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Omega-3 PUFA in Clinical Trials/Studies with Coronary Heart Disease (CHD) Risk Factor-Relevant Endpoints

The majority of basic and clinical studies as well as several clinical trials using various types of omega-3-rich foods or supplements^1-10 strongly suggest that there is an inverse correlation between omega-3 (n-3) consumption and risk of coronary heart disease.

Research showed that n-3 polyunsaturated fatty acids (PUFA) displayed potential beneficial effects on cardiovascular risk factors such as antiatherogenicity, antithrombotic and antiarrhythmic ^11-13 and hypotriglyceridemic¹ effects. The Chicago Western Electric Study⁵ (Chicago), entitled "Fish consumption and the 30-year risk of fatal myocardial infarction"; the GISSI-Prevention Trial¹⁰ (GISSI); entitled "Dietary supplementation with n-3 PUFA and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial" and the Lyon Diet Heart Trial⁸ (Lyon), entitled "Mediterranean diet , traditional risk factors and the rate of cardiovascular complications after myocardial infarct" are three representative studies discussed below. Table 1 summarizes the structure, organization and outcomes of the Chicago, GISSI and Lyon studies.

Comparison of the Chicago Western Electric Study, the GISSI-Prevention Trial
 and the Lyon Diet Heart Trial

TABLE 1

Major differences and similarities among the three studies in Table 1 are noted below:

Major differences:

1. Nature of the study or clinical trial, e.g., observational (Chicago) vs, interventional (GISSI and Lyon); stratified (Chicago) vs. Randomized (GSSI and Lyon); single center (Chicago and Lyon) vs. Multicenter (GSSI); retrospective (Chicago) vs. prospective (GSSI and Lyon).

2. The observational study (Chicago) had a 30-year long follow-up period vs. the interventional trials where therapy and follow-up lasted between 3 and 5 years.

3. Omega-3 PUFA was administered to the appropriate treatment groups in three different forms:

a. As amount of fish consumed per day (Chicago)

b. As supplemented fish oil in 1.0 g per day capsules containing eicosapentaenoic acid (EPA,

20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) in a 2:1 ratio (GISSI)

c. As a special Mediterranean diet (MedDiet), (Lyon) that was enriched in alpha-linolenic acid (ALN, 18:1n-9) and oleic acid (18:1n-9) the major component of olive oil.

In the Chicago study two nutritionists collected the information at the first and second examinations, 1 year apart with standard questionnaires on typical workday and weekend day eating patterns. Fish consumption was assessed as part of the overall eating pattern, and a profile was established that contained some 26 foods over a 28 day period. The food profile included soft drinks; coffee; milk (whole and skim); cream, cheese; eggs; ice cream, puddings or custards; soups; fish, beef; veal or lamb; pork; ham or bacon; liver; poultry; vegetables; breads and cereals; potatoes; fruits; pastries; sugar; butter; margarine and fried foods. Items were coded on a four point scale as 0 for none, 1 for low, 2 for moderate and 3 for high consumption. Fish consumption in 120 g units per 28 days was coded as 0 for none, 1 for less than 4 units, 2 for 4 to 8 units and 3 for more than 8 units. The profile obtained was a representative cross section of a Mid-Western American diet. The average omega-6 and omega-3 profiles of an American diet as assessed by an ongoing food survey of the U.S. Department of Agriculture for the 1994-1996 period, presented in Table 2, indicates that the ratio of omega-6 to omega-3 PUFA in a daily food intake is about 10:1. It also shows that the major polyunsaturated fat consumed by the American public is linoleic acid (LA, 18:3n-6) and that the daily intake of long-chain n-3 PUFA (EPA and DHA) is negligible. Given these intake levels, the study outcome of the cohort that consumed >35 g fish/day becomes even more significant. It should be noted that the 10:1 ratio for omega-6:omega-3 intake should be considered a low value. Larger intake of LA and lower intake of LNA, would raise this ratio even higher. A discussion of the association between high values for the omega-6:omega-3 ratio and increased risk of CHD, later in the Lyon study, will further clarify this issue.

