Arachidonic acid,prostaglandin E2 and leukotriene C4 levels in gingiva and submandibular salivary glands of rats fed diets containing n−3 fatty acids

The effect of dietary n−3 fatty acids on prostaglandin E₂ (PGE₂) and leukotriene C₄ (LTC₄) levels in rat salivary glands and gingiva was examined in two separate nutritional studies. In the first set of experiments, two groups of male weanling Sprague-Dawley rats were fed semipurified diets containing 10% corn oil (control group) or 10% menhaden oil (experimental group). Rats were killed after 8 wk on the diets; the fatty acid composition of total phospholipids and the concentrations of PGE₂ and its precursor, arachidonic acid, were measured in gingiva and submandibular salivary glands (SMSG). Dietary n−3 fatty acids were incorporated into the tissue phospholipids. Arachidonic acid levels were reduced by 56% in gingiva and SMSG of rats fed menhaden oil compared with the control rats fed the diet containing corn oil. The concentrations of PGE₂ in SMSG and gingiva of rats fed the diet containing menhaden oil were reduced by 74% and 83%, respectively. In a subsequent nutritional study, we tested whether the diet-induced reduction in tissue arachidonic acid levels would also result in a corresponding decrease in LTC₄ production. Three groups of rats were fed diets containing 5% corn oil (group 1), 4% ethyl ester concentrate of n−3 fatty acids plus 1% corn oil (group 2), or 5% ethyl ester concentrate of n−3 fatty acids (group 3). After 6 wk of feeding, gingiva and SMSG were analyzed for arachidonic acid content andin vitro production of LTC₄. Arachidonic acid content of total phospholipids was about 60% lower in gingiva and 69% lower in SMSG of rats fed the ethyl ester concentrate of n−3 fatty acids (groups 2 and 3) than those of the control group fed the corn oil diet (group 1). Upon incubation with calcium ionophore, gingiva and SMSG from rats fed the n−3 fatty acids rich diet produced significantly less TLC₄ than those from rats of the control group. Because PGE₂ and LTC₄ are believed to be important biochemical mediators of periodontal disease, one may speculate that a diet-induced reduction in their levels may have a beneficial effect upon the course of the disease. The function of salivary glands may also be altered because of the role of these eicosanoids in salivary secretions. Presented in part for the Hatton Award Competition at the American Association for Dental Research Meeting, San Francisco, California, March 15–19, 1989, and at the International Association for Dental Research Meeting, Acapulco, Mexico, April 17–21, 1991.     

Urinary prostaglandin E2 and vasopressin excretion in essential fatty acid-deficient rats: Effect of linolenic acid supplementation

Three groups of weanling male rats were fed on a fat-free diet for 13 weeks. One group received only the fat-free diet (FF rats), the other 2 groups received the fat-free diet and a daily supplement of 2 energy% ethyl linoleate ([n−6] rats), or 2 energy% ethyl linolenate ([n−3] rats). Urinary excretion of prostaglandin E₂ (PGE₂), immunoreactive arginine vasopressin (iA VP), and kallikrein were determined. PGE₂ was quantitated with a radioimmunoassay having 4.9% cross-reactivity with prostaglandin E₃ (PGE₃). After 4 weeks on the diet, water consumption and urinary iAVP excretion increased significantly in the FF rats and the (n−3) rats compared with the (n−6) rats. Urinary PGE₂ excretion was the same for all 3 groups during the first 10 weeks; thereafter it decreased in FF rats and (n−3) rats compared with the (n−6) rats. There was no difference in urinary PGE₂ excretion between the FF rats and the (n−3) rats, even though large differences were found in the percentage of arachidonic acid (20∶4[n−6]), icosapentaenoic acid (20∶5[n−3]), and icosatrienoic acid (20∶3[n−9]) of total kidney fatty acids as well as of kidney phosphatidylinositol fatty acids. Fractionation of urine extracts on high performance liquid chromatography with radioimmunoassay detection indicated that (n−3) rats excreted very little PGE₃, if any. Urine output followed the same pattern, as did urinary PGE₂ excretion. Urinary kallikrein was estimated at week 12 only. It was found to be significantly lower in FF rats and (n−3) rats. Increased water consumption and increased urinary iAVP excretion seem to be early symptoms (after 4 weeks) of EFA deficiency, whereas decreased urine output and decreased urinary PGE₂ excretion occur much later (after 10 weeks). Two energy% linolenate supplementation to a fat-free diet did not change the appearance of any of the measured EFA-deficiency symptoms except for a slightly improved growth rate. There was no evidence of a significant urinary PGE₃ excretion in spite of an extreme enrichment of kidney lipids with 20∶5(n−3). It is suggested that urinary PGE₂ is derived from precursors delivered from an arachidonic acid pool, which is rather resistant to restriction in dietary linoleate. Presented in part at the 23rd International Conference on Biochemistry of Lipids, Nyborg, Denmark, August 1981, and at the Vth International Conference on Prostaglandins, Florence, Italy, May 1982.     

