Oxidative degradation of model lipids representative for main paper pulp lipophilic extractives by the laccase–mediator system

Different model lipids-alkanes, fatty alcohols, fatty acids, resin acids, free sterols, sterol esters, and triglycerides-were treated with Pycnoporus cinnabarinus laccase in the presence of 1-hydroxybenzotriazole as mediator, and the products were analyzed by gas chromatography. The laccase alone decreased the concentration of some unsaturated lipids. However, the most extensive lipid modification was obtained with the laccase-mediator system. Unsaturated lipids were largely oxidized and the dominant products detected were epoxy and hydroxy fatty acids from fatty acids and free and esterified 7-ketosterols and steroid ketones from sterols and sterol esters. The former compounds suggested unsaturated lipid attack via the corresponding hydroperoxides. The enzymatic reaction on sterol esters largely depended on the nature of the fatty acyl moiety, i.e., oxidation of saturated fatty acid esters started at the sterol moiety, whereas the initial attack of unsaturated fatty acid esters was produced on the fatty acid double bonds. In contrast, saturated lipids were not modified, although some of them decreased when the laccase-mediator reactions were carried out in the presence of unsaturated lipids suggesting participation of lipid peroxidation radicals. These results are discussed in the context of enzymatic control of pitch to explain the removal of lipid mixtures during laccase-mediator treatment of different pulp types.     

Unsaturated lipids protect the integral membrane peptide gramicidin A from singlet oxygen

In contrast to expectations that unsaturated fatty acids contribute to oxidative stress by providing a source of lipid peroxides, we demonstrated the protective effect of double bonds in lipids on oxidative damage to membrane proteins. Photodynamic inactivation of gramicidin channels was decreased in unsaturated lipid compared to saturated lipid bilayers. By estimating photosensitizer (boronated chlorine e₆ amide) binding to the membrane with the current relaxation technique, the decrease in gramicidin photoinactivation was attributed to singlet oxygen scavenging by double bonds in lipids rather than to the reduction in photosensitizer binding. Gramicidin protection by unsaturated lipids was also observed upon induction of oxidative stress with tert-butyl hydroperoxide.     

The reaction of unsaturated fats with the acid hematein test

Summary The reaction of saturated and unsaturated oils and fatty acids was examined in vitro with the Baker's acid hematein test. It has been found that oils whose molecules contain fatty acid components of two or more double bonds give a positive reaction with the acid hematein technique. The intensity of the reaction runs parallel with the number of double bonds.     

Effects of various numbers and positions of cis double bonds in the sn-2 acyl chain of phosphatidylethanolamine on the chain-melting temperature

In an attempt to investigate systematically the effects of various single and multiple cis carbon-carbon double bonds in the sn-2 acyl chains of natural phospholipids on membrane properties, we have de novo synthesized unsaturated C20 fatty acids comprised of single or multiple methylene-interrupted cis double bonds. Subsequently, 15 molecular species of phosphatidylethanolamine (PE) with sn-1 C20-saturated and sn-2 C20-unsaturated acyl chains were semi-synthesized by acylation of C20-lysophosphatidylcholine with unsaturated C20 fatty acids followed by phospholipase D-catalyzed base-exchange reaction in the presence of excess ethanolamine. The gel-to-liquid crystalline phase transitions of these 15 mixed-chain PE, in excess H2O, were investigated by high resolution differential scanning calorimetry. In addition, the energy-minimized structures of these sn-1 C20-saturated/sn-2 C20-unsaturated PE were simulated by molecular mechanics calculations. It is shown that the successive introduction of cis double bonds into the sn-2 acyl chain of C(20):C(20)PE can affect the gel-to-liquid crystalline phase transition temperature, Tm, of the lipid bilayer in some characteristic ways; moreover, the effect depends critically on the position of cis double bonds in the sn-2 acyl chain. Specifically, we have constructed a novel Tm diagram for the 15 species of unsaturated PE, from which the effects of the number and the position of cis double bonds on Tm can be examined simultaneously in a simple, direct, and unifying manner. Interestingly, the characteristic Tm profiles exhibited by different series of mixed-chain PE with increasing degree of unsaturation can be interpreted in terms of structural changes associated with acyl chain unsaturation.     