Mean percentages of food energy, 1 Day

Sex and Age	Food Energy Intake	18:2	18:3	18:4	20:4	20:5	22:5	22:6	Total PUFA
All Indiv.	2,002 kcal	5.7	0.6	*	0.1	*	*	*	6.4

Omega 6: Omega-e ratio 9.6:1

*Values less than 0.05 but more than zero.

Mean percentages of PUFA intakes (in grams)

Omega 6: Omega-3 ratio 10:1

Mean percentages of total contributed by individual fatty acids

Omega 6: Omega-3 ratio 10:1

* Results from the 1994-1996 Continuing Survey of Food Intakes by Individuals. The data provides national probability estimates for the U.S. population (over 16,000 individuals participated in a survey known as What We Eat in America). Recall data represent weighted intakes based on respondents first of 2 days reported.

The investigators of the GISSI trial assessed the patient’s dietary habits with questionnaires. Fish (>1 serving/week), fruit (<1 serving/day), fresh vegetables (>1 serving/day) and olive oil consumption were followed every 6 months during the trial. The regimen of one capsule/day of fish oil containing about 850 mg of n-3 PUFA (EPA and DHA as ethyl esters in a 1:2 ratio) used in the trial was considered similar to a diet that contained about 100 g of fatty fish consumed every day. This amount is significantly lower than the pharmacological doses of fish oil used in mechanistic and animal studies. The moderate fish oils provided daily were considered in line with a need for long term supplementation. Although the value of the omega-6: omega-3 intake is not provided for this trial, it should approach the value of the typical Japanese diet (<4:1).

The MedDiet in the Lyon trial was modeled after the Cretan Diet discovered in the Seven Countries Study¹⁴. In this study, a 5-15 year follow-up concluded that, mortality rates from CHD in Southern Europe was two-to-threefold lower than in Northern Europe or the United States ^{15, 16} (see Table 3). It is interesting to note that in the Seven Countries Study the cohort from Crete presented a CHD mortality that was 95% less than in Northern European Countries and 98% less than the U.S. cohort. It is also of interest to note that the differences in cholesterol levels do not appear to account for these large differences in mortality by themselves.

Thus the diet for the experimental group of the Lyon Diet Heart Trial was constructed to contain a) more bread, b) more vegetables and legumes, c) more fish, d) less meat (beef, lamb, pork) and replaced with poultry, e) no day without fruit and f) no butter and cream. Butter and cream were replaced with a special, erucic acid-free canola oil-based margarine comparable with olive oil (high in oleic acid, 18:1n-9) enriched mostly with alpha-linolenic acid (LNA, 18:3n-3) and slightly with linoleic acid (LA, 18:2n-6). Its composition is 8.4% 16:0 (saturated fat), 6% 18:0 (saturated fat), 48% cis 18:1n-9, 5.4% trans 18:1n-9, 16.4% cis 18:2n-6 and 4.8% cis 18:3n-3. This margarine, which was used for food preparations and on bread contains an omega-6:omega-3 ratio of 3:1. Oils recommended for salads and food preparations were erucic acid-free rapeseed oil (relatively high in 18:3n-3) and olive oil (high in 18:1n-9). Moderate amounts of alcohol in form of red wine were allowed and recommended at meals. Counseling by dietitians was provided, and compliance with the diet was monitored by providing measured amounts of margarine at outpatient clinic bimonthly visits and by checking consumption at each visit to the clinic. Dietary surveys were conducted regularly through 24 hour recall and a food frequency questionnaire. The study showed that the approximately 300 patients randomized to the experimental group on the MedDiet maintained their diets during the average of 46 months follow-up period.

