxide anion (O₂ ^?-) and carbon-centered (R^?) radicals by Photosystem II (PSII). It is evidenced here that exposure of PSII membranes to high light (2,000 μmol photons m^?2 s^?1) or heat (47 °C) treatments prior to the illumination suppressed O₂ ^?- production, while R^? was formed. Formation of R^? in the both high light- and heat-treated PSII membranes was enhanced by DCMU. Removal of molecular oxygen by glucose/glucose oxidase/catalase system and O₂ ^?- scavenging by exogenous superoxide dismutase completely suppressed carbon-centered radical formation. It is proposed here that the oxidation of polyunsaturated fatty acids and amino acids by O₂ ^?- on the electron acceptor side of PSII results in the formation of R^?, known to initiate a cascade reaction leading to the lipid peroxidation and protein degradation, respectively.     

Perhydroxyl Radical (HO<Subscript>2</Subscript><Superscript>•</Superscript>) as Inducer of the Isoprostane Lipid Peroxidation in Mitochondria

The nonenzymatic isoprostane pathway of lipid peroxidation of polyunsaturated fatty acids results in formation of products, termed isoprostanes, which have very large positional and stereo isomerism, possess various biological activities, produce adducts with proteins, and thus contribute to pathogeneses of the agedependent diseases. However, it was unclear what mechanism drives this type of lipid autoxidation, and why the products have very large isomerism. We propose a mechanism when perhydroxyl radicals (HO₂^?) react with polyunsaturated fatty acids in the hydrophobic milieu of membranes. In the membrane HO₂^? initiates a chain of reactions with formation first H₂O₂, which undergoes homolytic fission producing two ^?OH radicals, thus very rapidly abstracting three H atoms from a polyunsaturated fatty acid. As a result, the HO₂^? molecule is converted to two molecules of water, and the molecule of a polyunsaturated fatty acid loses two double bonds, becomes highly unstable and undergoes peroxidation and random intramolecular re-arrangements causing a very large isomerism of the final products. The extremely high reactivity of ^?HО₂ with polyunsaturated fatty acids is the cause of very subtle and slow accumulation of damages in the membrane and membrane associated proteins, even though the concentration of ^?HО₂ relative to superoxide radical may be very low.     

Fat metabolism in higher plants. LXII. The pathway of ricinoleic acid catabolism in the germinating castor bean (Ricinus communis L.) and pea (Pisum sativum L.)

With cell-free extracts from both germinating peas and castor beans, O-¹⁴Cricinoleate (¹⁴C located at odd-numbered positions in the carbon chain) was β-oxidized at least to the C₁₀ level. With the pea system, formation of unsaturated hydroxy acid intermediates to the C₁₂ level occurred. Acetyl-CoA was the primary product of β-oxidation activity. Although the pathway beyond the C₁₂-intermediate level was not resolved conclusively, two alternative routes may exist in the castor bean system to convert 4-hydroxy-decanoic acid to 2-keto-octanoate, one involving 4-keto-decanoate, the other 2-hydroxy-octanoate. Subsequent degradation of the 2-keto-octanoate tentatively involves an α-oxidation step, releasing CO₂ and heptanoic acid. Further β-oxidation of the latter is envisaged to yield propionyl and acetyl CoA. All necessary enzymes for the catabolism of ricinoleic acid to propionate appear to be associated with the cytosomes.     

Cis-unsaturated fatty acids modulate the function of the cell-surface cAMP receptor of Dictyostelium discoideum

The binding of cAMP to the chemotactic cAMP receptor in intact Dictyostelium discoideum cells and isolated membranes is strongly inhibited by unsaturated fatty acids. In isolated membranes, cis-unsaturated fatty acids decreased the number of accessible cAMP binding sites, without significantly altering their affinity. Most potent were C₁₈ and C₂₀ cis-poly unsaturated fatty acids, like arachidonic acid, linoleic acid and linolenic acid. Trans-unsaturated fatty acid was less potent than its cis isomer, while saturated fatty acids did not affect the binding of cAMP to receptors at all. Oxidation reactions were not important for the effect of unsaturated fatty acids. When membranes were preincubated with millimolar concentrations of Ca²⁺, the effect of unsaturated fatty acids was strongly diminished. Mg²⁺ was ineffective. Ca²⁺, if presented after the incubation of membranes with unsaturated fatty acids, did not reverse the inhibitory effect. The specificity of the fatty acid effect, and the interference with Ca²⁺, but not Mg²⁺, suggest that the properties of the cAMP receptor are changed as a result of alterations in the lipid bilayer structure of the membrane.     

