EFA-deficient Rats Research Articles

Influences of dietary polyunsaturated or saturated fats and of concanavalin-a upon proliferation of spleen lymphoid cells from rats

The effects of dietary polyunsaturated and saturated fats upon several metabolic phenomena were studied in rats. These included phospholipid (PL) levels in unstimulated spleen lymphoid cells (SLC) and serum; in vitro proliferation of unstimulated SLC from the two groups of rats; the influence of mitogenic concanavalin-A (Con-A) on in vitro proliferation of SLC from the two groups of rats; and the influence of serum from the two groups of rats on SLC proliferation. Weanling male Long-Evans rats were fed ad libitum either a polyunsaturated fatty acid (PUFA)-adequate (safflower oil, SO) or PUFA-deficient (coconut oil, CNO) diet for 21 days. Growth was significantly less in the CNO group than in the SO group. Food intake was not different for the dietary groups. Unstimulated SLC and serum of CNO rats showed changes in fatty acid composition of PL typical of tissue PL in essential fatty acid-deficient (EFA-D) rats. Unstimulated SLC from the two groups cultured in medium containing serum from the same groups showed equivalent rates of in vitro proliferation. In vitro proliferations of SLC were influenced: by the dietary source of SLC but not by the dietary source of serum, used in the culture medium, when the cells were stimulated by suboptimal doses of Con-A; by the dietary source of serum but not by the dietary source of SLC when the cells were stimulated by the optimal dose of Con-A; and by an interaction between source of SLC and serum, when the cells were stimulated by a supra-optimal dose of Con-A. The present study indicates that in vitro proliferation of SLC from PUFA-adequate and PUFA-deficient rats is influenced by both the dietary source of serum used in the culture medium and the concentration of Con-A used for stimulation of the SLC.

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.

Effect of Essential Fatty Acid Deficiency on Acyl Group Composition, Membrane Fluidity and Adenylate Cyclase Activity in Plasma Membranes of Rat Submandibular Salivary Glands

The effects of a marginal and severe deficiency of essential fatty acids (EFA) on the fatty acid composition, fluorescence polarization of diphenyl-hexatriene (DPH) and adenylate cyclase activity were studied in plasma membranes of rat submandibular salivary glands (SMSG). Three groups of male weanling Sprague-Dawley rats were fed ad libitum EFA-deficient (EFAD), marginally EFA-deficient (MEFAD) or the control diets. Rats were killed after 8–17 wk of feeding and plasma membranes were prepared from the SMSG and the assayed for basal and fluoride-stimulated adenylate cyclase activity. In membranes prepared from EFAD and MEFAD rats, the fluoride-stimulated enzyme activity was 30–35% higher than that of the control group. Lower double-bond index of phospholipids in plasma membranes of the SMSG from EFAD and MEFAD rats was observed, suggesting a lower fluidity compared with the control group. Higher fluorescence polarization of DPH in membranes of EFAD and MEFAD rats was more evidence of lower fluidity. It is suggested that higher adenylate cyclase activity in SMSG of rats fed EFAD or MEFAD diets may be related to lower membrane fluidity. Fluoride-stimulated enzyme activity and fatty acid composition of membrane lipids were restored to normal values after feeding the control diets for 6 wk to the EFAD and MEFAD rats.

Urinary Excretion of Arginine-Vasopressin and Prostaglandin E2 in Essential Fatty Acid-Deficient Rats after Oral Supplementation with Unsaturated Fatty Acid Esters

Essential fatty acid-deficient rats were supplemented with 300 mg/d of pure fatty acid esters: oleate (O), linoleate (L), arachidonate (A), and columbinate (C) for 10 d. The 24-h urine collections from each animal, collected 3 d before supplementations and again the last 3 d of the 10-d supplementation period, were analyzed for volume, and by radioimmunoassay for arginine-vasopressin (AVP) and prostaglandin E2 (PGE2). Linoleate and arachidonate supplements both decreased the initial high urinary AVP excretion, whereas it was further increased by the oleate supplement. There was no effect of columbinate supplementation on urinary AVP excretion. Urinary PGE2 excretion was increased ca. twofold by both linoleate and oleate supplements, increased ca. fivefold by arachidonate supplementation but was unaffected by columbinate supplementation. There was no effect of any of the supplemented fatty acids on urine output. Fatty acid analysis of total kidney lipids revealed a low percentage of 20:3(n-9) in the rats supplemented with (n-6) fatty acid (L, A and C). The triene-tetraene ratio was 1.8 ± 0.6 (n = 6) in the kidneys of the oleate-supplemented rats. No relationship was found between urinary PGE2 excretion and the percentage of arachidonate or the ratio of 20:3 (n-9)/20:4(n-6) in total kidney lipids. It is suggested that increased urinary AVP excretion in EFA-deficient rats is mainly caused by a change in the renal excretatory mechanism of AVP rather than reflecting an increased plasma AVP concentration. Furthermore it is suggested that renal PGE2 synthesis in vivo is unaffected by high levels of 20:3(n-9) in kidney lipids.