Fatty acid analysis of plasma lipids was performed at each visit. After only 2 months of trial (at the first follow-up) plasma fatty acid levels of the experimental group showed statistically significant increases in 18:1n-9, 18:3n-3, 20:5n-3 and 22:6n-3 and decreases in 18:0, 18:2n-6 and arachidonic acid (20:4n-6, another prevalent and important omega-6 PUFA) levels vs. the control group. These changes brought about a lowering of the n-6:n-3 ratio in plasma of the experimental group vs. the control group from 11.6 to 9.95 during the same period.

Seven Countries Study: Comparison of Selected Cohorts

TABLE 3

From Renaud S. et al.¹⁶

Major similarities

1. Patient selection criteria were comparable in all three studies.

2. Primary endpoints are comparable. These included death from all causes, death due to MI, to CVD, to CHD, N-F, MI and N-F stroke.

3. Relative risks and risk ratio outcomes show reduced recurrence or occurrence of primary endpoints in all studies.

In the Chicago study all-cause and cardiovascular mortality and the combination of recurrent MI and cardiac death (CO1) were reduced (risk ratio for CO1 was 0.28).

In the GISSI trial it was shown that the magnitude of effectiveness of the n-3 PUFA treatment on occurrence of total death, N-F MI and N-F Stroke (combined primary endpoints) corresponds to a 10% relative decrease in risk in the 2-way analysis ( considering only the cohorts supplemented with fish oil vs. the controls).and 15% relative decrease in the 4-way analysis (considering all supplemented cohorts and controls ).

The magnitude of effectiveness of the n-3 PUFA treatment on occurrence of cardiovascular death, N-F MI and N-F Stroke (combined primary endpoints) reaches a 20% relative decrease in the 4-way analysis [showing the more realistic ("true") results].

It should be noted that Vitamin E supplementation had no added advantages for the primary endpoint groups analyzed. The Italian subjects included in the GISSI trial could have already been on a Mediterranean-type diet. Thus, the 300 mg vitamin E would not have had the same beneficial effect on CHD risk factors such as inhibition of LDL-cholesterol oxidation, in this population as was found in other studies.¹⁹

Since the subjects in all groups were treated with various drugs, including aspirin, the observed preventive outcomes for n-3 PUFA supplementation are largely independent and above those effected by the drug therapies.

The beneficiary effect of EPA and DHA in the cardiac signal and transduction pathway and its potential of reducing the incidence of instant cardiac death from ventricular fibrillation has been extensively documented in recent studies by Billman et al.,¹⁸.

The Lyon Heart Diet Study ^8,16, with its diet based on increased consumption of bread and cereals, vegetables and legumes, fruit, fish and edible vegetable fat, resulted in an adjusted, decreased risk of recurrence ratio of 0.28 (95% CI, p=0.0001), a highly significant reduction in combined cardiac death, and N-F MI in favor of the experimental group on the MedDiet in the CO 1 endpoints group.

Although the Lyon study did not find any correlation involving long-chain omega-3 PUFA (EPA or DHA) the study design did not emphasize their role in the Mediterranean diet. It should be noted, however, that the levels of alpha-linolenic acid EPA and DHA in plasma lipids increase in the experimental group, while the levels of n-6 acids (linoleic and arachidonic 20:4n-6) decrease. Dietary linolenic acid is the precursor of EPA and DHA in vivo. Nevertheless, given the slow rate

and low yield of the 18:3n-3 to 20:5n-3 in vivo transformation steps, both EPA and DHA can be supplemented by inclusion of fatty fish and some seafood varieties in diet by preparing food products with supplemented EPA and DHA or by taking moderate amounts of concentrated omega-3 oil supplements.. Alpha-linolenic acid (18:3n-3) was associated with improved prognosis in agreement with another recent study outcome ^17.

4. Adjustment of outcomes for CHD risk factors or confounding variables is comparable among the studies. These include plasma cholesterol levels (LDL) and total cholesterol, systolic blood pressure, blood leukocyte counts, smoking and cardiovascular medication.

