Scandinavian Cardiovascular Journal, 2013; 47: 377–382
ORIGINAL ARTICLE
Effects of trans fats on prostacyclin production
FRED A. KUMMEROW, MOHAMEDAIN MAHFOUZ, QI ZHOU & CHRISTOPHER MASTERJOHN Department of Comparative Biosciences, Burnsides Research Laboratory, College of Veterinary Medicine, University of Illinois, Urbana, IL, USA
Abstract Objectives. Prostacyclin is a prostanoid derived from arachidonic acid that prevents thrombosis and is thereby expected to protect against heart disease, while trans fats present in partially hydrogenated oils interfere with arachidonic acid metabolism. Therefore, the objective of the present study was to investigate how fats with different proportions of linoleic acid and trans-18:1 affect prostacyclin released by cultured endothelial cells, and to compare these proportions with those found in commercially available foods. Design. Soybean oil and hydrogenated soybean oil (coating fat) were mixed in different proportions to yield seven fat mixtures with proportions of linoleic acid ranging from 54.1% to 5.7% and trans-18:1 acid ranging from 0.4% to 43.9%. Human endothelial cells were cultured in each of the mixtures, and their phospholipid fractions were then separated and their fatty acids were analyzed by gas chromatography. The prostacyclin released by the cells was measured using RIA kits. Margarines and processed foods were purchased from the supermarket for comparison. Results. Our work revealed that as the percentage of trans fat was increased, the amount of prostacyclin released dose-dependently and significantly (P ⬍ 0.0001) decreased, the concentration of linoleic and arachidonic acid decreased in the membrane phospholipids while the concentration of trans 18:1 acids increased, the prostacyclin decreased by 35–98%. Supermarket margarines had levels of trans fats similar to those that suppressed prostacyclin by 35–54%. Most processed foods labeled as trans-free contained trans fats. Conclusions. Trans fatty acids suppress prostacyclin production at levels found in commercial margarines, and processed foods labeled as trans-free could contribute to this effect if consumed in multiple servings or in addition to foods containing larger amounts of trans fats. Key words: arachidonic acid, linoleic acid, prostacyclin, trans fats
Introduction Prostanoids are involved in a variety of physiological processes in atherosclerosis and thrombosis, such as leukocyte arterial cell adhesion, vasorelaxation, and platelet aggregation (1,2). Prostacyclin, which prevents blood clotting, is produced by arterial cells from arachidonic acid in all the vascular tissues (3). Platelet activation leads to the formation of thromboxane as a potent inducer of vasoconstriction and platelet adhesion. An upset of the balance between prostacyclin and thromboxane can promote thrombosis. A recent study showed that prostacyclin modulates platelets’ vascular interaction in vivo and limits the response to thromboxane (4). Free arachidonic acid released from the cell membrane phospholipids by the action of phospholipase
is metabolized to prostaglandin H2 by cyclooxygenase and peroxidase, which is in turn metabolized to prostacyclin-by-prostacyclin synthase. Therefore, the arachidonic acid level in the arterial membrane phospholipids is one of the controlling factors that regulates the amount of prostacyclin released by the arterial cell membrane. The arachidonic acid in the cell membrane is derived from the metabolic conversion of linoleic acid to arachidonic acid through the action of Δ6 and Δ5 desaturase enzymes in vivo. Because linoleic acid is an essential fatty acid, it must be provided in the diet; therefore, the level of linoleic acid in the diet will have a regulatory effect on the whole process starting from linoleic acid to arachidonic acid then to prostacyclin.
Correspondence: Dr. Fred A. Kummerow, Department of Comparative Biosciences, Burnsides Research Laboratory, College of Veterinary Medicine, University of Illinois, 1208 W. Pennsylvania Avenue, Urbana, IL 61801, USA. Tel: ⫹(217)344–6380. Fax: ⫹(217)333-7370. E-mail: [email protected] (Received 27 July 2013 ; revised 10 October 2013 ; accepted 11 October 2013) ISSN 1401-7431 print/ISSN 1651-2006 online © 2013 Informa Healthcare DOI: 10.3109/14017431.2013.856462
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Previous work Consumption of the hydrogenated fats available in the supermarket, such as margarine and partially hydrogenated soybean oil, which contain trans fatty acids, may also be considered another factor that can perturb the essential fatty acid metabolism and inhibit the linoleic acid conversion to arachidonic acid. In an in vitro study (5), we showed that trans isomeric 18:1 acids act as competitive inhibitors for the desaturase enzymes involved in the desaturation of linoleic acid by rat liver microsomes. In an in vivo study (6), we found that trans-18:1 acids in partially hydrogenated soybean oil have more inhibitory effect than saturated acids on linoleic acid metabolism to arachidonic acid even in presence of adequate amount of linoleic acid. In another study using swine as a model (7), we showed that dietary trans fatty acids in partially hydrogenated soybean oil perturbed the essential fatty acid metabolism, which led to changes of the phospholipid fatty acid composition in the aorta, the target tissue of atherogenesis, leading to more accumulation of linoleic acid and significant decrease of arachidonic acid in the aortic tissues phospholipids. The objective of the present study was to investigate how dietary fats with different levels of linoleic acid and trans-18:1 acids will affect the linoleic acid and arachidonic acid in cellular membrane phospholipids of human vascular endothelial cells in culture, and the resultant effects on the prostacyclin released by these cells. For comparison to the fat mixtures used to study the suppression of prostacyclin production, we report herein the concentrations of linoleic and trans fatty acids in four margarines purchased in the supermarket and 13 random food samples labeled as containing 0% trans fat.
Methods Soybean oil and partially hydrogenated soybean oil (coating fat) obtained from a local hydrogenation plant were mixed in different proportions to yield seven fat mixtures with linoleic acids ranging from 54.1% to 5.7% and trans-18:1 acids ranging from 0.4% to 43.9%. The free fatty acids of these fat mixtures were prepared by heating 10 g of each fat in 100 ml of 2.5% potassium hydroxide in ethanol under reflux condition under nitrogen. After heating for 1 h, the solution was diluted with water and extracted twice with hexane to remove any unsaponifiable materials, then acidified to pH 2.0 with cold 10% sulfuric acid in water. The free fatty acids were extracted using three 100 ml portions of hexane-diethyl ether (50:50, v/v). The organic extract was washed with water, dried over
anhydrous sodium sulfate and the solvent was removed under vacuum with a rotary evaporator. Methyl ester derivatives of fatty acids were prepared from free fatty acids using 4% sulfuric acid in methanol (8).