Conjugated linoleic acids alter bone fatty acid composition and reduce ex vivo prostaglandin E2 biosynthesis in rats fed n-6 or n-3 fatty acids

This study evaluated the effects of conjugated linoleic acids (CLA) on tissue fatty acid composition and ex vivo prostaglandin E₂ (PGE₂) production in rats given diets varying in n-6 and n-3 fatty acids. Four groups of rats were given a basal semipurified diet (AIN-93G) containing 70 g/kg of added fat for 42 d. The fat treatments were formulated to contain CLA (0 vs. 10 g/kg of diet) and n-6 (soybean oil having an n-6/n-3 ratio of 7.3) and n-3 fatty acids (menhaden oil+safflower oil having an n-6/n-3 ratio of 1.8) in different ratios in a 2×2 factorial design. Fatty acids in liver, serum, muscle, heart, brain, spleen, and bone (cortical, marrow, and periosteum) were analyzed by capillary gas-liquid chromatography. The various dietary lipid treatments did not affect growth; however, CLA improved feed efficiency. The CLA isomers were found in all rat tissues analyzed although their concentrations varied. Dietary CLA decreased the concentrations of 16∶1n−7, 18∶1, total monounsaturates and n−6 fatty acids, but increased the concentrations of n−3 fatty acids (22∶5n−3 and 22∶6n−3), and saturates in the tissues analyzed. Ex vivo PGE₂ production in bone organ culture was decreased by n−3 fatty acids and CLA. We speculate that CLA reduced the concentration of 18∶1 fatty acids by inhibiting liver Δ9-desaturase activity. The fact that CLA lowered ex vivo PGE₂ production in bone organ culture suggests that these conjugated fatty acids have the potential to influence bone formation and resorption.     

Effects of different polunsaturated fatty acids on growth-related early gene expression and cell growth

Effects of polyunsaturated fatty acids on expression of early-response genesc-fos and Egr-1 and induction of cell growth were assessed in Swiss 3T3 fibroblasts. Stimulation with arachidonic acid increased mRNA levels ofc-fos and Egr-1. This effect was inhibited by preincubation with cyclooxygenase inhibitors and restored by addition of prostaglandin E₂ (PGE₂), the predominant eicosanoid produced in Swiss 3T3 fibroblasts. Further signaling of PGE₂ was mediated by a protein kinase C-dependent pathway, since downregulation, or inhibition, of protein kinase C reduced increases in mRNA levels. Parallel to the stimulatory effects on mRNA levels, AA and PGE₂ also increased cell growth, as determined by uptake of [³H]-thymidine. In contrast to arachidonic acid, n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) did not increasec-fos and Egr-1 mRNA levels or cell growth. Furthermore, preliminary data indicate that EPA and DHA even reduce the stimulatory effect of AA, which is associated with reduced formation of PGE₂. In conclusion, our data indicate that AA increases expression of growth-related early genesc-fos and Egr-1 in Swiss 3T3 fibroblasts by its conversion to PGE₂ and subsequent activation of protein kinase C, whereas n-3 fatty acids do not activate this signaling cascade.     

