**The Role of Specific Components of a Plant-Based Diet in Management of Dyslipidemia and the Impact on Cardiovascular Risk**

#### **Elke A. Trautwein 1,\* and Sue McKay <sup>2</sup>**


Received: 4 August 2020; Accepted: 31 August 2020; Published: 1 September 2020

**Abstract:** Convincing evidence supports the intake of specific food components, food groups, or whole dietary patterns to positively influence dyslipidemia and to lower risk of cardiovascular diseases (CVD). Specific macro- and micro-components of a predominantly plant-based dietary pattern are vegetable fats, dietary fibers, and phytonutrients such as phytosterols. This review summarizes the current knowledge regarding effects of these components on lowering blood lipids, i.e., low-density lipoprotein cholesterol (LDL-C) and on reducing CVD risk. The beneficial role of a plant-based diet on cardiovascular (CV) health has increasingly been recognized. Plant-based dietary patterns include a Mediterranean and Nordic diet pattern, the dietary approaches to stop hypertension (DASH), and Portfolio diet, as well as vegetarian- or vegan-type diet patterns. These diets have all been found to lower CVD-related risk factors like blood LDL-C, and observational study evidence supports their role in lowering CVD risk. These diet patterns are not only beneficial for dyslipidemia management and prevention of CVD but further contribute to reducing the impact of food choices on environmental degradation. Hence, the CV health benefits of a predominantly plant-based diet as a healthy and environmentally sustainable eating pattern are today recommended by many food-based dietary as well as clinical practice guidelines.

**Keywords:** review; CVD; dyslipidemia; cardiovascular health; dietary fats; dietary fiber; phytosterols; plant-based diet; dietary pattern; sustainability

#### **1. Introduction**

Cardiovascular diseases (CVD), which include coronary heart disease (CHD), cerebrovascular disease (stroke), and peripheral vascular disease, are the most common non-communicable diseases globally, accounting for an estimated 31% of all deaths worldwide [1]. The underlying cause of CVD is atherosclerosis which is a progressive (and reversible) disease of the blood vessels. By managing risk factors associated with atherosclerosis it is possible to slow down (or reverse) the development of the disease and ultimately reduce the risk of CVD [2]. Underlying risk factors include elevated blood pressure, dyslipidemia, overweight/obesity, and type 2 diabetes mellitus (T2DM), as well as behavioral risk factors such as smoking, unhealthy diet, and physical inactivity [2]. Dyslipidemia is a metabolic abnormality leading to an increase in circulating concentrations of blood cholesterol (C) and triglycerides (TG). It is characterized by elevated low-density lipoprotein cholesterol (LDL-C), also known as hypercholesterolemia, and often combined with low concentrations of high-density lipoprotein cholesterol (HDL-C) and elevated TG, mainly as TG-rich lipoproteins (TRL) such as chylomicrons and very-low-density lipoprotein (VLDL).

Elevated LDL-C is a causal risk factor for CVD [3] and lowering LDL-C concentrations is the primary target for treatment and prevention of CVD [2]. Elevated TG, and especially TRL, are also treatment targets recommended in guidelines for the management of dyslipidemia [2].

Adopting a healthy diet and lifestyle should always be the cornerstone when seeking to lower LDL-C (and TG) concentrations [2]. Based on the severity of dyslipidemia and the total CVD risk score, additional pharmacological therapy may be recommended. With adequate changes in diet and lifestyle, about 80% of (premature) CVD mortality may be prevented [4].

The role of a healthy dietary pattern in the prevention of CVD has well been recognized [5–7]. Evidence supports the intake of specific nutrients, food groups, or certain dietary patterns to positively influence dyslipidemia and promote the prevention of CVD. A healthy dietary pattern is also a major determinant of environmental sustainability. Current food production practices contribute to environmental degradation in several ways, e.g., through greenhouse-gas emissions, fresh-water withdrawal, and land use [8]. Consequently, authorities are starting to adapt their food-based dietary guidelines to reflect a shift towards a predominantly plant-based diet. These recommendations aim to reduce the intake of animal-based foods for both health and environmental reasons [9,10].

Predominantly plant-based dietary patterns emphasize a higher intake of fruits, vegetables, legumes, whole-grain products, nuts, seeds, and vegetable oils and limited intake of foods from animal origin such as low- or non-fat dairy, lean meat, and fish [7]. Plant-based dietary patterns vary from flexitarian (with low consumption of meat) to pescatarian or lacto/ovo-vegetarian to vegan, where plant-based foods represent most or all the food in the diet.