Cell culture Eagle’s minimum essential medium, fetal bovine serum, and other reagents were purchased from Sigma (Saint Louis, MO). Human Endothelial SFM was purchased from Life Technologies Corporation (Carlsbad, CA). Human plasma fibronectin-purified protein, basic fibroblast growth factor, and epidermal growth factor were purchased from Millipore (Milford, MA). Human endothelial cells from umbilical vein were obtained from American Type Culture Collection (Manassas, VA). The cells were cultured in minimum essential medium supplemented with 20% fetal bovine serum in a 5% carbon dioxide incubator at 37°C. The cells were cultured in 75 cm2 or 25 cm2 flasks and were grown until they reached 30% confluence, then the test medium which contained 100 μM of the test fatty acids in human arterial serum free medium containing human plasma fibronectin purified (10 μg/ml), basic fibroblast growth factor (20 ng/ml) and epidermal growth factor (10 ng/ml) was added. The media were changed every 3 days. In order to study how the fatty acids modify the cellular membrane fatty acids and affect the level of arachidonic acid, the cells were cultured in a 75 cm2 flask in a medium containing the fatty acids at 100 μM concentration. All the fatty acids tested were added as sodium-salt-bovine serum albumin complex. In our experiment, there was no indication of endothelial damage. The cells grew at the normal rate, reached the same density as in control culture, adhered well to the surface, and appeared normal. Viability test was determined by trypan blue inclusion in order to determine the optimal nontoxic level of fatty acids. The cells that grew in 100 μM fatty acids supplemented medium excluded dye. We chose 100 μM concentration of fatty acids because it is below the toxic level and high enough to accelerate the uptake of the fatty acids by the cells, since the cellular uptake of fatty acids depends on their concentration in the medium. After incubation of the cells with the test medium for the designated time, the cells were rinsed thrice with ice-cold phosphate-buffered saline, trypsinized, collected, and suspended in 2 ml methanol then sonicated for 30 s, then another 3 ml of methanol and 10 ml chloroform were added for lipid extraction by Folch method (9). In order
Trans fats and prostacyclin to analyze the fatty acids of the cellular membrane, the phospholipid fraction of the cell lipid was separated from other portions of the lipid extract with a polysilicic acid impregnated glass fibers sheet (Gelman Science, Ann Arbor, MI) using a solvent system petroleum ether/diethyl ether/ acetic acid (80:20:1 v/v/v). The phospholipid fraction was then transesterified using boron fluoride methanol (10).
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Fatty acid analysis of commercial foods Four margarines labeled as containing trans fats and 13 samples of fats, oils, or processed foods were randomly selected and purchased from local supermarkets between the summers of 2011 and 2013. Fatty acids were analyzed by gas chromatography as described above.
Statistical analysis Fatty acid analysis by gas chromatography The cellular phospholipid fatty acids were separated by gas chromatography using Hewlett Packard Model 5890 Series II gas chromatograph (Chicago, IL) equipped with an all glass splitter and FID to separate methyl esters on Varian CP- select CB 200 m ⫻ 0.25 mm film thickness, fused silica capillary column (Varian, Walnut Creek, CA, Part # CP 7421). The oven temperature was programmed at 185°C for 60 min and then increased to 215°C at a rate of 3°C/minute for 50 min, using helium gas as carrier at 74 psi and split ratio 1:20, and injector and detector temperature at 250°C. Retention times, peak areas, and peak relative percentages were determined electronically using a Hewlett Packard 3390 Reporting Integrator. Identification of methyl esters of fatty acids was accomplished by comparing relative retention times with authentic standards (Nu-Check Prep, Elysion, MN) and comparing to the chromatogram of partially hydrogenated soybean oil fatty acids methyl ester provided with the column.
Measurements of prostacyclin released by cells The ability of the cells to form prostacyclin from the endogenous arachidonic acid was assessed using radioimmunoassay kit (Amersham, Arlington Heights, IL) after the cells were cultured with the test fatty acids for 6 days. At the end of the incubation time the test medium was removed and the monolayer of intact confluent endothelial cells in 25 cm2 flasks was washed with Tris–HCl buffer containing 140 mM sodium chloride, 15 mM Tris-HCl, and 5.5 mM glucose, pH 7.4. After washing, 2 ml of the fresh buffer was added to the monolayer, and the cells were incubated at 37°C for 90 min. The supernatant solution was centrifuged for 5 min at 2000 rpm and assayed for 6 Keto PGFiα, the stable metabolite of prostacyclin; after their dilution 1:10 with the assay buffer using radioimmunoassay kits. The cell monolayers were digested with 0.1 M sodium hydroxide and their protein was measured using bovine serum albumin as standard (11).
Data (means ⫾ SD) were analyzed by one-way ANOVA with Dunnett’s post-test to adjust for multiple comparisons, using GraphPad Prism (version 5, San Diego, CA, USA). All analyses were considered statistically significant at an α-level of P ⱕ 0.05.
Results Replacing soybean oil with coating fat dose-dependently decreased (P ⬍ 0.0001) the proportion of linoleic (18:2ω6) and arachidonic (20:4ω6) acids, but increased the proportion of trans 18:1 acids, in the membrane phospholipids of vascular endothelial cells (Table I). Consistent with these changes, coating fat also dose-dependently suppressed prostacyclin release (P ⬍ 0.0001; Table I). The lowest proportion of coating fat used (23% coating fat providing 13.4% trans fat) suppressed prostacyclin by 35% (P ⬍ 0.05), while pure coating fat (providing 43.9% trans fat) nearly abolished prostacyclin release, suppressing it by 98% (P ⬍ 0.05; Table I). The proportions of linoleic acid in four commercial margarines labeled as containing trans fat ranged from 25.6% to 40.7%, while the proportions of trans 18:1 acids ranged from 15.2% to 28.7% (Table II). These proportions are similar to those in the mixtures containing 23–50% coating fat used in the present study, which lowered prostacyclin release by 35–54% (P ⬍ 0.05, Table I). Thus, the proportions of fatty acids used to study prostacyclin suppression in the present report are relevant to those found in margarines currently available in the market. Of 13 food items that were labeled 0% trans fat, only two were free of all trans fats. Three contained amounts close to 0.25 g/serving (Table III). According to the Federal Food and Drug Administration (FDA), a food item that contains less than 0.5g/ serving can be labeled 0% trans fat (12). The FDA allows any amount of trans fat as long as it is stated on the label. Many commercial foods labeled as trans-free contain hidden sources of trans fats that could contribute to the suppression of prostacyclin production, especially if multiple servings are consumed, or if these foods are eaten in addition to foods
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F. A. Kummerow et al. Table I. Prostacyclin release (ng/mg) from vascular endothelial cells cultured with increased amounts of trans fat and decreased amounts of linoleic acid.