Influence of medium-chain triglycerides on lipid metabolism in the rat

Lipid metabolism was studied in rats fed diets containing corn oil, coconut oil, or medium-chain triglyceride (MCT), a glyceride mixture containing fatty acids of 8 and 10 carbons in length. The ingestion of MCT-supplemented, cholesterolfree diets depressed plasma and liver total lipids and cholesterol as compared with corn oil-supplemented diets. In rats fed cholesterol-containing diets, plasma cholesterol levels were not influenced by dietary MCT, but liver cholesterol levels were significantly lower than in animals fed corn oil. In vitro cholesterol synthesis from acetate-1-¹⁴C was lower in liver slices of rats that consumed MCT than in similar preparations from corn oil-fed rats. Studies of fatty acid carboxyl labeling from acetate-1-¹⁴C and the conversion of palmitate-1-¹⁴C to C₁₈ acids by liver slices showed that chain-lengthening activity is greater in the liver tissue of rats fed MCT than in the liver of animals fed corn oil. The hepatic fatty acid desaturation mechanisms, evaluated by measuring the conversion of stearate-2-¹⁴C to oleate, was also enhanced by feeding MCT. Adipose tissue of rats fed MCT converts acetate-1-¹⁴C to fatty acids at a much faster rate than does tissue from animals fed corn oil. Evidence is presented to show that the enhanced incorporation of acetate into fatty acids by the adipose tissue of rats fed MCT represents de novo synthesis of fatty acids and not chain-lengthening activity. Data are also presented on the fatty acid composition of plasma, liver, and adipose tissue lipids of rats fed the different fats under study.     

Essential fatty acid supplemented diet increases renal excretion of prostaglandin E2 and water in essential fatty acid deficient rats

Weanling male rats were fed an essential fatty acid (EFA)-deficient diet for 25 weeks and then switched to an EFA-supplemented diet for 3 weeks. Control rats received the EFA-supplemented diet for 25 weeks and then the EFA-deficient diet for 3 weeks. Throughout the last 19 weeks, the rats were housed in metabolic cages once a week for a 24-hr period. Urinary excretion of prostaglandin E₂ (PGE₂) was estimated by radioimmunoassay. Throughout a period of 12 weeks (weeks 13–24) water consumption increased ca. 60%, and urine output and PGE₂ excretion decreased ca. 45% and 70%, respectively, in the EFA-deficient rats. Feeding EFA-supplemented diet to the EFA-deficient rats for 3 weeks decreased the water consumption and raised the urine output to that observed in the controls. However, the urine output was corrected within 1 day whereas the water consumption was not corrected until the second measurement 8 days after the dietary change. The PGE₂ excretion increased more than 9-fold (from 18±8 ng/24 hr to 165±51 ng/24 hr) 1 day after EFA-supplementation, followed by a decrease to 86±29 ng/24 hr over the following 2 weeks. On the basis of the present data, it is suggested that EFA deficiency in rats causes diminished PGE₂ excretion, which can be normalized by EFA supplementation. The normalization of the urine flow may, in part, be caused by the concomitant considerable increase in endogenous PGE₂ synthesis. Presented in part at the International Symposium on Prostaglandins and the Kidney, Stuttgart, West Germany, July 1980.     

Dietary n-3 and n-6 PUFA Enhance DHA Incorporation in Retinal Phospholipids Without Affecting PGE<Subscript>1</Subscript> and PGE<Subscript>2</Subscript> Levels

The purpose of this study was to determine whether dietary n-3 and n-6 PUFA may affect retinal PUFA composition and PGE₁ and PGE₂ production. Male Wistar rats were fed for 3 months with diets containing: (1) 10% eicosapentaenoic acid (EPA) and 7% docosahexaenoic acid (DHA), or (2) 10% γ-linolenic acid (GLA), or (3) 10% EPA, 7% DHA and 10% GLA, or (4) a balanced diet deprived of EPA, DHA, and GLA. The fatty acid composition of retinal phospholipids was determined by gas chromatography. Prostaglandin production was measured by enzyme immunoassay. When compared to rats fed the control diet, the retinal levels of DHA were increased in rats fed both diets enriched with n-3 PUFA (EPA + DHA and EPA + DHA + GLA diets) and decreased in those supplemented with n-6 PUFA only (GLA diet). The diet enriched with both n-6 and n-3 PUFA resulted in the greatest increase in retinal DHA. The levels of PGE₁ and PGE₂ were significantly increased in retinal homogenates of rats fed with the GLA-rich diet when compared with those of animals fed the control diet. These higher PGE₁ and PGE₂ levels were not observed in animals fed with EPA + DHA + GLA. In summary, GLA added to EPA + DHA resulted in the highest retinal DHA content but without increasing retinal PGE₂ as seen in animals supplemented with GLA only.     