Extracellular secretion of free fatty acids by the chrysophyte Ochromonas danica under photoautotrophic and mixotrophic growth

Recently, microalgae have gained a lot of attention because of their ability to produce fatty acids in their surrounding environments. The present paper describes the influence of organic carbon on the different fatty acid pools including esterified fatty acids, intracellular free fatty acids and extracellular free fatty acids in Ochromonas danica. It also throws light on the ability of O. danica to secrete free fatty acids in the growth medium under photoautotrophic and mixotrophic conditions. Biomass production of photoautotrophically grown O. danica was higher than that of mixotrophically grown, where a cellular biomass formation of 1.8 g L^?1 was observed under photoautotrophic condition which was about five folds higher than that under mixotrophic conditions. Contrary, the esterified fatty acid content reached up to 99 mg g^?1 CDW under photoautotrophic conditions at the late exponential phase, while during mixotrophic conditions a maximum of 212 mg g^?1 CDW was observed at the stationary phase. Furthermore, O. danica cells grown under mixotrophic conditions showed higher intracellular free fatty acid and extracellular free fatty acid contents (up to 51 and 20 mg g^?1 CDW, respectively) than cells grown under photoautotrophic conditions (up to 26 and 4 mg g^?1 CDW, respectively). The intra- and extracellular free fatty acids consisted of a high proportion of polyunsaturated fatty acids, mainly C18:2n?6, C18:3n?3 and C20:4n?6.     

Decomposition of unsaturated phospholipid by iron-ADP-adriamycin co-ordination complex

The system, which contains NADPH, purified cytochrome P-450 reductase, and adriamycin, produces H₂O₂ and $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ in appreciable amounts with oxygen consumption and NADPH oxidation under aerobic conditions. Such an adriamycin-induced NADPH oxidation system, however, does not cause the decomposition of unsaturated fatty acids in microsomal phospholipid micelles, suggesting no direct participation of the active oxygen species and semiquinone radicals of adriamycin in lipid peroxidation. Adriamycin produces a co-ordination complex with Fe³⁺ and ADP, which, but no Fe³⁺-ADP complex, could be reduced by NADPH-cytochrome P-450 reductase at the expence of NADPH. The decomposition of unsaturated fatty acids in phospholipid micelles is achieved by the Fe³⁺-ADP-adriamycin complex and strikingly enhanced by enzymatically reduced iron-ADP-adriamycin complex.     

The effect of low temperatures on fatty acid biosynthesis in plants

1. Of three systems, bulb tissue, plant leaf tissue and intact green algal (Chlorella vulgaris) cells, only the former shows an increase in rate of formation of unsaturated fatty acids with decrease in temperature. 2. In bulb tissue the oxygen concentration is rate-limiting for synthesis of unsaturated fatty acids at temperatures down to 10°. 3. At elevated oxygen concentrations the formation of unsaturated fatty acids in bulb tissue increases with temperature. 4. The failure of photosynthetic tissues to respond to either lower temperatures or increased oxygen concentrations in the presence of light is attributed to photosynthetic production of excess of oxygen. This is supported by the fact that in the dark a potentiating oxygen effect on the formation of unsaturated fatty acids can be demonstrated. 5. The HCO₃⁻ ion concentration has a small effect on the formation of unsaturated fatty acids. 6. Elevated content of unsaturated acids at lower temperatures in plants is attributed to increases in oxygen concentration in solution.     

