Introduction

B-vitamins, notably folate, and omega-3 fatty acids are essential nutrients that have received a lot of interest in many nutritional observational studies published during the last couple of decades. Both nutrients have been fairly consistently associated with a protective effect against cardiovascular diseases (CVD) (Dolecek & Grandits, 1991; Chasan Taber et al, 1996; Morrison et al, 1996; Zeitlin et al, 1997; Albert et al, 1998; Folsom et al, 1998; Ford et al, 1998; Giles et al, 1998; Loria et al, 2000; Voutilainen et al, 2000; Hu et al, 2002).

Higher intakes, or plasma concentrations, of the B-vitamins folate and B₂, B₆, B₁₂ are associated with a lower plasma total homocysteine concentration (tHcy) (Selhub et al, 1993; Rasmussen et al, 1996, 2000; Bates et al, 1997; Shimakawa et al, 1997; Ubbink et al, 1998; de Bree et al, 2001; Jacques et al, 2001; Koehler et al, 2001; Saw et al, 2001). A lower tHcy concentration is considered beneficial, as elevations have been related to an increased risk of CVD (Boushey et al, 1995; Ueland et al, 2000; Ford et al, 2002; The Homocysteine Studies Collaboration, 2002; Wald et al, 2002). As a methyl-donor for the degradation of homocysteine, folate is the most important dietary determinant of the tHcy concentration. The role of other B-vitamins is limited to being cofactors of enzymes in homocysteine metabolism (Finkelstein, 1990).

The hypothesis that omega-3 fatty acids might protect against CVD, results from observations by Bang and Dyerberg, who linked a high consumption of fatty fish in Eskimos to the lower incidence of CVD in this population (Bang et al, 1980; Dyerberg & Bang, 1982). As fatty fish is especially a rich source of long-chain polyunsaturated omega-3 fatty acids, these nutrients were held responsible for the protective effect.

As causality cannot be inferred from the observational studies mentioned above, this review summarizes the currently available dietary intervention trials that investigated to what extent B-vitamins and omega-3 fish fatty acids may protect against CVD. Moreover, we investigated whether these two nutritional factors may have a synergistic effect on the protection of CVD.

Methods

A Pubmed search (http://www4.ncbi.nlm.nih.gov/entrez/query.fcgi), 1986 through January 2003, was used to identify intervention trials that reported on omega-3 fatty acids, B-vitamins or homocysteine. We used the following medical subject heading (MESH) word combinations: ["omega-3 fatty acids" or "fish oil"] and ["folic acid" or "vitamin B₂" or "vitamin B₆" or "vitamin B₁₂" or "homocysteine"]. In addition, reference lists of all identified articles were searched for additional relevant studies.

The quality of the identified studies was assessed with a score. For each of the following criteria, a study could receive 1 point if the criterion was fulfilled: (a) randomization of participants; (b) inclusion of a control group; (c) blinding of the subjects; (d) blinding of the researchers and care givers; (e) description of compliance assessment and of subjects who withdrew or dropped out. The lowest score was 0 and the highest score was 5.

Dietary intervention trials with B-vitamins to lower homocysteine in order to prevent CVD

Homocysteine is a sulfur-containing amino acid derived from the metabolism of the essential amino acid methionine, which is its only dietary precursor. For the degradation of homocysteine to either cysteine or methionine, the B-vitamins folate, B₂, B₆ and B₁₂ are indispensable as substrate donor (folate) or as coenzymes (vitamin B₂, B₆ and B₁₂), which is illustrated in Figure 1.

Figure 1.

Simplified version of the B-vitamin-dependent intracellular homocysteine metabolism. Homocysteine can be irreversibly degraded to cysteine through the transsulphuration. The enzymes in this pathway, cystathionine -synthase (CBS) and -cystathionase (C), are both dependent on pyridoxal-5'-phosphate, a biologically active form of vitamin B₆, as co-factor. Homocysteine can also be remethylated to methionine, by the enzyme methionine synthase (MS). This enzyme uses methylcobalamin (a biologically active form of vitamin B₁₂) as co-factor. The methyl group for the latter reaction is donated by 5-methyltetrahydrofolate. This form of folate is produced by the enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR). MTHFR in turn uses flavin adenine dinucleotide (a biologically active form of vitamin B₂) as co-factor (Finkelstein, 1990; Guenther et al, 1999).