In view of macronutrients, specific for a plant-based diet are the intake of more complex carbohydrates and plant-based proteins next to a lower total fat intake, especially fewer saturated and trans fats and more unsaturated fatty acids. Plant-based diets are also higher in dietary fiber (DF). In addition, they contain modest amount of phytosterols (PS) next to other bioactive plant-derived compounds often called phytonutrients, which are associated with positive health effects. This review focuses on PS because of their proven cholesterol-lowering effect which allows them to carry an authorized health claim. Other phytonutrients, such as polyphenols, lignans, and carotenoids, as well as vitamins like folic acid, are out of scope for this review.

This narrative review, based on evidence from reviews, systematic reviews, meta-analyses, and randomized controlled trials (RCTs) on CVD risk factors (LDL-C and on clinical endpoints) focuses on the beneficial effects of specific macro- and micro-components of a plant-based diet (vegetable fats, DF, and PS) in the management of dyslipidemia and the role of plant-based dietary patterns in cardiovascular (CV) health and briefly discusses aspects of environmental sustainability. Dietary fatty acids, DF, and PS have been reviewed in detail because of the proven evidence for cholesterol-lowering and CVD risk benefits and because they are specifically referred to in dietary recommendations for CVD prevention [2]. Literature databases like MEDLINE were used as the main sources for searching relevant literature.

While health benefits of these individual dietary components have been addressed in specific reviews and meta-analyses, the information provided in this review addresses them in the context of a plant-based diet/dietary pattern and contributes to a better understanding of the increasingly important role of a plant-based diet in the prevention of CVD. In the following sections, specific components of a plant-based diet in the management of dyslipidemia and prevention of CVD are discussed in detail.

#### **2. Dietary Fats**

#### *2.1. Dietary Sources and Fat Quality*

There is general consensus that fat quality, i.e., the fatty acid composition of dietary fat, is more important than fat quantity (total amount of fat) in the management of dyslipidemia and the prevention of CVD. The fat quality of foods is determined by the content of different fatty acids. Fat from animal origin such as fatty meat, butter, full-fat dairy, as well as the tropical oils coconut and palm oil, are typically rich in saturated fatty acids (SAFA). Meat and dairy foods are the main food sources of SAFA in Western diets [11]. In contrast, plant-based fats, i.e., vegetable oils, are generally rich in unsaturated fatty acids. Unsaturated fatty acids can be monounsaturated (MUFA), such as oleic acid and polyunsaturated (PUFA). Plant-based sources of PUFA are predominately n-6 (omega-6) fatty acids such as linoleic acid and some n-3 (omega-3) fatty acids such as α-linoleic acid. Very long-chain n-3 fatty acids such as eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) come from marine sources (fish, fish oil, and algae). Trans fatty acids (TFA), unsaturated fatty acids with double bonds in the trans configuration, are naturally found in butter, full-fat dairy, and meat from ruminants like beef, sheep, and goat. Other sources of TFA are partially hydrogenated vegetable oils. TFA are commonly found in commercial baked goods like pastries, convenience foods, and battered or deep-fried foods. TFA from partially hydrogenated oils have been removed from many foods because of their adverse effects on blood lipids and CVD risk [12], leading to a substantial decrease in TFA intake, which is typically now less than 1% of energy [13]. TFA and SAFA raise LDL-C but TFA also lowers HDL-C concentrations and therefore has the most unfavorable effects amongst dietary fatty acids [12].

Based on dietary intake data, SAFA consumption is still higher and PUFA consumption lower than recommended [11,14]. For CVD prevention, it is recommended that a decrease in SAFA intake should be accompanied by an increase in PUFA intake, but this is not always achieved at a population level [14].

#### *2.2. The Role of Saturated, Monounsaturated, and Polyunsaturated Fatty Acids on Blood Lipids and CVD Risk*

Replacing SAFA with unsaturated fatty acids in the diet lowers LDL-C without affecting HDL-C and TG. The LDL-C lowering effect is larger when SAFA is replaced by PUFA compared to MUFA [15–17]. Replacing SAFA with carbohydrates, i.e., consuming a low-fat, high carbohydrate diet, lowers both LDL-C and HDL-C and raises fasting TG, and therefore does not improve the overall blood lipid profile [15,17]. The most beneficial effect on blood lipids is therefore achieved by replacing SAFA with unsaturated fats. Supplemental intake of the very long-chain n-3 PUFA (EPA and DHA), from fish oil have no substantial effect on LDL-C but dose-dependently lower TG concentrations [13].

