Physiologic effects of columbinic acid and its metabolites on rat skin

Columbinic acid and its cyclooxygenase and lipoxygenase metabolites were applied topically to the skin of essential fatty acid-deficient rats and their transepidermal water loss was measured. Despite the ability of the major lipoxygenase metabolite to reverse the scaly dermatitis, neither metabolite of columbinate was able to normalize the epidermal water loss, but the parent acid lowered the measurements in a dose-dependent fashion. These results indicate that some essential fatty acids have important functions as such, and not merely as precursors for other, biologically active, oxygenated metabolites.     

Effect of essential fatty acid deficiency on the epidermal sphingolipids of the rat

The epidermal sphingolipids from rats maintained on either a rat stock diet or a fat-free diet have been analyzed. Thin-layer chromatographic analyses have revealed glucosylceramides, acylglucosylceramides and four fractions of ceramides, one of which proved to be an acylceramide. The relative amounts of the glucosylceramides, acylglucosylceramides and acylceramides were increased in the essential fatty acid-deficient epidermis while one ceramide fraction was diminished. The other two ceramide fractions remained unchanged. The acylceramides and acylglucosylceramides from normal rat epidermis both contained long-chain omega-hydroxy acids in amide linkage to sphingosine bases and high proportions of linoleic acid in ester linkage. The linoleate, which is known to be crucial for the formation and maintenance of the epidermal water barrier, was replaced by oleate in the essential fatty acid-deficient rats.     

Apparent in vivo retroconversion of dietary arachidonic to linoleic acid in essential fatty acid-deficient rats

Essential fatty acid-deficient rats were fed ethyl [U-14C]arachidonate (308 dpm/nmol) and when a decrease in the transepidermal water loss was seen, the epidermal sphingolipids, acylglucosylceramide and acylceramide were isolated. [14C]Linoleic acid (approx. 130 dpm/nmol) was present in both lipid classes, while the substrate was only detected in the former. These results intimate that in vivo retroconversion of arachidonic to linoleic acid can be induced in the rat.     

Leukotriene B formation by neutrophils from essential fatty acid-deficient rats

Analysis of neutrophil phospholipids from rats fed an essential fatty acid-deficient diet revealed a 33% reduction in arachidonate and a 90% reduction in linoleate compared to neutrophil phospholipids of rats fed a normal diet. The neutrophil phospholipids from rats fed the essential fatty acid-deficient diet also contained significant amounts of 5,8,11-eicosatrienoate, a fatty acid not found in the neutrophils of rats fed a normal diet. Analysis of the production of leukotrienes of the B series by ionophore-stimulated neutrophils from rats fed an essential fatty acid-deficient diet revealed a 87% reduction in leukotriene B4 compared to neutrophils from rats fed a normal diet even though the arachidonate content was reduced by only 34%. Essential fatty acid-deficient neutrophils converted endogenous 5,8,11-eicosatrienoic acid to leukotriene A3 and its nonenzymatic degradation products, but little or no leukotriene B3 was formed. Neutrophils from rats fed a normal diet incubated with ionophore and exogenous 5,8,11-eicosatrienoate also produced leukotriene A3 and its nonenzymatic degradation products but little or no leukotriene B3. Exogenous 5,8,11-eicosatrienoate incubated with ionophore-stimulated normal neutrophils caused a dose-dependent inhibition of leukotriene A hydrolase resulting in diminished production of leukotriene B4 from endogenous arachidonate. Assays of leukotriene A hydrolase in the 10,000 X g supernatant fraction of a homogenate of RBL-1 cells revealed that a lipoxygenase metabolite of 5,8,11-eicosatrienoate rather than 5,8,11-eicosatrienoate itself is the inhibitor of leukotriene A hydrolase. Thus the finding that leukotriene B4 production by neutrophils from essential fatty acid-deficient rats is diminished out of proportion to the decrease in arachidonate content appears to be due to inhibition of leukotriene A hydrolase by a lipoxygenase metabolite.     

Essential fatty acid deficiency and adrenal cortical function in vitro.