Comparative studies of the responses of two strains of rats to an essential fatty acid deficient diet

1. 1. A comparative study of two strains of rats to an EFA deficient diet was conducted. Parameters of insulin status in BHE and Sprague-Dawley rats were measured. No differences in growth were observed. 2. 2. The strains differed in their hepatic and adipose tissue response to insulin stimulation of glucose oxidation and conversion to fatty acids. 3. 3. Hepatic tissue from EFA deficient BHE rats converted more glucose to fatty acid under the influence of insulin than their controls while diet had no effect on glucose oxidation. 4. 4. Hepatic tissue from EFA deficient Sprague-Dawley rats oxidized more glucose than their controls but diet did not affect fatty acid synthesis. 5. 5. A reverse of these strain and diet differences was observed in adipose tissue. 6. 6. These results suggest that the genetic heritage of the rat may determine the type of response to EFA deficiency.

Urinary prostaglandin E 2 excretion in EFA-deficient rats after ten days supplementation of ethyl arachidonate, ethyl linoleate, ethyl oleate and methyl columbinate

Urinary prostaglandin E 2 excretion in EFA-deficient rats after ten days supplementation of ethyl arachidonate, ethyl linoleate, ethyl oleate and methyl columbinate

Incorporation into the tissues and turnover of arachidonic acid after administration to normal and essential fatty acid deficient rats

A comparison was made of the incorporation into the tissues and metabolism of [1- 14C] arachidonic acid (AA) after i.v. administration to normal and EFA-deficient rats. At different times, ultra-thin whole body sections were prepared and the distribution of the radioactivity determined by autoradiograms. After 5 min, a considerable incorporation occurs in the following organs: subcutaneous and perispinal fat, liver, heart muscle, kidney and adrenal. The EFA deficient rats show a similar distribution but the radioactivity is longer retained. The total amount of radioactivity in the heart, liver, kidney and adrenal was measured at different times. A decline occurs in the heart, and an increase in the adrenal. In the urine, the highest amount of radioactivity is excreted on the first day. The excretion is lower in the EFA-deficient rats. Small amounts of radioactive metabolites with the chromatographic characteristics of PGE 2 and 13,14 dihydro-15ketoPGE 2 were isolated from urine. The amounts of 14Co 2 produced were determined after the administration of [1- 14C) AA. Half times were: 39 ± 2.9 min in the EFA-deficient and 28 ± 2.8 min in the normal rats. In the heart, AA is incorporated into phospholipids and neutral lipids. The following percentages were determined: phosphatidylinositol: 6.9 ± 0.6%, phosphatidylcholine: 44 ± 4.1%, phosphatidylethanolamine: 10.0 ± 1.0%, neutral lipids: 9.3 ± 1.6%. Several explanations can be given for the higher requirements of some tissues for AA. It could be, that this substance is used in the formation of particular membranes with a high AA content. Differences in the amounts of metabolites produced may also play a role.

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.

Essential function of linoleic acid esterified in acylglucosylceramide and acylceramide in maintaining the epidermal water permeability barrier. Evidence from feeding studies with oleate, linoleate, arachidonate, columbinate and α-linolenate

Essential fatty acid-deficient rats were supplemented with 300 mg per day of pure fatty acid esters: oleate (O), linoleate (L), arachidonate (A), and columbinate (C) for 10 days. During this period, the rats in groups L, A, and C all showed a decrease in their initially high trans-epidermal water loss, a classical essential fatty acid-deficiency symptom, to a level seen in non-deficient rats (group N). The trans-epidennal water loss in rats of group O was unaffected by the supplementation. Fatty acid composition of two epidermal sphingolipids, acylglucosylceramide and acylceramide, from the skin were determined. The results indicate that re-establishment of a low trans-epidennal water loss was associated with incorporation of linolenate into the two epidermal sphingolipids. Supplementation with columbinate resulted in relatively high amounts of this fatty acid in the investigated epidermal sphingolipids. Analysis of pooled skin specimens from a previous study in which weanling rats were fed a fat-free diet and supplemented orally with pure α-linolenate for 13 weeks (Hansen H.S. and Jensen B. (1983) Lipids 18, 682–690) revealed very little polyunsaturated fatty acid in the two sphingolipids. These rats showed increased evaporation which was comparable to that of essential fatty acid-deficient rats. We interpret these results as strong evidence for a very specific and essential function of linoleic acid in maintaining the integrity of the epidermal water permeability barrier. This function of linoleate is independent of its role as precursor for arachidonate and icosanoids.