In view of effects on CVD risk and clinical endpoints, both observational, i.e. prospective cohort studies and RCTs with clinical endpoints, have shown that reducing SAFA intake lowers the risk of CVD and CHD events. There is clear evidence that partial replacement of SAFA with unsaturated fatty acids, especially vegetable oil PUFA (mainly n-6 linoleic acid and the plant-based n-3 fatty acid α-linolenic acid) lowers the risk of CVD, mainly the risk of CHD [13,17]. There is insufficient or limited evidence for effects on CVD or CHD risk when SAFA is replaced by MUFA, carbohydrates, or proteins, and when the replacement nutrient is not specified [13]. Replacing 5% of total energy intake (% TE) of SAFA with PUFA was found to lower the risk of CHD by about 10%, while evidence is inconclusive due to lack of adequate studies to quantify the CHD risk benefit of MUFA [13,17]. More recently, data from two large cohort studies found a significantly lower CHD risk when 5% TE intake from SAFA, TFA, and refined carbohydrates was replaced by MUFA from plant sources (vegetable oils, nuts, and seeds), while replacement with MUFA from animal sources (red and processed meats and dairy foods) was not associated with lower CHD risk [18]. In addition, data from two other cohort studies has shown that replacing 5 g/day of dietary fats like margarine, mayonnaise, butter or dairy fat with a higher intake of olive oil, which is rich in MUFA, was associated with a 5–7% lower risk of CHD and total CVD [19]. The Prevención con Dieta Mediterránea (PREDIMED) trial also studied the effect of consuming MUFA, in the form of olive oil, as part of a Mediterranean (MED)-type diet on CVD outcomes. The MED-type diet supplemented with either extra-virgin olive oil or with nuts significantly lowered major CVD events by around 30% compared to a low-fat diet [20]. It should, however, be noted that these dietary patterns differ in more aspects than just dietary fat and fatty acid intake.

Even though marine n-3 PUFA are not the focus of this review, observational studies have consistently shown that higher intakes of fish, fish oil, and EPA and DHA is associated with lower risk of CVD, especially CHD risk, in the general population [13,21]. While evidence from RCTs for the primary prevention of CVD is weak, higher intakes of EPA and DHA have been shown to lower CVD risk in RCTs in secondary prevention [13,21].

An analysis based on prospective cohort studies has further shown that replacing SAFA with PUFA, MUFA, or whole-grain (high-quality) carbohydrates was associated with a lower CHD risk, while replacing SAFA with refined carbohydrates and sugars (low-quality carbohydrates) was not associated with CHD risk [22]. Furthermore, a higher PUFA intake was associated with a lower CHD risk compared to higher intakes of refined carbohydrate and sugar. These data support that PUFA-rich plant-based fats as well as whole grain products as part of a predominantly plant-based diet are beneficial for CV health.

For management of dyslipidemia not all plant-based fats are beneficial, especially not those that are rich in SAFA such as coconut oil. Despite the widespread popularity of coconut oil and its alleged health benefits, its consumption significantly raises LDL-C compared with other vegetable oils as summarized in a recent meta-analysis [23]. Thus, coconut oil should not be recommended as a healthy vegetable oil for CVD risk reduction, despite its HDL-C-raising effect [23,24].

In addition to the beneficial effects on dyslipidemia, emerging data also show that unsaturated fatty acids, especially n-6 PUFA, favorably affect blood glucose and insulin resistance, therefore helping to reduce the risk of T2DM, a risk factor for CVD [13,17].

