Patient survival

To the best of our knowledge, no observational study performed to date has investigated associations between marine n-3 PUFAs and patient survival in organ transplantation. The insights provided by RCTs focusing on the effects of marine n-3 PUFAs in transplant recipients are limited by the small sample size and relatively short follow-up (42, 53-72, 123-129, 168), and meta-analyses of RCTs in RTRs could not evaluate effects on patient survival due to a low event rate (73, 74). Although causal inference cannot be made due to the observational study design, the large number of patients and relatively long follow-up time in the present study provide an opportunity to evaluate associations between plasma marine n-3 PUFA levels and mortality. Our findings suggest a beneficial effect of marine n-3 PUFAs on overall and cardiovascular mortality in RTRs.

In non-transplant populations, most epidemiological studies report beneficial associations between plasma marine n-3 PUFA level or fish consumption and either cardiovascular morbidity or mortality or both (9, 88-100). Although there is a tendency towards more negative associations in populations with high fish consumption, beneficial effects of fish consumptions are also reported in populations with low fish consumption (Table 3). In contrast, reports from large RCTs have shown mixed results, with mostly moderate beneficial or neutral effects, especially in recent years (78, 109-121). There might be several reasons for the discrepant results:

1) Interventional studies are less likely to be hampered by systematic errors than

observational studies, including potential confounder effects of a more health dietary pattern

and / or life-style associated with higher fish consumption. Cohort and periodic effects on cardiovascular morbidity and mortality rates associated with changes in dietary pattern have been known for decades (2). In Norway, increased consumption of saturated fatty acids after the 2^nd World War led to an increased MI incidence (169). Dietary changes after migration from Japan to the US or Greenland to Denmark were also associated increased morbidity (6, 8, 9, 170). Although the refered studies are not sufficient scientific evidence for a causal relationship between marine n-3 PUFAs and cardiovascular health (7), they show that dietary patterns are associated with cardiovascular risk. A traditional Japanese diet is not only characterized by a high consumption of marine n-3 PUFAs, but also a relatively low intake of energy and macronutrients compared to a typical Western diet, which clearly also influence cardiovascular morbidity risk (Figure 4) (145). In some countries, fish is expensive and hence fish consumption is associated with educational level and / or socioeconomic class that might confound an association between marine n-3 PUFAs and health. However, in Norway, fish consumption show regional differences and is mostly related to access to fresh fish (150).

Nonetheless, confounder effects from dietary patterns and life-style factors associated with fish consumption is likely to influence results in epidemiological studies, with no exception made for the present study.

2) Some of the RCTs mentioned in Table 3 had obvious methodological limitations that might limit their ability to detect either beneficial or adverse effects of marine n-3 PUFAs. Most RCTs used low-dose marine n-3 PUFA supplementation. The background consumption of marine n-3 PUFAs in a Norwegian cohort levels with the supplemental dose used in several RCTs (114, 115, 153). On the other hand, no clear dose response relationship has been established for marine n-3 PUFAs (52).

3) Divergent results from early and recent studies might be due to improved cardioprotective treatment regimens in recent years, including optimal anti-thrombotic, anti-hypertensive and statin therapy as standard care in patients at high risk of cardiovascular events (171). The Nurses’ Health Study found a negative association between fish consumption and stroke incidence in persons who did not receive aspirin therapy, whereas no association was found in aspirin users (100). The Alpha Omega study reported a reduction in major cardiovascular events after marine n-3 PUFA supplementation in patients who did not receive statin therapy, while not in statin users (116). On the other hand, the large Japan Eicosapentaenoic acid Lipid

Intervention Study (JELIS) in hypercholesterolemic patients reported beneficial effects on cardiovascular mortality after addition of EPA supplements to statin therapy (78).

4) Some interventional studies were hampered by short follow-up time, low sample size or patient populations not at high risk of the primary outcome variable (e.g. cardiovascular mortality) (118, 120, 121). The Norwegian Diet and Omega-3 Interventional Trial (DOIT) study in a general population of elderly men, randomly assigned patients to receive diet counseling and high-dose marine n-3 PUFA or corn oil supplements (121). The number of deaths were lower in the marine n-3 PUFA group (14 deaths versus 24 deaths, HR 0.54, p=0.06). Due to the sample size (n=563), the DOIT study was only powered to investigate effects on cardiovascular risk markers, not mortality. Hopefully, the ongoing OMEMI study (ClinicalTrials.gov number NCT 01841944) in MI survivors may provide important

information about the effects of marine n-3 PUFA supplementation on cardiovascular events in a Norwegian population.

To date, the largest interventional study focusing on the effects of marine n-3 PUFA supplementation was performed in Italian MI survivors in the Gruppo Italiano per lo Studio della Soprawivenza nell’Infarcto Miocardio Prevenzione (GISSI-Prevenzione) Trial. This study found reduced cardiovascular mortality in patients who received marine n-3 PUFA supplements compared with vitamin E or controls (111). They used low-dose supplementation (0.9 g/day), but the consumption of fish and other seafood is quite high in Mediterranean area, especially in Italy, France and Spain (Figure 4) (143), so the effective dose of EPA and DHA for patients who received marine n-3 PUFA supplements was assumed to be around 1.2 – 1.4 g/day compared with 0.3 – 0.5 g/day in the control group.

The Diet and Reinfarction Trial (DART) was performed in the UK in the late 80’s, and reported a modest beneficial effect of dietary advice (high fish consumption) or marine n-3 PUFA supplementation (110). Burr and colleagues performed the DART 2 study in the same participants, evaluating long-term results of dietary advice or supplements given 15 years earlier (109). Fish consumption is lower in the UK than in most Western European countries (149). Therefore, many of the participants in the DART 2 study had probably lowered their intake of marine n-3 PUFAs after the trial had finished. DART 2 found no long-term effect of the intervention given 15 years earlier (109). The heterogeneity in meta-analyses on RCTs focusing on the effects of marine n-3 PUFA supplements on cardiovascular morbidity and mortality is high and results should be interpreted with caution (103-107). Nevertheless, the

benefits of marine n-3 PUFA supplementation on top of optimal medical care for prevention of cardiovascular morbidity and mortality remains unclear.

We found a strong negative association between plasma marine n-3 PUFA levels and the risk of deaths due to stroke. A modest reduction of stroke incidence with higher fish consumption has been reported from observational studies in non-transplant populations (172). The GISSI-Prevenzione study did not find beneficial effects of marine n-3 PUFAs on the incidence of fatal or non-fatal strokes (111) and a recent meta-analysis of RCTs concluded with no effect of marine n-3 PUFA supplementation on stroke incidence (172). No previous study has assessed associations between marine n-3 PUFAs and the incidence of fatal and non-fatal strokes in organ transplantation or in renal medicine. RTRs and especially patients with ESRD suffer a high stroke risk (22, 173).

Interventional studies in chronic kidney disease, focusing on the effects of marine n-3 PUFAs are scarce, all of them are quite small and they rarely report data on mortality, nor were they powered to detect any differences in mortality rates between the interventional and placebo group (174). In patients treated with hemodialysis, high-dose marine n-3 PUFA

supplementation had no impact on mortality, but the incidence of MI was significantly reduced in the intervention group compared with controls (4 events versus 13 events), which also explained a lower incidence of major cardiovascular events in the intervention group (7 events versus 17 events) (122). A matched case control study from a large American hemodialysis cohort reported a lower risk of SCD with higher plasma marine n-3 PUFA levels. The odds of SCD were five times higher in patients belonging to the lower marine n-3 PUFA quartile compared with the upper (79).