6.3 Strenghts and limitations
6.3.1 Study design and study population
A major strength of this study is the randomized controlled study design with parallel groups, which have allowed to investigate the causal effects of differentn-3 PUFAs on the fatty acid composition of blood lipid fractions and lipoprotein metabolism. The parallel study design might also be preferable to the crossover design for studying the effects ofn-3 PUFAs. These fatty acids, especially DHA, are cleared slowly from plasma and tissues (15,47), and therefore they may have long-term carryover effects. Furthermore, the food-based study design permitted study of the effects ofn-3 PUFAs with amounts achievable from dietary sources, whereas in supplemental studies, intakes of especially EPA and DHA have often been considerably larger.
Another strength is that this study included both men and women. However, the sample size was too small to investigate the gender-specific effects within the groups.
Furthermore, subjects had impaired glucose metabolism. Therefore, the results of this study may not apply to healthy, lean individuals. Moreover, subjects were not allowed to usen-3 PUFA supplements, but they were instructed not to change their habitual fish intake during the run-in period. This together with the fact that subjects had high habitual fish consumption, and consequently high baseline n-3 PUFA status, may have diluted the effects in this study. Moreover, due to blood sampling only at baseline and at the end of the study and lack of follow-up after the intervention, we were not able to assess the rate of changes in the investigated variables during the study or after it.
Another limitation in this study is that power calculations were based on differences in DHA in plasma PL, and it is possible that there was not enough power to see all changes in these secondary outcome variables. However, it would have been impossible to use most of these secondary outcome variables for the power calculations due to lack of previous data.
6.3.2 Methods Analytical methods
Fatty acid analyses were performed by methods that have been in long-term use and are considered high-quality (88,209). Fatty acid compositions of separate lipid fractions were analyzed, which may be preferable over whole blood or total plasma fatty acids since changes in fatty acids of individual lipid classes may have considerable influence on the fatty acid composition of whole blood or total lipids (9,64). The stability of fatty acids during storage may have affected the results.
89 However, it has been shown that storage at –80 °C up to 10 years does not significantly affect the fatty acid profiles of serum PL, CE or TG (263).
Another strength in this doctoral thesis is the use of an NMR-based metabolomic approach, which has been found to be a robust and reliable method for investigating changes in dietary intervention studies. Moreover, the NMR-based metabolomics method requires minimal sample preparation and has high reproducibility as compared with other available methods, such as density ultracentrifugation and gradient gel electrophoresis (264,265). NMR also provides a complete lipoprotein subclass profile, which allows to study the changes in all individual lipoprotein subclasses.
The lipoprotein functions assessed in this doctoral thesis have been detected in both human and experimental atherosclerosis (8,174,266). However, the results should be interpreted with caution because in vitro treatments do not necessarily mimic physiological conditions. Furthermore, it would have been interesting to also study the role of individual lipoprotein particles (with isolated lipoproteins) in lipoprotein-proteoglycan interactions.
Other methods
A significant strength of this study is good compliance with the study diets which was monitored with 4-day food records three times during the study, consumption records regarding the intakes of CSO and fish and several biomarkers,i.e. fatty acid composition of EM and plasma PL, CE and TG. Dietary records were checked by a nutritionist at return, but inaccuracies due to under- or overreporting, altered food intake behaviors during the recording period and limitations in the food composition databases cannot be ruled out. Furthermore, baseline food records from only four days may not have been long enough to accurately assess then-3 PUFA intake (219).
Another limitation in this study is that we were not able to assess the dietary intake of DPA and its association with circulating DPA because the AivoDiet nutrient calculation software, and the food composition database on which it is based, do not provide information about the DPA content of food.
Several possible confounding factors that have been found to affect plasman-3 fatty acid proportions (79) or lipoprotein subclasses (133,134) were considered in the statistical analyses (Studies I and II), but the possibility of residual confounding cannot be ruled out. Furthermore, due to the lack of genetic information, we could not control for genetic factors that may have had an impact on the fatty acid compositions, conversion of ALA to long-chainn-3 PUFAs and lipoprotein particles (75,78,136,137).