Linoleic acid (18:2ω6) and trans 18:1 in the fat used
Percent fat used
Soybean oil Coating fat 18:2ω6 Trans 18:1 100 77 50 33 25 9 0
0 23 50 67 75 91 100
54.1 42.1 27.5 19.4 14.4 7.4 5.7
Fatty acids in the phospholipid fraction of the EC incubated with 100 uM of fatty acids of the fat (percentage) 18:2ω6 12 ⫾ 1.3 9.6 ⫾ 0.6 9.1 ⫾ 1.3* 6.6 ⫾ 0.7* 5.1 ⫾ 0.5* 3.9 ⫾ 0.5* 1.9 ⫾ 0.7*
0.4 13.4 24.3 31.8 36.0 40.5 43.9
20:4ω6
Trans 18:1
Prostacyclin release ng/mg Cell protein
9.0 ⫾ 1.2 0.9 ⫾ 0.6 7.4 ⫾ 0.6 6.6 ⫾ 0.1* 6.3 ⫾ 0.9* 6.2 ⫾ 0.1* 5.1 ⫾ 0.1* 8.0 ⫾ 0.3* 3.9 ⫾ 0.4* 8.0 ⫾ 0.0* 2.0 ⫾ 0.2* 13.1 ⫾ 1.5* 1.3 ⫾ 0.1* 13.5 ⫾ 0.8*
38.7 ⫾ 6.4 25.1 ⫾ 5.2* 21.0 ⫾ 3.2* 15.5 ⫾ 0.8* 15.7 ⫾ 1.3* 4.1 ⫾ 0.4* 0.6 ⫾ 0.1*
Data (n ⫽ 2–4) are presented as means ⫾ SD. One-way ANOVA main effects of fat used are statistically significant (P ⬍ 0.0001) for 18:2ω6, 20:4ω6, trans 18:1, and prostacyclin release. *Significantly different (P ⬍ 0.05) from 100% soybean oil control using Dunnett’s test to adjust for multiple comparisons.
containing margarine and shortening with greater amounts of trans fat. Thus, even processed foods labeled as 0% trans fat may provide hidden sources of trans fat that contribute to prostacyclin suppression.
Discussion Clearly, the presence of trans fat suppresses the production of prostacyclin. The trans fat present in the mixtures used increased its own concentration in membrane phospholipids at the expense of linoleic and arachidonic acids. Linoleic acid is a precursor to arachidonic acid, from which prostacyclin is derived, and would thus be expected to support the production of this critical prostanoid. The linoleic acid present in partially hydrogenated soybean oil, however, is clearly insufficient to overcome the suppressive effect of the trans fatty acids present in these hydrogenated oils. Herein, we show that margarines purchased in the supermarket provide linoleic and trans 18:1 fatty acids in proportions similar to those that suppressed prostacyclin in our study by 35–54%. Even processed foods labeled as containing 0% trans fat, moreover, contain additional trans fats that could contribute to the suppression of prostacyclin.
Prostacyclin is an important factor in cardiovascular health (13). It is a dominant prostaglandin produced by endothelial cells and is a potent vasodilator and inhibitor of platelet aggregation and leukocyte adhesion (14). It limits the response to thromboxane (4), which is a potent inducer of vasoconstriction and platelet adhesion in the arteries, and is partially responsible for the interruption of blood flow (15). Our data thus offer additional support for current concerns that trans fats contribute to heart disease. Previous in vitro studies reported an inhibition of Δ6 desaturase enzyme activity by trans 18:1 isomers (5,16), which act as competitive inhibitors for that enzyme. In vivo studies also indicated that isomeric trans 18:1 acids in hydrogenated fats have inhibitory effects on the desaturase enzymes and the metabolic conversion of linoleic acid to arachidonic acid (6,7,17). Therefore, the trans 18:1 isomers of fatty acids inhibit the metabolism of linoleic acid to arachidonic acid leading to its decreased levels in the cells (18,19). This fact is of importance with regard to the role of arachidonic acid as substrate to the synthesis of hemostatically active prostanoids such as prostacyclin. Therefore, the significant decrease of prostacyclin released by the cells incubated with trans 18:1 acids
Table II. Proportions of trans fat and linoleic acid in four store-bought margarines labeled as containing trans fats.
Labeled trans fat (g/serving) Actual trans fat (% Fatty acids) Actual linoleic acid (% Fatty acids)
Margarine #1
Margarine #2
Margarine #3
Margarine #4
2.5 22.3
1.5 23.2
2.5 28.7
1.5 15.2
25.6
27.7
29.9
40.7
Trans fats and prostacyclin Table III. Amounts of trans fat in 13 randomly selected processed foods labeled as containing zero trans fat.
Product
Labeled Trans Per Serving (g)
Actual Trans Per Serving (mg)
0 0 0 0 0 0 0 0 0 0 0 0 0
29 14 0 25 233 0 252 26 37 70 28 203 12
Canola oil #1 Canola oil #2 Cookies Corn chips Crackers #1 Crackers #2 Donuts Mayonnaise Potato chips Soybean oil Vegetable oil spread #1 Vegetable oil spread #2 Vegetable shortening
can be attributed to the inhibition of linoleic acid conversion to arachidonic acid which led to a lower level of arachidonic acid in the phospholipids. Since the availability of arachidonic acid as a precursor is a limiting factor for prostaglandin synthesis, small differences in the level of arachidonic acid as precursor can cause large variation in the amount of prostacyclin synthesized (20). Our data, moreover, suggest that reductions in the trans fat content of edible oils that occurred 45 years ago have contributed to the decline in heart disease mortality that has occurred since then, but still fall short of what eliminating these partially hydrogenated oils could accomplish. I received a letter from W.H. Meyer, a manager at Procter & Gamble, May 22, 1967, giving me data on the composition of the old types of shortenings being sold at that time. These are shown in Table IV. In 1967, I suggested to Dr. Campbell Moses, Medical Director of the American Heart Association, that he meet with the industry to reduce the amount of trans fat in the partially hydrogenated fat. In 1968, Dr. Moses, and the president of the Institute of Shortening and Edible Oils changed industry standards (21,22) to
Table IV. Composition of shortenings and margarines before 1968 (pre-1968) and after 1968 (post-1968) compared to butterfat and tallow (26). Shortening Fatty acid composition Saturated Monounsaturated Polyunsaturated Cis,-cis linoleic Trans acids
Margarine
Pre 1968
Post 1968
Pre 1968
Post 1968
27 61 12 8 30
25 47 28 24 20
22 62 16 8.4 43.9
21 50 29 27.3 27.7
Butter Tallow 61 31 4 ⬎3 2–4
46 47 4 ⬍3 5–10
Courtesy of W.H. Meyer, Manager at Procter & Gamble, 1967.