#### *2.3. Recommendations for Dietary Fat Intake*

Quality of dietary fat intake is key for the prevention of CVD more so than quantity. Recommendations for total fat intake typically range from 20 to 35% TE; intakes exceeding 35–40%TE should be avoided as they are usually associated with an increased intake of SAFA [25,26]. Reducing SAFA intake to <10% TE and replacing SAFA with unsaturated fatty acids (MUFA and PUFA) is the recommendation in most food-based dietary guidelines aiming to prevent CVD [2,27,28]. Some guidelines advise SFA intake <7% TE, especially in the presence of hypercholesterolemia (Table 1) [2]. TFA increases the risk of CVD [2,28,29], and intake should be avoided (Table 1). Intake of n-3 PUFA, especially EPA and DHA, is not part of dietary recommendations to lower TC and LDL-C concentrations. Nevertheless, some guidelines do recommend an intake of 200–500 mg/day of EPA and DHA as part of a heart healthy diet [21] or advise 1 to 2 portions of fatty fish per week [30]. For specific TG-lowering, high doses (2–4 g/day) of n-3 fatty acid supplements are recommended either alone or as an adjunct to other lipid-lowering therapy [2,21,30].

#### *2.4. Food-Based Dietary Guidelines and How a Plant-Based Diet Can Improve the Quality of Dietary Fat Intake*

Food-based dietary guidelines advise a more plant-based diet and recommend eating less foods of animal origin such as meat, butter, and full-fat milk and cheese [9]. They also advise switching to leaner meat and low-fat dairy products and replacing animal fats with vegetable oils and vegetable oil-based fats. As a result, pre-dominantly plant-based diets typically deliver less total fat, especially less SAFA and TFA, more unsaturated fats, and more plant-based proteins, DF, micronutrients (vitamins and minerals), and phytonutrients. Regular fish consumption, especially fatty fish, is also advised as a part of a plant-based, heart-healthy dietary pattern.


**Table1.**Impactofspecificdietarychangesinthemanagementofdyslipidemiawithfocusonloweringlow-densitylipoproteincholesterol(LDL-C)

(RCTs) or

meta-analyses;

 \*\* Data derived from a single RCT trial or from large

non-randomized

 studies.

#### **3. Dietary Fibers (DF)**

#### *3.1. Types of DF and Dietary Sources*

DF consists of a wide range of plant-based compounds (carbohydrate polymers with ≤10 monomeric units) that vary in their physical and chemical properties. They have no nutritional value as such but play an important role in the regulation of different physiological functions in the body. DF are typically categorized into (water) soluble (SF) and insoluble fibers (IF) and are not hydrolyzed by digestive enzymes and hence not fully digested in the human gut [31]. IF include cellulose, hemi-celluloses, and lignin. They affect gut function by facilitating transit time of foods, normalizing bowel movements, increasing stool bulk, and preventing constipation. SF include pectin, beta-glucans, gums such as guar or konjac mannan, and mucilages like psyllium. They dissolve in water and form viscous gels in the gut lumen and so partially delay or reduce the absorption of carbohydrates, dietary fats, and cholesterol. IF are found mainly in vegetables, potatoes, nuts, and whole grain products such as wheat bran; sources of SF are vegetables, legumes, fruits such as apples, pears, citrus fruits, and cereals like oat and barley.

In addition to gut health, DF intake provides several health benefits and observational evidence has shown that a higher DF intake is associated with lower risk of CVD, T2DM, obesity, and certain forms of cancer [32,33].

Commonly, Western populations do not reach the recommended DF daily intakes. Grain-based foods contribute most to dietary DF intake with bread being by far the largest grain source followed by breakfast cereals. Vegetables, potatoes, and fruits also contribute substantially to DF intake [34].

In the context of this review, the beneficial effects of DF, and more specifically selected SF, on the risk of CVD and underlying risk factors such as dyslipidemia are detailed.

#### *3.2. E*ff*ect of Specific DF on Blood Lipids and CVD Risk and Underlying Mechanism of Action*

The first meta-analysis by Brown et al. [35] summarized the cholesterol-lowering effects of SF, i.e., oat products, psyllium, pectin, and guar gum. SF intake between 2–10 g/day was associated with modest but statistically significant decreases in TC and LDL-C, with no significant difference between the SF. Since then, several meta-analyses as discussed below have summarized the cholesterol-lowering effects of specific SF such as beta-glucan from oats and barley, psyllium, and glucomannan. A recent review found that increased DF intake significantly lowered TC and LDL-C, modestly but significantly decreased HDL-C, and had no effect on TG concentrations [36]. Further, there was no evidence that the type of DF, i.e., SF or IF or the way DF are administered, i.e., via supplements or foods, influenced the cholesterol-lowering effect [36].

