Intake of Fish and Marine n-3 Polyunsaturated Fatty Acids and Risk of Cardiovascular Disease Mortality: A Meta-Analysis of Prospective Cohort Studies

Previous epidemiological studies have investigated the association of fish and marine n-3 polyunsaturated fatty acids (n-3 PUFA) consumption with cardiovascular disease (CVD) mortality risk. However, the results were inconsistent. The purpose of this meta-analysis is to quantitatively evaluate the association between marine n-3 PUFA, fish and CVD mortality risk with prospective cohort studies. A systematic search was performed on PubMed, Web of Science, Embase and MEDLINE databases from the establishment of the database to May 2021. A total of 25 cohort studies were included with 2,027,512 participants and 103,734 CVD deaths. The results indicated that the fish consumption was inversely associated with the CVD mortality risk [relevant risk (RR) = 0.91; 95% confidence intervals (CI) 0.85−0.98]. The higher marine n-3 PUFA intake was associated with the reduced risk of CVD mortality (RR = 0.87; 95% CI: 0.85–0.89). Dose-response analysis suggested that the risk of CVD mortality was decreased by 4% with an increase of 20 g of fish intake (RR = 0.96; 95% CI: 0.94–0.99) or 80 milligrams of marine n-3 PUFA intake (RR = 0.96; 95% CI: 0.94–0.98) per day. The current work provides evidence that the intake of fish and marine n-3 PUFA are inversely associated with the risk of CVD mortality.


Introduction
Cardiovascular diseases (CVD) are a group of disorders of the heart and blood vessels, including coronary heart disease, cerebrovascular disease, rheumatic heart disease and other conditions. The global CVD mortality increased 12.5% from 2005 to 2015. 17.9 million people died of CVD in 2015 [1]. In addition to drug treatment, the potential role of dietary components hasreceived increased attention. Previous studies have shown the effectiveness of healthy dietary patterns and components for the prevention of CVD and other diseases [2][3][4]. Fish is rich in various nutrients (e.g., protein, vitamin D and polyunsaturated fatty acids) and may have a beneficial role in preventing CVD events [5,6].
Marine n-3 polyunsaturated fatty acids (n-3 PUFA)-including eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA)-mainly exist in fatty fish. A high consumption of n-3 PUFA from fatty fish led to an increase in highdensity lipoprotein and a decrease in inflammation factors [7,8]. Besides, n-3 PUFA may improve heart rate and blood pressure through improving left ventricular diastolic filling or augmenting vagal tone [9].
Previous epidemiological studies have investigated the association of fish consumption with CVD mortality risk [10,11]. A recent meta-analysis of prospective observational studies revealed a negative association between fish intake and CVD mortality risk [12]. In recent years, another 11 prospective cohort studies investigated the association between   Tables 1 and 2. Among these articles, sixteen were from Europe and America, seven from Asia, one from Oceania and one from five continents. The range of the age was 18-84 years old. The population in the study included males and females. Besides, follow-up duration ranged from 5-30 years and the NOS quality score ranged from 6-9 points (Tables S1 and S2). Age, sex, study center, BMI, educational level, smoking status, alcohol intake, physical activity, urban or rural location, history of diabetes, cancer, use of statin or antihypertension medications, and intake of fruit, vegetables, red meat, poultry, dairy, and total energy Kobayashi 2019, Japan [13] 45-74 14   Age, sex, raceethnicity, marital status, education, body mass index, physical activity, smoking, alcohol intake, total energy intake, vegetables intake, dietary intake of arachidonic acid, aspirin use, use of non-aspirin nonsteroidal anti-inflammatory drugs, self-rated health, sigmoidoscopy, mammogram, prostate-specific antigen test, current use of cholesterol-lowering medication, history of cardiovascular disease, family history of heart attack, current use of blood pressure medication, percentage of calories derived from trans-fat, percentage of calories derived from saturated fat, years of estrogen therapy, and years of estrogen + progestin therapy etc. Age, total energy intake, income, occupation, education, comorbidity index, physical activity level, red meat intake, poultry intake, total vegetable intake, total fruit intake, smoking history, and alcohol consumption (among men only) Age, years of education, BMI, smoking, alcohol consumption, systolic blood pressure, total and HDL-cholesterol concentrations, physical activity, living alone, and energy intake n-3 PUFA, n-3 polyunsaturated fatty acid; CVD, cardiovascular disease; PCB, polychlorinated biphenyl; BMI, body mass index; HRT, hormone replacement therapy; MI, myocardial infarction.