Production of prostaglandins in homogenates of kidney medullae and cortices of spontaneously hypertensive rats fed menhaden oil

Menhaden oil (MO), whose polyunsaturated fatty acids consist mainly of (n−3) fatty acids, was fed to spontaneously hypertensive rats to determine the effect of (n−3) fatty acid on the in vitro production of prostaglandins produced from arachidonic acid (20∶4[n−6]). Capacity to form PGE₂ and PGF_2α was impaired in homogenates of kidney medullae and cortices from rats fed the MO diet compared to rats fed the control diet. The lower amounts of diene prostaglandins produced corresponded to the decrease in the amount of 20∶4 (n−6) in the tissue. Possibly changes produced in tissue lipids by dietary fatty acids affect prostaglandin production by reducing the availability of substrate in tissue lipids.     

Eicosanoid synthesis in 7,12-dimethylbenz(a)anthracene-induced mammary carcinomas in Sprague-Dawley rats fed primrose oil,menhaden oil or corn oil diet

The comparative effects of high-fat diets (20%, w/w) on eicosanoid synthesis during mammary tumor promotion in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rats were studied using diets containing 20% primrose oil (PO), 20% menhaden oil (MO) or 20% corn oil (CO). Sprague-Dawley rats fed the PO or MO diet had 21% or 24% fewer adenocarcinomas, respectively, than rats fed the CO diet. Histologically (i.e., mitotic figures, inflammatory cell infiltration and necrosis), the CO-fed rats exhibited the highest frequency of changes within tumors. Plasma fatty acid composition was significantly altered by diet, reflecting the composition of the oils which were being fed. Only the plasma of PO-fed rats contained detectable levels of gamma-linolenic acid (GLA). Arachidonic acid (AA) levels were significantly higher (p<0.05) in PO-fed than in CO- or MO-fed rats. MO-fed rats had significantly higher levels of plasma palmitic acid, while palmitoleic, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids were detected only in MO-fed rats. As expected, linoleic acid (LA) and AA levels were lower (p<0.05) in the MO-fed rats than in PO- or CO-fed groups. The plasma of the CO-fed rats contained significantly higher levels of oleic acid. Eicosanoid synthesis in mammary carcinomas of rats fed the 20%-fat diets was 2–10 times higher than in mammary fat pads of control rats. The synthesis of PGE₁ and LTB₄ was significantly (p<0.05) higher in PO-fed rats than in CO-fed or MO-fed rats, although PGE values were significantly (p<0.05) higher in CO-fed rats than in Mo or PO groups. The synthesis of eicosanoids in both mammary fat pads and mammary carcinomas of MO-fed rats was lower (p<0.05) than in tissues of rats fed either CO or PO diets due to less AA precursor being fed and/or to competition between n−6 and n−3 fatty acids for cyclooxygenase and lipoxygenase. The ratios of monoenoic to dienoic eicosanoids in both mammary fat pads and mammary carcinomas were higher in the PO group than in the MO or CO groups. These results suggest that inclusion of GLA (PO feeding) or EPA and DHA (MO feeding) in the diet may decrease malignancy by altering eicosanoid profiles.     

