Measurement of arachidonic acid liberation in thrombin-stimulated human platelets. Use of agents that inhibit both the cyclooxygenase and lipoxygenase enzymes

The formation of radiolabelled oxygenated products of arachidonic acid in thrombin-stimulated, [3H]arachidonic acid-prelabelled human platelets is inhibited in a concentration-dependent manner by BW 755C (3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline) or propyl gallate, both of which are combined inhibitors of lipoxygenase and cyclooxygenase. These compounds do not inhibit the thrombin-induced decrease in the radioactivity of platelet phospholipids but, instead, allow the accumulation of free radiolabelled arachidonic acid. Thrombin causes an increase in the levels of free, endogenous palmitic, stearic, oleic, linoleic and arachidonic acids of up to 10 nmol/10(9) platelets. In the presence of BW 755C or propyl gallate, further increases in the level of free arachidonic acid, of 20-50 nmol/10(9) platelets, occur. The enzyme inhibitors do not affect the accumulation of the other free fatty acids. The increase in arachidonic acid is optimal at 1 U/ml thrombin and 60% complete by 1 min at 37 degrees C. In the platelets from eight donors, the average increases in free fatty acids (in nmol/10(9) platelets) induced by 5 U/ml thrombin in 5 min at 37 degrees C in the presence of 100 microM BW 755C were 1 for linoleic acid, 3.6 for oleic acid, 4.5 for palmitic acid, 7.6 for stearic acid and 32.0 for arachidonic acid.     

A Novel Delta5-Desaturase-Defective Mutant of Mortierella alpina 1S-4 and Its Dihomo-gamma-Linolenic Acid Productivity

A novel Delta5-desaturase-defective mutant was derived from an arachidonic acid-producing fungus, Mortierella alpina 1S-4, after treating the parental spores with N-methyl-N'-nitro-N-nitrosoguanidine. The mutant produced only a trace (about 1%) amount of arachidonic acid, and the ratio of dihomo-gamma-linolenic acid (DGLA) to total fatty acids in each lipid class was markedly high, accounting for as much as 60% in phosphatidylcholine. Under submerged batch culture conditions, the mutant produced 2.4 g of DGLA per liter (43.3% of total fatty acids) when grown at 28 degrees C for 7 days in a 5-liter jar fermentor. The other major (more that 1%) fatty acids were palmitic acid (21.2%), stearic acid (9.6%), oleic acid (14.3%), linoleic acid (4.4%), and gamma-linolenic acid (5.8%). About 80 mol% of the DGLA produced was found in triacylglycerol.     

Production of Dihomo-gamma-Linolenic Acid by a Delta5-Desaturase-Defective Mutant of Mortierella alpina 1S-4

A mutant, which has low Delta5-desaturase activity, of an arachidonic acid-producing fungus, Mortierella alpina 1S-4, was shown to be a novel potent producer of dihomo-gamma-linolenic acid (DHGA). On submerged culture under optimal conditions for 6 days at 28 degrees C in a 10-liter fermentor, the mutant produced 3.2 g of DHGA per liter of culture broth (123 mg/g of dry mycelia), which accounted for 23.4% of the total mycelial fatty acids. Mycelial arachidonic acid amounted to only 19 mg/g of dry mycelia (0.5 g/liter of culture broth), which accounted for 3.7% of the total mycelial fatty acids. The other major mycelial fatty acids were palmitic acid (11.0%), stearic acid (12.8%), oleic acid (22.7%), linoleic acid (8.9%), gamma-linolenic acid (6.5%), and lignoceric acid (7.8%). More than 97 mol% of the DHGA produced was found in the triglyceride fraction irrespective of the growth temperature employed (12 to 28 degrees C).     

Characteristics of myocardial metabolism during induction and prolongation of artificial hypobiosis

Glucose, free fatty acids and lipid fatty acid spectrum were studied in arterial and right atrial blood and myocardium of 122 rats during induction and prolongation of artificial hypobiosis (3 and 24 hours) at body temperature of 30 and 20 degrees C. Prolongation of hypobiosis was shown to be accompanied by enhanced participation of free fatty acids in the myocardial energy metabolism. Lipid fatty acid spectrum in the myocardium was characterized by the decrease in linoleic and palmitooleic acid content and the increase in oleic, palmitic, stearic and arachidonic acid levels.     