Beta-glucan: Beta-glucan is a viscous SF found in oats, barley, edible mushrooms, and (baker's) yeast. A recent meta-analysis found that a median intake of 3.5 g/day of oat beta-glucan modestly lowered LDL-C by −0.19 mmol-L (95% CI: −0.23 to −0.14) or 4.2% and non-HDL-C by −0.20 mmol/L (95% CI: −0.26 to −0.15) or 4.8% compared with control diets [37]. An earlier meta-analysis found an LDL-C reduction of −0.25 mmol/L or 6% with a median daily intake of 5.1 g beta-glucan from oats [38].

Barley beta-glucan was also shown to lower blood cholesterol. A median intake of 6.5 and 6.9 g/day of barley beta-glucan, respectively, lowered LDL-C by −0.25 mmol/L (95% CI: −0.30 to −0.20) or 7% and non-HDL-C by −0.31 mmol/L (95% CI: −0.39 to −0.23) or 7% compared with control diets [39]. Similar effects were reported in a previous meta-analysis [39]. There was no clear dose–response relationship for barely beta-glucan [39], while a significant inverse association between dose and LDL-C lowering was found for oat-beta-glucan [37]. The established LDL-C lowering effect of beta-glucan, especially from oats, has resulted in approval of health claims for oat beta-glucan and its LDL-C lowering effect or CVD risk reduction benefit in Europe, USA, Canada, and Australia/New Zealand referring to an intake of at least 3 g/day of oat beta-glucan.

Psyllium: Psyllium is a viscous SF from the husk of the *Plantago ovata* seed and is a common fiber supplement. Two recent meta-analyses [40,41] found that daily intake of 10.2 g psyllium significantly lowered LDL-C by −0.28 mmol/L (95% CI: −0.21 to −0.31) [39] and -0.33 mmol/L (95% CI: −0.38 to −0.27) [40]; non-HDL-C was found to be lowered by −0.39 mmol/L (95% CI: −0.50 to −0.27) [41]. No clear dose–response relationship was observed, suggesting that the cholesterol-lowering benefit of psyllium intakes of ≥10 g/day will not result in bigger LDL-C lowering [41]. The recommended intake of psyllium for an optimal cholesterol-lowering and heart health benefit is 7 g/day SF from 10.2 g psyllium husk, based on the approved US FDA health claim.

Glucomannan: Glucomannan, also known as konjac mannan, is one of the most viscous DF. Its main source is the tuberous root of the konjac plant. Typically, glucomannan is consumed in the form of capsules next to some food formats like bars or biscuits. A recent meta-analysis found that daily intake of ~3 g glucomannan significantly lowered LDL-C by −0.35 mmol/L (95% CI: −0.46 to −0.25) or 10% and non-HDL-C by −0.32 mmol/L (95% CI: −0.46 to −0.19) or 7% [42]. There was no indication of a dose–response effect with glucomannan intakes between 2.0–15.1 g/day. An intake of 4 g/day glucomannan is the minimal dose of an approved health claim in Europe for maintaining a healthy blood cholesterol concentration.

Summarizing, an intake of 4–10 g/day of different types of SF is required to achieve a 5–10% reduction in LDL-C without substantially affecting HDL-C and TG concentrations.

SF with high viscosity and water-binding capacity form viscous gels in the intestinal lumen which decrease absorption of macronutrients as well as of cholesterol and bile acids and subsequent increased fecal excretion [32]. An impaired reabsorption and increased excretion of bile acids stimulates bile acids synthesis in the liver which consequently lowers circulating cholesterol concentrations in the blood. Furthermore, colonic fermentation of SF by gut bacteria produces short-chain fatty acids, and increased concentrations of circulating propionate may contribute to cholesterol lowering by decreasing cholesterol synthesis in the liver [32,43].

In view of the effects on CVD risk and clinical endpoints, observational studies have consistently shown that higher intakes of DF and dietary patterns high in DF are associated with a reduced risk of CVD as well as T2DM and obesity, also risk factors for CVD [32]. Meta-analyses of prospective cohort studies have shown a significant association between DF intake and lower risk of all-cause mortality [44] and mortality from CVD and CHD [43,44]. An additional DF intake of 7–10 g/day was inversely linked to a reduction in CVD mortality by 9% and CHD by 9–11% [44,45]. A recent umbrella review of systematic reviews and meta-analyses of observational studies concluded that there is convincing evidence that higher DF intake is associated with a lower risk of CVD, and particularly of coronary artery disease (CAD) and CVD-related death [33].