Full figure and legend (26K)

Currently, five dietary intervention trials examined the effects of B-vitamin supplementation on intermediary end points of CVD. None of these studies have included vitamin B₂ in their supplements. These studies are summarized in Table 1. Two trials indicate that when subjects with elevated tHcy concentrations are supplemented with a combination of folic acid (synthetic form of natural folate) and vitamin B₆, the risk of cardiovascular events becomes equal to the risk of similar patients with normal tHcy concentrations (de Jong et al, 1999; Vermeulen et al, 2000a). Another trial showed that a combination of folic acid, vitamin B₆ and B₁₂ reduced the rate of plaque growth in the carotid artery. This rate was compared to the rate of plaque growth in the same patients, but in the period before supplementation (Hackam et al, 2000). Due to the absence of randomization and a placebo-controlled group in all the three above-mentioned trials, it cannot be excluded that the risk reduction would have also happened without supplementation. Two trials (Vermeulen et al, 2000b; Schnyder et al, 2002) exclude this problem because of their randomized double-blind placebo-controlled design. Vermeulen et al observed that supplemental folic acid and vitamin B₆, in healthy siblings of patients with subclinical vascular disease, resulted in less abnormal exercise-induced coronary ischemia as detected by electrocardiography, as compared to the placebo supplemented group (Vermeulen et al, 2000b). The electrocardiographic changes in the exercise test are regarded as a surrogate measure for coronary atherosclerosis. A critical note, however, is the low positive predictive value of this test when used in a symptom-free population (Bostom & Garber, 2000). Furthermore, no effect on other intermediary end points of vascular disease were observed (ie ankle-brachial pressure index and duplex scanning of the carotid and femoral artery). The quantitative coronary angiography used by Schnyder et al to detect the extent of stenosis had an acceptable internal validity (intraobserver variability for degree of stenosis was 721%). With this method they showed that a combination of folic acid, vitamin B₆ and B₁₂, given for 6 months, reduced the need for revascularization after angioplasty 1 y after supplementation started. This indicates that a protective effect of B-vitamin supplementation was detectable 6 months after cessation of supplementation (Schnyder et al, 2002).

Table 1 - Intervention trials evaluating the effect of B-vitamins on the risk of cardiovascular diseases.

Full table

These results favor the hypothesis that higher intakes of B-vitamins, and subsequently lower tHcy concentrations, are causally associated with a decreased risk of coronary as well as cerebrovascular diseases in patients with CVD (de Jong et al, 1999; Hackam et al, 2000; Vermeulen et al, 2000a; Schnyder et al, 2002) and in healthy subjects (Vermeulen et al, 2000b). Yet, the lack of hard end point and appropriate study designs precludes a definitive conclusion.

The mechanism that underlies the protective effect of a higher B-vitamin intake on the prevention of CVD has not been unraveled completely. However, improving the endothelial function appears to be a very promising and plausible mechanism, which can explain the relation with coronary as well as cerebrovascular diseases (Brown & Hu, 2001; Cosentino et al, 2001). The main functions of the endothelium are to maintain blood circulation and fluidity, regulate vascular tone and modulate leukocyte and platelet adhesion and leukocyte transmigration (Brown & Hu, 2001). Nitric oxide synthase is a key regulatory system of endothelial cells (McDowell & Lang, 2000); it synthesizes nitric oxide NO that regulates vessel tone, inhibits platelet activation, adhesion and aggregation, limits smooth muscle cell proliferation and modulates endothelial–leukocyte interaction (Thambyrajah & Townend, 2000). High tHcy concentrations are associated with a lower availability of NO as homocysteine may: (a) directly damage endothelial cells; (b) form reactive oxygen species (ROS) that in turn can damage the endothelial cells; and/or (c) inhibit glutathione peroxidase, which is an important enzyme protecting the endothelial cell against oxidative stress (Starkebaum & Harlan, 1986; Jia & Furchgott, 1993; Stamler et al, 1993; Loscalzo, 1996; Upchurch et al, 1996, 1997).

