Eating Leafy Greens May Reduce Risk of Atherosclerosis

Frequent vegetable consumption is generally considered part of a healthy diet due to proposed predictions in risk of chronic disease. In addition to their antioxidant properties, vegetables are the source of >80% of dietary nitrate, which can be converted in vivo to nitric oxide, improving endothelial function. A recent study by Blekkenhorst et al. found high intake of dietary nitrate from vegetable sources to reduce risk of mortality from atherosclerotic vascular diseases [1]. After discussing these findings within wider literature, it could be concluded that consumption of 3-5 servings of vegetables per day and regularly choosing nitrate-rich sources such as spinach or beetroot may reduce risk of atherosclerosis.

Atherosclerosis, a condition characterised by the accumulation of fatty material within arteries [2], affects at least 2.6 million people in the UK [3]. The resulting arterial narrowing can lead to coronary heart disease (CHD), stroke and peripheral artery disease. Endothelial dysfunction is considered an early step in the initiation of atherosclerosis [4] as dysregulation of vascular homeostatic mechanisms [4] impairs endothelium-dependent vasodilation [5] and induces a pro-inflammatory, proliferative, pro-oxidant and pro-coagulative state [6].

Frequent consumption of vegetables is often associated with reduced risk of atherosclerotic vascular disease (ASVD) as antioxidants reduce oxidative stress [7]. However, their high nitrate content has also been hypothesised to be a significant protective factor against chronic disease [8]. It is attributed to the fixation of atmospheric nitrogen by bacteria, which produces nitrate to be taken up by plants and used in amino acid synthesis [9], but the content in vegetables can be variable, being influenced by environmental, agricultural and genetic factors such as humidity, temperature, water content, sunlight and nitrogen fertiliser use [10]. Nonetheless, they are said to provide >80% of dietary nitrate [11].

Approximately 75% of ingested nitrate is excreted in urine [12], however the remaining 25% undergoes bioconversion in vivo to form nitrite, a precursor for NO. Small quantities of nitrate are converted by nitrate reductase enzymes in tissues [8], but the majority is delivered to the mouth by enterosalivary circulation where commensal gram negative bacteria on the posterior third of the tongue [10] use it as an alternative electron acceptor in energy production [13]. The nitrite produced is either swallowed and protonated in the acidic stomach to form NO [14], or re-enters circulation and is subsequently reduced by deoxyhaemoglobin, deoxymyoglobin or xanthine oxidoreductase [8]. This increase in the bioavailability of NO is thought to improve endothelial function [15] and therefore be significant in the pathogenesis of cardiovascular diseases  [11].

Despite the potential benefits of high dietary nitrate consumption, the World Health Organisation’s (WHO) acceptable daily intake (ADI) is set at 3.7mg/kg body weight/day [13] due to the much debated concern regarding the formation of carcinogenic nitrosamines by reaction of nitrites with dietary amines [16]. A recent study by Blekkenhorst et al. investigated the association between dietary nitrate, particularly that from vegetable sources, and mortality from atherosclerotic vascular disease [1]. This review will discuss their conclusions within wider research to determine whether high intake of dietary nitrate, primarily from vegetables, improves endothelial function and reduces risk of mortality from atherosclerotic diseases, to assess whether the WHO guidelines allow for adequate quantities of nitrate to be obtained from the diet to offer confer vascular benefits.

Method

Study population

Participants were women aged ≥70 years from the Calcium Intake Fracture Outcome Study (CAIFOS).

Assessment of ASVD mortality

Cause of mortality within the 15 years of follow-up was assessed using the coded death certificate.

Dietary intake assessment

A self-administered FFQ at baseline assessed diet. A databased provided information on the nitrate content of vegetables. The amount of vegetable consumed (g/day) was multiplied by the median nitrate value (mg/g), before the sum of dietary nitrate from all vegetables was calculated. Total nitrate intake was determined by multiplying the amount of the food (g/day) by the mean nitrate value (mg/g). The sum of nitrate values from all foods, including vegetables, was calculated. Nitrate intake from drinking water was negligible so excluded.