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lower the trans fatty acids and increase the essential fatty acid composition of margarine and shortening (Table IV). After this ruling, the percentage of trans fatty acids in margarine decreased from 40% to 27%, while the percentage of linoleic acid increased from 11% to 25%. The analyzed margarine samples in the present study (Table II) had proportions of linoleic and trans fatty acids similar to those reported in 1968, suggesting that the industry standards established at that time are still being met by current producers. In the present study, reducing the proportion of trans fat from approximately 40% to 24%, similar to the 1968 reduction in trans fats, decreased the inhibition of prostacyclin production from 89% to 54%. Even reducing trans fat further to 13% only decreased the inhibition to 35%. As was stated in the Introduction (7), this situation in vivo has been found to be the case in piglets born to swine fed partially hydrogenated fat. These piglets had significant concentration of linoleic acid, less arachidonic acid, and deposits of trans fatty acid in their arteries. Piglets born to swine fed only butterfat had no trans fat in their arteries. Our data suggest that any and all reductions in trans fats from partially hydrogenated oils should prove beneficial for heart health, but completely eliminating them from the diet would prove most beneficial. Heart disease has declined dramatically since 1968 (23), yet remains the leading cause of death in the United States, causing almost 600,000 deaths in 2011 (24). 325,000 of these deaths are from sudden cardiac death, according to Jain (25). In conclusion, trans fat inhibits the production of prostacyclin by increasing its own incorporation into membrane phospholipids at the expense of linoleic and arachidonic acids. This would be expected to contribute to heart disease, especially in conjunction with dietary oxysterols and other factors that contribute to thromboxane production. While the 1968 reduction in the trans fat content of margarine and shortening have contributed to the reduction in heart disease mortality seen during that time period, if our data can be generalized to the in vivo situation they suggest that this reduction was not enough and that trans fats from partially hydrogenated oils should be eliminated from the diet. Even processed foods labeled as 0% trans fats often contain hidden trans fats that may contribute to this effect, providing an additional reason to support diets that emphasize whole foods over processed foods to reduce the risk of heart disease.
Declaration of interest: The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.
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References 1. Fitzgerald GA, Patrono C. The coxibs, selective inhibition of cyclooxygenase-2. N. Engl J Med. 2001;345:433–42. 2. Linton MF, Fazio S. Cyclooxygenase-2 and atherosclerosis. Curr Opin Lipidol. 2002;13:497–504. 3. Vane JR, Bakhle YS, Botting RM. Cyclooxygenases 1 and 2. Ann Rev Pharmacol Toxicol. 1998;38:97–120. 4. Cheng Y, Austin SC, Rocca B, Koller BH, Coffman TM, Grosser T, et al. Role of prostacyclin in the cardiovascular response to thromboxane A2. Science. 2002;296:539–41. 5. Mahfouz MM, Johnson S, Holman RT. The effect of isomeric trans-18:1 acids on the desaturation of palmitic, linoleic, and eicosa 8, 11, 14-trienoic acids by rat liver microsomes. Lipids. 1980;15:100–7. 6. Mahfouz MM, Smith T, Kummerow FA. Effect of dietary fats on desaturase activities and the biosynthesis of fatty acids in rat liver microosomes. Lipids. 1984;19:214–22. 7. Kummerow FA, Zhou Q, Mahfouz MM, Smiricky MR, Grieshop CM, Schaffer DJ. Trans fatty acids in hydrogenated fat inhibited the synthesis of the polyunsaturated fatty acids in the phospholipids of arterial cells. Life Sci. 2004; 74:2707–23. 8. DeSchrijver R, Privett GS. Interrelationship between dietary trans fatty acids and the 6- and 9-desaturases in the rat. Lipids. 1982;17:27–34. 9. Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1957;226:497–509. 10. Morrison WR, Smith LM. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron-fluoridemethanol. J Lipid Res. 1964;53:600–8. 11. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72: 248–54. 12. FDA. Food labeling: trans fatty acids in nutrition labeling, nutrient content claims, and health claims. Final rule. Fed Regist. 2003;68:41433–1506. 13. Mahfouz MM, Kummerow FA. Oxysterols and TBARS are among the LDL oxidation products which enhance
14.
15.
16.
17.
18. 19.
20.
21.
22. 23. 24. 25.
26.
thromboxane A2 synthesis by platelets. Prostaglandins Other Lipid Mediat. 1998;56:197–217. Kummerow FA, Mahfouz MM, Zhou Q. Trans fatty acids in partially hydrogenated soybean oil inhibit prostacyclin release by endothelial cells in presence of high level of linoleic acid. Prostaglandins Other Lipid Mediat. 2007;84:138–53. Mahfouz MM, Kummerow FA. Oxidized low-density lipoprotein (LDL) enhances thromboxane A(2) synthesis by platelets, but lysolecithin as a product of LDL oxidation has an inhibitory effect. Prostaglandins Other Lipid Mediat. 2000;62:183–200. Rosenthal MD, Doloresco MA. The effects of trans fatty acids on fatty acyl delta 5 desaturation by human skin fibroblasts. Lipids. 1984;19:869–74. Lawson LD, Hill EG, Holman RT. Suppression of arachidonic acid in lipids of rat tissues by dietary mixed isomeric cis and trans octadecenoates. J Nutr. 1983;113:1827–35. Gurr MI. Trans fatty acid and health- an update. Lipid Technol. 1990;2:105–7. Kinsella JE, Bruckner G, Mai J, Shimp J. Metabolism of trans fatty acids with emphasis on the effects of trans, transoctadecadienoate on lipid composition, essential fatty acid, and prostaglandins: an overview. Am J Clin Nutr. 1981; 34:2307–18. Hwang DH, Kinsella JE. The effects of trans linoleic acid on the concentration of prostaglandins and their precursors in rat. Prostaglandins. 1979;17:543–59. Kummerow FA. Viewpoint on the Report of the National Cholesterol Education Program Expert Panel on the Detection, Evaluation and Treatment of High Blood Cholesterol in Adults. J Am Coll Nutr. 1993;12:2–13. Moses C. Medical Director of the AHA. Dallas, TX: American Heart Association; 1968. Friedewald WT, Thom TJ. Decline of coronary heart disease mortality in the United States. Isr J Med Sci. 1986;22:307–12. Hoyert DL, Xu, J. Deaths: preliminary data for 2011. Natl Vital Stat Rep. 2012;61:1–65. Jain M. Explaining why so many cases of cardiac arrest strike in the morning. Presented at the national meeting of the American Chemical Society 2013; Sept. 6–11. Meyer WH. Letter to F. A. Kummerow, 22 May 1967. TS.