Effects of linoleic acid and mitogenic stimulation on the fatty acid composition of human lymphocytes

Perturbation of the fatty acid composition of human lymphocytes in vitro was investigated by addition of linoleic acid complexed to bovine serum albumin (BSA-LA) and by mitogenic stimulation with phytohaemagglutinin (PHA). BSA-LA resulted in a 45% increase in linoleic acid in phosphatidylethanolamine (PE) and over 100% in phosphatidylcholine (PC) in peripheral blood cells. Supplementation with BSA-LA in PHA-stimulated lymphocytes produced even greater changes: 100% increase in linoleic acid content for PE and over 300% for PC. There was a large decrease in oleic acid: 40% for PE and almost 100% in PC. Significant decreases in arachidonic acid occurred in both phospholipid fractions. PHA alone also altered membrane phospholipid fatty acid composition, with reductions in palmitic, stearic and linoleic acid for PE and increases in oleic acid and arachidonic acid (almost 100%). For PC, there were large decreases in stearic (40%), linoleic (30%) and arachidonic (40%) acids, together with an increase in oleic acid (65%). Cells supplemented with linoleic acid grown in the presence of PHA, compared with those grown in linoleic acid-supplemented medium alone, showed a 40% decrease in palmitic acid and a 55% increase in arachidonic acid in PE. For PC, there were large decreases in stearic acid (40%) and arachidonic acid (57%). Antibody-induced redistribution of surface molecules ('capping') was inhibited by some 14% after incubation with BSA-LA. However, no consistent alterations in PHA-induced cell proliferation were observed. These data suggest that profound alterations of membrane fatty acid composition occur spontaneously during the mitotic cycle, and may be further induced by experimental manipulation, without gross perturbation of cell function.     

Fatty acid specificity of acyl-CoA synthetase in rat glomeruli

The fatty acid specificity of acyl-CoA synthetase in rat glomeruli for physiologically and pathologically important long-chain fatty acids was studied. The apparent Michaelis constants (Km) for substrate fatty acids increased in the order, linolenic less than linoleic less than eicosapentaenoic less than arachidonic less than oleic less than palmitic acid. The maximum velocities with these fatty acids decreased in the order, oleic greater than linoleic greater than palmitic (approximately equal to) linolenic greater than arachidonic greater than eicosapentaenoic acid. The syntheses of radioactive arachidonyl-CoA and palmitoyl-CoA from radioactive arachidonic and palmitic acid, respectively, were both inhibited by all fatty acids mentioned above including the substrate fatty acids, their inhibitory effects being inversely correlated with their apparent Km values. These results suggest that the enzyme in glomeruli has a unique specificity for fatty acids and that there is no arachidonic acid-specific acyl-CoA synthetase in glomeruli. The possible contribution of the glomerular enzyme with this specificity to the abnormal fatty acid levels in diabetic animals is discussed.     

Selective retention of n-3 and n-6 fatty acids in human milk lipids in the face of increasing proportions of medium chain-length (C10-14) fatty acids

This paper reports the results of our analysis of the impact high levels of de novo fatty acids have on the proportions of essential and non-essential fatty acids in human milk lipids. The data for seven fatty acids (linoleic, alpha-linolenic, arachidonic (AA), docosahexaenoic (DHA), palmitic, stearic and oleic) were derived from several studies conducted in Nigeria. The proportion by weight of each of these fatty acids was plotted versus the proportion of C10-14 fatty acids. As the proportion of C10-14 fatty acids increased from 15 to 65%, there was not a proportional decrease in the percentages of all seven fatty acids, but, instead, preferential incorporation of the essential fatty acids, AA and DHA into the triacylglycerol component of the milk. At the same time, the proportions of stearic and oleic acid declined by 69% and 86%, respectively. However, the proportions of linoleic acid, palmitic acid, DHA, AA and alpha-linolenic acid, in milk lipids decreased by only 44%, 40%, 39%, 28% and 2.3%, respectively. These observations indicate that as the contribution of C10-14 fatty acids increases, essential fatty acids are preferentially incorporated into milk triacylglycerols at the expense of oleic acid and stearic acid.     