DF intake from cereals was significantly associated with lower CVD risk [43,44], while results for other fiber sources seem less conclusive. Kim and Je found that DF intake from legumes also showed an inverse association with CVD risk, while DF intake from vegetables and fruits failed to show an association [44]. Conversely, Threapleton et al. report an inverse association of vegetable fiber intake and CVD risk [44]. In particular, IF intake seems to be linked with lower risk of CVD while such a benefit was not convincingly seen for SF intake [44,45]. A recent prospective French cohort study supports that a higher intake of DF is associated with a decrease in CVD incidence and mortality. Both SFs and IFs were associated with lower risk of T2DM, while SFs were also associated with lower CVD risk. Amongst different DF sources, DFs from fruits were inversely associated with CVD risk [46]. Noteworthy, there are no RCTs on the effects of specific DF such as beta-glucan that have been shown that lowering LDL-C affects CVD outcomes.

#### *3.3. Food-Based Dietary Guideline Recommendations for DF Intake*

Recommendations for DF intake typically refer to total fiber intake. A healthy diet should contain more than 25 g/day of DF and most European countries recommend 25 and 30 g/day [34]. In the EFSA scientific opinion on dietary reference values, a DF intake of 25 g/day would be adequate for normal laxation in adults while intakes higher than 25 g/day would be necessary to reduce risk of CHD, T2DM, and to improve weight maintenance [34].

Usually, no recommendations are made for intakes of specific fiber types such as SF. Nevertheless, in view of approved health claims for maintaining or lowering blood cholesterol concentrations, daily intakes of 3 g beta-glucan from oats, oat bran, barley, or barley bran, 6 g pectin, 10 g guar gum, and 4 g glucomannan could be recommended. The 2019 ESC/EAS guidelines for the management of dyslipidemia recommend a DF intake of 25–40 g/day, including ≥7–13 g of SF, preferably from wholegrain products, e.g., oats and barley (Table 1) [2].

Adopting a predominantly plant-based diet helps to achieve the daily recommended intake of DF; in particular, vegan and vegetarian-type diets are rich in DF from various plant-based sources such as whole grains and seeds, legumes, vegetables and fruits, and nuts.

#### **4. Phytosterols**

#### *4.1. Dietary Sources*

Phytosterols (PS), comprising plant sterols and stanols, are compounds similar in structure and function to cholesterol. Principal PS are sitosterol, campesterol, and stigmasterol and their saturated counterparts sitostanol and campestanol. They occur naturally in all plant-based foods and are found in vegetable oils (especially unrefined oils), vegetable oil-based margarines, seeds, nuts, cereal grains, legumes, vegetables and fruits next to various foods and food supplements with added PS [47]. Daily intakes of PS with habitual diets typically range between 200 and 400 mg [48]. Consuming diets that emphasise plant-based foods results in higher PS intakes. With vegetarian- or vegan-type diets, PS intake can increase up to 600 mg/day [49]. With a MED-type diet such as the PREDIMED diet [20] or the dietary approaches to stop hypertension (DASH) diet [50], PS intakes of 500–550 mg/day can be achieved. Higher PS intakes such as a recommended intake of 2 g/day for LDL-C lowering [2] can only be achieved by consuming food products enriched with PS such as fat-based spreads and margarines, dairy-type foods like milk, yogurt, and yogurt drinks, or food supplements with added PS. A PS intake of about 2 g/day can be realized with the Portfolio diet through the consumption of a daily serving of a PS-added margarine next to consuming SF, soy, and other vegetable proteins and nuts, i.e., almonds [51].

#### *4.2. Cholesterol-Lowering E*ffi*cacy and Underlying Mechanisms of Action*

The TC and especially LDL-C lowering properties of PS in humans were discovered in the early 1950s [52]. Several meta-analyses have summarized their LDL-C lowering efficacy based on numerous RCTs [53–56]. The most recent meta-analysis including 124 clinical studies (with 201 study arms) concluded that PS intake significantly lowers LDL-C in a dose-dependent manner by 6–12% with intakes of 0.6–3.3 g/day without affecting HDL-C. [56]. PS intakes exceeding 3 g/day have only little additional benefit as the LDL-C lowering effect is expected to taper off because the underlying mechanism of action—inhibition of cholesterol absorption—is a saturable process. Nevertheless, RCTs with PS intakes greater than 4 g/day are limited; thus, it remains speculative whether the dose-response relationship would continue with higher PS intakes [56]. PS intake was also found to lower atherogenic apo-lipoproteins (apo) such as apo-B and apo-E and to increase anti-atherogenic apo-lipoproteins like apo-AI and apo-CII as summarized in a recent meta-analysis [57].