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ORIGINAL ARTICLE
Effects of trans fats on prostacyclin production
FRED A. KUMMEROW, MOHAMEDAIN MAHFOUZ, QI ZHOU & CHRISTOPHER MASTERJOHN Department of Comparative Biosciences, Burnsides Research Laboratory, College of Veterinary Medicine, University of Illinois, Urbana, IL, USA
Abstract Objectives. Prostacyclin is a prostanoid derived from arachidonic acid that prevents thrombosis and is thereby expected to protect against heart disease, while trans fats present in partially hydrogenated oils interfere with arachidonic acid metabolism. Therefore, the objective of the present study was to investigate how fats with different proportions of linoleic acid and trans-18:1 affect prostacyclin released by cultured endothelial cells, and to compare these proportions with those found in commercially available foods. Design. Soybean oil and hydrogenated soybean oil (coating fat) were mixed in different proportions to yield seven fat mixtures with proportions of linoleic acid ranging from 54.1% to 5.7% and trans-18:1 acid ranging from 0.4% to 43.9%. Human endothelial cells were cultured in each of the mixtures, and their phospholipid fractions were then separated and their fatty acids were analyzed by gas chromatography. The prostacyclin released by the cells was measured using RIA kits. Margarines and processed foods were purchased from the supermarket for comparison. Results. Our work revealed that as the percentage of trans fat was increased, the amount of prostacyclin released dose-dependently and significantly (P ⬍ 0.0001) decreased, the concentration of linoleic and arachidonic acid decreased in the membrane phospholipids while the concentration of trans 18:1 acids increased, the prostacyclin decreased by 35–98%. Supermarket margarines had levels of trans fats similar to those that suppressed prostacyclin by 35–54%. Most processed foods labeled as trans-free contained trans fats. Conclusions. Trans fatty acids suppress prostacyclin production at levels found in commercial margarines, and processed foods labeled as trans-free could contribute to this effect if consumed in multiple servings or in addition to foods containing larger amounts of trans fats. Key words: arachidonic acid, linoleic acid, prostacyclin, trans fats
Introduction Prostanoids are involved in a variety of physiological processes in atherosclerosis and thrombosis, such as leukocyte arterial cell adhesion, vasorelaxation, and platelet aggregation (1,2). Prostacyclin, which prevents blood clotting, is produced by arterial cells from arachidonic acid in all the vascular tissues (3). Platelet activation leads to the formation of thromboxane as a potent inducer of vasoconstriction and platelet adhesion. An upset of the balance between prostacyclin and thromboxane can promote thrombosis. A recent study showed that prostacyclin modulates platelets’ vascular interaction in vivo and limits the response to thromboxane (4). Free arachidonic acid released from the cell membrane phospholipids by the action of phospholipase
is metabolized to prostaglandin H2 by cyclooxygenase and peroxidase, which is in turn metabolized to prostacyclin-by-prostacyclin synthase. Therefore, the arachidonic acid level in the arterial membrane phospholipids is one of the controlling factors that regulates the amount of prostacyclin released by the arterial cell membrane. The arachidonic acid in the cell membrane is derived from the metabolic conversion of linoleic acid to arachidonic acid through the action of Δ6 and Δ5 desaturase enzymes in vivo. Because linoleic acid is an essential fatty acid, it must be provided in the diet; therefore, the level of linoleic acid in the diet will have a regulatory effect on the whole process starting from linoleic acid to arachidonic acid then to prostacyclin.
Correspondence: Dr. Fred A. Kummerow, Department of Comparative Biosciences, Burnsides Research Laboratory, College of Veterinary Medicine, University of Illinois, 1208 W. Pennsylvania Avenue, Urbana, IL 61801, USA. Tel: ⫹(217)344–6380. Fax: ⫹(217)333-7370. E-mail: [email protected] (Received 27 July 2013 ; revised 10 October 2013 ; accepted 11 October 2013) ISSN 1401-7431 print/ISSN 1651-2006 online © 2013 Informa Healthcare DOI: 10.3109/14017431.2013.856462
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Previous work Consumption of the hydrogenated fats available in the supermarket, such as margarine and partially hydrogenated soybean oil, which contain trans fatty acids, may also be considered another factor that can perturb the essential fatty acid metabolism and inhibit the linoleic acid conversion to arachidonic acid. In an in vitro study (5), we showed that trans isomeric 18:1 acids act as competitive inhibitors for the desaturase enzymes involved in the desaturation of linoleic acid by rat liver microsomes. In an in vivo study (6), we found that trans-18:1 acids in partially hydrogenated soybean oil have more inhibitory effect than saturated acids on linoleic acid metabolism to arachidonic acid even in presence of adequate amount of linoleic acid. In another study using swine as a model (7), we showed that dietary trans fatty acids in partially hydrogenated soybean oil perturbed the essential fatty acid metabolism, which led to changes of the phospholipid fatty acid composition in the aorta, the target tissue of atherogenesis, leading to more accumulation of linoleic acid and significant decrease of arachidonic acid in the aortic tissues phospholipids. The objective of the present study was to investigate how dietary fats with different levels of linoleic acid and trans-18:1 acids will affect the linoleic acid and arachidonic acid in cellular membrane phospholipids of human vascular endothelial cells in culture, and the resultant effects on the prostacyclin released by these cells. For comparison to the fat mixtures used to study the suppression of prostacyclin production, we report herein the concentrations of linoleic and trans fatty acids in four margarines purchased in the supermarket and 13 random food samples labeled as containing 0% trans fat.
Methods Soybean oil and partially hydrogenated soybean oil (coating fat) obtained from a local hydrogenation plant were mixed in different proportions to yield seven fat mixtures with linoleic acids ranging from 54.1% to 5.7% and trans-18:1 acids ranging from 0.4% to 43.9%. The free fatty acids of these fat mixtures were prepared by heating 10 g of each fat in 100 ml of 2.5% potassium hydroxide in ethanol under reflux condition under nitrogen. After heating for 1 h, the solution was diluted with water and extracted twice with hexane to remove any unsaponifiable materials, then acidified to pH 2.0 with cold 10% sulfuric acid in water. The free fatty acids were extracted using three 100 ml portions of hexane-diethyl ether (50:50, v/v). The organic extract was washed with water, dried over
anhydrous sodium sulfate and the solvent was removed under vacuum with a rotary evaporator. Methyl ester derivatives of fatty acids were prepared from free fatty acids using 4% sulfuric acid in methanol (8).