A higher B-vitamin intake, especially folic acid, will lower the tHcy concentration (Clarke & Armitage, 2000) and may thereby improve the endothelial function. Numerous studies investigated the effect of folic acid supplementation on endothelial dysfunction. From these studies it becomes clear that folic acid improves or restores endothelium-dependent vasodilatation, and may decrease the chance of thrombosis by reducing levels of coagulation factors in healthy subjects and in patients with high tHcy concentrations (Brown & Hu, 2001). The observed benefit is probably largely explained by the lowering of tHcy concentrations. However, independent effects of folic acid have also been reported (Verhaar et al, 1998): an intervention study in 10 patients with familial hypercholesterolemia and 10 matched controls showed that the impaired endothelium-dependent vasodilatation seen in these patients could be reversed by infusion of 5-methyltetrahydrofolate, the natural form of folic acid (Verhaar et al, 1998). A tHcy-independent beneficial effect of 5-methyltetrahydrofolate on the endothelial function in diabetic rats has recently also been shown (De Vriese et al, 2002).

Finally, potential mechanisms to explain the beneficial effect of folates on the endothelial function may involve protection against ROS (Verhaar et al, 1998), regeneration of the cofactor for NO synthase, that is, tetrahydrobiopterin (BH₄)(Verhaar et al, 1999), or stimulation of NO synthase (Stroes et al, 2000).

Dietary intervention trials with long-chain polyunsaturated fatty acids in order to prevent CVD

More than 40 different fatty acids are found in nature. Among these, three classes can be distinguished, (a) the saturated fatty acids (SFA) without double bonds between the carbon atoms, (b) the unsaturated fatty acids with one double bond: mono unsaturated fatty acids (MUFAs) and (c) the unsaturated fatty acids with two or more double bonds, the polyunsaturated fatty acids (PUFAs). The four main dietary unsaturated fatty acids are known as omega-3, omega-6, omega-7 and omega-9, according to the position of the first double bond calculated from the methyl group end. Omega-3 and omega-6 fatty acids are essential nutrients for humans, that is, humans cannot synthesize them, they have to be provided by the diet. They are the predominant fatty acids in phospholipids and cholesteryl esters, which are the main component of the membranes surrounding cells and intracellular organelles such as mitochondria. In the omega-3 class, alpha-linolenic acid (C18:3) is the parent substance of the long-chain fatty acids abundantly present in fatty fish: eicosapentaenoic acid (EPA, C20:5) and docosahexanoic acid (DHA, C22:5). For the omega-6 class, linoleic acid (C18:2) is the dietary precursor of the longer chain omega-6 fatty acids like arachidonic acid (C20:4) (Passmore & Eastwood, 1986).

Since the 1980s, interest in the possible cardio-protective effects of fish oils rich in omega-3 has grown markedly (Bang et al, 1980; Dyerberg & Bang, 1982). Since then, many intervention trials with intermediary end points of CVD, like restenosis, coronary atherosclerosis and lower limp atherosclerosis (reviewed in Bucher et al, 2002) have been conducted. However, also trials with hard end points have been performed and these are summarized in Table 2. Two trials tried to modify the diet of the patients with a myocardial infarction by giving dietary advice; the Diet and Reinfarction Trial (DART) (Burr et al, 1989a) and the Lyon Diet Heart Study (de Lorgeril et al, 1999). In the DART trial, the group that received advice to eat at least two portions of fish per week (which was estimated to equal an intake in EPA of 2.3–2.4 g/week) showed a marked reduction in all cause mortality compared to the group of patients who did not receive this advice. The reduction was almost entirely due to a reduction in ischemic heart diseases. In the Lyon Diet Heart Study, the patients who were advised to consume a Mediterranean diet (low in total and saturated fats, high in omega-3 fatty acids from marine and plant origin, and high in fresh fruits, vegetables and legumes) showed a striking reduction in all CVD events compared to the group that received the advice to consume a prudent Western diet. A huge disadvantage of these kinds of dietary trials is that dietary advice cannot be provided double-blind, even single-blind is difficult to achieve. If the intervention is not provided in a double-blinded fashion, the participants or the researchers of the study may (un-)consciously influence the results. Furthermore, it is difficult to assess whether participants comply to the given dietary advice. Finally, the observed protective effect may also be due to dietary factors other than long-chain omega-3 fatty acids of which the intake might have changed over the intervention period. Indeed, analysis of plasma fatty acids in the Lyon Diet Heart Study showed that only alpha-linolenic acid (C18:3), and not long-chain omega-3 (fish) fatty acids, was significantly associated with an improved prognosis for cardiac deaths and nonfatal acute myocardial infarction.

Table 2 - Secondary prevention trials on the effect of omega-3 fatty acids on the risk of cardiovascular diseases as primary end point.