Differential regulation by fatty acids of protein histidine phosphorylation in rat pancreatic islets

Long-chain fatty acids (e.g. arachidonic acid) have been implicated in physiological control of insulin secretion. We previously reported histidine phosphorylation of at least two islet proteins (e.g., NDP kinase and the beta subunit of trimeric G-proteins), and suggested that such a signalling step may have regulatory roles in beta cell signal transduction, specifically at the level of G-protein activation. Since our earlier findings also indicated potential regulation by long-chain fatty acids of islet G-proteins, we undertook the current study to verify putative regulation, by fatty acids, of protein histidine phosphorylation of NDP kinase and Gbeta subunit in normal rat islets. The phosphoenzyme formation of NDP kinase was stimulated by various fatty acids in the following rank order: linoleic acid > arachidonic acid > oleic acid > palmitic acid = stearic acid = control. Furthermore, the catalytic activity of NDP kinase was stimulated by these fatty acids in the rank order of: oleic acid > arachidonic acid > linoleic acid > palmitic acid = stearic acid = control. Arachidonic acid methyl ester, an inactive analog of arachidonic acid, did not significantly affect either the phosphoenzyme formation or the catalytic activity of NDP kinase. Interestingly, arachidonic acid exerted dual effects on the histidine phosphorylation of beta subunit; it significantly stimulated the phosphorylation at 33 microM beyond which it was inhibitory. Together, these findings identify additional loci (e.g., NDP kinase and Gbeta subunit) at which unsaturated, but not saturated, fatty acids could exert their intracellular effects leading to exocytotic secretion of insulin.     

Sex differences in plasma cholesterol-esterifying activity in rats

Esterification of free cholesterol was investigated after incubation at 37 degrees C of plasma from immature and adult rats of both sexes kept on stock, fat-free, or cholesterol-supplemented diets. According to measurements of the decrease in free cholesterol, plasma from the fat-deficient rats showed the highest cholesterol-esterifying activity. Esterification was higher in the mature female rats than in the mature males on stock or cholesterol-containing diets, although no sex differences were observed in the sexually immature young or in the fat-free animals. There were no sex differences in the fatty acid composition of the plasma sterol esters, phospholipids, and triglycerides in the immature animals, but arachidonic acid increased at the expense of linoleic acid in the sterol ester fraction in the adult female (not, however, in the adult male). In the phospholipid fraction the higher ratio of palmitic to stearic acids in the male was confirmed. There was an increase in linoleic acid in all three plasma lipid fractions of the mature male after cholesterol feeding. It is suggested that cholesterol may inhibit the conversion of linoleate to arachidonate. During the incubation of plasma, there was little change in the distribution of fatty acids except for a decrease in palmitoleate, and increases in C(20) tri- and tetraenoic acids, in the sterol esters of mature female rats on the stock ration and the fat-free diet. These C(20) acids decreased concomitantly in the phospholipid fraction, as the transesterification reaction mechanism proposed by earlier workers would predict.     

Macrophages transfer [14C]-labelled fatty acids to pancreatic islets in culture

Macrophages are able to produce, export, and transfer fatty acids to lymphocytes in culture. The purpose of this study was to examine if labelled fatty acids could be transferred from macrophages to pancreatic islets in co-culture. We found that after 3 h of co-culture the transfer of fatty acids to pancreatic islets was: arachidonic > oleic > linoleic = palmitic. Substantial amounts of the transferred fatty acids were found in the phospholipid fraction; 87.6% for arachidonic, 59.9% for oleic, 53.1% for palmitic, and 36.9% for linoleic acids. The remaining radioactivity was distributed among the other lipid fractions analysed (namely polar lipids, cholesterol, fatty acids, triacylglycerol and cholesterol ester), varying with the fatty acid used. For linoleic acid, a significant proportion (63.1%) was almost equally distributed in these lipid fractions. Also, it was observed that transfer of fatty acids from macrophages to pancreatic islets is time-dependent up to 24 h, being constant and linear with time for palmitic acid and remaining constant after 12 h for oleic acid. These results lead us to postulate that in addition to the serum, circulating monocytes may also be a source of fatty acids to pancreatic islets, mainly arachidonic acid.     