Conclusions

1. Varied approaches utilized by the Chicago study, and by the GISSI and Lyon trials resulted in comparable endpoint outcomes, all pointing strongly toward an inverse relationship between omega-3 in the diet and risk of CHD,

2. It appears that a modified Mediterranean diet (MedDiet), modeled after a diet from the island of Crete and based on high oleic and alpha linolenic acid content, provides increased protection from CHD,

3. In line with the original study of Bang, Dyerberg, and Hjorne ²⁰, who reported for the first time a possible connection between the almost complete lack of CHD among the Greenland Eskimos and the high concentration of long-chain n-3 PUFA (EPA and DHA) that their diet contained, the Chicago study and the GISSI trial showed that high level fish consumption (>35 g/day) and supplementation of EPA and DHA (about 850 mg/day as EPA and DHA in a 1:2 ratio) are both beneficial in reducing the occurrence or recurrence of fatal and non-fatal coronary events.

4. The above clinical studies and trials also pinpoint the importance of a low omega-6 to omega-3 ratio in the maintenance of a healthy cardiovascular system.

5. Although we are still far from understanding the mechanisms through which omega-3 fatty acids exert their positive effects on lowering the risk of CHD, the amassed clinical information justify the acknowledgement of their positive effects in protecting the cardiovascular health of American public at large.

6. Although there were no significant deleterious or toxic effects reported in these trials, long-term administration of pharmacological amounts of fish oils should be considered only under supervision of a physician.

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Recommendations for Adequate Intakes (AI) of PUFA for Adults

Participants of the NIH International Workshop on the Essentiality of, and Recommended Dietary Intakes for Omega-6 and Omega-3 Fatty Acids presented and discussed clinical nutritional study outcomes in the established fields of infant nutrition and cardiovascular disease and the new and promising area of mental health. The international nature and outstanding professional level of the participants rendered the group and its conclusions highly competent.^{21 ,22} A summary of the recommendations for adults is presented in Table 4..

The group agreed that sufficient scientific evidence has been amassed in the infant nutrition and cardiovascular areas to recommend daily adequate intakes (AIs) of omega-6 and omega-3 essential fatty acids for adult diets and for infant formulas/diets. The group also discussed the

health effects of trans-fatty acids and recommended an upper limit for daily AI of trans-fatty acids in adults, In a similar fashion, an upper limit for saturated fatty acids was recommended for adults.

Table 4, AIs for Adults, details the recommendation for linoleic acid (LA) (or 18:2n-6, the essential polyunsaturated omega-6 fatty acid) for alpha-linolenic acid (LNA) (or 18:3n-3, the essential polyunsaturated omega-3 fatty acid), and for eicosapentaenoic acid (EPA 20:5-n-3) and docosahexaenoic acid (DHA 22:6n-3), which are the penta- and hexapolyunsaturated long chain omega-3 fatty acids derived through metabolic processes from LA.

The group consensus was that due to the low yield of the LNA to EPA to DHA pathway, it is advisable to supplement EPA and DHA resulting from the above pathway with naturally occurring EPA and DHA (from fish, other foods, or nutritional supplements). It should be noted that beside LA, the group did not recommend AIs for arachidonic acid (AA, 20:4n-6), a metabolic transformation product of LA. The reason for this is twofold: 1) The LA to AA pathway provides higher yields of elongated and further desaturated products, and 2) In the general population the direct consumption of AA is much higher than EPA or DHA since AA is one of the major polyunsaturated fatty acids in red meat (beef, lamb, or pork).

In terms of daily AIs recommended for LA (upper limit) vs. LNA+DHA and EPA, in other words the omega-6:omega-3 ratio, is 2.3:1. The omega-6:omega-3 ratio in adult plasma varies between 10.0 and 27.0, indicating that the major source of fat consumed in today’s Western diet is LA. Hence, the outcome of the recommendation is that monounsaturated fatty acids should constitute the predominant fatty acids consumed in our daily diet.