Adrenocortical cells were prepared from rats maintained on essential fatty acid-deficient diets and control litter mates. Cells from control rats had high concentrations of essential fatty acids in the cholesteryl ester fraction of which approximately 22% was arachidonate. In contrast, cells from EFA-deficient rats had only 2.5% arachidonate in the cholesteryl esters, even though the total esterified cholesterol level was comparable to that of controls. In place of the essential fatty acids, the cholesteryl esters of these cells were rich in 20:3(n--9) and 22:3(n--9). When cells from EFA-deficient rats were incubated with ACTH or dibutyryl cyclic AMP, the output of corticosterone was the same as in controls. Also sterol esters were hydrolyzed to the same extent as in controls despite the unusual composition of the fatty acid esters. The phospholipids in both control and EFA-deficient cells contained high levels of arachidonate but were not hydrolyzed in either type of cell during incubation with ACTH or dibutyryl cyclic AMP. The results indicate that high levels of the prostaglandin precursors, namely linoleate and arachidonate, are not a sine qua non for the steroidogenic action of ACTH or cyclic AMP.     

Metabolism of linoleic acid and other essential fatty acids in the epidermis of the rat

Essential fatty acids are absolutely necessary for maintaining the proper condition of the water barrier (stratum compactum) in the skin. Even direct topical application of linoleic acid or any other Z,Z-(n-6, n-9)-fatty acid to the skin restores the barrier in essential fatty acid-deficient animals. In order to investigate the mechanism by which these polyunsaturated fatty acids exert their activity, radioactively labelled fatty acids were applied to the skin of the live animal and the epidermal lipids were analysed after 1-4 days. Much radioactivity was incorporated into two peculiar lipids, viz. acyl ceramide and acyl acid, which are characteristic of the barrier, in which linoleate was esterified to the end-position of very-long-chain (C30-34) unsaturated omega-hydroxy fatty acids. Strong evidence was obtained which showed that these lipids carry linoleate into the barrier layer where it is converted, probably by lipoxygenase(s), into a series of peroxidated lipids. The lipoxygenase inhibitor, eicosatetraynoic acid, prevents both oxygenation of the polyunsaturated fatty acid and the formation of a healthy skin. This peroxidation may supply the mediators which induce the proper differentiation of the epidermal cells into an effective stratum compactum and a horny layer.     

Effect of sex and gonadal hormones on rat plasma lipids during the development of an essential fatty acid deficiency

1. Male, female and castrated rats treated with oestradiol (30mug./week) or testosterone (2mg./week) were given an essential fatty acid-deficient diet containing 10% of hydrogenated coconut oil for 9 weeks. The concentrations and fatty acid composition of plasma phospholipids, cholesteryl esters and triglycerides were determined. 2. Between the second and third weeks of the deficiency, concentrations of plasma cholesteryl esters, phospholipids and triglycerides decreased, then remained relatively constant. There were no significant differences between males and females, but oestradiol caused a significant rise in plasma phospholipids and triglycerides as compared with testosterone-treated animals. 3. During the first 2 weeks of the deficiency, linoleic acid in the plasma lipids of all groups decreased to low concentrations and changed very little thereafter. 4. Female rats maintained higher percentages and concentrations of arachidonic acid and stearic acid in plasma phospholipids and arachidonic acid in cholesteryl esters than did males. Males had higher proportions of eicosatrienoic acid and oleic acid. There was no sex difference in the fatty acid composition of plasma triglycerides. 5. Oestradiol-treated rats had concentrations of cholesteryl and phospholipid arachidonate comparable with those of female rats and higher than the testosterone-treated group. Eicosatrienoic acid in the oestradiol-treated rats was high and resembled that of the male rats, apparently because of the higher concentration of plasma phospho lipids in this group. 6. Supplementation of the essential fatty acid-deficient rats with linoleate restored plasma cholesteryl and phospholipid linoleate and arachidonate nearly to normal concentrations in a single day. The increase in arachidonic acid in these fractions was accompanied by a similar quantitative decrease in eicosatrienoic acid. 7. These sex differences appear to be related to the smaller size of the female rat and to a more direct influence of oestradiol on the formation or maintenance of phospholipids rich in arachidonic acid.     