90
CONCLUSIONS
ALA, EPA and DHA were incorporated into blood lipid fractions in diet-dependent manner, which confirms the findings of previous studies. However, results of this doctoral thesis also provide insight into the metabolism ofn-3 fatty acids showing thatn-3 PUFA content of blood lipid fractions are closely related. Moreover, EM, PL, CE and TG seem to respond similarly to the intake of long-chainn-3 PUFAs from fish and ALA from CSO. Dietary intakes of ALA, EPA and DHA also correlate with their respective proportions in a similar manner in different lipid fractions. Together these results suggest that these lipid fractions can be used interchangeably as biomarkers of dietaryn-3 PUFA intake in studies of similar length.
Diet is one of the modifiable lifestyle factors in the prevention of CVD. The results of this study offer practical dietary approaches for the prevention of CVD in high-risk subjects. Fatty fish intake four times a week was found to alter the size and composition of HDL toward larger and lipid-rich particles, whereas ALA intake of approximately 10 g per day from CSO decreased the IDL particle concentration. The changes in HDL particles may be associated with the atheroprotective properties of HDL. Furthermore, the decrease in IDL particle concentration after CSO intake may favorably modify the risk of CVD. Although lean fish intake was not found significantly affect the lipoprotein subclass distribution, complementary effects of other bioactive components in fish on lipoprotein particles cannot be ruled out.
The present study also contributes to the limited number of studies on effects of diet and especiallyn-3 PUFAs on pro- and anti-atherogenic lipoprotein functions.
We found that intake of CSO decreases the binding of lipoproteins to aortic proteoglycans by decreasing serum apoB-containing lipoprotein concentrations.
However, the intake of fish or CSO had no effect on LDL aggregation, activation of endothelial cells or cholesterol efflux capacity of HDL. Lack of effects on these lipoprotein functions may be related to the high long-chainn-3 PUFA status of the subjects at the baseline of this study. Therefore, studies in the future should include subjects with low long-chain n-3 PUFA status. Alternatively, in populations with high fish consumption, studies should include a sufficiently long run-in period during which the intake of fish should be limited, and fish oil supplements should not be allowed.
In conclusion, in this doctoral thesis we have shown that easily achieved dietary modifications beneficially affect the lipoprotein subclass profile and one of the key mechanisms in early atherosclerosis despite the high habitual fish intake of the subjects in this study. Furthermore, ALA and long-chainn-3 PUFAs seem to differ in their effects on lipoprotein particles. Consequently, this suggests that ALA possesses activity in lipoprotein metabolism independent of its conversion to long-chainn-3 PUFAs. Thus, these findings highlight that n-3 PUFAs of both marine and plant origin are important for the cardiovascular health.
91
REFERENCES
1. Roth GA, Johnson C, Abajobir A, et al. Global, Regional, and National Burden of Cardiovascular Diseases for 10 Causes, 1990 to 2015.J Am Coll Cardiol 2017;
70: 1–25.
2. Rippe JM. Lifestyle Strategies for Risk Factor Reduction, Prevention, and Treatment of Cardiovascular Disease.Am J Lifestyle Med 2019; 13: 204–212.
3. Mozaffarian D, Appel LJ, Van Horn L. Components of a cardioprotective diet:
new insights.Circulation 2011; 123: 2870–91.
4. Schwab U, Lauritzen L, Tholstrup T, et al. Effect of the amount and type of dietary fat on cardiometabolic risk factors and risk of developing type 2 diabetes, cardiovascular diseases, and cancer: a systematic review.Food Nutr Res 2014; 58: 25145.
5. National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) final report.
Circulation 2002; 106: 3143–421.
