Interactions of saturated, n-6 and n-3 polyunsaturated fatty acids to modulate arachidonic acid metabolism

Anti-thrombotic effects of omega-3 (n-3) fatty acids are believed to be due to their ability to reduce arachidonic acid levels. Therefore, weanling rats were fed n-3 acids in the form of linseed oil (18:3n-3) or fish oil (containing 20:5n-3 and 22:6n-3) in diets containing high levels of either saturated fatty acids (hydrogenated beef tallow) or high levels of linoleic acid (safflower oil) for 4 weeks. The effect of diet on the rate-limiting enzyme of arachidonic acid biosynthesis (delta 6-desaturase) and on the lipid composition of hepatic microsomal membrane was determined. Both linseed oil- or fish oil-containing diets inhibited conversion of linoleic acid to gamma-linolenic acid. Inhibition was greater with fish oil than with linseed oil, only when fed with saturated fat. delta 6-Desaturase activity was not affected when n-3 fatty acids were fed with high levels of n-6 fatty acids. Arachidonic acid content of serum lipids and hepatic microsomal phospholipids was lower when n-3 fatty acids were fed in combination with beef tallow but not when fed with safflower oil. Similarly, n-3 fatty acids (18:3n-3, 20:5n-3, 22:5n-3, and 22:6n-3) accumulated to a greater extent when n-3 fatty acids were fed with beef tallow than with safflower oil. These observations indicate that the efficacy of n-3 fatty acids in reducing arachidonic acid level is dependent on the linoleic acid to saturated fatty acid ratio of the diet consumed.     

An assessment of the ability of yeast cells to incorporate photolabile fatty acids into their membrane phospholipids in vivo

The photolabile fatty acids 12-azidooleic, 12-(4-azido-2-nitrophenoxy)oleic, 12-azidolauric and 12-(4-azido-2-nitrophenoxy)lauric are readily taken up in vivo by an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae. A low level of the two lauric acid derivatives and none of the two oleic acid derivatives are incorporated into membrane phospholipids. Under certain conditions of growth in the presence of 12-(4-azido-2-nitrophenoxy)oleic acid, the nitrophenylazide group is metabolized to a product that lacks the photolabile azido group.     

Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa

Intact human sperm incorporated radiolabelled fatty acids into membrane phospholipids when incubated in medium containing bovine serum albumin as a fatty acid carrier. The polyunsturated fatty acids were preferentially incorporated into the plasmalogen fraction of phospholipid. Uptake was linear with time over 2 hr; at this time sufficient label was available to determine the loss of fatty acids under conditions of spontaneous lipid peroxidation. Loss of the various phospholipid types, the loss of the various fatty acids from these phospholipids, and the overall loss of fatty acids were all first order. The loss of saturated fatty acids was slow with first order rate constant k₁ = 0.003 hr^?1; for the polyunsaturated fatty acids, arachidonic and docosahexaenoic acids, k₁ = 0.145 and 0.162 hr^?1, respectively. The rate of loss of fatty acids from the various phospholipid types was dependent on the type, with loss from phosphatidylethanolamine being the most rapid. Among the phospholipid types, phosphatidylethanolamine was lost at the greatest rate. Analysis of fatty acid loss through oxidation products was determined for radiolabelled arachidonic acid. Under conditions of spontaneous lipid peroxidation at 37°C under air in the absence of albumin, free arachidonic acid was found in the medium, along with minor amounts of hydroxylated derivative. All the hydroperoxy fatty acid remained in the cells. In the presence of albumin, all the hydroperoxy fatty acid was found in the supernatant bound to albumin; none could be detected in the cells. Albumin is known as a very potent inhibitor of lipid peroxidation in sperm; its action may be explained, based on these results, as binding the damaging hydroperoxy fatty acids. These results also indicate that a phospholipase A₂ may act in peroxidative defense by excising a hydroperoxy acyl group from phospholipid and providing the hydroperoxy fatty acid product as substrate to glutathione peroxidase. This formulation targets hydroperoxy fatty acid as a key intermediate in peroxidative degradation. © 1995 wiley-Liss, Inc.     

Human fetal and adult spleen phospholipids and their fatty acid composition

Total phospholipid contents and the individual phospholipid components of human adult and fetal spleens from 17--18 and 23--24 week's pregnancies composition of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, diphosphatidylglycerol and phosphatidic acid studied in human adult, 17--18 and 23--24 week fetal spleens.     