Modulation of chloroplast membrane lipids by homogeneous catalytic hydrogenation

A method is reported for the modification of lipids in situ in chloroplast membrane by which a homogeneous, water-soluble catalyst Pd(QS)2 (QS, sulphonated alizarine; C14H6O7NaS) is incorporated into the thylakoids of isolated chloroplast. The catalyst itself did not affect the photosynthetic activity but caused an extensive loss of unsaturated fatty acids in the presence of hydrogen gas. The polyunsaturated fatty acids were hydrogenated at a faster rate than the monoenoic acids. During hydrogenation the orientational ordering of membrane lipids, as measured with the C-12 positional isomer of spin-labelled stearic acid, displayed a slight increase in agreement with the alterations in membrane composition. Progressive saturation of double bonds of lipids primarily inhibits electron transport between the photosystems followed by the inhibition of electron flow around photosystem II. Photosystem I electron transport was not inhibited even by 50% fatty acid hydrogenation. We suggest that using Pd(QS)2 catalyst for thylakoid hydrogenation offers an excellent technique to study the role of various unsaturated fatty acids in the regulation of membrane fluidity and photosynthetic processes.     

A system for manipulating the membrane Fatty Acid composition of soybean cell cultures by adding tween-Fatty Acid esters to their growth medium : basic parameters and effects on cell growth

The development of a system for modifying the membrane fatty acid composition of cultured soybean cells (Glycine max [L.] Merr.) is described. Tween-fatty acid esters carrying specific fatty acids were synthesized and added to the medium of suspension cultures. Cells transferred large quantities of exogenous fatty acids from Tweens to all acylated membrane lipids; up to 50% of membrane fatty acids were exogenously derived. C15 to C20 saturated fatty acids and C16, C18, and C20 unsaturated fatty acids with either cis or trans double bonds were incorporated into lipids. Cells elongated saturated fatty acids of C16 or less, and unsaturated fatty acids with cis double bonds were further desaturated. No other types of modifications were observed. Growth ceased in cells treated with excessive concentrations of Tween-fatty acid esters, but frequently not for several days. Cessation of cell growth was correlated with changes in membrane fatty acid composition resulting from incorporation of large amounts of exogenous fatty acids into membrane lipids, although cells tolerated large variations in fatty acid composition. Maximum tolerable Tween concentrations varied widely according to the fatty acid supplied. Potential uses of this system and implications of the observed modifications on the pathway of incorporation are discussed.     

13C-NMR of double and triple bond carbon atoms of unsaturated fatty acid methyl esters

The carbon magnetic resonance spectra of many fatty acid methyl esters with cis and trans double bonds and triple bonds at various positions and in many different combinations have been investigated.The influence of the ester group on double and triple bonds in the fatty acid chain depends strongly on the positions of these bonds. For a given position the influence is constant, even if one or more other double or triple bonds are present.Together with the evaluated chemical shift parameters for the effects of double and triple bonds on each other, complete assignments are possible and spectra of various types of unsaturated esters can be predicted with high accuracy (±0.1 ppm).     

Spectrophotometric assay of long-chain unsaturated and hydroxy fatty acids in concentrated sulfuric acid

A spectrophotometric method has been developed for the determination of long-chain unsaturated and hydroxy fatty acids in concentrated sulfuric acid. The assay is based on the absorbance produced in the 290 to 300-nm range from their reaction with sulfuric acid at 100°C. α,β-Unsaturated aliphatic acids give absorption bands at 235–240 nm and thus can be easily differentiated from unsaturated fatty acids having the double bond(s) at positions not conjugated with the carboxyl group. A certain minimum chain length is required for full development of the absorption band at 300 nm. Position and intensity of the so-formed absorption band is independent on the position and number of the double bonds or hydroxyl groups. Carboxyl groups are not essential, as unsaturated hydrocarbons and higher alcohols likewise react with sulfuric acid to produce the absorbing species at 300 nm, providing a minimum chain length of 5 carbon atoms is present. The nature of the absorbing species at 300 nm is discussed.     