Fatty acid-specific, regiospecific, and stereospecific hydroxylation by cytochrome P450 (CYP152B1) from Sphingomonas paucimobilis: Substrate structure required for α-hydroxylation

Fatty acid α-hydroxylase from Sphingomonas paucimobilis is an unusual cytochrome P450 enzyme that hydroxylates the α-carbon of fatty acids in the presence of H₂O₂. Herein, we describe our investigation concerning the utilization of various substrates and the optical configuration of the α-hydroxyl product using a recombinant form of this enzyme. This enzyme can metabolize saturated fatty acids with carbon chain lengths of more than 10. The K _m value for pentadecanoic acid (C₁₅) was the smallest among the saturated fatty acids tested (C₁₀–C₁₈) and that for myristic acid (C₁₄) showed similar enzyme kinetics to those seen for C₁₅. As shorter or longer carbon chain lengths were used, K _m values increased. The turnover numbers for fatty acids with carbon chain lengths of more than 11 were of the same order of magnitude (10³ min⁻¹), but the turnover number for undecanoic acid (C₁₁) was less. Dicarboxylic fatty acids and methyl myristate were not metabolized, but monomethyl hexadecanedioate and ω-hydroxypalmitic acid were metabolized, though with lower turnover values. Arachidonic acid was a good substrate, comparable to C₁₄ or C₁₅. The metabolite of arachidonic acid was only α-hydroxyarachidonic acid. Alkanes, fatty alcohols, and fatty aldehydes were not utilized as substrates. Analysis of the optical configurations of the α-hydroxylated products demonstrated that the products were S-enantiomers (more than 98% enantiomerically pure). These results suggested that this P450 enzyme is strictly responsible for fatty acids and catalyzes highly stereo- and regioselective hydroxylation, where structure of ω-carbon and carboxyl carbon as well as carbon chain length of fatty acids are important for substrate-enzyme interaction.     

Antimicrobial agents derived from fatty acids

The author reviews his research, since 1966, for the ideal germicide. The relationship between structure of fatty acids, their corresponding esters, and antimicrobial activity is presented. Saturated fatty acids have their highest activity when the chain length is twelve carbons (C₁₂) long; monounsaturated fatty acids reach their peak with palmitoleic acid (C_16∶1); the most active polyunsaturated fatty acid is linoleic.Trans isomers are not active against microorganisms. The esterification of fatty acids to monohydric alcohols leads to inactive derivatives, whereas esterification to polyhydric alcohols increases biological activity. Examples of glycerol and sucrose esters are reviewed. In general, the lauroyl derivatives are the most active. A few examples of esters as active pharmacological agents against organisms causing bovine mastitis are presented as well as the use of monolaurin (Lauricidin^®) as cosmetic and food preservatives. The safety and efficacy of fatty acid esters as potential germicides offer new and expanded roles for oleochemicals.     

Identification and quantification of prostaglandin E3 in renal medullary tissue of three strains of rats fed fish oil

Three strains of rats were fed a fish oil diet to verify their ability to incorporate and convert dietary eicosapentaenoic acid (20∶5ω3) into trienoic prostaglandins. Our results show that such conversion indeed occurs in kidney medullae homogenates. Specifically, the presence of prostaglandin E₃ (PGE₃) was established by gas chromatographic-mass spectrometric (GC-MS) analysis. That compound was conclusively identified by comparison of fragment ions and their relative intensities with those obtained from authentic PGE₃. Further evidence was provided by studying the recovery of exogenously added PGE₃. The crude ethyl acetate extracts of the medullary homogenates were methylated and cleaned up by liquid-gel chromatography with Lipidex-5000 prior to conversion to PGB₃ for GC-MS analysis. The PGE₃ was quantified by selected ion monitoring (SIM) with [3,3,4,4-²H₄PGE₂ as internal standard. The levels of PGE₃ were similar, about 3 ng/mg of wet tissue, in the 3 strains of rats. Identical in vivo conversion of the 20∶5ω3 fatty acid to PGE₃ could not be positively established by analysis of pooled urine specimens.     

Spoilage of coconut oil purification and properties of a fungal lipase that attacks coconut oil

A lipase was isolated from a strain ofAspergillus flavus which attacked coconut kernel and oil with the liberation of free fatty acids. The enzyme was purified 109-fold by ammonium sulphate precipitation, diethyl aminoethyl-cellulose and Sephadex G-200 chromatography. The optimum pH of the enzyme reaction was 6.2. The action of the enzyme on pure triglycerides was studied. The triglycerides of the shorter chain fatty acids were more rapidly hydrolyzed, while hydrolysis of tristearin was not detected under the conditions of assay. K _m for trilaurin and trimyristin were 9.09×10⁻⁴ and 1.42×10⁻³ M, respectively. Para-chloromercuricbenzoate was an inhibitor. Thin layer chromatography and gas liquid chromatography of the esterified products of enzymatic hydrolysis of coconut oil showed the presence of oleate, palmitate, myristate, laurate, caprate, caproate and caprylate but not stearate, although stearate was present in coconut oil.     