The effect of endogenous essential and nonessential fatty acids on the uptake and subsequent agonist-induced release of arachidonate

We have demonstrated that the uptake and agonist-induced release of a pulse of arachidonate are influenced by the size and composition of preexisting endogenous fatty acid pools. EFD-1 cells, an essential fatty acid-deficient mouse fibrosarcoma cell line, were incubated with radiolabeled (14C or 3H] arachidonate, linoleate, eicosapentaenoate (EPA), palmitate, or oleate in concentrations of 0-33 microM for 24 h. After 24 h, the cells were pulsed with 0.67 microM radiolabeled (3H or 14C, opposite first label) arachidonate for 15 min and then stimulated with 10 microM bradykinin for 4 min. Because EFD-1 cells contain no endogenous essential fatty acids, we were able to create essential fatty acid-repleted cells for which the specific activity of the newly constructed endogenous essential fatty acid pool was known. Loading the endogenous pool with the essential fatty acids arachidonate, eicosapentaenoate, or linoleate (15-20 nmol of fatty acid incorporated/10(6) cells) decreased the uptake of a pulse of arachidonate from 200 to 100 pmol/10(6) cells but had no effect on palmitate uptake. The percent of arachidonate incorporated during the pulse which was released upon agonist stimulation increased 2-fold (4-8%) as the endogenous pool of essential fatty acids was increased from 0 to 15-20 nmol/10(6) cells. This 8% release was at least 3-fold greater than the percent release from the various endogenous essential fatty acid pools. In contrast, loading the endogenous pool with the nonessential fatty acids oleate or palmitate to more than 2-3 times their preexisting cellular level had no effect on the uptake of an arachidonate pulse. Like the essential fatty acids, increasing endogenous oleate increased (by 2-fold) the percent release of arachidonate incorporated during the pulse, whereas endogenous palmitate had no effect on subsequent agonist-induced release from this arachidonate pool. These studies show that preexisting pools of essential and nonessential fatty acids exert different effects on the uptake and subsequent releasability of a pulse of arachidonate.     

Arachidonic acid and linoleic acid supplementation increase prostanoid production in rats fed a butter-enriched diet

Male Sprague Dawley rats were fed a butter-enriched diet (50% fat) for 2 weeks and then supplemented orally with either 90 mg of ethyl arachidonate or ethyl linoleate daily for 2 weeks. For comparative reasons, one group of animals was fed standard laboratory rat chow for 4 weeks. Aortic prostacyclin (PGI2) production, platelet aggregation and thromboxane A2 (TXA2) production and plasma and aortic phospholipid (PL) fatty acids were measured. When compared to butter-fed rats, aortic PGI2 production, collagen-induced platelet aggregation and TXA2 production were significantly increased in rats supplemented with ethyl arachidonate to levels similar to those seen in chow-fed rats. Ethyl linoleate supplementation also tended to increase aortic PGI2 production, collagen-induced platelet aggregation and TXA2, but not to the same extent. These changes were accompanied by increases in the level of arachidonic acid and linoleic acid in aortic and plasma PL and a decrease in the level of eicosapentaenoic acid (EPA) and docsahexaenoic acid (DHA). These data indicate that supplementation with small doses of preformed arachidonic acid was more effective than supplementation with its precursor, linoleic acid, in reversing the effects on prostanoid production and phospholipid fatty acid composition in rats fed diets enriched with butter.     

Fatty acid desaturation and microsomal lipid fatty acid composition in experimental hypothyroidism.

We have studied the influence of experimental hypothyroidism in the rat on the synthesis of unsaturated fatty acids and on liver microsomal lipid fatty acid composition. Hypothyroid rats demonstrated an 80% decrease in delta 9 (stearate) desaturation and a 43% decrease in delta 6 (linoleate) desaturation. Liver microsomal fatty acid composition was altered in the hypothyroid animals with a significantly decreased proportion of arachidonate and increased proportions of linoleate, eicosa-8,11,14-trienoate, eicosapentaenoate and docosahexaenoate. The bulk of these changes occurred in both of the two major phospholipid components, phosphatidylcholine and phosphatidylethanolamine. All of the changes were corrected by treatment of the hypothyroid rat with 25 micrograms of tri-iodothyronine/100 g body wt. twice daily. The diminished delta 9 desaturation did not lead to any changes in fatty acid composition. The increased linoleate and decreased arachidonate levels may be due to the diminished delta 6 desaturase activity, the rate-controlling step in the conversion of linoleate into arachidonate. The increases in the proportions of the other polyunsaturated fatty acid components cannot be explained by changes in the synthesis of unsaturated fatty acids, but are probably due to diminished utilization of these fatty acids.     

