Uploaded byMary Rodavich
Mary Rodavich - WVU Master's Defense Presentation
The study examined the effects of non-marine sources of EPA or DHA (algae oil and yeast oil) versus fish oil on body weight and serum lipids in mice. Mice fed fish oil had significantly lower serum total cholesterol, non-HDL cholesterol, and triglycerides compared to other diet groups. Fish oil also increased EPA and DHA levels in the liver to a greater extent than algae oil or yeast oil. The results suggest that fish oil may be more effective than some non-marine sources at reducing cardiovascular disease risk factors.
Mary Rodavich - WVU Master's Defense Presentation
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- 2. • Introduction &Background • Objective • Methods • Results • Conclusion • Implications • Future Research • Questions
- 3. • Cardiovascular disease (CVD): leading cause of death in the U.S. • 1 out of 4 deaths in West Virginia are due to heart disease 3 http://www.cdc.gov/dhdsp
- 4. Non-Modifiable Modifiable Age Hypertension Family History Type 2 Diabetes Gender Abdominal Obesity • Males >45 Physical Inactivity • Females >55 Smoking Diet Omega-3 fatty acids High blood LDL cholesterol Low blood HDL cholesterol 4
- 5. • LDL: “Bad” Cholesterol Protein • Cholesterol transport to tissues LDL CVD Cholesterol Phospholipid • HDL: “Good” Cholesterol Triglyceride • Reverse cholesterol transport to liver for recycling and disposal HDL CVD 5
- 6. Ω6 Ω3 linoleic acid* α-linolenic acid (ALA)* arachidonic acid (ARA) eicosapaentanoic acid (EPA) docosahexaenoic acid Pro-inflammatory (DHA) Increases CVD risk Anti-inflammatory Decreases CVD risk 6
- 7. Mechanism not completelyknown • Reduces susceptibility of the heart to ventricular arrhythmia • Antithrombogenic • Hypotriglyceridemic • Retards growth of atherosclerotic plaque • Reducing adhesion molecule expression and platelet-derived growth factor • Anti-inflammatory • Promotes nitric oxide-induced endothelial relaxation • Mildly hypotensive 7 Kris-Etherton PM (2002)
- 8. 0.05-4% 8-10% HOW MUCH DO WE NEED?
- 9. • Ω6:Ω3 Ratio • Optimal 4:1 • Western Diet 15:1 • Recommendation • 0.3-0.5 g/day EPA + DHA • 0.8-1.1 g/day ALA • Most DO NOT meet these recommendations • SUPPLEMENTATION may be beneficial 9 Kris-Etherton PM (2002)
- 10. NON-MARINE MARINE SOURCES SOURCES Tuna Flaxseeds Sardines Walnuts Salmon Soybean Oil Mackerel Canola Oil Menhaden Hempseed Herring Butternuts Rainbow Trout Algae Oil Halibut Yeast Oil Cod Haddock Catfish Flounder/Sole Oyster Lobster Alaskan King Crab 10 Shrimp
- 11. 1. Safety • Toxic mercury, polychlorinated biphenyls (PCBs) • Highest levels of mercury: shark, swordfish, king mackerel, tilefish, tuna 2. Ethical & personal reasons • Veganism • Vegetarianism 11
- 12. • Introduction &Background • Objective • Methods • Results • Conclusion Objective • Implications • Future Research • Questions 12
- 13. The objective ofthis study was to examine the effects of non-marine sources of EPA or DHA vs. fish oil on body weight and serum lipids in mice. 13
- 14. • Introduction &Background • Objective • Methods Methods • Results • Conclusion • Implications • Future Research • Questions 14
- 15. • 100 male ICR mice (8 wks old) • Modified AIN-93G diet, 12% lipid EPA DHA Diet (g/kg (g/kg diet) diet) Soy Oil (SO) 0 0 Fish Oil (FO) 7.03 12.64 Algae Oil (AO) 0 12.64 Yeast Oil (YO) 7.03 0 15 Algae Oil + Yeast Oil
- 16. DHA EPA • Provided by Martek Biosciences Corp. • Produced from C. Cohnii algae • Commercially available as life’sDHA • We previously compared AO to FO at equal DHA concentrations • AO less effective at reducing serum lipids in mice 16 Pietrofesa RA (2010), Shelton AG (2011)
- 17. EPA DHA • Purchased from New Harvest • Derived from a strain of yeast called Yarrowia lipolytica • One study showed TC and non-HDL levels were both significantly lower in rats in the medium (488 mg/kg diet) and high (976 mg/kg diet) EPA YO groups compared to control 17 Mackenzie SA (2010)
- 18. 2 4 weeks weeks 7 Day Experimental Experimental Acclimatio Diets Diets n Period 18