Relative utilization of fatty acids for synthesis of ketone bodies and complex lipids in the liver of developing rats

The regulation of hepatic ketogenesis, as related to the metabolism of fatty acids through oxidative and synthetic pathways, was studied in developing rats. [1-¹⁴C] palmitate was used as a substrate to determine the proportions of free fatty acids utilized for the production of ketone bodies, CO₂ and complex lipids. Similar developmental patterns of hepatic ketogenesis were obtained by measuring the production of either [¹⁴C]acetoacetate from exogenous [1-¹⁴C] palmitate or the sum of unlabeled acetoacetate and β-hydroxybutyrate from endogenous fatty acids. The production of total ketone bodies was low during the late fetal stage and at birth, but increased rapidly to a maximum value within 24 hr after birth. The maximal ketogenic capacity appeared to be maintained for the first 10 days of life.¹⁴CO₂ production from [1-¹⁴C] palmitate increased by two- to fourfold during the suckling period, from its initial low rate seen at birth. The capacity for synthesis of total complex lipids was low at birth and had increased by day 3 to a maximal value, which was comparable to that of adult fed rats. The high lipogenic capacity lasted throughout the remaining suckling period. When ketogenesis was inhibited by 4-pentenoic acid, the rate of synthesis of complex lipids did not increase despite an increase in unutilized fatty acids. During the mid-suckling period, approximately equal amounts of [1-¹⁴C] palmitate were utilized for the synthesis of ketone plus CO₂ and for complex lipid synthesis. By contrast, in adult fed rats, the incorporation of fatty acids into complex lipids was four times higher than that of ketone plus CO₂. These observations suggest that stimulated hepatic ketogenesis in suckling rats results from the rapid oxidation of fatty acids and consequent increased production of acetyl CoA, but not from impaired capacity for synthsis of complex lipids.     

Prostaglandin and acyl chain effects on glutamate dehydrogenase activity

Prostaglandins A₁ (PGA₁), A₂, B₁, B₂, E₁, E₂, F_1α, F_2α, and 19 esterified natural fatty acids were tested as effectors of beef liver glutamate dehydrogenase (L-glutamate: NAD(P)⁺, oxidoreductase [deaminating], EC 1.4.1.3). All prostaglandins tested are found to activate the enzyme initially, but only PGA₂>PGB₂≥PGA₁ cause a subsequent time-dependent loss (not inhibition) of NADH oxidation activity. Both PGA₁ and PGA₂ desensitize glutamate dehydrogenase to allosteric activation by ADP, whereas PGA₂ and PGB₂ desensitize to allosteric inactivation by GTP. Preincubation of enzyme with diethylstilbestrol prevents the initial activation by the PG. Of the methyl esters, only prostaglandin precursors inactivated the enzyme. Simultaneous desensitization to the ADP and GTP allosteric effects resulted. Multiple esterification to glycerol or phospholipids enhanced the action of linoleoyl and diminished the action of linolenoyl chains. Preincubation of the PGA with glutathione or cysteine prevents the inactivation; i.e., the sulfhydryl binding region of the prostaglandin must be free for enzyme to be inactivated. Sulfhydryl reagents also protect the enzyme from the effects of the unsaturated acyl chains, and pHMB mimics acyl protection against GTP allosteric inactivation. Where the lipid effector is active against sulfhydryl groups, the desensitizations to the ADP and GTP allosteric effectors are reciprocal. The initial activation, subsequent inactivation and desensitization to ADP and GTP are all characteristic of binding in the estrogen-specific effector site, suggesting this site as the target for PG and acyl action. In the PGA₂ activation, the effect is found to be amplified by the cooperativity of the enzyme at 1 PG molecule/6 molecules of GDH. We conclude from the action of the PG and structural analogs that the initial activation of glutamate dehydrogenase is caused by α,β-unsaturated monoketo cyclopental structures. GTP inhibition is blocked primarily by diketo structures which eventually inactivate the enzyme. ADP activation is blocked by sulfhydryl binding of the unsaturated cyclopental keto structure of the PG. Appearance of a 270 nm absorbance simultaneous to the acyl effects on the enzyme suggests that conjugated unsaturations are responsible for the precursor's qualitatively similar action to that of the PG.     