Cell culture Eagle’s minimum essential medium, fetal bovine serum, and other reagents were purchased from Sigma (Saint Louis, MO). Human Endothelial SFM was purchased from Life Technologies Corporation (Carlsbad, CA). Human plasma fibronectin-purified protein, basic fibroblast growth factor, and epidermal growth factor were purchased from Millipore (Milford, MA). Human endothelial cells from umbilical vein were obtained from American Type Culture Collection (Manassas, VA). The cells were cultured in minimum essential medium supplemented with 20% fetal bovine serum in a 5% carbon dioxide incubator at 37°C. The cells were cultured in 75 cm2 or 25 cm2 flasks and were grown until they reached 30% confluence, then the test medium which contained 100 μM of the test fatty acids in human arterial serum free medium containing human plasma fibronectin purified (10 μg/ml), basic fibroblast growth factor (20 ng/ml) and epidermal growth factor (10 ng/ml) was added. The media were changed every 3 days. In order to study how the fatty acids modify the cellular membrane fatty acids and affect the level of arachidonic acid, the cells were cultured in a 75 cm2 flask in a medium containing the fatty acids at 100 μM concentration. All the fatty acids tested were added as sodium-salt-bovine serum albumin complex. In our experiment, there was no indication of endothelial damage. The cells grew at the normal rate, reached the same density as in control culture, adhered well to the surface, and appeared normal. Viability test was determined by trypan blue inclusion in order to determine the optimal nontoxic level of fatty acids. The cells that grew in 100 μM fatty acids supplemented medium excluded dye. We chose 100 μM concentration of fatty acids because it is below the toxic level and high enough to accelerate the uptake of the fatty acids by the cells, since the cellular uptake of fatty acids depends on their concentration in the medium. After incubation of the cells with the test medium for the designated time, the cells were rinsed thrice with ice-cold phosphate-buffered saline, trypsinized, collected, and suspended in 2 ml methanol then sonicated for 30 s, then another 3 ml of methanol and 10 ml chloroform were added for lipid extraction by Folch method (9). In order
Trans fats and prostacyclin to analyze the fatty acids of the cellular membrane, the phospholipid fraction of the cell lipid was separated from other portions of the lipid extract with a polysilicic acid impregnated glass fibers sheet (Gelman Science, Ann Arbor, MI) using a solvent system petroleum ether/diethyl ether/ acetic acid (80:20:1 v/v/v). The phospholipid fraction was then transesterified using boron fluoride methanol (10).
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Fatty acid analysis of commercial foods Four margarines labeled as containing trans fats and 13 samples of fats, oils, or processed foods were randomly selected and purchased from local supermarkets between the summers of 2011 and 2013. Fatty acids were analyzed by gas chromatography as described above.
Statistical analysis Fatty acid analysis by gas chromatography The cellular phospholipid fatty acids were separated by gas chromatography using Hewlett Packard Model 5890 Series II gas chromatograph (Chicago, IL) equipped with an all glass splitter and FID to separate methyl esters on Varian CP- select CB 200 m ⫻ 0.25 mm film thickness, fused silica capillary column (Varian, Walnut Creek, CA, Part # CP 7421). The oven temperature was programmed at 185°C for 60 min and then increased to 215°C at a rate of 3°C/minute for 50 min, using helium gas as carrier at 74 psi and split ratio 1:20, and injector and detector temperature at 250°C. Retention times, peak areas, and peak relative percentages were determined electronically using a Hewlett Packard 3390 Reporting Integrator. Identification of methyl esters of fatty acids was accomplished by comparing relative retention times with authentic standards (Nu-Check Prep, Elysion, MN) and comparing to the chromatogram of partially hydrogenated soybean oil fatty acids methyl ester provided with the column.
Measurements of prostacyclin released by cells The ability of the cells to form prostacyclin from the endogenous arachidonic acid was assessed using radioimmunoassay kit (Amersham, Arlington Heights, IL) after the cells were cultured with the test fatty acids for 6 days. At the end of the incubation time the test medium was removed and the monolayer of intact confluent endothelial cells in 25 cm2 flasks was washed with Tris–HCl buffer containing 140 mM sodium chloride, 15 mM Tris-HCl, and 5.5 mM glucose, pH 7.4. After washing, 2 ml of the fresh buffer was added to the monolayer, and the cells were incubated at 37°C for 90 min. The supernatant solution was centrifuged for 5 min at 2000 rpm and assayed for 6 Keto PGFiα, the stable metabolite of prostacyclin; after their dilution 1:10 with the assay buffer using radioimmunoassay kits. The cell monolayers were digested with 0.1 M sodium hydroxide and their protein was measured using bovine serum albumin as standard (11).
Data (means ⫾ SD) were analyzed by one-way ANOVA with Dunnett’s post-test to adjust for multiple comparisons, using GraphPad Prism (version 5, San Diego, CA, USA). All analyses were considered statistically significant at an α-level of P ⱕ 0.05.
Results Replacing soybean oil with coating fat dose-dependently decreased (P ⬍ 0.0001) the proportion of linoleic (18:2ω6) and arachidonic (20:4ω6) acids, but increased the proportion of trans 18:1 acids, in the membrane phospholipids of vascular endothelial cells (Table I). Consistent with these changes, coating fat also dose-dependently suppressed prostacyclin release (P ⬍ 0.0001; Table I). The lowest proportion of coating fat used (23% coating fat providing 13.4% trans fat) suppressed prostacyclin by 35% (P ⬍ 0.05), while pure coating fat (providing 43.9% trans fat) nearly abolished prostacyclin release, suppressing it by 98% (P ⬍ 0.05; Table I). The proportions of linoleic acid in four commercial margarines labeled as containing trans fat ranged from 25.6% to 40.7%, while the proportions of trans 18:1 acids ranged from 15.2% to 28.7% (Table II). These proportions are similar to those in the mixtures containing 23–50% coating fat used in the present study, which lowered prostacyclin release by 35–54% (P ⬍ 0.05, Table I). Thus, the proportions of fatty acids used to study prostacyclin suppression in the present report are relevant to those found in margarines currently available in the market. Of 13 food items that were labeled 0% trans fat, only two were free of all trans fats. Three contained amounts close to 0.25 g/serving (Table III). According to the Federal Food and Drug Administration (FDA), a food item that contains less than 0.5g/ serving can be labeled 0% trans fat (12). The FDA allows any amount of trans fat as long as it is stated on the label. Many commercial foods labeled as trans-free contain hidden sources of trans fats that could contribute to the suppression of prostacyclin production, especially if multiple servings are consumed, or if these foods are eaten in addition to foods
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F. A. Kummerow et al. Table I. Prostacyclin release (ng/mg) from vascular endothelial cells cultured with increased amounts of trans fat and decreased amounts of linoleic acid.