Purification and characterization of intracellular lipase from the polyunsaturated fatty acid-producing fungus Mortierella alliacea

Previous studies on an arachidonic acid-producing fungus, Mortierella alliacea YN-15, suggested that its intracellular lipase plays an important role in the metabolism of exogenous and storage lipids. The lipase purified in this study through acetone precipitation and three-step chromatography was estimated to be about 11 kDa in size by SDS-PAGE and mass spectrometry, and it tended to form large aggregates in aqueous solution. The purified lipase retained its activity over wide ranges of pH (2-12) and temperature (20-80 °C). Its activity was enhanced by the Ca(2+) ion and reduced by some heavy metal ions, such as Zn(2+) and Hg(2+), and diethylpyrocarbonate. Among the various substrates tested, monoacylglycerols containing long-chain unsaturated fatty acids and phosphatidylcholine were preferentially hydrolyzed over triacylglycerols and fatty acid methyl esters. The lipase strongly hydrolyzed the sn-1/3 ester bonds and weakly hydrolyzed the sn-2 ester bonds of triolein, and it also catalyzed the acylglycerol synthesis reaction in a solvent-free two-phase system. The results indicate that triacylglycerol may be formed via 2-monoacylglycerol. Thus, the highly stable M. alliacea lipase may be useful for the synthesis of structured lipids, particularly acylglycerols containing functional unsaturated fatty acids at the sn-2 position.     

Bromine-Sudan Black: a general stain for lipids including free cholesterol

Synopsis Preliminary bromination (with bromine water) increases the intensity of staining of tissue lipids with Sudan Black and certain other dyes. The mechanism appears to be due to the formation of sudanophilic bromo-derivatives of cholesterol and to the retention of certain other lipids, notably phosphatidyl choline and free fatty acids, during staining. The advantage of the bromine-Sudan Black method is that all tissue lipids are stained, except saturated fatty acids, saturated triglycerides and perhaps saturated cholesterol esters. In practice, such lipids rarely, if ever, occur alone, and normally are admixed with their stainable unsaturated counterparts.     

Homogeneous catalytic hydrogenation of lipids in the photosynthetic membrane: Effects on membrane structure and photosynthetic activity

We have carried out a series of experiments in which the lipid composition of the photosynthetic membrane has been altered by the homogeneous catalytic hydrogenation of the unsaturated fatty acid residues of membrane lipids. The modified membrane was investigated by electron microscopy, electron-spin resonance and fluorescence polarization methods. Alteration in the functional characteristics of the hydrogenated membrane was monitored by the measurement of photophosphorylation and electron-transport activities. The following results were found. (a) Saturation of 10% of the fatty acyl double bonds induced a definite decrease in the dimension of both thylakoids and loculi. Microdensitometry showed that these structural changes arose from a thickening of the single membranes with a simultaneous decrease in the spacing between membranes. These changes might be accounted for by the alignment of the hydrocarbon chains of saturated lipids and the increased hydrophobicity of the membranes. (b) The orientational pattern of chlorophyll-a molecules was not altered by saturating up to 50% of fatty acyl double bonds in membrane lipids, indicating that the energy-transfer processes amongst the chlorophyll molecules remained functional after hydrogenation. (c) Saturation of double bonds of lipids inhibited whole electron transport prior to the inhibition of Photosystem II and Photosystem I activity, which may suggest that the unsaturation level of fatty acids plays a crucial role by ensuring the lateral mobility of plastoquinone between Photosystem II and Photosystem I.     