6. Krauss RM. Lipoprotein subfractions and cardiovascular disease risk. Curr Opin Lipidol 2010; 21: 305–311.
7. Borén J, Williams KJ. The central role of arterial retention of cholesterol-rich apolipoprotein-B-containing lipoproteins in the pathogenesis of atherosclerosis: a triumph of simplicity.Curr Opin Lipidol 2016; 27: 473–83.
8. Oörni K, Pentikäinen MO, Ala-Korpela M, et al. Aggregation, fusion, and vesicle formation of modified low density lipoprotein particles: molecular mechanisms and effects on matrix interactions.J Lipid Res 2000; 41: 1703–14.
9. Hodson L, Skeaff CM, Fielding BA. Fatty acid composition of adipose tissue and blood in humans and its use as a biomarker of dietary intake.Prog Lipid Res 2008; 47: 348–380.
10. Serra-Majem L, Nissensohn M, Øverby NC, et al. Dietary methods and biomarkers of omega 3 fatty acids: A systematic review.Br J Nutr 2012; 107:
S64–76.
11. Arab L. Biomarkers of fat and fatty acid intake.J Nutr 2003; 133 Suppl: 925S–
932S.
12. Cholewski M, Tomczykowa M, Tomczyk M. A Comprehensive Review of Chemistry, Sources and Bioavailability of Omega-3 Fatty Acids.Nutrients 2018;
10: e1662.
13. Calder PC. Mechanisms of Action of (n-3) Fatty Acids.J Nutr 2012; 142: 592S–
599S.
14. Burdge GC, Calder PC. Conversion of -linolenic acid to longer-chain polyunsaturated fatty acids in human adults.Reprod Nutr Dev 2005; 45: 581–
92 597.
15. Arterburn L.M., Hall E.B., Oken H. Distribution, interconversion, and dose response of n-3 fatty acids in humans.Am J Clin Nutr 2006; 83: 1467S–1476S.
16. Wood KE, Mantzioris E, Gibson RA, et al. The effect of modifying dietary LA and ALA intakes on omega-3 long chain polyunsaturated fatty acid (n-3 LCPUFA) status in human adults: a systematic review and commentary.
Prostaglandins Leukot Essent Fatty Acids 2015; 95: 47–55.
17. Rodriguez A, Sarda P, Nessmann C, et al. Delta6- and delta5-desaturase activities in the human fetal liver: kinetic aspects.J Lipid Res 1998; 39: 1825–32.
18. Barceló-Coblijn G, Murphy EJ. Alpha-linolenic acid and its conversion to longer chain n-3 fatty acids: Benefits for human health and a role in maintaining tissue n-3 fatty acid levels.Prog Lipid Res 2009; 48: 355–374.
19. Mozaffarian D, Wu JHY. Omega-3 fatty acids and cardiovascular disease:
effects on risk factors, molecular pathways, and clinical events.J Am Coll Cardiol 2011; 58: 2047–67.
20. Vidgren HM, Agren JJ, Schwab U, et al. Incorporation of n-3 fatty acids into plasma lipid fractions, and erythrocyte membranes and platelets during dietary supplementation with fish, fish oil, and docosahexaenoic acid-rich oil among healthy young men.Lipids 1997; 32: 697–705.
21. Mozaffarian D, Wu JHY. (n-3) Fatty Acids and Cardiovascular Health: Are Effects of EPA and DHA Shared or Complementary?J Nutr 2012; 142: 614S–
625S.
22. Metherel AH, Irfan M, Klingel SL, et al. Compound-specific isotope analysis reveals no retroconversion of DHA to EPA but substantial conversion of EPA to DHA following supplementation: a randomized control trial.Am J Clin Nutr 2019; 110: 823–831.
23. Gillingham LG, Harding S V, Rideout TC, et al. Dietary oils and FADS1-FADS2 genetic variants modulate [13C] -linolenic acid metabolism and plasma fatty acid composition.Am J Clin Nutr 2013; 97: 195–207.