Regulation of epidermal sphingolipid synthesis by permeability barrier function

A mixture of sphingolipids, cholesterol, and free fatty acids forms the intercellular membrane bilayers of the stratum corneum which are presumed to regulate epidermal barrier function. Prior studies have shown that both cholesterol and fatty acid synthesis are rapidly regulated by epidermal barrier requirements. In contrast, the importance of sphingolipids in barrier function has not been directly demonstrated. Here, we have assessed both sphingolipid synthesis by [3H]H2O incorporation and serine palmitoyl transferase (SPT) activity in relation to modulations in barrier function. Incorporation of [3H]H2O into sphingolipids increased after barrier disruption with acetone, with maximal increase (170%) occurring 5-7 h after treatment (P less than 0.005). As barrier function returned to normal over 24 h, incorporation of tritium into sphingolipids normalized. SPT activity also increased after barrier disruption, peaking at 6 h (150%) (P less than 0.05), and returning towards normal by 24 h. Artificial restoration of the barrier with a water vapor-impermeable membrane prevented the increases in both [3H]H2O incorporation into sphingolipids and enzyme activity. Finally, SPT activity was increased in two other models of barrier dysfunction, cellophane tape-stripping and essential fatty acid deficiency. Occlusion normalized SPT activity in both of these models as well. These studies: a) demonstrate a distinctive, delayed increase in epidermal sphingolipid synthesis in response to barrier requirements that contrasts with the immediate responses of cholesterol and fatty acid synthesis; and b) suggest that sphingolipids are important for the maintenance of the epidermal permeability barrier.     

Effect of essential fatty acid deficiency on release of triglycerides by the perfused rat liver

Studies are reported on release of triglycerides during perfusion of livers of male Sprague-Dawley rats fed a fat-free diet or diets containing hydrogenated coconut oil or corn oil. Perfusions were carried out with Krebs-Ringer bicarbonate buffer containing albumin with and without infusion of oleate or linoleate. Infusion with sodium oleate or linoleate caused an accumulation of triglycerides in the livers of the corn oil-fed animals and stimulated the release of triglycerides into the perfusing medium. In similar experiments with essential fatty acid-deficient animals, which were fed fat-free diets or diets containing hydrogenated coconut oil, there was no increase in secretion of triglycerides into the perfusate, and the amount of triglyceride which accumulated in the liver was greater than in the livers of the control (corn oil-fed) animals. Tracer experiments with oleate-1-(14)C or linoleate-1-(14)C also showed that with livers of essential fatty acid-deficient animals, secretion of triglyceride into the perfusate was not stimulated by infusion of fatty acids into the perfusing medium. It is concluded that impairment of the secretion of triglycerides is a factor in the accumulation of fat in the livers of essential fatty acid-deficient animals.     

Effect of essential fatty acid deficiency on activity of liver plasma membrane enzymes in the rat

Liver plasma membranes (LPM) were isolated from rats fed an essential fatty acid-supplemented diet (+EFA) or from rats fed an essential fatty acid-deficient diet (-EFA). The proportions of linoleate and arachidonate in membrane total fatty acids in the ?EFA preparations were one-half or less than the values for the +EFA preparations. Basal, F^?, or glucagon-stimulated adenylate cyclase activities were significantly lower in EFA-deficient livers than in nondeficient ones. Addition of GTP significantly enhanced glucagon-stimulated adenylate cyclase in both groups, but extent of stimulation above basal was greater in EFA-deficient livers. Portal vein injection of glucagon in vivo resulted in significantly higher cAMP formation in +EFA livers than in ?EFA livers. When glucagon was used in vitro at 1–1,000 nM, stimulation of adenylate cyclase remained lower in EFA-deficient membranes, but extent of stimulation above basal activity was larger in ?EFA membranes than in +EFA. Total Na⁺, K⁺ (Mg²⁺)-ATPase from EFA-depleted LPM exhibited significantly higher values of apparent K_m and V_max. 5′-Nucleotidase activity, in contrast, was considerably decreased in EFA-deficient rats. These findings show that, in animals, changes in unsaturated fatty acid composition can affect the properties of membrane-bound enzymes. These alterations could be due to changes in membrane physical properties and/or prostaglandin formation.     