Uptake and metabolic conversion of saturated and unsaturated fatty acids in Hep2 human larynx tumor cells

Research on fatty acid metabolism in cultured human larynx tumor cells Hep2 was carried out.The cells were incubated with either a saturated (palmitic) or a polyunsaturated (linoleic, alpha-linolenic and eicosatrienoic (n-6)) radioactive fatty acid (0.66 pM, 24 h). The best incorporation capacity was observed in the linoleic acid followed by alpha-linolenic, palmitic and eicosatrienoic acids. All fatty acids tested were anabolized to higher derivatives within their own family. Palmitic acid was primarily monodesaturated rather than elongated, proving to have a very active A9 desaturase activity.With respect to polyunsaturated acid metabolism, the conversion of alpha-linolenic acid to higher homologs, although better than linoleic acid, occurred far less efficiently than that observed in other non-highly undifferentiated human tumor cells. This impairment in higher polyunsaturated fatty acid biosynthesis, reflected in the low levels of arachidonic acid in the fatty acid composition, would not reside in the A5 desaturation step since Hep2 cells can readily convert eicosatrienoic acid into arachidonic acid. Considering the potential regulatory role of specific polyunsaturated fatty acids in the cell proliferative control, the knowledge of the metabolism of fatty acids in this human tumor cell would be important for designing future experiments in order to clarify the mechanism involved in balance, proliferation and cell death.     

Changes in the fatty acid profiles of red blood cell membrane phospholipids in human neonates during the first month of life

We have studied the changes in the fatty acid profiles of red blood cell membrane phospholipids in 47 infants who were exclusively fed human milk from birth to 1 month of life. Twenty blood samples were obtained from cord, 15 at 7 days and 12 at 30 days after birth. Membrane phospholipids were obtained from erythrocyte ghosts by thin-layer chromatography and fatty acid composition was determined by gas liquid chromatography. Phosphatidylcholine showed the most important changes during early life; stearic, w6 eicosatrienoic and arachidonic acids decreased whereas oleic and linoleic acids increased. In phosphatidylethanolamine, palmitic and stearic acid declined and oleic, linoleic and docosahexenoic acids increased with advancing age. Small changes were noted for individual fatty acids in phosphatidylserine. In sphingomyelin stearic acid increased from birth to 1 month and linoleic, arachidonic and nervonic acids decreased. Total polyunsaturated fatty acids of the w6 series greater than 18 carbon atoms increased with advancing age in phosphatidylethanolamine and decreased in choline and serine phosphoglycerides and in sphingomyelin. Long chain fatty acids derived from linoleic acid decreased in phosphatidylcholine but increased in ethanolamine and serine phosphoglycerides. The different behavior in the changes observed in fatty acid patterns for each erythrocyte membrane phospholipid may be a consequence of its different location in the cell membrane bilayer and specific exchange with plasma lipid fractions.     

Gestational hyperlipidemia in the rat is characterized by accumulation of n - 6 and n - 3 fatty acids, especially docosahexaenoic acid.

We have evaluated the relative and quantitative changes in long-chain fatty acids in maternal liver, serum, carcass and conceptus (fetuses plus placentae) during pregnancy in the rat, to ascertain whether previous concern over lower proportions of n - 6 and n - 3 fatty acids in maternal serum could be indicative of suboptimal n - 6 or n - 3 fatty acid status. Gestational hyperlipidemia was characterized by proportional decreases in linoleic, stearic and arachidonic acids but increases in palmitic and docosahexaenoic acids. However, the quantitative amount (microgram/ml) of linoleic, arachidonic and docosahexaenoic acids in serum lipids actually increased 2-5-fold from mid-pregnancy to term. Compared to non-pregnant rats, gestational hyperlipidemia was also associated with a lower proportion but similar quantity of linoleic acid in maternal carcass and adipose stores. We conclude that gestational hyperlipidemia in the rat is characterized by a relative but not quantitative decrease in whole-body stores of n - 6 fatty acids and a marked proportional and quantitative increase in docosahexaenoic acid in maternal organs and in the conceptus.     