Effects of the catalytic hydrogenation of microsomal lipids upon four enzyme activities involved in oleic acid desaturation

Catalytical hydrogenation of the unsaturated fatty acyl residues of microsomal lipids was realized for different times. Progress of the reaction was followed by calculating the progressive loss of double-bonds in 100 initial acyl residues (percentage of hydrogenation). The maximum loss observed was 45% after 60 min.The drop in polyunsaturated faty acid content was coupled with an increase in the amount of stearic acid and oleic acid.The order parameter of microsomal lipids, measured by ESR, increased parallely to the reduction of double bonds. Maximum hydrogenation of microsomal lipids strongly (200–250%) stimulated microsomal NADH-ferricyanide reductase activity. NADH-cytochrome c reductase, lysophosphatidylcholine-acyl-transferase and oleoyl-phosphatidylcholine desaturase were inhibited (40%, 100% and 100% respectively). These modifications of enzyme activities are discussed in conjunction with the changes observed in membrane fluidity, following hydrogenation of microsomal lipids     

The nature of mycobacterial acid-fastness.

Phenol is not essential to acid-fast staining, for it will occur in the absence of phenol where such lipoid-soluble basic dyes as night blue, Victoria blue B or Victoria R are used; it is essential for acid-fast staining with water soluble basic dyes such as basic fuchsin. When phenol is added to the staining solution, such water soluble basic dyes behave in effect like their lipid-soluble counterparts. The loss of mycobacterial acid-fastness with carbol-fuchsin after bromination or chromation indicates that this phenomenon is related to the presence of unsaturated lipids in the bacterial cells. Within the cells these acid-fast lipids are bound in such a way that they are easily removed from all mycobacteria by hot dilute HCl; from leprosy bacilli alone they are easily removed with hot pyridine. From the results of various blocking reactions it appears that carboxyl and especially hydroxyl groups of these cellular lipids are essential to the acid-fast reaction of mycobacteria.     

The Nature of Mycobacterial Acid-Fastness

Phenol is not essential to acid-fast staining, for it will occur in the absence of phenol where such lipoid-soluble basic dyes as night blue, Victoria blue B or Victoria R are used; it is essential for acid-fast staining with water soluble basic dyes such as basic fuchsin. When phenol is added to the staining solution, such water soluble basic dyes behave in effect like their lipid-soluble counterparts. The loss of mycobacterial acid-fastness with carbolfuchsin after bromination or chromation indicates that this phenomenon is related to the presence of unsaturated lipids in the bacterial cells. Within the cells these acid-fast lipids are bound in such a way that they are easily removed from all mycobacteria by hot dilute HCl; from leprosy bacilli alone they are easily removed with hot pyridine. From the results of various blocking reactions it appears that carboxyl and especially hydroxyl groups of these cellular lipids are essential to the acid-fast reaction of mycobacteria.     

Modulation of lipid-induced ER stress by fatty acid shape

Exposure of pancreatic β cells to long-chain saturated fatty acids (SFA) induces a so-called endoplasmic reticulum (ER) stress that can ultimately lead to cell death. This process is believed to participate in insulin deficiency associated with type 2 diabetes, via a decrease in β-cell mass. By contrast, some unsaturated fatty acid species appear less toxic to the cells and can even alleviate SFA-induced ER stress. In the present study, we took advantage of a simple yeast-based model, which brings together most of the trademarks of lipotoxicity in human cells, to screen fatty acids of various structures for their capacity to counter ER stress. Here we demonstrate that the tendency of a free fatty acid (FFA) to reduce SFA toxicity depends on a complex conjunction of parameters, including chain length, level of unsaturation, position of the double bonds and nature of the isomers (cis or trans). Interestingly, potent FFA act as building blocks for phospholipid synthesis and help to restore an optimal membrane organization, compatible with ER function and normal protein trafficking.     

Studies to determine the role rates of chain elongation and desaturation play in regulating the unsaturated fatty acid composition of rat liver lipids.

Rat liver microsomes were used to measure the rates of chain elongation and desaturation of acids in the linoleate, oleate and palmitoleate biosynthetic pathways. These studies were designed to determine whether there is a relationship between rates of conversion and the types of unsaturated fatty acids found in rat liver lipids. In some cases rates of conversion correlate well with the types of unsaturated fatty acid found inrat liver lipids. In other cases, rates of conversion must be correlated with other controls such as competitive interactions, retroconversion, and specificities for incorporating given acids into lipids in order to explain the unsaturated fatty acid composition of rat liver lipids. The roles and interrelationships of these various metabolic processes are discussed relative to the control of polyunsaturated fatty acid biosynthesis.     