Full table

Intervention studies with long-chain omega-3 fish fatty acids provided in a supplement offer the possibility to ascribe an observed effect to the content of the supplement, provided that the diet, lifestyle and CVD risk factors remain constant over the intervention period. Supplements with fish oil were given daily to patients with a suspected acute myocardial infarction during 1 y in the randomized double-blind placebo-controlled Indian Experiment of Infarct Survival (Singh et al, 1997). Compared to the placebo group, the fish oil supplemented group showed a reduction in all cardiac events of 30%. This reduction was not statistically significant in the group receiving omega-3 fatty acids from plant origin; the relative risk for all cardiac events was 0.81 with a 95% confidence interval of 0.30–1.12. Yet, the proportion of all cardiac events was statistically significantly lower in both intervention groups compared to the placebo group (fish oil: 24.5% and plant oil: 28 vs 34.7%, P<0.01) (Singh et al, 1997). The results of this small study were reproduced in the much larger GISSI trial (Gissi Prevenzione, 1999). Compared to the Indian trial, the dose of omega-3 fatty acids of fish origin was lower and the follow-up time was longer. The results showed a 20% reduction in CVD events compared to the groups that did not receive the omega-3 fish fatty acids. Yet the GISSI trial was not placebo-controlled, which may have favored a positive result. Nevertheless, this fact seemed not to have had a large influence on the results, as no positive effect was observed in the vitamin E treatment group, unless the investigators on forehand expected that the omega-3 fish fatty acids would be more effective than vitamin E (Gissi Prevenzione, 1999). A recent randomized double-blind controlled trial, with 300 subjects with an acute myocardial infarction, showed no protective effects on any cardiac events (cardiac death, resuscitation, recurrent myocardial infarction, unstable angina pectoris, revascularization), after supplementation with omega-3 fatty acid in a dose four times higher as used in the GISSI trial (Nilsen et al, 2001). The authors postulate that if subjects already have a high intake of omega-3 fatty acids, there may be no additional cardio-protective effect of extra omega-3 fatty acids. As the habitual fish consumption in this Norwegian population was already high (at least two portions a week), both the intervention as well as the control (corn oil) group had a high intake of omega-3 fatty acids. This may explain the null finding of the intervention with respect to cardiac events. Yet the subjects supplemented with omega-3 fatty acids showed a decrease in serum lipids (Nilsen et al, 2001).

In conclusion, the results of these intervention studies favor a protective effect of a higher intake of omega-3 fish fatty acids on cardiovascular events in coronary and cerebral arteries.

Omega-3 fish fatty acids are thought to prevent CVD by a variety of actions. Experimental studies with omega-3 fatty acids in the form of fish oil capsules have shown that they lower triglyceride levels, with little effect on LDL and HDL cholesterol (Harris, 1997; Bucher et al, 2002). The mechanism behind this decrease is however not yet unraveled. Furthermore, omega-3 fish fatty acids are direct substrates for the production of eicosanoides, which explains many of their CVD protective effects. Eicosanoides, including prostaglandins, thromboxane and prostacyclin, have an important role in regulating platelet aggregation and blood pressure (von Schacky, 2000). In platelets, thromboxane A2, a highly potent vasoconstrictor and a platelet aggregator, is synthesized by cyclooxygenase from arachidonic acid (omega-6). Prostacyclin (or prostaglandin I2) is the main product synthesized by cyclooxygenase from arachidonic acid in endothelial cells and counterbalances the effect of thromboxane A2 by vasodilatation and antiplatelet aggregation. When EPA and DHA are included in the diet, they compete with the action of arachidonic acid in several ways: (a) they inhibit the synthesis of arachidonic acid from linoleic acid; (b) they compete with arachidonic acid for a position in the membrane phospholipids, thereby reducing the concentration of plasma and cellular arachidonic acid; and (c) EPA competes with arachidonic acid as the substrate for cyclooxygenase. The latter inhibits the formation of thromboxane A2 in platelets, because thromboxane A3 is synthesized from EPA instead. Thromboxane A3 has no vasoconstrictor and platelet aggregator effects. Furthermore, in endothelial cells the production of prostacyclins is not markedly inhibited. Thus, the net result is a change in hemostatic balance towards a more vasodilatory state with less platelet aggregation (Leaf & Weber, 1988). Human intervention trials also indicated that fish omega-3 fatty acids may reduce the formation of plasma proteins like thrombomodulin and von Willebrand factor, and cellular adhesion molecules like E-selectin, produced by endothelial cells. These compounds are markers of endothelial function and are involved in platelet adhesion during thrombosis (reviewed in Brown & Hu, 2001; Bucher et al, 2002). Finally, there are many studies that indicate that omega-3 fish fatty acids may have an antiarrhythmic effect, potentially because they modulate sodium and calcium currents in the myocytes (Nair et al, 1997; Kang & Leaf, 2000).