Molybdenum-induced alteration of fatty acids of thylakoid membranes contributed to low temperature tolerance in wheat

The fact that molybdenum (Mo) applications can alleviate low temperature stress (LTS) in plants has been widely reported, but the underlying mechanisms are not fully understood. The effects of Mo (0 and 0.15 mg kg^?1) on photosynthetic pigments, fatty acids and the chlorophyll–protein complex of wheat seedlings in pot culture were investigated at 0, 2, 4 and 6 days of LTS. Chlorophyll a and b, total chlorophyll and carotenoid contents were significantly enhanced by Mo application. The palmitic acid (C16:0) content and total saturated fatty acids (TSFA) were drastically decreased in wheat cultivar 97003 at 2 and 4 days of LTS and in wheat cultivar 97014 at 2 days of LTS. The linolenic acid (C18:3) content and total unsaturated fatty acids (TUSFA) were significantly increased in both cultivars at 2 and 4 days of LTS. The palmitoleic acid (C16:1) content was also drastically increased in cultivar 97003 at 2 days of LTS, suggesting that Mo induced a greater production of unsaturated fatty acids or the conversion of TSFA to TUSFA. The ratio of unsaturated to saturated fatty acids and the index of unsaturated fatty acids in the thylakoid membranes were enhanced with supplemental Mo, suggesting that Mo might improve the degree of unsaturation. However, no significant differences were observed in the chlorophyll–protein complexes between the +Mo and ?Mo treatments. These results indicated that the alteration of fatty acids induced by Mo application in the thylakoid membranes of wheat contributed to LTS tolerance.     

Synthesis of Long-Chain Acyl-CoA in Chloroplast Envelope Membranes

The chloroplast envelope is the site of a very active long-chain acylcoenzyme A (CoA) synthetase. Furthermore, we have recently shown that an acyl CoA thioesterase is also associated with envelope membrane (Joyard J, PK Stumpf 1980 Plant Physiol 65: 1039-1043). To clarify the interacting roles of both the acyl-CoA thioesterase and the acyl-CoA synthetase, the formation of acyl-CoA in envelope membranes was examined with different techniques which permitted the measurement of the actual rates of acyl-CoA formation. Using [¹⁴C]ATP or [¹⁴C]oleic acid as labeled substrates, it can be shown that the envelope acyl-CoA synthetase required both Mg²⁺ and dithiothreitol. Triton X-100 slightly stimulated the activity. The specificity of the acyl-CoA synthetase was determined either with [¹⁴C]ATP or with [³H]CoA as substrates. The results obtained in both cases were similar, that is, as substrates, the unsaturated fatty acids were more effective than saturated fatty acids, the velocity of the reaction increased from lauric acid to palmitic acid, and the maximum velocity was obtained with unsaturated C₁₈ fatty acids.     

Free Radical Scavenging by Myocardial Fatty Acid Binding Protein

Recent investigations have indicated the presence of a fatty acid binding protein (FABP) in mammalian heart. This protein binds free fatty acids and their esters with high affinity, however, its physiological role remains unknown. Since FABP constitutes a significant amount of cystolic protein, it is likely that it would be a target for free radical attack. To test this hypothesis, FABP was examined for scavenging against free radicals such as the superoxide anion (O^?₂,). hydroxyl radical (OH') and hypochlorite radical (OCl') which may be present in an ischemic reperfused heart. Our results suggest that FABP scavenges O^?₂, OH' and OCl' as indicated by the FABP inhibition of O^?₂-dependent reduction of cytochrome c, OH'-dependent hydroxybenzoic acid formation and OCl'-mediated chemiluminescence response. FABP was found to be a more potent scavenger of these free radicals compared to bovine serum albumin. Furthermore, FABP was more effective in scavenging OH' than O^?₂, and inhibited OH' mediated lipid peroxidation process. These results indicate that FABP can scavenge free radicals which may be present in an ischemic/reperfused heart and, thus, may play a significant physiological role in the heart during ischemia and reperfusion.     