Linoleic acid (18:2ω6) and trans 18:1 in the fat used
Percent fat used
Soybean oil Coating fat 18:2ω6 Trans 18:1 100 77 50 33 25 9 0
0 23 50 67 75 91 100
54.1 42.1 27.5 19.4 14.4 7.4 5.7
Fatty acids in the phospholipid fraction of the EC incubated with 100 uM of fatty acids of the fat (percentage) 18:2ω6 12 ⫾ 1.3 9.6 ⫾ 0.6 9.1 ⫾ 1.3* 6.6 ⫾ 0.7* 5.1 ⫾ 0.5* 3.9 ⫾ 0.5* 1.9 ⫾ 0.7*
0.4 13.4 24.3 31.8 36.0 40.5 43.9
20:4ω6
Trans 18:1
Prostacyclin release ng/mg Cell protein
9.0 ⫾ 1.2 0.9 ⫾ 0.6 7.4 ⫾ 0.6 6.6 ⫾ 0.1* 6.3 ⫾ 0.9* 6.2 ⫾ 0.1* 5.1 ⫾ 0.1* 8.0 ⫾ 0.3* 3.9 ⫾ 0.4* 8.0 ⫾ 0.0* 2.0 ⫾ 0.2* 13.1 ⫾ 1.5* 1.3 ⫾ 0.1* 13.5 ⫾ 0.8*
38.7 ⫾ 6.4 25.1 ⫾ 5.2* 21.0 ⫾ 3.2* 15.5 ⫾ 0.8* 15.7 ⫾ 1.3* 4.1 ⫾ 0.4* 0.6 ⫾ 0.1*
Data (n ⫽ 2–4) are presented as means ⫾ SD. One-way ANOVA main effects of fat used are statistically significant (P ⬍ 0.0001) for 18:2ω6, 20:4ω6, trans 18:1, and prostacyclin release. *Significantly different (P ⬍ 0.05) from 100% soybean oil control using Dunnett’s test to adjust for multiple comparisons.
containing margarine and shortening with greater amounts of trans fat. Thus, even processed foods labeled as 0% trans fat may provide hidden sources of trans fat that contribute to prostacyclin suppression.
Discussion Clearly, the presence of trans fat suppresses the production of prostacyclin. The trans fat present in the mixtures used increased its own concentration in membrane phospholipids at the expense of linoleic and arachidonic acids. Linoleic acid is a precursor to arachidonic acid, from which prostacyclin is derived, and would thus be expected to support the production of this critical prostanoid. The linoleic acid present in partially hydrogenated soybean oil, however, is clearly insufficient to overcome the suppressive effect of the trans fatty acids present in these hydrogenated oils. Herein, we show that margarines purchased in the supermarket provide linoleic and trans 18:1 fatty acids in proportions similar to those that suppressed prostacyclin in our study by 35–54%. Even processed foods labeled as containing 0% trans fat, moreover, contain additional trans fats that could contribute to the suppression of prostacyclin.
Prostacyclin is an important factor in cardiovascular health (13). It is a dominant prostaglandin produced by endothelial cells and is a potent vasodilator and inhibitor of platelet aggregation and leukocyte adhesion (14). It limits the response to thromboxane (4), which is a potent inducer of vasoconstriction and platelet adhesion in the arteries, and is partially responsible for the interruption of blood flow (15). Our data thus offer additional support for current concerns that trans fats contribute to heart disease. Previous in vitro studies reported an inhibition of Δ6 desaturase enzyme activity by trans 18:1 isomers (5,16), which act as competitive inhibitors for that enzyme. In vivo studies also indicated that isomeric trans 18:1 acids in hydrogenated fats have inhibitory effects on the desaturase enzymes and the metabolic conversion of linoleic acid to arachidonic acid (6,7,17). Therefore, the trans 18:1 isomers of fatty acids inhibit the metabolism of linoleic acid to arachidonic acid leading to its decreased levels in the cells (18,19). This fact is of importance with regard to the role of arachidonic acid as substrate to the synthesis of hemostatically active prostanoids such as prostacyclin. Therefore, the significant decrease of prostacyclin released by the cells incubated with trans 18:1 acids
Table II. Proportions of trans fat and linoleic acid in four store-bought margarines labeled as containing trans fats.
Labeled trans fat (g/serving) Actual trans fat (% Fatty acids) Actual linoleic acid (% Fatty acids)
Margarine #1
Margarine #2
Margarine #3
Margarine #4
2.5 22.3
1.5 23.2
2.5 28.7
1.5 15.2
25.6
27.7
29.9
40.7
Trans fats and prostacyclin Table III. Amounts of trans fat in 13 randomly selected processed foods labeled as containing zero trans fat.
Product
Labeled Trans Per Serving (g)
Actual Trans Per Serving (mg)
0 0 0 0 0 0 0 0 0 0 0 0 0
29 14 0 25 233 0 252 26 37 70 28 203 12
Canola oil #1 Canola oil #2 Cookies Corn chips Crackers #1 Crackers #2 Donuts Mayonnaise Potato chips Soybean oil Vegetable oil spread #1 Vegetable oil spread #2 Vegetable shortening
can be attributed to the inhibition of linoleic acid conversion to arachidonic acid which led to a lower level of arachidonic acid in the phospholipids. Since the availability of arachidonic acid as a precursor is a limiting factor for prostaglandin synthesis, small differences in the level of arachidonic acid as precursor can cause large variation in the amount of prostacyclin synthesized (20). Our data, moreover, suggest that reductions in the trans fat content of edible oils that occurred 45 years ago have contributed to the decline in heart disease mortality that has occurred since then, but still fall short of what eliminating these partially hydrogenated oils could accomplish. I received a letter from W.H. Meyer, a manager at Procter & Gamble, May 22, 1967, giving me data on the composition of the old types of shortenings being sold at that time. These are shown in Table IV. In 1967, I suggested to Dr. Campbell Moses, Medical Director of the American Heart Association, that he meet with the industry to reduce the amount of trans fat in the partially hydrogenated fat. In 1968, Dr. Moses, and the president of the Institute of Shortening and Edible Oils changed industry standards (21,22) to
Table IV. Composition of shortenings and margarines before 1968 (pre-1968) and after 1968 (post-1968) compared to butterfat and tallow (26). Shortening Fatty acid composition Saturated Monounsaturated Polyunsaturated Cis,-cis linoleic Trans acids
Margarine
Pre 1968
Post 1968
Pre 1968
Post 1968
27 61 12 8 30
25 47 28 24 20
22 62 16 8.4 43.9
21 50 29 27.3 27.7
Butter Tallow 61 31 4 ⬎3 2–4
46 47 4 ⬍3 5–10
Courtesy of W.H. Meyer, Manager at Procter & Gamble, 1967.