- 19. • Body weight weekly • Fresh feed 3 times/wk • Total Average Daily Feed Intake (TADFI) • Tissues collected & weighed: • Serum • Liver • Retroperitoneal (RP) and epididymal (EPI) fat pads • Body fat index (%) = (EPI wt + RP wt) X 100 total body weight 19
- 20. Retroperitoneal (RP) fat pads Epididymal (EPI) fat pad 20
- 21. Serum lipids bycolorimetric kits • Total cholesterol (TC) • HDL cholesterol • Non-HDL cholesterol = TC – HDL cholesterol • Triglycerides (TG) 21
- 22. Liver mRNA expressionby real-time PCR 1. 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) 2. apolipoprotein B100 (ApoB100) 3. acyl-CoA:cholesterol acyltransferase (ACAT) 4. diglyceride acyltransferase (DGAT) 5. LDL receptor (LDLR) • Normalized to acidic ribosomal protein (ARP) • housekeeping gene 22
- 23. Acyl-CoA Synthase DAG Acetyl- Fatty Acid Acyl- CoA HMG-CoA CoA DGAT Reductase 1 Triglycerid Mevalonate e ACA Cholestero Cholestero T l esters l Liver Cell ApoB100 LDL Blood 23
- 24. Liver fatty acidanalysis by gas chromatography • Fatty acids extracted by modified method by Bligh and Dyers • Fatty acids methylated by method by Park and Goins • Fatty acids were identified using retention times and peak area based on mixed fatty acid standards 24 Bligh & Dyers (1959), Park & Goins (1994)
- 25. • An individual mouse was the experimental unit for all analyses • SAS software (Statistical Analysis System) • Data were analyzed by analysis of variance (ANOVA) using a fixed model testing the main effects of diet and block within time period • P < 0.05 significant 25
- 26. • Introduction &Background • Objective • Methods • Results • Conclusion Results • Implications • Future Research • Questions 26
- 27. Diet Measuremen Week SO FO AO YO AO+YO SEM P-Value t 2 37.21 37.62 37.73 35.79 37.98 0.608 0.109 FBW (g) 4 39.86 40.08 39.68 40.15 40.92 1.019 0.928 2 2.94 2.54 2.94 2.52 3.18 0.198 0.098 BFI (%) 4 4.68 3.39 3.93 3.63 4.07 0.445 0.316 2 6.25 6.05 5.17 5.80 5.43 0.282 0.065 TADFI (g) 4 5.75 5.67 5.91 5.76 5.10 0.296 0.362 RP Weight 2 0.25 0.21 0.29 0.22 0.29 0.0248 0.098 (g) 4 0.66 0.28 0.38 0.35 0.41 0.122 0.271 EPI Weight 2 0.85 0.74 0.83 0.69 0.93 0.065 0.119
- 28. SO FO AO YO AO+YO P < 0.05 Relative Liver Weights (g/gbw) 0.06 z a bc ab abc y y y y c 0.05 0.04 0.03 0.02 0.01 0 2 Wk 4 Wk Data are presented as mean + SEM. Different letters within week indicate significant differences between treatments (p < 0.05). 28
- 29. • Typically, increased liver weights associated with: • Excess lipid storage in the liver • Disease (steatosis, non-alcoholic liver disease, cirrhosis) • Hepatic total lipid content: • 2 weeks - No significant differences • 4 weeks – SO significantly greater compared to all other diet treatments • Hepatic TG content: • No significant differences at any time point • The increased liver weights in the FO-fed mice cannot be attributed to hepatic total lipid or TG content 29 Ketz JS, Barnes KM (unpublished results)
- 30. SO FO AO YO AO+YO Serum Total Cholesterol P < 0.05 120 z a z 100 bc ab y 80 cd y y mg/dL de 60 40 20 0 2 Wk 4 Wk Data are presented as mean + SEM. Different letters within week indicate significant differences between treatments (p < 0.05). 30
- 31. SO FO AO YO AO+YO Serum Non-HDL Cholesterol P < 0.05 80 z 70 a 60 zy a 50 mg/dL y y 40 b b b y 30 20 10 0 2 Wk 4 Wk Data are presented as mean + SEM. Different letters within week indicate significant differences between treatments (p < 0.05). 31
- 32. SO FO AO YO AO+YO P < 0.05 Serum Triglycerides 80 z a a 70 ab zy zy ab zy 60 50 y mg/dL b 40 30 20 10 0 2 Wk 4 Wk Data are presented as mean + SEM. Different letters within week indicate significant differences between treatments (p < 0.05). 32
- 33. No significant differencesin ACAT, ApoB100, DGAT, or LDLR SO FO AO YO AO+YO P < 0.05 HMG-CoA Reductase 2.5 z Expression Ratio yz 2 HMGCR/ARP a xy 1.5 ab x xy bc bc 1 c 0.5 0 2 Wk 4 Wk Data are presented as mean + SEM. Different letters within week indicate significant 33 differences between treatments (p < 0.05).