Increased concentration of vasopressin in plasma of essential fatty acid-deficient rats

The effect of essential fatty acid deficiency (EFA-D) on the plasma concentration of arginine-vasopressin (AVP) and the urinary AVP excretion was investigated. Weanling rats were fed a fat-free diet (FF-rats). Control rats received the same diet in which 6% by wt. of sucrose was replaced by arachis oil. After 4–6 weeks of feeding, urine and plasma were analysed for AVP, osmolality, sodium and potassium. When compared to control rats FF-rats had decreased urine volume (6.0±1.6 ml/24 hr versus 11.7±3.2 ml/24 hr), increased urine osmolality (2409±691 mOsm/kg versus 1260±434 mOsm/kg), increased urinary AVP excretion (5.52±1.62 ng/24 hr versus 2.19±1.20 ng/hr and increased plasma AVP (2.55±0.45 pg/ml versus 1.95±0.25 pg/ml. There was no difference between the groups in plasma osmolality. The triene/tetraene ration was 0.78+0.15 (n=12) in total kidney lipids of the FF-rats. The triene/tetraene ratio was positively correlated with urinary AVP excretion. It is concluded that FF-rats had elevated plasma AVP concentration and disturbed water balance, both of which probably were provoked by an increased transepidermal water loss.

Effects of essential fatty acid deficiency and indomethacin on histologic, ultrastructural, and phagocytic responses of hepatic macrophages to glucan.

Glucan administration in the rat induces a hyperplasia and hypertrophy of the reticuloendothelial system (RES) and a concomitant leukocytosis. Increased phagocytic function and lysozymal immunoreactivity of macrophages are also characteristic of the glucan effect. The potential role of arachidonic acid metabolites in mediating this hepatic inflammatory response induced by the RES stimulant glucan was assessed in the present study by two experimental approaches. In one study, rats were depleted of arachidonic acid by rendering them deficient in essential fatty acids (EFA). In another study, rats were pretreated with the fatty acid cyclooxygenase inhibitor indomethacin. Both treatment interventions markedly attenuated the hepatic Kupffer cell proliferative and granulomatous response to glucan and the associated leukocytosis. Lysozyme immunoreactivity of the Kupffer cells and rates of colloidal carbon clearance (T/2), however, were enhanced by the above treatments. Supplementation of EFA-deficient rats with ethyl arachidonate restored their glucan response to an extent that was not significantly different from nondeficient rats. Marked hepatic proliferative responses were apparent only in those treatment groups characterized by leukocytosis, which suggests that extrahepatic recruitment is an important component of the glucan response in normal, nutritionally adequate rats. Collectively these data suggest that arachidonic acid metabolites may play a role in modulating this extrahepatic recruitment and the associated cellular proliferative and granulomatous responses following glucan administration of the rat.

The Effect of a Single Oral Dose of Ethyl Linoleate on Urinary Prostaglandin E2 Excretion in Essential Fatty Acid-Deficient Rats

The effects of a single oral dose of ethyl linoleate on urinary prostaglandin E2 (PGE2) excretion and urine output were investigated in essential fatty acid (EFA)-deficient rats. Weanling male rats were fed a fat-free diet. After 13 wk of feeding, eight rats received an oral dose of 400 mg of ethyl oleate. Seven days later the same eight rats received 400 mg of ethyl linoleate. The oleate dosage served as control. Another seven EFA-deficient rats received an oral dose of 100 mg of ethyl linoleate. The 24-h urine collections from each animal were analyzed for PGE2 by radioimmunoassay. Within 24 h the oleate dose resulted in a 1.3-fold increase in urinary PGE2 excretion. The 400-mg dose of ethyl linoleate induced a 2- to 3-fold increase in urinary PGE2 excretion during 7 d. The 100-mg dose of ethyl linoleate resulted in a 1.3- to 1.5-fold increase for 2 d. Neither the ethyl oleate dose nor the low dose of ethyl linoleate had any effect on urine output, whereas the high dose of ethyl linoleate induced a slow increase during the following 6 d. The present results show that a single dose of 400 mg of ethyl linoleate increased urinary PGE2 excretion and urine output in EFA-deficient rats. However, these two parameters do not seem to be correlated. The amount of urinary PGE2 excreted in excess of baseline urinary PGE2 excretion accounted on a molar basis for less than 1 ppm of the administered dose of ethyl linoleate.