Animal intervention trials indicating a metabolic link between B-vitamins and omega-3 fish fatty acids

Involvement of vitamin B₆ in the metabolism of long-chain PUFAs

Already in 1963, Mueller and Iacono postulated that vitamin B₆ deficiency inhibits the conversion of alpha-linolenic acid (omega-3) and linoleic acid (omega-6) into longer chain PUFAs (Mueller & Iacono, 1963). Researchers from Italy investigated whether this adverse effects could be counteracted by fish oil (Bergami et al, 1999). In their experiment, 72 male Wistar rats were divided into six dietary groups with varying concentrations of fish oil and vitamin B₆. The diets were provided during 6 weeks. In the rats fed a diet with a normal fat content but with insufficient vitamin B₆, the amount of EPA, DHA and arachidonic acid in the plasma total lipid fraction decreased, whereas the amount of linoleic acid increased, as compared to the rats fed a diet with a normal fat content and sufficient vitamin B₆. In the rats fed a diet with fish oil and insufficient vitamin B₆, the above-mentioned changes in arachidonic and linoleic acids were the same; however, the extra fish oil prevented a decrease in the amount of EPA and DHA, as compared to the rats fed a normal fat diet insufficient in vitamin B₆ (Bergami et al, 1999). Another study that used a smaller number of male Wistar rats (n=12) confirmed these findings; a vitamin B₆-deficient diet (n=6) for 5 weeks resulted in a lower EPA and DHA concentration in the plasma total lipid fraction compared to the control diets (n=24). Furthermore, the concentration of DHA also decreased in the (a) total lipid fraction, (b) phosphatidylethanolamine fraction, and (c) phosphatidylcholine fraction of liver microsomes. On the other hand, the concentration of alpha-linolenic acid and EPA increased in several compartments of the liver microsomes (Tsuge et al, 2000).

The above results suggest that a vitamin B₆ deficiency impairs omega-3 metabolism from alpha-linolenic acid to EPA and DHA, with the most pronounced effect onthe production of DHA (Tsuge et al, 2000). Adding fish oil to a vitamin B₆ deficient diet seems to counteract some of these negative effects (Bergami et al, 1999). However, it may also have a detrimental effect: peroxidative stress increased in the heart of Wistar rats (n=8) after a period of low vitamin B₆ availability and the presence of fish oil with a high omega-3 content, compared to rats on a normal vitamin B₆ diet and/or fed with vegetable oil (n=24) (Cabrini et al, 2001).

Involvement of folate in the metabolism of long chain PUFAs

In an animal experiment with 18 Sprague–Dawley rats, Durand et al, 1996) showed that depletion of folate (250 g/kg/day for 6 weeks) compared to a control diet (750 g/kg/day) was associated with a marked fall in long-chain omega-3 fatty acids in plasma and platelets. The lower concentration of omega-3 PUFAs in the platelets may have been responsible for the increased platelet aggregation seen in these folate deficient mice (Durand et al, 1996). The authors explain their finding by an increased lipid peroxidation. The folate depletion resulted in a higher tHcy concentration, this in turn can lead to the formation of homocysteine-related ROS (Starkebaum & Harlan, 1986), which may oxidize the double bonds of the PUFAs. Following this experiment, Pita and Delgado (2000) investigated whether extra folate would positively affect the plasma and tissue fatty acid composition. During 15 days, 22 male Wistar rats were given either folate, provided as 5-methyltetrahydrofolate (0.15 ml 1 mg/ml), or saline. After this period, an increased concentration of omega-3 PUFAs was observed in the plasma lipid fractions and in the erythrocyte, platelet and intestinal phospholipids (Pita & Delgado, 2000). The authors propose that the extra folate results in an increased availability of methionine, through an augmented remethylation of homocysteine to methionine, for which 5-methyltetrahydrofolate is the methyl donor (Figure 1). This may stimulate the metabolism of linoleic acid in favor of long-chain omega-6 and omega-3 fatty acids in liver phospholipids, as reported earlier (Sugiyama et al, 1998).