- 34. SO FO AO YO AO+YO Liver EPA P < 0.0001 8 z 7 a 6 % Fatty Acids b y y 5 c 4 3 d x 2 x 1 e 0 2 Wk 4 Wk Retroconversion DHA to EPA Data are presented as mean + SEM. Different letters within week indicate significant differences between treatments . 34
- 35. SO FO AO YO AO+YO Liver DHA a z P < 0.0001 25 20 y b b x % Fatty Acids 15 c w 10 d v 5 0 2 Wk 4 Wk Conversion EPA to DHA Data are presented as mean + SEM. Different letters within week indicate significant differences between treatments . 35
- 36. Why did theAO and YO diets have less EPA and DHA incorporation into the liver? • Excretion in the feces • DHA: Less excretion in the FO-fed mice • EPA: No differences 36 (Ketz JS, Barnes KM, unpublished results)
- 37. SO FO AO YO AO+YO Liver Ω3:Ω6 Ratio P < 0.0001 3.5 z 3 2.5 % Fatty Acids a 2 1.5 b y y y y 1 bc d cd 0.5 0 2 Wk 4 Wk Data are presented as mean + SEM. Different letters within week indicate significant differences between treatments . 37
- 38. • Introduction &Background • Objective • Methods • Results • Conclusion Conclusion • Implications • Future Research • Questions 38
- 39. • FO consumption more beneficial than AO or YO at: • Reducing serum lipids • Reducing serum TGs. • Increasing incorporation of EPA and DHA into the liver • Increasing Ω3: Ω6 ratio • Down-regulating hepatic HMGCR mRNA expression 39
- 40. • Not all sources of Ω3 fatty acids have equal benefits on cardiovascular health • May effect consumer’s choice of Ω3 fatty acid supplements 40
- 41. • Comparing different sources of marine origin vs. non-marine origin • Variety of other hepatic genes involved in lipogenesis • Incorporation EPA and DHA into various other tissues in addition to the liver • Human studies 41
- 42. Thesis Committee Members Kimberly M. Barnes, PhD, Chair Janet C. Tou, PhD Marie Krause, PhD WVU Davis College Siri Ippagunta, PhD & John Ketz Thank You! Melissa Olfert, DrPH, MS, RD, LD All other faculty Fellow peers and dietetic interns My Family & Friends My parents - Laura and Ted My siblings - Brian, Kelly, and Janie 42
- 44. • Kris-Etherton PM, Harris WS, Appel LJ. American Heart Association. Nutrition Committee. 2002. Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease. Circulation. 106(21):2747-57. • Centers for Disease Control and Prevention. Heart Disease Facts and Statistics. http://www.cdc.gov/dhdsp. • Pietrofesa RA, Azain MJ, Barnes KM. 2010. Lipidemic and cholesterolemic effects of feeding an algal source of docosahexaenoic acid to mice. FASEB J. 24:Abst#939.8. • Shelton AG, Pietrofesa RA, Barnes KM. 2011. Effect of high docosahexaenoic acid-algal oil on body fat and serum lipids in mice. FASEB J. 25:Abst#586.5. • MacKenzie SA, Belcher LA, Sykes GP, Frame SR, Mukerji P, Gillies PJ. 2010. Safety assessment of EPA-rich oil produced from yeast: Results of a 90-day subchronic toxicity study. Regul Toxicol Pharmacol. 58(3):490-500. • Bligh EG, Dyer WJ. 1959. A rapid method of total lipid extraction and purification. Can J Biochem Phys. 37: 911-917. • Park PW, Goins RE. 1994. In Situ Preparation of Fatty Acid Methyl Esters for Analysis of Fatty Acid Composition in Foods. Journal of Food Science, 59: 1262–1266. 44
Editor's Notes
- #9 ALA and is found primarily in plant-based foods.ALA is converted to EPA by the introduction of a double bond by Δ6-desaturase to stearidonic acid (18:4); this is the rate limiting step of the omega-3 pathway. Then, two carbons are added by elongase, followed by the action of Δ5-desaturase to form EPA (20:5). The next step is the conversion of EPA to DHA. This involves the addition of two carbons by elongase followed by the addition of another two carbons by elongase.The next step is desaturation by Δ6-desaturase to form a double bond, followed by β-oxidation which loses two carbons in order to form DHA (22:6). The conversion of ALA to EPA and ALA to DHA is not very efficient by the human body. The body only converts between approximately 8-10% of ALA to EPA and 0.05% to 4% of ALA to DHA. Therefore, it is more efficient to consume those PUFA’s as whole sources of EPA and DHA rather than through ALA.