The metabolic transformations of columbinic acid and the effect of topical application of the major metabolites on rat skin.

The metabolism of columbinic acid by various fatty acid oxidizing enzyme systems was studied. A cyclooxygenase product, 9-hydroxy-(5E,10E,12Z)-octadecatrienoic acid, was formed nearly quantitatively by ram seminal vesicle microsomes and in small amounts by washed human platelets. The major lipoxygenase product from washed human platelets, soybean lipoxygenase, and neonatal rat epidermal homogenate was 13-hydroxy-(5E,9Z,11E)-octadecatrienoic acid, although lesser quantities of other isomers differing in the double bond configurations were also identified by ultraviolet spectrophotometry and gas chromatography-mass spectroscopy. Topical application of the major lipoxygenase product to paws of essential fatty acid-deficient rats resulted in nearly as complete resolution of the scaly dermatitis as did the application of columbinic acid itself; the cyclooxygenase product was not at all effective.

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.

The Role of Essential Fatty Acids and Prostaglandins in Cercarial Penetration (Schistosoma mansoni)

We used rat skin membranes to test the putative role of prostaglandins (PG) and essential fatty acids (EFA) in the penetration response of Schistosoma mansoni cercariae. To examine the effects of EFA on cercarial penetration an EFA-deficient rat model was used. Dams were fed an EFA-deficient diet during lactation and the pups were weaned to this diet. Cercarial penetration of EFA-deficient rat skin membranes was not reduced from control levels until 12 wk on the diet. At this time a decrease of 64.3% was observed. This decrease remained constant up to 16 wk, after which the study was terminated. Other normal rats were treated with 20 mg/kg ibuprofen, a PG inhibitor, to examine the role of PG in the penetration response. Treated rat skin contained a mean of 2 micrograms ibuprofen per 30 mm3 of skin (25-mm skin disc) at 1.5 hours post-injection. Skin from treated rats inhibited penetration by over 81%. These studies indicate that skin EFA and PG may have a critical role in the completeness of penetration by cercariae through the skin, although it is not clear whether cercarial or host PG are involved in the penetration response.

Kinin effects on chloride secretion do not require eicosanoid synthesis.

The actions of bradykinin on colonic epithelia from essential fatty acid-deficient (EFAD) rats has been examined. Electrogenic chloride secretion as short circuit current (SCC) and release of immunoreactive prostaglandin E2 (iPGE2) and i 6-keto PGF1 alpha have been measured. Resting release of prostanoids was significantly less in EFAD than in control tissues. Bradykinin, in a maximally effective concentration, produced no increase in prostanoid release in EFAD tissues in contrast to controls, while the SCC response was 55% of that in controls. In EFAD tissues the SCC response to bradykinin was the same whether or not the cyclooxygenase inhibitor piroxicam was present. EFAD tissues were not more sensitive to prostaglandins than control tissues. We conclude that while prostaglandin release contributes to the totality of the response to bradykinin, the latter's effect on electrogenic chloride secretion does not require the obligatory production of arachidonic acid metabolites.

Effects of Dietary Vitamin E on Serum and Pulmonary Fatty Acids and Prostaglandins in Rats Fed Excess Linoleic Acid

The interrelationships of dietary vitamin E and essential fatty acids and their effects on serum and pulmonary prostaglandin (PG) synthesis and fatty acid precursors were examined. In a preexperimental period, male weanling rats were depleted of essential fatty acids (EFA) by feeding on a hydrogenated coconut oil diet. At the end of 45 days, average serum triene:tetraene ratio for the EFA-deficient rats was 0.76. After a refeeding period with a 20% safflower oil diet and 0, 1 or 50 mg of dl-α-tocopheryl acetate daily, serum and pulmonary fatty acid profiles and PG synthesis were determined. A trend to growth depression on the high vitamin E diet was observed. Vitamin E supplementation seemed to have no significant effect on fatty acid composition or synthesis of PGE1, PGE2, PGF2α or PGI2 in lung. This may be due to the small lipid content and presumed inability of lung to accumulate excess vitamin E. Lung may, therefore, be resistant to such dietary manipulations. Serum PG synthesis was not affected by vitamin E dose, although the C20:4ω6/C18:2ω6 ratio in serum was significantly lowered on the high vitamin E diet.