Polyunsaturated fatty acids,vitamine E,and the proliferation of aortic smooth muscle cells

Smooth muscle cell cultures were obtained from the aortas of prepubertal guinea pigs. Cell proliferation in these cultures was inhibited by 8,11,14-eicosatrienoic acid, 5,8,11,14-eicosatetraenoic acid, and their prostaglandin E derivatives, PGE₁ and PGE₂. Prostaglandin F derivatives, PGF_1α and PGF_2α, stimulated cell proliferation. Cell proliferation was also inhibited by 5,8,11-eicosatrienoic acid and 11,14,17-eicosatrienoic acid. The monoene and diene precursors of the triene acids, 9-octadecenoic acid and 9,12-octadecadienoic acid, did not inhibit cell, proliferation. Indomethacin alone had no effect on cell proliferation, and indomethacin did not suppress the inhibition of cell proliferation with a triene acid. The antioxidant α-naphthol alone stimulated cell proliferation and suppressed prostaglandin E formation. α-Naphthol in the presence of either triene or tetraene acids also stimulated cell proliferation and suppressed prostaglandin E formation. The antioxidants butylated hydroxy toluene and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid either alone or in the presence of triene and tetraene acids stimulated cell proliferation and had no effect on prostaglandin E formation. Vitamin E either alone or in the presence of triene or tetraene acids stimulated cell proliferation and had no effect on prostaglandin E formation. More prostaglandin E was formed from 8,11,14-eicosatrienoic acid than from 5,8,11,14-eicosatetraenoic acid in the presence of antioxidants. Vitamin E suppressed the inhibitory effects of both PGE₂ and palmitic acid on cell proliferation. The cyclic nucleotide phosphodiesterase inhibitors, caffeine and papaverine, suppressed the stimulatory effect of vitamin E on cell proliferation and enhanced the inhibitory effect of a triene acid on cell proliferation. Substrate and inhibitor specificities are consistent with the oxidative regulation of cell proliferation through the formation of hydroperoxy fatty acids. We propose that hydroperoxy fatty acids may regulate both cyclase and cyclic nucleotide phosphodiesterase enzymes through sulfhydryl-disulfide interconversions. We suggest that this regulatory mechanism may help to explain the acculation of 5,8,11-eicosatrienoic acid in essential fatty acid deficiency, the effects of antioxidants on cell proliferation, and one of the several effects of polyunsaturated fatty acids in proliferative disorders such as cancer and atherosclerosis.     

Comparison of dialysis,thin layer and silicic acid column chromatography for prostaglandin isolation from biological material

Chemical and biochemical methods assaying the total content of neutral lipids during some conventional procedures for purification and isolation of prostaglandins from biological fluids revealed all fractions to be contaminated with fatty acids, cholesterol, cholesterol esters, tri-, di- and/or mono-glycerides. It was demonstrated that it was not possible to make pure prostaglandin fractions by simple dialysis. On the basis of these findings a new preparative schedule involving either dialysis or column chromatography on silicic acid, combined with preparative thin layer chromatography, made it possible to isolate substantially pure PGE₂ from a synthetic biological system involving arachidonic acid as a precursor and the microsomal synthesizing system from sheep vesicular glands. Sheep vesicular glands have been used for biosynthesis of prostaglandins (PG) (1). For the study of the metabolism of various prostaglandins, labeled PGE₁ has been prepared from 8, 11, 14-eicosatrienoic acid labeled at various positions (2). Recently a method employing arachidonic acid and sheep vesicular glands for the formation of PGE₂ has been patented. The method claims a high purity (98%) with quick recovery of PGE₂ from the incubation mixture. The essential feature of the method is the use of a dialysis step which is said to be superior to the time-consuming chromatographic procedures (3). In our recent studies on the metabolism of essential fatty acids, we have found it impossible to prepare chemically pure prostaglandins by the above mentioned methods, since cholesterol, cholesterol esters, fatty acids and glycerides were found to contaminate the final products. The present paper therefore gives some data on these contaminants, and on this basis a new approach to the isolation of chemically pure prostaglandin (PGE₂) has been developed.     