24. Burdge G. Alpha-linolenic acid metabolism in men and women: nutritional and biological implications.Curr Opin Clin Nutr Metab Care 2004; 7: 137–44.
25. Micha R, Khatibzadeh S, Shi P, et al. Global, regional, and national consumption levels of dietary fats and oils in 1990 and 2010: A systematic analysis including 266 country-specific nutrition surveys.BMJ 2014; 348: 1–20.
26. Saini RK, Keum Y-S. Omega-3 and omega-6 polyunsaturated fatty acids:
Dietary sources, metabolism, and significance — A review.Life Sci 2018; 203:
255–267.
27. Burdge GC, Finnegan YE, Minihane AM, et al. Effect of altered dietary n-3 fatty acid intake upon plasma lipid fatty acid composition, conversion of [13C]alpha-linolenic acid to longer-chain fatty acids and partitioning towards beta-oxidation in older men.Br J Nutr 2003; 90: 311–21.
28. Davis BC, Kris-Etherton PM. Achieving optimal essential fatty acid status in vegetarians: current knowledge and practical implications.Am J Clin Nutr 2003;
93 78: 640S–646S.
29. Surette ME, Edens M, Chilton FH, et al. Dietary echium oil increases plasma and neutrophil long-chain (n-3) fatty acids and lowers serum triacylglycerols in hypertriglyceridemic humans.J Nutr 2004; 134: 1406–11.
30. Tahvonen RL, Schwab US, Linderborg KM, et al. Black currant seed oil and fish oil supplements differ in their effects on fatty acid profiles of plasma lipids, and concentrations of serum total and lipoprotein lipids, plasma glucose and insulin.J Nutr Biochem 2005; 16: 353–359.
31. Passi S, Cataudella S, Di Marco P, et al. Fatty acid composition and antioxidant levels in muscle tissue of different Mediterranean marine species of fish and shellfish.J Agric Food Chem 2002; 50: 7314–22.
32. Strobel C, Jahreis G, Kuhnt K. Survey of n-3 and n-6 polyunsaturated fatty acids in fish and fish products.Lipids Health Dis 2012; 11: 1–10.
33. Sun Q, Ma J, Campos H, et al. Blood concentrations of individual long-chain n-3 fatty acids and risk of nonfatal myocardial infarction.Am J Clin Nutr 2008; 88:
216–23.
34. Miller E, Kaur G, Larsen A, et al. A short-term n-3 DPA supplementation study in humans.Eur J Nutr 2013; 52: 895–904.
35. Childs CE, Romeu-Nadal M, Burdge GC, et al. Gender differences in the n-3 fatty acid content of tissues.Proc Nutr Soc 2008; 67: 19–27.
36. Gil A, Gil F. Fish, a Mediterranean source of n-3 PUFA: benefits do not justify limiting consumption.Br J Nutr 2015; 113: S58–S67.
37. Schuchardt JP, Hahn A. Bioavailability of long-chain omega-3 fatty acids.
Prostaglandins Leukot Essent Fat Acids 2013; 89: 1–8.
38. Fleming JA, Kris-Etherton PM. The evidence for -linolenic acid and cardiovascular disease benefits: Comparisons with eicosapentaenoic acid and docosahexaenoic acid.Adv Nutr 2014; 5: 863S–76S.
39. Gabbs M, Leng S, Devassy JG, et al. Advances in Our Understanding of Oxylipins Derived from Dietary PUFAs.Adv Nutr 2015; 6: 513–40.
40. Innes JK, Calder PC. Omega-6 fatty acids and inflammation. Prostaglandins, Leukot Essent Fat Acids 2018; 132: 41–48.
41. Karvonen HM, Aro A, Tapola NS, et al. Effect of alpha-linolenic acid-rich Camelina sativa oil on serum fatty acid composition and serum lipids in hypercholesterolemic subjects.Metabolism 2002; 51: 1253–60.