Transformation of an arachidonic acid hydroperoxide into epoxyhydroxy and trihydroxy fatty acids by liver microsomal cytochrome P-450

In the absence of NADPH, the addition of an arachidonic acid hydroperoxide, 15-hydroperoxyeicosa-5,8,11,13-tetraenoic acid, to liver microsomes, prepared from phenobarbital-treated rats, resulted in the formation of two major metabolites and several minor products, some of which have been purified by reverse-phase high-performance liquid chromatography. We propose the structures of the two major products to be 13-hydroxy-14,15-epoxyeicosa-5,8,11-trienoic acid and 11,14,15-trihydroxyeicosa-5,8,12-trienoic acid based on spectral characteristics and mass spectral analysis of derivatives of the compounds. A potential heterolytic cleavage product, 15-hydroxyeicosa-5,8,11,13-tetraenoic acid, was not a product of the reaction. Ferric cytochrome P-450 catalyzed the formation of these products as shown by the inability of boiled microsomes to support the reaction, the inhibition of epoxyhydroxy and trihydroxy fatty acid formation by imidazole derivatives which bind tightly to the ferric heme iron of cytochrome P-450, and the inability of carbon monoxide (which binds to ferrous P-450) and free iron chelators (EDTA and diethylenetriaminepentaacetic acid) to inhibit product formation. These results show that liver microsomal cytochrome P-450, in addition to its role in the NADPH-dependent metabolism of arachidonic acid, can utilize a hydroperoxide to produce an interesting series of potentially important arachidonic acid metabolites.     

Activation of D-beta-hydroxybutyrate apodehydrogenase using molecular species of mixed fatty acyl phospholipids

D-beta-Hydroxybutyrate apodehydrogenase is a lipid-requiring enzyme with a specific requirement of lecithin for enzymatic function. The purified enzyme which is devoid of lipid can be reactivated with lecithin or mixtures of natural phospholipid-containing lecithin. However, it is mitochondrial phospholipid which activates the enzyme optimally and with kinetic parameters similar to that of the native membrane-bound enzyme. Mitochondrial phospholipid consists of three classes of phospholipid (lecithin:phosphatidylethanolamine:diphosphatidylglycerol in a ratio of approximately 2:2:1 by phosphorus); each class consists of a multiplicity of different molecular species due to diversity in the fatty acyl substituents. In this study, we have synthesized defined molecular species of mixed fatty acyl phospholipids to evaluate whether multiplicity of phospholipid molecular species are essential for optimal reactivation. We find that: 1) ternary mixtures of single molecular species of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylpropan-1,3-diol in the liquid crystalline state mimic the optimal reactivation of the enzyme obtained with mitochondrial phospholipids; 2) although some negatively charged phospholipid appears necessary for optimizing the efficiency of activation, diphosphatidylglycerol can be replaced by phosphatidylpropan-1,3-diol, another negatively charged phospholipid; and 3) biphasic Arrhenius plots can be correlated with the liquid crystalline and gel states of the phospholipid.     

Incorporation of radioiodinated fatty acids into cardiac phospholipids of normoxic canine myocardium

The aim of this study was to assess the phospholipid distribution of radioiodinated 17-iodoheptadecanoic acid (IHDA), 15-(p-iodophenyl)pentadecanoic acid (p-IPPA) and 15-(p-iodophenyl)-3,3-dimethylpentadecanoic acid (DMIPPA) under normoxic conditions and to compare these data with the fatty acid composition of the phospholipid classes. After simultaneous i.v. injection of the radioiodinated fatty acids (1-123-IHDA; 1-131-p-IPPA; 1-125 DMIPPA) in open-chest dogs seven myocardial biopsies were taken over 40 min (n = 26). After lipid extraction of the biopsies the organic phase was analyzed for both neutral and polar lipids by two different TLC systems. The following polar lipid fractions were analyzed: lysophopshatidylcholine (LPC), sphingomyelin (SPH), phosphatidy1choline (PC; lecithin), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG; cardiolipin) and neutral lipids. Fractions were counted in a gamma well counter and corrected for cross-over and recovery. Results of the polar phospholipids analysis showed that IHDA has the highest incorporation into the phospholipids. The IHDA was mainly incorporated into PI (45.6%) followed by PC (30.9%), PE (14.0%) and PS (5.6%). The p-IPPA was predominantly incorporated incorporated into PC (37.2%), followed by PS (20.1%) and PE (13.7%). In contrast to IHDA, incorporation of p-IPPA into PI was small (6.4%). The DMIPPA analogue was incorporated into phopsholipids to only a very small degree, compared to IHDA and p-IPPA. PS (27.4%) was the only considerable phospholipid fraction into which DMIPPA was incorporated.The results clearly demonstrated that these radioiodinated fatty acid analogues have entirely different patterns of phospholipid incorporation. Major resemblances have been found between the incorporation into phospholipids of IHDA and the phospholipid distribution of the natural counterpart: stearic acid. The p—IPPA phospholipid incorporation only partly resembles the phospholipid distribution of palmitic acid. DMIPPA is because of its modified structure, incorporated into phospholipids to a low extent, mainly into PS. (Mol Cell Biochem116: 79–87, 1992)     