- #16 After a 7 day acclimation period to the control diet, mice were blocked by body weight and randomly assigned to one of five dietary treatment groupsMice had free access to diets and waterThe AO and YO containing diets were formulated to provide the level of DHA and/or EPA provided by the FO diet
- #17 Subsequently, we determined that to equal the serum total cholesterol in the FO-fed mice, 3.5 - 5 times as much dietary DHA from AO needed to be fed. This may be due to the fact that less DHA is incorporated into liver and adipose tissue lipids in AO-fed mice compared to FO-fed mice Therefore, AO appears less effective than fish oil at reducing serum lipids in mice.However, most studies do not compare AO to FO
- #18 New on the market with little research, however a recent study concluded that the yeast oil produced little adverse ortoxicity effects.
- #24 HMGCR: rate-limiting step in cholesterol synthesisACAT: esterifies cholesterol for storageDGAT: formation of TGsLDLR:The number and activity of LDL receptors is a determinant of the level of LDL-C in the blood. When LDL binds to its LDL receptor, endocytosis is initiated and the LDL as well as the receptor are taken into the cell (known as receptor-mediated endocytosis). ApoB100: ligand for activation of the LDL-R
- #29 First let me set up and explain this graph because I will be using similar graphs throughout this presentation.At weeks 2 and 4, FO-fed mice had significantly greater relative liver weights compared to SO-fed miceWhen there is an increase in liver weight, we expect to see an increase in lipid in the liver. However there were no differences in total lipid or TGs in the liver.
- #31 As expected,FO and AO+YO-fed mice had significantly less serum total cholesterol compared to SO-fed mice at 2 and 4 weeksAt 2 and 4 weeks, AO and YO-fed mice were intermediate in lowering TC
- #32 At 2 wks, the YO-fed mice had similar non-HDL levels as SO-fed miceAt 4 wks, FO, YO, and AO+YO fed mice had sign less non-HDL levels than SO-fed miceThere were no differences in HDL cholesterol between any of the diets compared to SO. For this reason, most of the differences in TC were due to differences in non-HDL cholesterol.
- #33 FO-fed mice had significantly less serum TGs compared to the SO-fed mice at 2 weeks but not at 4 weeksHowever, FO-fed mice had significantly less serum TGs at 4 weeks compared to YO-fed mice
- #34 In an effort to understand the decreases in serum lipids, we measured liver mRNA expression of genes involved in lipid synthesis.There were no significant difference in ACAT, ApoB100, DGAT, or LDLR expressionHowever, there were significant differences in HMG-CoA Reductase expression. FO-fed mice had significantly reduced HMG-CoA Reductase expression compared to SO at both 2 and 4 weeksBecause HMGCR is the rate-limiting step in cholesterol synthesis. Therefore, this may explain the reductions we found in serum TC.Due to minimal differences in mRNA expression, we determined that the changes in serum lipids were not due to changes in transcription. Therefore, we measured the incorporation of these fatty acids into the liver.
- #35 At 2 and 4 weeks, FO-fed mice had significantly greater hepatic EPA thanall other diet treatments.At 2 weeks, AO fed mice had significantly greater EPA levels compared to SO-fed mice. This was interesting because the AO diet was formulated to provide almost no EPA. However, these results may have been due to the retro conversion of DHA to EPA.At 4 weeks, AO-fed mice did not differ in EPA compared to SO-fed mice. YO and AO+YO-fed mice were similar in EPA, but still less that FO-fed mice.
- #36 At 2 and 4 weeks, all diet treatments had significantly greater DHA concentrations than SO-fed mice. FO-fed mice had significantly greater DHA concentrations than SO-fed mice at 2 and 4 weeks.The AO+YO-fed mice had greater DHA than SO-fed mice, but not as much as the FO-fed mice.The YO diet was formulated to provide almost no DHA. Despite this, YO-fed mice still had sign. greater DHA levels at 2 and 4 weeks. This may be due to conversion of EPA to DHA.
- #38 Often, the ratio of omega-3 to omega-6 fatty acids is more important than the absolute concentrations of each. Therefore we calculated liver n3:n6 ratios. A higher ratio is said to be more heart healthy.At weeks 2 and 4, FO-fed mice had significantly greater omega-3 to omega-6 ratios