Absorption and metabolism of 1-14C-methyl linoleate hydroperoxide

The absorption and metabolism of 1-¹⁴C-methyl linoleate hydroperoxide by rats was investigated. After intubation with 2 mg of peroxide, peak¹⁴CO₂ production occurred at 90 min and 25% of the dose was expired in 24 hr. Fortyfive per cent stil remained in the gastrointestinal tract after 24 hr, most of it bound to the stomach epithelium in the form of intact peroxide. Lymph was collected from the thoracic duct 2 hr after intubation and examined for labeled metabolites. Seven per cent of the radioactivity in the lymph was present in a free 1-¹⁴C-hydroxy fatty acid and 31% in its methyl ester. Fifty-seven per cent occurred in lymph triglycerides where it was equally distributed between a 1-¹⁴C-trienoic fatty acid and an unidentified 1-¹⁴C-oxy acid. The radioactivity in liver lipids was associated mainly with randomly labeled normal fatty acids. No¹⁴C-hydroxy acids were detected in liver lipids and no evidence was obtained for the absorption of unchanged peroxide. The hydroxy and trienoic acids appear to be formed during absorption by a reduction-dehydration reaction sequence.     

Fatty acid biosynthesis and incorporation into lipid classes in seeds and seed tissues of flax

Fatty acid metabolism in developing flaxseeds was studied by incubating whole seeds or isolated seed tissues in buffered solutions of 1-¹⁴C-acetate, 2-¹⁴C-malonate and¹⁴CO₂. Lipid classes were separated by thin layer chromatography, and fatty acid labeling in phospholipids, diglycerides and triglycerides was determined by combined thin layer and gas liquid chromatographic techniques. Incorporation of¹⁴C from acetate into embryo lipids was very rapid with phospholipids and 1,2-diglycerides becoming highly labeled in treatment times as short as 5 min. Triglycerides were labeled more slowly. Phospholipid radioactivity was largely associated with the phosphatidyl choline fraction. Oleic acid had the highest specific activity of all major fatty acids in short treatment periods. This was followed in decreasing order of activity by palmitic, linoleic, stearic and linolenic acids. As the treatment period was lengthened to 90 min or longer, linoleic and linolenic activities were markedly increased. Use of malonate or CO₂ rather than acetate as the substrate increased the labeling of the saturated acids. Incorporation of¹⁴C from acetate into lipids of endosperm tissues and whole flax seeds was slower than incorporation into embryo lipids. Stearate had the highest specific activity of the fatty acids in endosperm and whole seeds. Presented in part at the AOCS Meeting in New York, October 1968.     

Isomeric monoethylenic fatty acids in herring oil

Monoethylenic fatty acids from herring oil were concentrated by chromatography by chromatography on silver nitratesilicic acid columns. Examination of consecutive fractions by open tubular gas chromatography confirmed the preferential elution of longer chain length esters and of esters within one chain length with the double bond closer to the terminal methyl group. Isomeric monoethylenic fatty acids with double bonds in the positions closer to the carboxyl group than the approximate midpoint of the even-numbered fatty acid chains could not be adequately separated by gas chromatography and were determined by ozonolysis. The isomers observed are consistent with primary formation from saturated acids through the action of an enzyme specifically removing hydrogen atoms in positions Δ⁹ and Δ¹⁰ relative to the carboxyl group. Chain extension of particular monoethylenic isomers by two carbon atoms in the C₂₀ and longer chain lengths is apparently influenced by the position of the double bond. This work was carried out in partial fulfillment of MSc requirements at Dalhousie University.