Statistical analysis

Differences between baseline characteristics and tertile of vegetable nitrate intake were assessed. The association between nitrate intake per standard deviation and ASVD mortality was analysed using Cox proportional hazards regression, with adjustment for known risk factors for CVD. Nitrate intake was modelled as categorical variables to test for a dose-response relationship, and as continuous variables for a trend.

Sensitivity analyses excluded those taking medication containing organic nitrates, those with ASVD related deaths in the first 24 months, adjusted for dietary quality, and for individual dietary confounders. The difference between vegetable nitrate intake at baseline, 5 years and 7 years of follow-up was tested and changes accounted for in a multi-variable adjusted model. Finally, the association between vegetable nitrate intake and both all-cause mortality and non-ASVD mortality was assessed.  

Results

Subject characteristics

1226 participants were included in the study. 238 participants died of an ASVD-related cause during follow-up. The mean nitrate intake from vegetables was 67.0mg/day and from all foods was 79.4mg/day. Vegetables contributed 84% of total nitrate intake, with the highest nitrate values being from lettuce and salad greens, spinach, celery, beetroot and potatoes.

Results of ASVD mortality

There was a statistically significant association between vegetable nitrate intake per SD (29.3mg/day) and ASVD mortality in the unadjusted, age and energy adjusted, and multivariable-adjusted models. Trends between quantity of vegetable nitrate intake and ASVD mortality were significant in the unadjusted and multivariable-adjusted models.

There was a significant association between total nitrate intake from all foods per SD (31.1mg/day) and ASVD mortality in all three models, but no association between nitrate intake from non-vegetable sources and ASVD mortality.

Results of sensitivity analyses

The association between nitrate intake from vegetables and ASVD mortality remained significant after excluding participants taking medication containing organic nitrates and those that died within the first 24 months of follow-up. A strong positive correlation was observed between nitrate intake from vegetables and total vegetable intake.

Adjustment for dietary quality attenuated the association between nitrate intake from vegetables and ASVD mortality, but adjustment for individual dietary factors did not. Considering vegetable nitrate intake at 5 and 7 years of follow up as well as baseline did not change the association. There was a statistically significant association between vegetable nitrate intake and all-cause mortality but not non-ASVD mortality. 

Discussion

The study by Blekkenhorst et al. concluded that there was 21% lower risk of ASVD mortality with every 30mg/day higher intake of dietary nitrate from vegetables [1]. Although previous studies seem to have not investigated ASVD mortality as a primary outcome, vascular compliance has been found to improve in response to supplementation with inorganic nitrate [17], reflecting greater arterial elasticity and the ability for the vessel wall to passively expand and contract with changes in blood pressure (BP). This limits endothelial damage, which would otherwise cause predominance of a vasoconstrictive state [5] and narrowing of arteries. Additionally, the reduction in vascular stiffness observed by Rammos et al. was accompanied by decreased systolic blood pressure (SBP) in subjects with mild hypertension [18]. To reflect increases in dietary nitrate intake from vegetables, beetroot juice has frequently been used as a supplement in research, resulting in an observed reduction in SBP of 4.4mmHg and DBP of 1.1mmHg in healthy individuals [19], and 8mmHg and 3-5mmHg in those with hypertension [20]. This favourable change in BP prevents excessive stress being put on blood vessel walls, which increases their susceptibility to atherosclerotic plaque formation [21].

The mechanism of action of dietary nitrates firstly involves bioconversion to nitrites and reduction to NO, as previously described. The NO generated by the enterosalivary nitrate-nitrite-NO pathway supplements endogenous production by endothelial NO synthase (eNOS), which catalyses the oxidation of L-arginine by molecular oxygen [8]. Hypoxic conditions, such as those that feature in atherosclerosis and ischaemia, limit eNOS derived NO synthesis [13], yet the nitrate-nitrite-NO pathway is able to supply NO in diminished oxygen conditions [8]. This, and the direct induction of vasodilation by nitrite [13], may be the cause of greater beneficial vascular effects from dietary nitrate often being observed in those already with endothelial dysfunction [10].