The recommendations of Table 4 are consistent with the elements of a modified Mediterranean diet. Such a diet was also described in a recently published book entitled The Omega Plan ²³ .

Adequate Intakes (AI) of PUFA for Adults*
Recommendations of an Expert Working Group (WG) on Omega-6 and Omega-3 PUFA Intakes.

TABLE 4

*For pregnant and lactating women ensure 300 mg/d of DHA
**Except for dairy products, other foods under natural conditions do not contain trans-FA. Therefore the WG does not recommend trans-FA to be in the food supply.
***The WG recommends that the majority of FA ingested be from monounsaturates.

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The Hypotriglyceridemic Effect of Long-Chain Omega-3 Fatty Acids

The GISSI Prevention trial ¹⁰ reported a statistically significant lowering of triacylglyceride (TAG) levels in the plasma of the subjects supplemented with fish oils. Several other studies indicate an inverse relationship between plasma TAG concentrations and omega-3 consumption.

A few examples of n-3 PUFA clinical supplementation studies in triglyceridemic subjects are mentioned below:

1. A review ²⁴ of 72 placebo-controlled human studies which supplemented a range of 1.0 to 7.0g. EPA and DHA daily for at least 2 weeks reduce plasma TAG concentrations by 25-30%.
2. A background diet rich in n-3 PUFA reduced postprandial response to a saturated fat test meal in comparison with response to a saturated fat meal lacking the n-3 PUFA-rich background diet^25,26.
3. Using lower doses of n-3 PUFA for longer times resulted in an equivalent hypotriacyglycerolemic effect ^{27,28 .}
4. 7.0g/d n-3 PUFA significantly reduced fasting plasma TAG concentration by 21.2% and the postprandial TAG response by 32% ²⁹. (This shows that dietary intake of n-3 PUFA as a supplement has a positive effect on TAG metabolism). ²⁹

The above data strongly suggests the possibility of an inverse relationship between n-3 PUFA and risk of CHD.