Cholesterol, phospholipids, and fatty acids of normal immature neutrophils: comparison with acute myeloblastic leukemia cells and normal neutrophils.

The lipid composition of immature myeloid cells from the bone marrow of normal persons and myeloblasts from patients with acute myeloblastic leukemia was studied and compared with the lipid composition of normal mature human neutrophils. Total cholesterol, phospholipid, and fatty acid composition was determined on each cell type. The leukemic cells showed decreased total cholesterol and cholesterol-to-phospholipid ratio, increase phosphatidylcholine and phosphatidylinositol, decreased phosphatidylethanolamine, and an increased percentage of unsaturated fatty acids when compared to normal mature neutrophils. A nearly identical pattern was seen in the normal immature myeloid precursors from normal bone marrow. We conclude that the altered lipid composition of acute myeloblastic leukemia cells is related to unexplained factors related to cell age and not to malignancy per se.     

Calcium dependency of arachidonic acid incorporation into cellular phospholipids of different cell types.

Ca2+ -independent phospholipase A2 (iPLA2) is involved in the incorporation of arachidonic acid (AA) into resting macrophages by the generation of the lysophospholipid acceptor. The role of iPLA2 in AA remodeling in different cells was evaluated by studying the Ca2+ dependency of AA uptake from the medium, the incorporation into cellular phospholipids, and the effect of the iPLA2 inhibitor bromoenol lactone on these events. Uptake and esterification of AA into phospholipids were not affected by Ca2+ depletion in human polymorphonuclear neutrophils and rat fibroblasts. The uptake was Ca2+ independent in chick embryo glial cells, but the incorporation into phospholipids was partially dependent on extracellular Ca2+. Both events were fully dependent on extra and intracellular Ca2+ in human platelets. In human polymorphonuclear neutrophils, the kinetics of incorporation in several isospecies of phospholipids was not affected by the absence of Ca2+ at short times (<30 min). The involvement of iPLA2 in the incorporation of AA from the medium was confirmed by the selective inhibition of this enzyme with bromoenol lactone, which reduced < or =50% of the incorporation of AA into phospholipids of human neutrophils. These data provide evidence that suggests iPLA2 plays a major role in regulating AA turnover in different cell types.     

Production of the polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid by the fungus Pythium ultimum

Several strains of species of the fungal genus Pythium, and of Phytophthora cinnamomi, were screened for content of the polyunsaturated fatty acids (PUFAs) arachidonic acid (AA) and eicosapentaenoic acid (EPA). The aim of the investigation was to establish alternative sources of these PUFAs, which are of importance in human nutrition. As a relatively prolific producer of EPA and AA, P. ultimum strain #144 was selected for a study of conditions that enhance their production over baseline levels that are present in the fungus when cultured for 6 d at 25 degrees C with rotary shaking (120 r.p.m.) in Vogel's medium containing sucrose as the carbon substrate. The levels of AA and EPA under these conditions were 133 +/- 27 and 138 +/- 25 mg l-1 (n = 5), respectively. Maximal production of these fatty acids was accomplished by the following sequence of steps. (1) Incubate the cultures for 6 d after inoculation under the conditions described above. Then (2) add glucose to the cultures (2%, w/v, final concentration) and incubate for a further 6 d at 13 degrees C. Under these conditions, the AA content of the mycelium was 205% higher than baseline levels and the EPA content was 198% higher. (3) Allow the cultures to remain stationary for 10 d which increases the AA content to 253% above baseline levels and the EPA content by 236%. Using such a procedure, 322 mg AA l-1 and 383 mg EPA 1-1 were produced.