NO has a fundamental role in vascular homeostasis by the initiation of cGMP-mediated intracellular signals in vascular smooth muscle cells [13], resulting in muscle relaxation and vasodilation. NO also inhibits platelet aggregation and adhesion, suppresses proliferation of vascular smooth muscle cells, and reduces leukocyte adhesion and migration, and activity of inflammatory markers [8], all of which are important factors in the pathogenesis of atherosclerotic diseases. Further to this, it has been found that NO reduces oxidative tissue damage and inflammation when blood supply returns to tissues following ischaemia, known as ischaemia-reperfusion injury, and that it decreases intimal hyperplasia, or the thickening of the tunica intima associated with atherosclerosis [10].

Despite the biological plausibility of the beneficial vascular effects of high nitrate intake, no anti-atherosclerotic effect was observed in LDL knockout mice following a prolonged period of nitrate supplementation [16], questioning whether there is a feedback that down-regulates eNOS activity or induces greater clearance of nitrate and nitrite in urine. Nonetheless, a similar dose-dependent increase in plasma nitrite concentration has been reported in response to both supplemented inorganic nitrate and beetroot juice [20], and consumption of beetroot juice has been shown to counteract endothelial dysfunction associated with ingestion of a mixed meal [22], attenuating the adverse effect of dietary fat on NO bioavailability and vasodilation [23]. This could suggest that inorganic nitrate supplementation does not induce the same response as nitrate from vegetables, potentially due to the enhancement of NO generation from nitrite by vitamin C [11] and the protection of NO from oxidation by polyphenols [13], both of which are components found in vegetables.

Impacts

The proposed biological mechanisms by which nitrate may prevent atherosclerosis, and the beneficial effects on vascular compliance and BP observed in the wider literature, have been discussed. From this, it could be said that dietary nitrate has the potential to reduce risk of mortality from ASVD, as concluded by Blekkenhorst et al.  [1]. It was suggested in this study that an increase in intake of nitrate containing vegetables of 10-30g/day, depending on nitrate content, may offer a significant risk reduction, although the magnitude of the effect is likely to be dependent on basal eNOS activity [1]. This amount should be easily achievable by the general population, equating to 1-2 servings per day. For maximum effect, the optimum choices would be green vegetables such as spinach and lettuce, which have high nitrate concentrations in their leaves, or beetroot, which stores nitrate in its roots [9].

The implications of processing should be considered when selecting and preparing vegetables. Those that are organically grown are likely to have a lower nitrate content due to the lack of use of nitrogen-based fertilisers. Additionally, nitrate is water soluble so washing and cooking can be detrimental, with boiling causing up to 75% losses [24]. Peeling potatoes also removes high quantities due to nitrate being in highest concentration just below the skin and, finally, nitrate content reduces during storage as a consequence of endogenous nitrate reductase activity and/or bacterial contamination [24].

As previously mentioned, there are concerns regarding high dietary nitrate intake in terms of cancer development. However, it is thought that a typical diet including a high quantity of vegetables is not likely to exceed the WHO ADI unless all the vegetables consumed fall within the category of highest nitrate content. Therefore, it could be thought safe and appropriate to advise that individuals should ensure they eat 3-5 servings of vegetables per day [25]. The strong correlation between nitrate intake and vegetable consumption observed by Blekkenhorst et al. [1] suggests this would result in high dietary nitrate intake, however, it would be essential to select a variety, including nitrate-rich sources such as spinach or beetroot, to obtain sufficient dietary nitrate for the maintenance and improvement of endothelial function and prevention of atherosclerosis.

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