Hydroperoxy-arachidonic acid mediated n-hexanal and (Z)-3- and (E)-2-nonenal formation in Laminaria angustata

In higher plants, C6 and C9 aldehydes are formed from C18 fatty acids, such as linoleic or linolenic acid, through formation of 13- and 9-hydroperoxides, followed by their stereospecific cleavage by fatty acid hydroperoxide lyases (HPL). Some marine algae can also form C6 and C9 aldehydes, but their precise biosynthetic pathway has not been elucidated fully. In this study, we show that Laminaria angustata, a brown alga, formed C6 and C9 aldehydes enzymatically. The alga forms C9 aldehydes exclusively from the C20 fatty acid, arachidonic acid, while C6 aldehydes are derived either from C18 or from C20 fatty acid. The intermediates in the biosynthetic pathway were trapped by using a glutathione/glutathione peroxidase system, and subjected to structural analyses. Formation of (S)-12-, and (S)-15-hydroperoxy arachidonic acids [12(S)HPETE and 15(S)HPETE] from arachidonic acid was confirmed by chiral HPLC analyses. These account respectively for C9 aldehyde and C6 aldehyde formation, respectively. The HPL that catalyzes formation of C9 aldehydes from 12(S)HPETE seems highly specific for hydroperoxides of C20 fatty acids.     

The effect of sn-2 fatty acid substitution on phospholipase C enzyme activities.

The human monocyte cell line U937 expresses phospholipase A2 and phospholipase C activities and produces eicosanoids. The phospholipase C (PLC) activity exhibits substrate preference for phosphatidyl-choline (PC), rather than phosphatidylinositol or phosphatidylethanolamine. In order to characterize the PLC activity found in these cells, the effects of substitution of the sn-2 fatty acid on this activity were examined. PC substrates with palmitic acid (PC-2P), oleic acid (PC-2O), arachidonic acid (PC-2A) and linoleic acid (PC-2L) at the sn-2 position were used. The sn-1 fatty acid was palmitic acid. PC-2L and PC-2A with the longer-chain less-saturated fatty acids linoleic acid and arachidonic acid esterified at sn-2 were found to be better substrates for PLC activity than PC-2P or PC-2O in these cells. This preference was maintained even when substrate phospholipid was solubilized in non-ionic, anionic, cationic and zwitterionic amphiphiles. Furthermore, when a 500-fold excess of 1,2-diolein or 1,2-dipalmitin was added to the reaction, the specificity of the PLC activity for PC-2A and PC-2L remained unchanged. When similar experiments were performed with phosphatidylinositol as a substrate, we did not observe any effect when the sn-2 position was altered. These data show that the fatty acid constituent at the sn-2 position affects the observed PLC activity when phosphatidylcholine, but not phosphatidylinositol, is used as a substrate by these cells.     

Studies on temperature adaptation in Tetrahymena. Positional distribution of fatty acids and species analysis of phosphatidylethanolamine from Tetrahymena pyriformis grown at different temperatures.

Phosphatidylethanolamine of 15 degrees C-grown Tetrahymena pyriformis (NT-I) cells contains more polyunsaturated fatty acids than 39.5 degrees C-grown cells. This increase in unsaturation is due to an increase in linoleic (C18 : 2) and linolenic (C18 : 3) acids, and a decrease in myristic (C14 : 0), palmitic (C16 : 0), palmitoleic (C16 : 1) and heptadecanoic (C17 : 0) acids. Compared with 39.5 degrees C-grown cells, the proportion of palmitic acid (C16 : 0) decreased in the 1-position as does at the 2-position in 15 degrees C-grown cells. On the contrary, there is a significant increase in linoleic (C18 : 2 delta 9, 12) and gamma-linolenic (gamma-C18 : 3) acids in the 1- and 2-positions, respectively. Phosphatidylethanolamine has been subfractionated into seven different diglyceride species. In 15 degrees C cells, the amounts of fractions 2 (1-linolenoyl-2-linoleoyl) and 3 (1-linolenoyl-2-palmitoleoyl, 1-linolenoyl-2-oleoyl) increased while there was a great decrease in subfraction 7 (1-myristoyl-2-palmitoleoyl, 1-palmitoyl-2-palmitoleoyl). Since subfractions 1 and 2 contain over 70% linoleic (C18 : 2) and linolenic (C18 : 3) acids, these fractions might be composed mainly of 1-linolenoyl-2-linolenoyl and 1-linolenoyl-2-linoleoyl molecular species at 15 degrees C. These data support evidence that phosphatidylethanolamine would play a principal role as an acceptor of acyl chains for temperature acclimation.     