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List of References

References on n-3 PUFA basic and clinical studies/trials

1. Burr, M.L., Fehily, A.M., Gilbert, J.F., et al.; Effects of changes in fat, fish, and fibre intakes on death and myocardial reinfarction: diet and reinfarction trial (DART), Lancet 1989,ii: 757-61.
2. Ascherio, A., Rimm, E.B., Stampfer, M.J., et al.; Dietary intake of marine n-3 fatty acids, fish intake and the risk of coronary disease among men (The Health Professionals Study),N Engl J Med 1995; 332: 977-982.
3. Albert, C.M., Hennekens, C.H., O’Donnell, C.J, et al.; Fish consumption and risk of sudden cardiac death (U.S. Physicians’ Health Study), JAMA 1998; 279: 23-28.
4. Kromhout, D., Bosschieter,E.B., de Lezenne;. The inverse relationship between fish consumption and 20-year mortality from coronary heart disease (Zutphen Study), N Engl J Med 1985; 312: 1205-1209.
5. Daviglus, M.L., Stamler, J., Orencia, A.J., et al.; Fish consumption and the 30-year risk of fatal myocardial infarction (Western Electric Study), N Engl J Med 1997; 336:1046-53.
6. Dolecek, T.A.; Epidemiological evidence of relationships between dietary polyunsaturated fatty acids and mortality in the Multiple Risk Factor Intervention Trial Proc, Soc Exp Biol Med 1992; 200: 177-182.
7. Rodriguez, B.L., Sharp, D.S., Abbott, R.D, et al.; Fish intake may limit the increase in risk of coronary heart disease morbidity and mortality among heavy smokers: The Honolulu Heart Program Circulation 1996; 94: 952-956.
8. DeLorgeril, M., Salen, P., Martin, J-L, et al.; Mediterranean diet, traditional risk factors, and the rate of cardiovascular complications after myocardial infarction: final report of the Lyon Diet Heart Study Circulation 1999; 99: 779-785.
9. Singh, R.B., Rastogi, S.S., Verma, R., et al.; Randomised controlled trial of cardioprotective diet in patients with recent acute myocardial infarction: results of one year follow up (The Indian Trial), BMJ 1992; 304: 1015-19.
10. Marchioli R., et al., Dietary Supplementation with n-3 polyunsaturated Fatty Acids and Vitamin E After Myocardial Infarction: Results of the GISSI-Prevenzione Trail. Lancet, 1999; 354: 447-455.
11. Simopoulos, AP., Omega-3 fatty acids in health and disease and growth and development: Am J Clin Nutr 1991;54: 438-53
12. Simopoulos, AP.; N-3 fatty acids in the management of cardiovasucular disease. Can J Physiol Pharmacology 1997;75: 234-39
13. Marchioli; R., DiPasquale, A., per: Ricercatori GISSI-Prevenzione G Ital Cardiol 1993;23: 933-69.
14. Keys, A., Coronary Heart disease in seven countries. Circulation 1970; 41(suppl1): 1-211.
15. Keys ,A., Menotti A, Aravanis C, et al.; The Seven Countries Study: 2289 death in 15 years. J. Prev Med 1984; 13:141-54.
16. Renaud S., DeLorgeril et al., Mediterranean alpha linolenic acid-rich diet in the secondary prevention of coronary heart disease. Lancet. 1994; 343: 1454-9.
17. Ascherio A., et al.; Dietary fat and risk of coronary heart disease in man: cohort follow up study in the United States. BMJ 1996; 313: 84-90.
18. Billman G.E., Kang J.X., Leaf A., Prevention of Ischemia-induced cardiac sudden death by n-3 polyunsaturated fatty acids. Lipids 1997; 1161-1168 and references therein.
19. Princen, H.M.G., van Duyvenvoorde, W., Brytenhek, R., et al.; Supplementation with low doses of vitamin E protects LDL from lipid peroxidation in men and women, Artherioscl. Throm. Biol, 1994, 15(3): 325-32.
20. Bang, H.O., Dyerberg, J., and Hjorne, N.; The composition of foods consumed by Greenland Eskimos, Acta Med. Scand. 1976; 200: 67-73.
21. Simopoulos, A.P., Leaf, A., Salem, N. Jr.; Essentiality of and recommended dietary intakes for omega-6 and omega-3 fatty acids. Ann Nutr Metab 1999; 43: 127-130.
22. Simopoulos, A.P., Leaf, A., Salem, N. Jr.; Workshop on the essentiality of and recommended dietary intakes for omega-6 and omega-3 fatty acids. Food Australia 1999; 5: 332-333.
23. 23 The Omega.Plan. A.P.Simopoulos and Jay Robinson. HarperCollins, N.Y., 1999.
24. Harris, W.S., n-3 Fatty acids and lipoproteins: Comparison of results from human and animal studies. Lipids, 1996; 31: 243-252.
25. Harris, W.S., Connor, W.E., Alam, N., and Illingworth, R.D., . Reduction of postprandial triglyceridemia in humans by dietary n-3 fatty acids. J.Lipid Res 1998; 291: 1451-60
26. Weintraub, M.S., Zechner, R., Brown, A., Dietary polyunsaturated fats of the W-6 and W-3 series reduce postprandial lipoprotein levels. et al., J Clin Invest 1988; 82:1884-93.
27. Williams, C.M., Moore, F., Morgan, L., and Wright J., Effects on n-3 fatty acids on postprandial triacylglycerol and hormone concentrations in normal subjects. Br J Nutr 1992; 68: 655-66.
28. Agren JJ, Hanninen O, Julkunen A, Fogelholm L, Vidgren H, Schwab U, Pynnonen O & Unsitupa M. Fish diet, fish oil and docosahexaenoic acid rich oil lower fasting and postprandial plasma lipid levels.
29. Roche, H.M., and Gibney, M.J., Eur J Clin Nutr 1996; 50: 617-24.

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