Photogenerated reagents for membrane labeling. 2. Phenylcarbene and adamantylidene formed within the lipid bilayer

Phenylcarbene and adamantylidene have been generated photochemically from the corresponding diazirines within lipid bilayers. Reasonable yields of labeled fatty acid side chains have been observed. The products have been characterized by gas chromatography-mass spectrometry and derive both from the insertion of the carbene into carbon-hydrogen bonds of saturated fatty acids and from the addition of the carbene to the carbon-carbon double bonds of unsaturated fatty acids. In contrast to the results found using phenylnitrene, the lipid labeling by carbene is not reduced by the water-soluble scavenger glutath ione. Carbenes generated from diazirines are evidently superior reagents for the photolabeling of lipids and should be useful for identifying the intrinsic hydrophobic sections of membrane proteins.     

Targeted mutagenesis of acyl-lipid desaturases in Synechocystis: evidence for the important roles of polyunsaturated membrane lipids in growth, respiration and photosynthesis.

Acyl-lipid desaturases introduce double bonds (unsaturated bonds) at specifically defined positions in fatty acids that are esterified to the glycerol backbone of membrane glycerolipids. The desA, desB and desD genes of Synechocystis sp. PCC 6803 encode acyl-lipid desaturases that introduce double bonds at the delta12, omega3 and delta6 positions of C18 fatty acids respectively. The mutation of each of these genes by insertion of an antibiotic resistance gene cartridge completely eliminated the corresponding desaturation reaction. This system allowed us to manipulate the number of unsaturated bonds in membrane glycerolipids in this organism in a step-wise manner. Comparisons of the variously mutated cells revealed that the replacement of all polyunsaturated fatty acids by a monounsaturated fatty acid suppressed growth of the cells at low temperature and, moreover, it decreased the tolerance of the cells to photoinhibition of photosynthesis at low temperature by suppressing recovery of the photosystem II protein complex from photoinhibitory damage. However, the replacement of tri- and tetraunsaturated fatty acids by a diunsaturated fatty acid did not have such effects. These findings indicate that polyunsaturated fatty acids are important in protecting the photosynthetic machinery from photoinhibition at low temperatures.     

The Effect of Formaldehyde on Tissue Lipids and on Histochemical Reactions for Carbonyl Groups

Histcchemical and chemical evidence indicates that formaldehyde combines with unsaturated lipids at the double bond. The resulting complex contains a free carbonyl group which probably originates from the formaldehyde. The reaction occurs over a wide pH range, and takes place in the absence of oxygen or moisture. The reaction product is visualized by the Schiff reagent, and by the Ashbel-Seligman procedure. In the plasmal procedure, when performed on formalin-treated material, the reaction has the same significance as the pseudo-plasmal reaction, i.e. it denotes the presence of double bonds. The Ashbel-Seligman technic seems to be more sensitive to this complex than the Schiff reagent and shows it more markedly than it does the true plasmals and the atmospherically oxidized unsaturated compounds.     

Superoxide dismutase deficiency and the toxicity of the products of autooxidation of polyunsaturated fatty acids in yeast

Mutants of Saccharomyces cerevisiae, deficient in cytosolic superoxide dismutase and catalase activities were used to study the role of various oxygen species in the process of lipid peroxidation in yeast cells. Lipid peroxidation does not occur normally in yeast, because this organism is unable to form fatty acids with more than one double bond, whereas under physiological conditions, only fatty acids with at least two double bonds undergo this process. The fatty acid content of cellular lipids was modified by growing the cells in anoxia in the presence of oleic or linolenic acid. Toxic effects of oxygen were observed almost exclusively in those cells of yeast mutants deficient in superoxide dismutase, which contain linolenic acid in cellular lipids. Hypersensitivity of the mutant cells, however, results mainly from toxic effects of the products of autooxidation of extracellular fatty acids. These facts suggest that superoxide dismutases are in some way involved in preventing toxic effects of the products of lipid peroxidation and to some extent prevent the process of lipid peroxidation.