Polyunsaturated fatty acids inhibit mouse hepatic glucocorticoid receptor activation in vitro

The effect of saturated fatty acids (SFAs) stearic and palmitic acids and polyunsaturated fatty acids (PUFAs) oleic, linoleic and arachidonic acids was studied on in vitro heat activation of mouse hepatic glucocorticoid receptor (GR) complex, as assessed by binding to DNA-cellulose and purified nuclei. Significant dose-dependent inhibition of heat activation of hormone-receptor complex by the PUFAs was observed. Linoleic and arachidonic acids were found to be more potent (caused approximately 70% inhibition maximally at 160 microM) inhibitors of GR heat activation, compared to oleic acid (approximately 38% inhibition at 40 microM). However, stearic and palmitic acids were unable to modulate GR heat activation, suggesting that the unsaturated moieties in PUFAs are possibly the important determinants of receptor activation. Thus, our study shows an inhibitory effect of PUFAs on in vitro hepatic GR activation.     

Hydrogenation of polyunsaturated fatty acids by human colonic bacteria

Emulsions of the fatty acids linoleic (C18:2 n-6), alpha-linolenic (C18:3 n-3) and arachidonic acid (C20:4 n-6) were incubated for 4 h under anaerobic conditions with human faecal suspensions. Linoleic acid was significantly decreased (P < 0.001) and there was a significant rise (P < 0.05) in its hydrogenation product, stearic acid. Linolenic acid was also significantly decreased (P < 0.01), and significant increases in C18:3 cis-trans isomers (P < 0.01) and linoleic acid (P < 0.05) were seen. With each acid, there were non-significant increases in acids considered to be intermediates in biohydrogenation. The study provides evidence that bacteria from the human colon can hydrogenate C18 essential polyunsaturated fatty acids. However, with arachidonic acid there was no evidence of hydrogenation.     

Fatty acids profile of Spirulina platensis grown under different temperatures and nitrogen concentrations

The influence of culture temperature and the concentration of sodium nitrate (NaNO3) on the gas-chromatographic profile of the fatty acids of the filamentous cyanobacterium Spirulina platensis was evaluated. We found that temperature was the most important factor and that the greatest amount of gamma-linolenic acid (GLA) was obtained at 30 degrees C, the fatty acid profile of the Spirulina cultivated showing that (in order of abundance) palmitic, linolenic and linoleic acids were most prevalent.     

Long-chain acyl CoA synthetase in microsomes from rat brain gray matter and white matter

Long-chain acyl coenzyme A (CoA) synthetase in homogenates and microsomes from rat brain gray and white matter was studied. The formation of the thioesters of CoA was studied upon addition of [1-¹⁴C]-labeled fatty acids. The maximal activities were seen with linoleic acid, followed by arachidonic, palmitic, and docosahexaenoic acids in both gray and white matter homogenates and microsomes. The specific activities in microsomes were 3–5 times higher than in homogenates. The presence of Triton X-100 in the assay system enhanced the activity of long-chain acyl CoA synthetase in homogenates. The effect was more pronounced in palmitic and docosahexaenoic acid activation. The apparentK _m values andV _max values for palmitic and docosahexaenoic acids were much lower than for linoleic and arachidonic acids. The presence of Triton X-100 in the medium caused a definite decrease in the apparentK _m and Vmax values for all the fatty acid except palmitic acid in which case the reverse was true. There were no significant differences observed in the kinetic measurements between gray and white matter microsomes. These findings are similar to those resulting from the known interference of Triton X-100 in the measurement of kinetic variables of long-chain acyl CoA synthetase of liver microsomes. In this work, no correlation was observed between the fatty acid composition of gray and white matter and the capacity of these tissues for the activation of different fatty acids.