PS are effective in both healthy and diseased individuals and their LDL-C lowering benefit has been demonstrated in adults and children with familial hypercholesterolemia, in patients with T2DM, and individuals with the metabolic syndrome [58]. Furthermore, PS are shown to be effective in various types of food formats such as fat-based foods like spreads and margarines, dairy-type foods, and food supplements including capsules and tablets, thereby offering a variety of choices to achieve the recommended daily PS intake for a cholesterol-lowering benefit [47]. Intake occasion and frequency are critical factors for an optimal LDL-C lowering efficacy. Thus, PS should be consumed with a (main) meal and twice daily [47].

Meta-analyses have also found a significant TG-lowering effect whereby the effect was more pronounced in individuals with higher baseline TG concentrations [59]. Hence, PS offer a dual blood lipid benefit especially in individuals with dyslipidemia such as patients with T2DM or the metabolic syndrome [59].

Partial inhibition of intestinal absorption of (dietary and biliary) cholesterol is the key mechanism for the cholesterol-lowering effect of PS, with several underlying mechanisms including displacing cholesterol from mixed micelles due to limited capacity to embody sterols, interfering with transport-mediated processes of sterol uptake and stimulating cholesterol excretion via the transintestinal excretion [60]. An intake of 2 g/day of PS reduces cholesterol absorption by 30–40%, resulting in a subsequent 10% lowering of circulating LDL-C [61].

PS intake can also be a useful adjunct to lipid-lowering medication. Statins inhibit hepatic cholesterol synthesis and PS inhibit intestinal cholesterol absorption; thus, combining PS and statins leads to an additive LDL-C lowering effect [61,62]. Additional effects on LDL-C lowering have also been reported when combining PS with fibrates or with ezetimibe [61,63].

Although there are no RCTs showing the effects of long-term PS intake on CVD outcomes, e.g., CV events; it seems reasonable that PS intake may lower CVD risk based on the established LDL-C lowering effect.

#### *4.3. Recommendations for PS Intake*

Based on the proven plasma LDL-C lowering effect and the absence of adverse effects, consumption of 2 g/day PS as an adjunct to a healthy diet is one of the recommended dietary interventions for the management of dyslipidemia (Table 1) [2,64,65]. Foods with added PS may be considered i) for individuals with high serum cholesterol at intermediate or low global CVD risk who do not (yet) qualify for drug treatment, ii) as adjunct to drug (statin) therapy, in high- to very high-risk patients who fail to achieve LDL-C target goals or could not be treated with statins, and iii) in adults and children (>6 years) with familial hypercholesterolaemia, in line with current guidelines [2,61].

#### **5. Combinations of Natural Lipid-Lowering Compounds as Part of a Heart-Healthy Diet**

Combining PS (2 g/day) with different types of SF, e.g., 3 g/day oat beta-glucan has been shown to lead to additional LDL-C lowering when combined in a single food [66]. Further, a combination of 10 g/day psyllium and 2.6 g/day PS added to cookies leads to a substantial reduction in LDL-C [67].

In addition, the combination of 3.3 g/day PS with a healthy diet (low in total and saturated fat) shows an additive effect of PS to that of the diet alone [68]. By combining PS with other plant-based, cholesterol-lowering compounds or foods, further reductions in LDL-C can be achieved as observed with the Portfolio diet. This plant-based dietary pattern combines (per 2000 kcal energy intake) 20 g/day of viscous DF from oats, barley, psyllium, eggplant, okra, apples, oranges, or berries, about 50 g/day of plant protein from soy products or pulses (beans, peas, chickpeas, and lentils), 42 g/day of nuts (tree nuts such as almonds and peanuts), and 2 g/day PS in the form of a PS-enriched margarine [51,69]. Under controlled settings, an LDL-C lowering effect of 30% could be achieved with the Portfolio diet, an effect comparable to that achievable with a low-dose statin [51]. Based on a meta-analysis, the Portfolio dietary pattern significantly lowers LDL-C by -0.73 mmol/L (95% CI: -0.89 to -0.56) or by ~17%. Non-HDL-C, apo B, TC, TG, as well as systolic and diastolic blood pressure, were also significantly lowered [69].