Nutrients 2021, 13, x FOR PEER REVIEW 13 of 20 negative association between marine n-3 PUFA and the risk of CVD mortality was altered from 0.87 (0.85-0.89) to 0.84 (0.81-0.87) by deleting this study. Subgroup analyses displayed a significant negative association among the Americas, and Asian and European countries compared with Oceania countries (Table 3). No publication bias was found (Egger's test: p = 0.722; funnel plot: Figure S4). Figure 3b showed the linear and non-linear dose-response analysis between marine n-3 PUFA intake and CVD mortality risk. Eight prospective cohort studies met the requirements of dose-response analysis [18,25,27,34,[36][37][38][39], and the curvilinear correlation presented a downward trend of CVD deaths with the increase of n-3 PUFA intake (p non-linearity < 0.001). Linear dose-response analysis suggested that an increase of 80 milligrams of n-3 PUFA per day was associated with a 4% lower risk of CVD mortality (95% CI: 0.94-0.98; p trend< 0.001).

Discussion
To our knowledge, the current work is the first meta-analysis of prospective observational studies for associating marine n-3 PUFA intake and CVD mortality risk. This study showed a significant inverse association between fish, marine n-3 PUFA intake and CVD mortality risk. Nonlinear dose-response relationship found that an increase of 20 g of fish intake or 80 milligrams of marine n-3 PUFA intake per day was associated with a 4% reduction in risk of CVD mortality.
In accordance with the previous study, the fish consumption was inversely associated with the CVD mortality risk in the current meta-analysis [12]. Bechthold et al.'s study also suggested a negative association between fish consumption and the risk of CVD [41]. Several studies showed no association between the fish intake and the risk of CVD [42,43]. Differences in preparation and type of fish might explain the observed difference. The progress of frying deteriorates oils through oxidation and hydrogenation, leading to an increase of trans fatty acids [44]. Trans fatty acids can aggravate inflammation and endothelial dysfunction, increasing the risk of CVD mortality [45]. Fish high in salt during cooking can increase the risk of CVD through increasing production of reactive oxygen species and oxidative stress, which contribute to impaired vascular function [46,47]. Fish can be divided into lean, medium-fatty or fatty fish with less than 2 g, 2-8 g and more than 8 g fat per 100 g in its body tissue [48]. Fatty fish diets significantly decreased the serum concentrations of triacylglycerol, apolipoprotein B, apolipoprotein CII and apolipoprotein CIII, which were known CVD risk markers [49]. Fishes also contain vitamin D, proteins, minerals and taurine which may decrease markers of inflammation and improve vascular function by increasing adiponectin levels [50]. In the subgroup of adjustment for diabetes, fish intake was associated with a reduction in the rate of major CVD mortality that approached significance (RR = 0.93; 95% CI: 0.85-1.01). Previous study has showed that supplementation of fish can decrease the CVD mortality risk in a diabetic population [51], the possible reason being that diabetes is a significant risk factor for CVD mortality [52]. EPA and DHA derived from fish can activate the G protein-coupled receptor 120 to reverse insulin resistance [53]. n-3 PUFA supplementation can protect against CVD in patients with diabetes [54].
In most studies where fish exits as an exposure variable, the observed benefits could often be attributed to the presence of fatty acids [55,56]. The long chain n-3 PUFA-namely, EPA and DHA-are naturally presented not only in fatty fish, but also in lean fish [57,58]. n-3 PUFA supplementation can decrease the risk of CVD [59,60]. The plasma level of EPA and DHA in humans may increase after intake of fish to improve the composition of lipoprotein cholesterol as cardiovascular markers affecting the risk of CVD [61,62]. However, previous study showed that low-dose supplementation with EPA and DHA did not significantly reduce the rate of CVD events [63]. This possible reason may be related to presence or absence of a history of CVD. The patients in the trial were all myocardial infarction patients for 4 years before enrollment. 85% of the patients were receiving statins. Patients with CVD who are receiving good clinical treatment showed low risk of future cardiovascular events [64]. Therefore, we wanted to observe the effect of the long chain n-3 PUFA on CVD mortality through the long-term duration.