Reactivities of highly unsaturated fatty acids for the hydrogenation on nickel catalyst

The reactivities of the C₂₀ highly unsaturated fatty acids (20:5, 20:4, 20:3 etc.…) during heterogeneous hydrogenation on nickel catalyst have been studied using a computer program. Three parameters are required to determine the hydrogenation rate constant of the different fatty acid classes; the C₂₀ fatty acid composition of the starting oil, the C₂₀ fatty acid composition of a partially hydrogenated oil (PHO) and the time of reaction. It is shown that, in order to minimize the experimental errors, the PHO must be selected in such a way that the induction period is over and that this oil still contains appreciable amounts of 20:5 and 20:4.Very little difference was found for the reactivities of the 20:5, 20:4 and 20:3 acids. The major difference among unsaturated fatty acids was found to be between the 20:2 and 20:1 isomers for a hydrogenation effected according to common commercial practices.The computer program is a general one also useful for the prediction of the fatty acid composition of slightly hydrogenated oils, but is not suitable for oils hydrogenated to very low iodine values, or for those containing high proportions of trans fatty acids.     

Photosynthetic studies with mesophyll protoplasts from Notonia grandiflora, a crassulacean acid metabolism plant

A method is described for rapid enzymatic isolation of mesophyll protoplasts and cells from the crassulacean acid metabolism (CAM) plant Notonia grandiflora DC. The mesophyll protoplasts exhibited high rates of ¹⁴CO₂ fixation both in the light (45 μmol of CO₂ fixed mg^?1 Chl h^?1) and in the dark (20 μmol of CO₂ fixed mg^?1 Chl h^?1). The protoplasts also showed O₂ evolution (40 μmol of O₂ evolved mg^?1 Chl h^?1) without added bicarbonate. Exogenously added bicarbonate had no stimulating effect on the O₂ evolution. Analyses of early photosynthetic products in the light showed the formation of both C₃ and C₄ acids. Aspartate was found to be a predominant photosynthate.     

Metabolism of endogenous lipid by the perfused rat kidney

It has been demonstrated that in vivo, exogenous [¹⁴C] palmitate is rapidly taken up and incorporated into phospholipid, neutral lipid and free fatty acid fractions of the kidney. During subsequent perfusion in an in vitro system, the amount of isotope decreases most rapidly in the neutral lipid (triglyceride) fraction. Net loss of chemical fatty acids cannot be detected after 2 hr. perfusion. The primary source of ¹⁴CO₂ produced appears to be fatty acids from either neutral lipid or phospholipids. Since loss of ¹⁴C from neutral lipids is independent of O₂ and substrates, regulation of fatty acid oxidation must be beyond triglyceride lipase.     

Regulation of the synthesis of unsaturated fatty acids by growth temperature in Bacillus subtilis

Bacillus subtilis synthesizes, almost exclusively, saturated fatty acids, when grown at 37° C. When cultures were transferred from 37° C to 20° C, a chloramphenicol- and rifampicin-sensitive synthesis of a C-16 unsaturated fatty acid was observed. Synthesis of this compound reached a plateau after 5 h at 20° C, reaching levels of 20% of the total fatty acid content. [¹⁴C]-labelled fatty acids attached as thioesters to acyl-carriers compounds, such as coenzyme A (CoA) or acyl-carrier protein (ACP) synthesized de novo by glycerol-requiring auxotrophs deprived of glycerol to arrest phospholipid synthesis, could not be desaturated at 20° C. Desaturation of these fatty acids was readily observed when glycerol was restored to the cultures allowing resumption of transfer of acyl-moieties from acyl-thioesters to phospholipid. It was also observed that depletion of the pools of CoA and ACP by starvation of pantothenate auxotrophs had no effect on the observed synthesis of unsaturated fatty acid at 20° C. The overall results indicate that synthesis of unsaturated fatty acids in B. subtilis is a cold-inducible process and that phospholipids are obligate intermediates in this fatty acid desaturation pathway.     