Fat absorption in essential fatty acid deficiency: a model experimental approach to studies of the mechanism of fat malabsorption of unknown etiology

Male rats were made deficient in essential fatty acids by feeding them a fat-free diet supplemented with 4% tripalmitin for 8-12 wk from the time of weaning. After feeding 0.5 ml of [(14)C]triolein or [(3)H]oleic acid, 72-hr stool recoveries of radioactivity were significantly greater in deficient rats than in chow-fed controls. Essential fatty acid deficiency did not reduce the absorptive capacities for triolein or for a medium-chain fat, trioctanoin, measured after 3 and 2 hr of maximal-rate duodenal infusion. In everted jejunal slices from essential fatty acid-deficient rats, uptake of micellar [(14)C]oleic acid at 0-1 degrees C was similar to that of controls, but the rate of incorporation of fatty acid into triglyceride after rewarming to 37 degrees C was significantly reduced. The specific activities of the microsomal esterifying enzymes, acyl CoA:monoglyceride acyltransferase and fatty acid CoA ligase in jejunal mucosa were 30% lower in essential fatty acid-deficient rats. However, the total microsomal enzyme activity adjusted to constant weight did not differ significantly in deficient rats compared with controls. After intraduodenal perfusion of triolein, accumulation of lipid in the intestinal wall was increased in the deficient rats. Because over 90% of the absorbed mucosal lipid was present as triglyceride, essential fatty acid deficiency appears to affect the synthesis or release of chylomicron lipid from the intestine. Analysis of regions of intestine showed that this delay in transport was most marked in the midportion of the small intestine.     

THE RELEASE OF BRAIN FREE FATTY ACIDS DURING ISCHAEMIA IN ESSENTIAL FATTY ACID-DEFICIENT RATS

—The release of free fatty acids and especially of free arachidonic acid occurring in rat brain during ischaemia has been studied in essential fatty acid-deficient animals. Free fatty acid levels are lower in essential fatty acid-deficient brains before and especially after 5 min of ischaemia. The percentage of arachidonic acid, in respect of total free fatty acids, is similar in both control and essential fatty acid-deficient brains before ischaemia (0 min), but is greatly reduced in deficient brains in respect of controls after ischaemia (5 min). Total levels of free arachidonic acid are thus greatly reduced after ischaemia in the brain of essential fatty acid-deficient rats. Focussed microwave irradiation of the brain, a technique which instantaneously kills the animals, and which was used for the determination of brain basal levels of free fatty acids and free arachidonic acid, does not per se modify brain lipid and fatty acid composition.     

Calcium, collagen dose, gender and fasting affect the response of rat platelet thromboxane formation to extremes in dietary linoleate

Platelet thromboxane synthesis in response to supplemental linoleate in the diet has been very inconsistent. The objective of this study was to investigate potential confounding factors known to affect platelet thromboxane synthesis. Citrated whole blood was recalcified with varying Ca2+ concentrations and challenged with low or high dose collagen preparations to induce extreme ranges of thromboxane synthesis from endogenous arachidonate pools by rat platelets. Male and female weanling rats were fed 0.0, 1.0 or 23 energy percent linoleate for 11 to 13 weeks. Fasting tended to enhance thromboxane synthesis. Both fasted and fed females showed slightly faster rates of thromboxane synthesis than males. Essential fatty acid deficiency depressed (P less than 0.01) thromboxane synthesis; the degree of this depression was inversely related to the level of recalcification (68% for 0.0 mM Ca2+, 36% for 2.5 mM Ca2+ and 20% for 5.0 mM Ca2+) when challenged with the high dose collagen. Essential fatty acid deficiency depressed platelet phospholipid arachidonate concentration 26%. Only blood from fed females stimulated with a mild challenge responded to excess dietary linoleate, and a 62% (not statistically significant) depression in TX synthesis was observed and this was associated with a decrease in platelet phospholipid arachidonate concentration.     