In this meta-analysis, we also found a negative association between the marine n-3 PUFA intake and the CVD mortality risk. In previous studies, the results were not consistent [65]. A randomized controlled trial (RCT) showed that n-3 PUFA supplementation (866 mg/d) for 3.5 years could reduce CVD mortality risk [66]. In contrast, the RCT with one-year n-3 PUFA supplementation (850 mg/d) suggested no association [67]. Although some randomized controlled trials (RCTs) had been published, the follow-up duration were short with most studies ranged from 1-5 years [66][67][68]. Hoverer, the cohort studies included in this meta-analysis have longer follow-up duration ranged from 5-29 years. CVD is a chronic disease with a long disease course. Longer follow-up duration was more in line with the nature of the CVD disease. The possible mechanisms were as follows. First, the plasma n-3 PUFA increased with the frequency and the amount of dietary n-3 PUFA intake [69,70]. A higher circulating n-3 PUFA may alter the cell membrane fluidity which modulates protein function and signaling. The dimerization and recruitment of toll-like receptor-4 may be disrupted to down-regulate the expression of nuclear factor-kappaB reducing the inflammatory responses, with the enrichment of n-3 PUFA [71]. Second, n-3 PUFA may inhibit oxidative stress through the nuclear factor E2-related factor 2/heme oxygenase-1 signaling pathway. 4-hydroxy-2E-hexenal, the product of n-3 PUFA peroxidation, will dissociate Nrf2 from Keap1 and react with the cysteine residues of Keap1 [72]. Then, Nrf2 can translocate into the nucleus and bind to antioxidant responsive element to increase the expression of HO-1 [73]. HO-1 is a representative antioxidant enzyme that can confer cytoprotection on a wide variety of cells against oxidative damage [72]. Third, n-3 PUFA may reduce the hepatic very low-density lipoprotein production rate to decrease the plasma triglyceride levels through affecting fatty acid desaturases, fatty acid elongases and peroxisomal βgene expression and fatty acid beta-oxidation [74,75]. In addition, long-chain n-3 PUFA may play an important role in improving the endothelial function, lowering circulating markers of endothelial dysfunction, such as E-selectin, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 [76][77][78].
The dose-response analysis showed that the risk of CVD mortality decreased with the increase of fish consumption from zero to 40 g/d. The adjusted RR reached a steady value when fish consumption increased beyond 40 g/d. Therefore, we believe that 40 g/d is the ideal dose for preventing CVD mortality. This is basically consistent with the average fish intake of the population of Europe and America [23,30]. However, the average intake of people in Japan is higher than this level [13].
This study has several strengths. First, compared with the previous meta-analysis [12], this study included additional 11 studies to investigate the association between the fish consumption and the CVD mortality risk, which may have a higher statistical power. Second, this meta-analysis was first to investigate the association between marine n-3 PUFA intake and CVD mortality risk with prospective cohort studies. Third, most studies had a long follow-up duration (9-30 years). CVD is a chronic disease and longer followup duration can better explain the association between fish, marine n-3 PUFA and CVD mortality risk.
The limitations should be acknowledged. First, several deep-sea fishes may be contaminated, while only one article reported whether fishes had pollutants or not [28]. Second, it is hard to standardize the fish and marine n-3 PUFA consumption due to the details of measurement methods not being available. Thus, we chose RR (95% CI) of the highest versus lowest fish and marine n-3 PUFA intake category and CVD mortality risk.

Conclusions
This meta-analysis indicated that the fish and marine n-3 PUFA intake were inversely associated with reduced risk of CVD mortality. This finding has important public health implications in terms of the prevention of CVD mortality. Since the biomarkers of fish and n-3 PUFA within an individual are important for food absorption, further research needs to be performed in biomarkers.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/nu13072342/s1, Figure S1: Sensitivity analysis with respect to fish intake and CVD mortality risk. Figure S2: Funnel plot of the RR of 18 articles on fish intake and CVD mortality risk. Figure S3: Sensitivity analysis with respect to marine n-3 PUFA intake and CVD mortality risk. Figure S4: Funnel plot of the RR of 10 articles on marine n-3 PUFA intake and CVD mortality risk. Table S1: Quality assessment of studies investigating fish intake and CVD mortality risk. Table S2: Quality assessment of studies investigating marine n-3 PUFA intake and CVD mortality risk.