42. Finnegan YE, Minihane AM, Leigh-Firbank EC, et al. Plant- and marine-derived n-3 polyunsaturated fatty acids have differential effects on fasting and postprandial blood lipid concentrations and on the susceptibility of LDL to oxidative modification in moderately hyperlipidemic subjects.Am J Clin Nutr 2003; 77: 783–95.
43. Kew S, Banerjee T, Minihane AM, et al. Lack of effect of foods enriched with plant- or marine-derived n 3 fa y acids on human immune function.Am J Clin Nutr 2003; 77: 1287–1295.
44. Wallace FA, Miles EA, Calder PC. Comparison of the effects of linseed oil and
94
different doses of fish oil on mononuclear cell function in healthy human subjects.Br J Nutr 2003; 89: 679–89.
45. Wilkinson P, Miller GJ, Millward DJ, et al. Influence of -linolenic acid and fish-oil on markers of cardiovascular risk in subjects with an atherogenic lipoprotein phenotype.Atherosclerosis 2005; 181: 115–124.
46. Harper CR, Edwards MJ, DeFilippis AP, et al. Flaxseed oil increases the plasma concentrations of cardioprotective (n-3) fatty acids in humans.J Nutr 2006; 136:
83–7.
47. Cao J, Schwichtenberg KA, Hanson NQ, et al. Incorporation and clearance of omega-3 fatty acids in erythrocyte membranes and plasma phospholipids.Clin Chem 2006; 52: 2265–72.
48. Schwab US, Callaway JC, Erkkilä AT, et al. Effects of hempseed and flaxseed oils on the profile of serum lipids, serum total and lipoprotein lipid concentrations and haemostatic factors.Eur J Nutr 2006; 45: 470–7.
49. Goyens PLL, Spilker ME, Zock PL, et al. Conversion of alpha-linolenic acid in humans is influenced by the absolute amounts of alpha-linolenic acid and linoleic acid in the diet and not by their ratio.Am J Clin Nutr 2006; 84: 44–53.
50. Zhao G, Etherton TD, Martin KR, et al. Dietary alpha-linolenic acid inhibits proinflammatory cytokine production by peripheral blood mononuclear cells in hypercholesterolemic subjects.Am J Clin Nutr 2007; 85: 385–91.
51. Barceló-Coblijn G, Murphy EJ, Othman R, et al. Flaxseed oil and fish-oil capsule consumption alters human red blood cell n-3 fatty acid composition: a multiple-dosing trial comparing 2 sources of n-3 fatty acid.Am J Clin Nutr 2008;
88: 801–9.
52. Dodin S, Cunnane SC, Mâsse B, et al. Flaxseed on cardiovascular disease markers in healthy menopausal women: a randomized, double-blind, placebo-controlled trial.Nutrition 2008; 24: 23–30.
53. Rajaram S, Haddad EH, Mejia A, et al. Walnuts and fatty fish influence different serum lipid fractions in normal to mildly hyperlipidemic individuals: a randomized controlled study.Am J Clin Nutr 2009; 89: 1657S–1663S.
54. Egert S, Lindenmeier M, Harnack K, et al. Margarines Fortified with -Linolenic Acid, Eicosapentaenoic Acid, or Docosahexaenoic Acid Alter the Fatty Acid Composition of Erythrocytes but Do Not Affect the Antioxidant Status of Healthy Adults.J Nutr 2012; 142: 1638–1644.
55. Zong G, Demark-Wahnefried W, Wu H, et al. Effects of flaxseed supplementation on erythrocyte fatty acids and multiple cardiometabolic biomarkers among Chinese with risk factors of metabolic syndrome.Eur J Nutr 2013; 52: 1547–51.
56. Chiang Y-L, Haddad E, Rajaram S, et al. The effect of dietary walnuts compared to fatty fish on eicosanoids, cytokines, soluble endothelial adhesion molecules and lymphocyte subsets: a randomized, controlled crossover trial.