The Effect of Essential Fatty Acid Deficiency on Basal Respiration and Function of Liver Mitochondria in Rats

Rats were fed a diet poor (0.05%) in essential fatty acids (EFA) with hydrogenated coconut oil as fat component, or a control diet containing 4% of the total energy intake in the form of linoleic acid. Effects of dietary EFA deficiency were investigated during a period of 2–30 weeks. Growth retardation becomes significant after 4 weeks of deficiency and attains about 25% when the deficiency is maintained for longer than 12 weeks. Respiration, body weight and age of EFA-deficient rats and controls are in a nonlinear relationship. Basal respiration in relation to the body weight is significantly increased by EFA deficiency; it is unchanged when related to total animals under the employed experimental conditions. Oxidative phosphorylation in isolated liver mitochondria is unaffected by EFA deficiency, i.e., the increased metabolic rate of EFA-deficient rats, related to body weight, cannot be explained from impaired functional integrity of the inner mitochondrial membrane. Respiratory chain enzyme activities in mitochondria from heart and skeletal muscle and specific amounts of mitochondria in these tissues are unchanged by EFA deficiency.

Humoral Immunity in Essential Fatty Acid-Deficient Rats and Mice: Effect of Route of Injection of Antigen

Weanling male Lewis Mai F rats and A/J mice were fed semi-purified diets either adequate or deficient in essential fatty acids (EFA) for 50–60 days. After death livers were excised, the lipids were extracted, and the fatty acid profile was determined. Groups of rats and mice were immunized by injection with sheep red blood cells (sRBC) either i.v. or i.p. One group of rats received an injection of sRBC plus Bordetella pertussis organisms. The plaque-forming cell response (PFC) of all groups was determined. Samples of mouse spleens were analyzed for prostaglandin F2α. EFA-deficient rat and mice liver fatty acid profiles showed elevated levels of ω7 and ω9 fatty acids and decreased ω6 fatty acids. The fatty acid profiles of mice differed quantitatively from the rats. As determined by the ratios of 18:0 to 18:2ω6 and 18:2ω6 to 20:4ω6 mice showed a higher Δ9 and a lower Δ6 desaturase activity. When the antigen was injected i.v. the EFA-deficient animals of both species showed an increased PFC response compared to controls, but when it was injected i.p. there was no difference between dietary groups. The PFC response in rats receiving B. pertussis increased dramatically but the difference between dietary groups was abrogated. As had been previously shown in rats the increase in PFC response in the mice immunized by the i.v. route correlated with a decreased synthesis of PGF2α by the spleen.

The potential role of thromboxane and prostacyclin in endotoxic and septic shock

The potential role of thromboxane (TxA 2), a platelet aggregator and vasoconstrictor, and prostacyclin (PGI 2) a platelet anti-aggregator and vasodilator, in endotoxic and septic shock was investigated. Early endotoxic shock in the rat is associated with marked elevations of plasma TxB 2 (the stable metabolite of TxA 2) and lesser increases in plasma 6-keto-PGF 1α (the stable metabolite of PGI 2). Selective inhibition of TxA 2 synthesis by several different chemical classes of Tx synthetase inhibitors was beneficial in endotoxic shock. In contrast, shock induced by acute intra-abdominal sepsis in the rat was characterized by high levels of plasma 6-keto-PGF 1α, which exceeded plasma TxA 2 six- to eight fold at most time intervals studied. Tx synthetase inhibitors were not protective in this model of acute sepsis, but treatment with fatty acid cyclo-oxygenase inhibitors, an antibiotic (gentamicin), or reduction in arachidonic acid metabolism by essential fatty acid (EFA) deficiency significantly prolonged survival time. An important aspect of the latter study is that decreased arachidonic acid metabolism was an effective adjunct to antibiotic therapy. Conjoint administration of gentamicin in EFA-deficient rats or with indomethacin synergistically improved long-term survival, a result that was not evident with single treatment interventions. In addition to experimental studies, plasma TxB 2 levels were measured during clinical sepsis. These studies demonstrated that plasma TxB 2 levels were elevated tenfold in patients dying of septic shock compared with septic survivors or nonseptic controls. These composite experimental and clinical observations suggest that arachidonic acid metabolites play a role in the pathogenesis of endotoxic and septic shock.