Schistosoma mansoni: eicosanoid production by cercariae

Cercariae of Schistosoma mansoni are stimulated to penetrate skin by certain free fatty acids. The cercariae have an active arachidonate cascade, presumably using host skin essential fatty acids as cascade precursors. Exposing cercariae to 3.3 mM linoleate for 1, 10, and 60 min resulted in production of a wide variety of eicosanoids. Using high-performance liquid chromatography, eicosanoids coeluting with prostaglandin E2, D2, and A2, leukotriene B4, and 5-hydroxyeicosatetraenoic acid standards were identified, as well as unidentified peak positions. Radioimmunoassay confirmed the presence of immunoreactive prostaglandin E1, and E2, and 5- and 15-hydroxyeicosatetraenoic acids in cercarial extracts. No eicosanoid production occurred when cercariae were exposed to 3.3 mM oleate and 1 or 330 microM linoleate. Both high-performance liquid chromatography and radioimmunoassay data indicated that cercariae regulate the production of eicosanoids through time. It is postulated that arachidonate metabolism and subsequent eicosanoid production are required for successful cercarial penetration.     

Fatty acid changes in liver and plasma lipid fractions after safflower oil was fed to rats deficient in essential fatty acids

1. Fatty acid patterns of liver and plasma triglycerides, phospholipids and cholesteryl esters were determined at intervals during 24hr. after essential fatty acid-deficient rats were given one feeding of linoleate (as safflower oil). 2. Liver triglyceride, phospholipid and cholesteryl ester fatty acid compositions did not change up to 7hr. after feeding. Between 7 and 10hr., linoleic acid began to increase in all fractions, but arachidonic acid did not begin to rise in the phospholipid until 14-19hr. after feeding. 3. Oleic acid and eicosatrienoic acid in liver phospholipid began to decline at about the time that linoleic acid increased, i.e. about 9hr. before arachidonic acid began to increase. 4. Changes in linoleic acid, arachidonic acid and eicosatrienoic acid in phosphatidylcholine resembled those of the total phospholipid. Phosphatidylethanolamine had a higher percentage content of arachidonic acid before the linoleate was given than did phosphatidylcholine, and after the linoleate was given the fatty acid composition of this fraction was little changed. 5. The behaviour of the plasma lipid fatty acids was similar to that of the liver lipids, with changes in linoleic acid, eicosatrienoic acid and arachidonic acid appearing at the same times as they occurred in the liver. 6. The results indicated that linoleic acid was preferentially incorporated into the liver phospholipid at the expense of eicosatrienoic acid and oleic acid. The decline in these fatty acids apparently resulted from their competition with linoleic acid for available sites in the phospholipids rather than from any direct replacement by arachidonic acid.     

Aging influence on delta-6-desaturase activity and fatty acid composition of rat liver microsomes

The activity of delta-6-desaturase (D6D) in liver microsomes and fatty acid composition of microsomal lipids of rats of different ages were studied. The D6D activity was similar in suckling rats and in weaning rats. However, the enzyme showed a significantly decreased activity in oldest animals, and a linear correlation was found between the D6D activity and the animal age. The fatty acid composition data on total lipids of liver microsomes were consistent with the age-dependent changes in fatty acid desaturase activity. The major changes occurred in the linoleate and arachidonate fractions; the 20:4/18:2 ratio in liver microsomes decreased together with D6D activity during aging. The loss of D6D activity may be a key factor in aging through altering lipid membrane composition.     

Novel Plant Growth Inhibitors and an Insect Antifeedant from Chrysanthemum coronarium (Japanese Name: Shungiku)

The effect of overnight fasting on the dietary protein-dependent change in the fatty acid composition of tissue lipids was studied in rats fed with casein or soybean protein (20%) diets containing 5 or 2% corn oil. The activity of the Δ6-desaturase of liver microsomes, a key enzyme of linoleate metabolism to arachidonate, was depressed significantly by overnight fasting, and the protein effect disappeared, irrespective of the level of dietary fat. The proportion of linoleate in liver phosphatidylcholine was decreased, whereas that of arachidonate was increased after overnight fasting in rats fed with a low fat diet, resulting in an elevation of the linoleate desaturation index. Although the effect of fasting became obscure on a high fat diet, the protein effects were maintained even after fasting. A similar trend was also observed in various lipid fractions. Thus, the effect of dietary protein on the polyunsaturated fatty acid profile was not modulated by overnight fasting, particularly when a minimal amount of linoleic acid was supplied.