Prostaglandins Leukot Essent Fatty Acids 2012; 87: 111–7.
57. Dittrich M, Jahreis G, Bothor K, et al. Benefits of foods supplemented with
95 vegetable oils rich in -linolenic, stearidonic or docosahexaenoic acid in hypertriglyceridemic subjects: a double-blind, randomized, controlled trail.
Eur J Nutr 2015; 54: 881–93.
58. Potischman N, Freudenheim JL. Biomarkers of Nutritional Exposure and Nutritional Status: An Overview.J Nutr 2003; 133: 873S–874S.
59. Silva V, Barazzoni R, Singer P. Biomarkers of fish oil omega-3 polyunsaturated fatty acids intake in humans.Nutr Clin Pract 2014; 29: 63–72.
60. Ratnayake WMN, Galli C. Fat and Fatty Acid Terminology, Methods of Analysis and Fat Digestion and Metabolism: A Background Review Paper.Ann Nutr Metab 2009; 55: 8–43.
61. Brennan L, Hu FB. Metabolomics-Based Dietary Biomarkers in Nutritional Epidemiology-Current Status and Future Opportunities. Mol Nutr Food Res 2019; 63: e1701064.
62. Calder PC. Functional Roles of Fatty Acids and Their Effects on Human Health.
J Parenter Enter Nutr 2015; 39: 18S–32S.
63. Venäläinen T, Schwab U, Ågren J, et al. Cross-sectional associations of food consumption with plasma fatty acid composition and estimated desaturase activities in Finnish children.Lipids 2014; 49: 467–79.
64. Brenna JT, Plourde M, Stark KD, et al. Best practices for the design, laboratory analysis, and reporting of trials involving fatty acids.Am J Clin Nutr 2018; 108:
211–227.
65. Browning LM, Walker CG, Mander AP, et al. Incorporation of eicosapentaenoic and docosahexaenoic acids into lipid pools when given as supplements providing doses equivalent to typical intakes of oily fish.Am J Clin Nutr 2012;
96: 748–58.
66. Walker CG, Browning LM, Stecher L, et al. Fatty acid profile of plasma NEFA does not reflect adipose tissue fatty acid profile.Br J Nutr 2015; 114: 756–762.
67. Browning LM, Walker CG, Mander AP, et al. Compared with daily, weekly n-3 PUFA intake affects the incorporation of eicosapentaenoic acid and docosahexaenoic acid into platelets and mononuclear cells in humans.J Nutr 2014; 144: 667–72.
68. Raatz SK, Rosenberger TA, Johnson LK, et al. Dose-dependent consumption of farmed Atlantic salmon (Salmo salar) increases plasma phospholipid n-3 fatty acids differentially.J Acad Nutr Diet 2013; 113: 282–7.
69. Katan MB, Deslypere JP, van Birgelen AP, et al. Kinetics of the incorporation of dietary fatty acids into serum cholesteryl esters, erythrocyte membranes, and adipose tissue: an 18-month controlled study.J Lipid Res 1997; 38: 2012–22.
70. Elvevoll EO, Barstad H, Breimo ES, et al. Enhanced incorporation of n-3 fatty acids from fish compared with fish oils.Lipids 2006; 41: 1109–14.
71. Harris WS, Pottala J V, Sands SA, et al. Comparison of the effects of fish and fish-oil capsules on the n 3 fatty acid content of blood cells and plasma phospholipids.Am J Clin Nutr 2007; 86: 1621–5.
72. Rice HB, Bernasconi A, Maki KC, et al. Conducting omega-3 clinical trials with
96
cardiovascular outcomes: Proceedings of a workshop held at ISSFAL 2014.
Prostaglandins, Leukot Essent Fat Acids 2016; 107: 30–42.
73. Yuzyuk T, Lozier B, Schwarz EL, et al. Intra-individual variability of long-chain
73. Yuzyuk T, Lozier B, Schwarz EL, et al. Intra-individual variability of long-chain