Metabolic links between vitamin B₂ and B₁₂ and long-chain PUFAs have not been reported as far as we know.

Human intervention trials indicating a metabolic link between B-vitamins or homocysteine and omega-3 fish fatty acids

Table 3 shows the available human trials that measured the tHcy concentration before and after intervention with fish oil. The first published study on this topic showed that the tHcy concentration decreased after a 6-week intervention with fish oil (Olszewski & McCully, 1993). However, the design of this study was poor (no randomization, no blinding) and was not clearly described. These results were not reproduced in other well-designed intervention studies with similar study populations (Brude et al, 1999; Grundt et al, 1999; Nenseter et al, 2000) (all in Table 3). Furthermore, Haglund et al (1993) observed only a decrease in the tHcy concentration in their crossover trial when the study group received fish oil with added B-vitamins, and not when the subjects received fish oil only. In another crossover intervention trial of the same investigators, the tHcy concentration decreased in both periods that the subjects received fish oil or fish oil with evening primrose oil, yet all subjects were also supplemented with folic acid and vitamin B₆ (Haglund et al, 1998). Thus, the effect on the tHcy concentration in both studies of Haglund et al is likely the effect of the additional folic acid and vitamin B₆. Furthermore, the tHcy concentration of 19 Greenland Inuits (12 females, 7 males) was not statistically significantly different from that of 29 Danish subjects (19 females, 10 males), even though the Greenland Inuits consumed a traditional diet rich in fish omega-3 fatty acids (Moller et al, 1997). Thus, currently there is no convincing proof that supplementation with fish oils may decrease the tHcy concentration.

Table 3 - Human intervention studies with omega-3 PUFA and the effect on the homocysteine concentration.

Full table

Only one study observed an increase of the tHcy concentration after giving participants fish oil (Piolot et al, 2003). The used dose of fish oil (6 g) and the duration of the trial (8 weeks) were comparable to those of other studies and can thus not explain this unexpected result. The authors hypothesize that an increase in fish oil may have led to an increase in NO. NO may inhibit methionine synthase, and as indicated in Figure 1, this will result in a decreased conversion of homocysteine to methionine (Piolot et al, 2003). Further studies need to indicate whether the results of this study are a chance finding or that they can be reproduced.

One study indicated a potential synergy between fish oil, folic acid and vitamins B₆ (Haglund et al, 1993). The atherogenic index (ie (total cholesterol-HDL cholesterol)/HDL cholesterol) decreased with 12% during supplementation with only fish oil, whereas it decreased with 24% during supplementation with a combination of fish oil, folic acid and vitamin B₆ (difference between the two treatments: P<0.05). Furthermore, a 9% greater decrease in plasma fibrinogen concentration (6% with the fish oil only supplementation, 15% in the fish oil with folic acid and vitamin B₆, P<0.05) was observed. Finally, the effect on the concentration of triglycerides, HDL cholesterol, glucose and on fibrinolysis was also more beneficial, although not statistically significant, in comparison with the fish oil supplement only (Haglund et al, 1993).

Another type of experiment showed that supplemental folic acid (5000 g/day) given for 4 weeks to Japanese patients with continuous ambulatory peritoneal dialysis (CAPD), significantly increased the concentration of dihomo-gamma-linolenic acid (DGLA) and arachidonic acid (both omega-6 fatty acids) in 11 CAPD patients with hyperhomocysteinemia (defined as a tHcy concentration >35 mol/l) to the level in CAPD patients without hyperhomocysteinemia (n=12) (Hirose et al, 1998). As fish oil alone did not improve the lipid profile in CAPD patients from Germany (Holdt et al, 1996) (Table 3), the Japanese results imply that a combination of fish oil and folic acid may beneficially affect the long-chain omega-6 fatty acid profile of CAPD patients.

Conclusions

Although the results of the published intervention trials are promising, there is no definitive proof that supplementation with B-vitamins or omega-3 fish fatty acids will lead to a reduced CVD morbidity and/or mortality. The currently available intervention trials did either not have a study design that allows this conclusion (de Jong et al, 1999; Gissi Prevenzione, 1999; Hackam et al, 2000; Vermeulen et al, 2000a, 2000b) or the results need to be reproduced before they can be regarded as definitive (Singh et al, 1997; Schnyder et al, 2002). In addition, all trials with B-vitamins evaluated the effect on intermediate end points. Secondary intervention trials with hard end points and B-vitamin supplementation have recently started (Clarke & Armitage, 2000), but not all of these trials use a combination of B-vitamins and most trials use pharmacological doses. The latter has the disadvantage that the results will be difficult to translate into dietary advice.