Fatty acids of rice coleoptiles in air and anoxia

The metabolism of lipids, like that of other components, was adversely and strongly affected when rice (Oryza sativa L.) coleoptiles were grown anaerobically. In aerobic coleoptiles, the amounts of total fatty acid, phospholipid, and total lipid per coleoptile increased by 2.5- to 3-fold between days three and seven, whereas under anoxia, the increases were all less than 60%. The total amount of lipid at day seven in anoxia was less than 30% of that in air. In air, the total fatty acid content at day three was 25 nanomoles per coleoptile and this increased to over 71 nanomoles per coleoptile at day seven. All acids except 18:0 showed substantial increases. In anoxia, the corresponding values for total fatty acids were 24 nanomoles and 27 nanomoles. The small increases were confined to the saturated fatty acids; no significant increase occurred in unsaturated fatty acids. A minor fatty acid constituent (16:1) increased from 0.09 to 1.99 nanomoles per coleoptile between days three and seven in air. This component was never observed in any fatty acid preparation from anaerobic coleoptiles. The major phospholipids under all conditions were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid. A small amount of unidentified phosphoester, not present on thin layer chromatography plates from aerobic coleoptiles, was seen in extracts of anaerobic coleoptiles. The fatty acyl substituents of each of the phospholipids were analyzed at days three and seven in coleoptiles grown aerobically and in anoxia. Each phospholipid had its own distinctive fatty acid composition which remained fairly constant under all treatments; 16:0 and 18:2 were the most abundant fatty acids in every phospholipid class. In air, the percentages of total fatty acids that were in the phospholipids were 86% on day three and 87% on day seven. In anoxia, the values at the corresponding ages were 47 and 57%. Since no net synthesis of unsaturated fatty acids occurred in anaerobic conditions, the small increase in total unsaturated acids in the phospholipids between days three and seven must have occurred at the expense of fatty acids preexisting in the neutral lipid. No unusual pathways of biosynthesis or unusual precursors are required to explain the presence of unsaturated fatty acids in the rice coleoptile. The present study and results of experiments where coleoptiles were fed [¹⁴C]acetate (BB Vartapetian et al. 1978 Plant Sci Lett 13:321-328) clearly show that unsaturated fatty acid synthesis in rice coleoptiles requires O₂, as it does in other plants.     

Lipoxygenase-mediated production of superoxide anion in senescing plant tissue

Lipoxygenase activity and superoxide (O^.?₂) production by microsomal membranes and cytosol from bean cotyledons increased in parallel as senescence progressed. Superoxide production was heat denaturable and dependent on the availability of linoleate, the substrate for lipoxygenase. The specific inhibitor of lipoxygenase, U28938, caused a parallel reduction in enzyme activity and the formation of O^?₂. These observations demonstrate that lipoxygenase activity mediates the formation of superoxide anion, and support the contention that membrane senescence is attributable to a sequence of reactions in which lipasederived fatty acids are utilized by lipoxygenase to generate O^?₂ and hydroperoxides.     

Flg22‐triggered oxylipin production in Pyropia haitanensis

This study provides evidence that flg22, the most conserved 22‐amino acid peptide in the N‐terminal part of bacterial flagellin can trigger the defense responses of Pyropia haitanensis (Bangiales, Rhodophyta). The defense responses are a chain of events including release of H₂O₂ and free unsaturated fatty acids C20:4, consumption of C18:3, and the chemical or enzymatic oxidation of both C20 and C18 polyunsaturated fatty acids. Oxidized C20 and C18 fatty acids lead to the production of corresponding hydroperoxy and hydroxylated derivatives, such as 9‐hydroperoxy octadecadienoic acid, 8‐hydroperoxy eicosapentaenoic acid, and 8‐hydroxyl eicosapentaenoic acid, which could be further oxidatively metabolized to yield saturated aldehydes and ketone. Changes of three typical hormones jasmonate, methyl jasmonate, and salicylic acid were observed. Contrary to the increase of jasmonate and methyl jasmonate, salicylic acid was decreased. The expression of key enzymes of oxylipin pathway PhLOX and PhLOX2 were upregulated. However, some defense and antioxidant related genes including PhHsp 70, Phsod , and PhRboh were downregulated markedly at the early stage of flg22 challenge. Overall, our results imply that red algae have evolved a similar defense response and may share the conservative‐recognizing receptor for flg22 as in higher plants.