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lower the trans fatty acids and increase the essential fatty acid composition of margarine and shortening (Table IV). After this ruling, the percentage of trans fatty acids in margarine decreased from 40% to 27%, while the percentage of linoleic acid increased from 11% to 25%. The analyzed margarine samples in the present study (Table II) had proportions of linoleic and trans fatty acids similar to those reported in 1968, suggesting that the industry standards established at that time are still being met by current producers. In the present study, reducing the proportion of trans fat from approximately 40% to 24%, similar to the 1968 reduction in trans fats, decreased the inhibition of prostacyclin production from 89% to 54%. Even reducing trans fat further to 13% only decreased the inhibition to 35%. As was stated in the Introduction (7), this situation in vivo has been found to be the case in piglets born to swine fed partially hydrogenated fat. These piglets had significant concentration of linoleic acid, less arachidonic acid, and deposits of trans fatty acid in their arteries. Piglets born to swine fed only butterfat had no trans fat in their arteries. Our data suggest that any and all reductions in trans fats from partially hydrogenated oils should prove beneficial for heart health, but completely eliminating them from the diet would prove most beneficial. Heart disease has declined dramatically since 1968 (23), yet remains the leading cause of death in the United States, causing almost 600,000 deaths in 2011 (24). 325,000 of these deaths are from sudden cardiac death, according to Jain (25). In conclusion, trans fat inhibits the production of prostacyclin by increasing its own incorporation into membrane phospholipids at the expense of linoleic and arachidonic acids. This would be expected to contribute to heart disease, especially in conjunction with dietary oxysterols and other factors that contribute to thromboxane production. While the 1968 reduction in the trans fat content of margarine and shortening have contributed to the reduction in heart disease mortality seen during that time period, if our data can be generalized to the in vivo situation they suggest that this reduction was not enough and that trans fats from partially hydrogenated oils should be eliminated from the diet. Even processed foods labeled as 0% trans fats often contain hidden trans fats that may contribute to this effect, providing an additional reason to support diets that emphasize whole foods over processed foods to reduce the risk of heart disease.
Declaration of interest: The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.
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References 1. Fitzgerald GA, Patrono C. The coxibs, selective inhibition of cyclooxygenase-2. N. Engl J Med. 2001;345:433–42. 2. Linton MF, Fazio S. Cyclooxygenase-2 and atherosclerosis. Curr Opin Lipidol. 2002;13:497–504. 3. Vane JR, Bakhle YS, Botting RM. Cyclooxygenases 1 and 2. Ann Rev Pharmacol Toxicol. 1998;38:97–120. 4. Cheng Y, Austin SC, Rocca B, Koller BH, Coffman TM, Grosser T, et al. Role of prostacyclin in the cardiovascular response to thromboxane A2. Science. 2002;296:539–41. 5. Mahfouz MM, Johnson S, Holman RT. The effect of isomeric trans-18:1 acids on the desaturation of palmitic, linoleic, and eicosa 8, 11, 14-trienoic acids by rat liver microsomes. Lipids. 1980;15:100–7. 6. Mahfouz MM, Smith T, Kummerow FA. Effect of dietary fats on desaturase activities and the biosynthesis of fatty acids in rat liver microosomes. Lipids. 1984;19:214–22. 7. Kummerow FA, Zhou Q, Mahfouz MM, Smiricky MR, Grieshop CM, Schaffer DJ. Trans fatty acids in hydrogenated fat inhibited the synthesis of the polyunsaturated fatty acids in the phospholipids of arterial cells. Life Sci. 2004; 74:2707–23. 8. DeSchrijver R, Privett GS. Interrelationship between dietary trans fatty acids and the 6- and 9-desaturases in the rat. Lipids. 1982;17:27–34. 9. Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1957;226:497–509. 10. Morrison WR, Smith LM. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron-fluoridemethanol. J Lipid Res. 1964;53:600–8. 11. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72: 248–54. 12. FDA. Food labeling: trans fatty acids in nutrition labeling, nutrient content claims, and health claims. Final rule. Fed Regist. 2003;68:41433–1506. 13. Mahfouz MM, Kummerow FA. Oxysterols and TBARS are among the LDL oxidation products which enhance
14.
15.
16.
17.
18. 19.
20.
21.
22. 23. 24. 25.
26.
thromboxane A2 synthesis by platelets. Prostaglandins Other Lipid Mediat. 1998;56:197–217. Kummerow FA, Mahfouz MM, Zhou Q. Trans fatty acids in partially hydrogenated soybean oil inhibit prostacyclin release by endothelial cells in presence of high level of linoleic acid. Prostaglandins Other Lipid Mediat. 2007;84:138–53. Mahfouz MM, Kummerow FA. Oxidized low-density lipoprotein (LDL) enhances thromboxane A(2) synthesis by platelets, but lysolecithin as a product of LDL oxidation has an inhibitory effect. Prostaglandins Other Lipid Mediat. 2000;62:183–200. Rosenthal MD, Doloresco MA. The effects of trans fatty acids on fatty acyl delta 5 desaturation by human skin fibroblasts. Lipids. 1984;19:869–74. Lawson LD, Hill EG, Holman RT. Suppression of arachidonic acid in lipids of rat tissues by dietary mixed isomeric cis and trans octadecenoates. J Nutr. 1983;113:1827–35. Gurr MI. Trans fatty acid and health- an update. Lipid Technol. 1990;2:105–7. Kinsella JE, Bruckner G, Mai J, Shimp J. Metabolism of trans fatty acids with emphasis on the effects of trans, transoctadecadienoate on lipid composition, essential fatty acid, and prostaglandins: an overview. Am J Clin Nutr. 1981; 34:2307–18. Hwang DH, Kinsella JE. The effects of trans linoleic acid on the concentration of prostaglandins and their precursors in rat. Prostaglandins. 1979;17:543–59. Kummerow FA. Viewpoint on the Report of the National Cholesterol Education Program Expert Panel on the Detection, Evaluation and Treatment of High Blood Cholesterol in Adults. J Am Coll Nutr. 1993;12:2–13. Moses C. Medical Director of the AHA. Dallas, TX: American Heart Association; 1968. Friedewald WT, Thom TJ. Decline of coronary heart disease mortality in the United States. Isr J Med Sci. 1986;22:307–12. Hoyert DL, Xu, J. Deaths: preliminary data for 2011. Natl Vital Stat Rep. 2012;61:1–65. Jain M. Explaining why so many cases of cardiac arrest strike in the morning. Presented at the national meeting of the American Chemical Society 2013; Sept. 6–11. Meyer WH. Letter to F. A. Kummerow, 22 May 1967. TS.
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