Thus, taking all this information together, there is a need for a large randomized double-blind placebo-controlled intervention trial evaluating the effect of supplementation with a combination of B-vitamins and omega-3 fish fatty acids in nutritional doses on hard cardiovascular end points. We have started such a study in March 2003: the SUpplementation with FOLate and vitamin B₆ and B₁₂ and OMega-3 fatty acids (SU.FOL.OM3) study.

The SU.FOL.OM3 study is a randomized double-blind placebo-controlled secondary prevention trial with a 2 2 factorial design. During 5 y, all subjects will receive either folate (in the natural 5-methyltetrahydrofolate form, 560 g/day), vitamin B₆ (3 mg/day) and vitamin B₁₂ (20 g/day), or fish oils (600 mg EPA:DHA 2:1), or both, or a complete placebo treatment. A schematic description of the study is given in Figure 2. The end points of this study will on the one hand be recurrent CVD events (hard end points) and on the other hand intermediary end points (in satellite protocols) such as structural and functional arterial parameters (eg intima media thickness), hemostatic factors and also cognitive function.

Figure 2.

Schematic overview of the SU.FOL.OM3 study.

Full figure and legend (45K)

The hypothesis is to observe a risk reduction of recurrent coronary and cerebrovascular events due to each intervention of 10%, with a power of 90% and an alpha of 5%. This means a risk of 0.9 in the groups that receive B-vitamins and a placebo, and in the group that received omega-3 fatty acids and a placebo, and a risk of 0.8 in group that receives both B-vitamins and omega-3 fatty acids as compared to the risk of the placebo group. The baseline risk of the complete placebo group is estimated to be 0.087. With these parameters, an inclusion period of 1 y and a follow up of 5 y we calculated that, without taking interaction into account, 2376 subjects are necessary. To make up for subjects who withdraw or who are lost to follow-up, the total number of CVD patients that will be included is set at 3000.

With respect to synergy between B-vitamins and omega-3 fish fatty acids, this would be most likely if there is a metabolic link between the two types of nutrients. There is currently no evidence that fish oils have a tHcy-lowering effect above the effect of the B-vitamins. Yet, deficiencies of vitamin B₆ as well as of folate may influence the omega-3 fatty acid status. Most likely, such a deficiency will have the same effect in humans. The question is, whether a link also exists when the intake of B-vitamins is sufficient or more than sufficient. In other words, if sufficient defines a B-vitamin intake according to the dietary recommendations, does an intake higher than the recommendations lead to a better omega-3 long-chain fatty acid status? This seemed to be the case in CAPD patients for the omega-6 fatty acid status (Hirose et al, 1998), but there are no direct data for omega-3 fatty acids. Nevertheless, indirect evidence comes from another human study showing that metabolic markers of CVD (eg total and HDL cholesterol, triglycerides) improved to a larger extent with a combination of fish oil and B-vitamins compared to fish oil alone (Haglund et al, 1993). Yet these results were produced in very small selective study populations and thus require confirmation.

The SU.FOL.OM3 study with its 2X2 factorial design has an ideal design to study a potential synergy between B-vitamins and omega-3 fish fatty acids. However, the current evidence for synergy is not solid enough to test this interaction for the hard end points of the study. To test such an interaction with sufficient power would double the number of subjects that need to be included and would enormously increase the costs of our study. Yet, the number of subjects planned to participate in satellite protocols with intermediary end points will be sufficient to test potential interactions. For example, for the protocol evaluating the effect of B-vitamin and/or omega-3 fatty acid supplementation on structural and functional arterial parameters, the number of subjects needed is based on an alpha of 5%, a power of 90%, a relative risk of the control treatment relative to the experimental treatment of 1.2, a median time without vascular problems on the placebo treatment of 1 y, an inclusion period of 1 y, an additional follow-up time after the recruitment period of 5 y, and a ratio of control to experimental patients of 1. Without a test for interaction we need 653 patients, to take the potential interaction into account we will double this number (n=1306). With these type of satellite protocols we hope to shed more light on the combined function of B-vitamins and omega-3 fatty acids in a couple of years.

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