A dual blood lipid lowering benefit of lowering both LDL-C and TG concentrations was achieved with the intake of 2 g/day PS and a minimum of 1 g/day EPA/DHA from fish oil [70].

#### **6. The Impact of Plant-Based Dietary Patterns on CV and Planetary Health**

#### *6.1. Dietary Patterns and CV Health*

Historically, dietary recommendations for CVD prevention focused on single nutrients like lowering SAFA or increasing DF intake, or on specific foods like eating more fish, whole grains, nuts, fruits, and vegetables, and less meat. Today, the focus is on the role of dietary patterns for the management of dyslipidemia and lowering CVD risk [71,72]. A healthy dietary pattern is typically described as a predominantly plant-based diet. Observational study evidence supports the beneficial effect of single plant-based foods as well as fatty fish, and the adverse effects of animal-based foods, on CVD risk [73,74]. Since foods are typically consumed in combinations, it is more reasonable to look at health benefits of a complete dietary pattern.

Several dietary patterns have been found to lower CVD outcomes and risk factors such as blood lipids, i.e., TC and LDL-C or blood pressure. These include traditional diets prevailing in the MED and in Nordic countries [75–79], dietary patterns intended to control CVD risk factors like the DASH diet [50] and Portfolio diet [51] as well as vegetarian- or vegan-type dietary patterns. A common characteristic of these dietary patterns is that they emphasize plant-based foods with reduced animal food consumption.

The MED dietary pattern refers to the traditional diet of Greece, Crete, and Southern Italy with (virgin) olive oil as the main dietary fat source as a key characteristic [72,75,78]. Further characteristics are summarized in Table 2. The Nordic diet emphasizes the intake of locally grown vegetables like cabbage and potatoes, whole grains and cereals such as oats, rye, and barley, locally grown seasonal fruits such as berries, next to rapeseed oil and fatty fish (Table 2) [72,77,78]. The DASH diet emphasizes amongst others higher intakes of vegetables, fruits and fat-free or low-fat dairy foods (Table 2) [50,79]. The Portfolio diet is a plant-based diet that emphasises the intake of four known cholesterol-lowering foods, i.e., viscous DF, plant proteins from soy and legumes, nuts, and PS (Table 2) [51,69]. The healthy vegetarian dietary pattern is based on a variety of vegetables, legumes, soy products, fruits, and whole grains, occasionally dairy foods, eggs, and fish, but no meat and poultry. A strict vegan dietary pattern excludes all animal-based foods (Table 2) [72,73,80].

#### *6.2. E*ff*ect on Blood Lipids*

Both observational studies and RCTs have found that dietary patterns emphasizing consumption of plant-based foods have beneficial effects on blood lipids, especially on TC and LDL-C.

Next to lowering systolic and diastolic blood pressure, for which the DASH diet was originally designed, the DASH diet was also found to lower TC and LDL-C. An umbrella review of systematic reviews and meta-analyses concluded that TC was lowered by −0.20 mmol/L (95% CI: −0.31, −0.10) and LDL-C by −0.10 mmol/L (95% CI: −0.20 to −0.01) without affecting HDL-C and TG [79,81]. The observed cholesterol-lowering benefit of the DASH diet may be attributable to the high intake of DF from the consumption of fruits, nuts, legumes, and whole grains, and the lower intake of saturated fat.

The Portfolio diet combining four recognized cholesterol-lowering foods/food components with a background diet low in total fat (≤30% TE) and saturated fat (<7% TE) and low in dietary cholesterol (<200 mg/day) led to clinically relevant benefits in lowering cholesterol and other CVD risk factors. A meta-analysis of RCTs has shown that TC was lowered by −0.81 mmol/L (95% CI: −0.98 to −0.64) or 12% and LDL-C by -0.73 mmol/L (95% CI: -0.89 to -0.56) or 17% [69]. Non-HDL-C was lowered by -0.83 mmol/L (95% CI: −1.03 to −0.64) or 14% and TG by −0.28 mmol/L (95% CI: −0.42 to −0.14 mmol/L) or 16%; all compared to a low total fat, low saturated fat, and low cholesterol diet. The LDL-C lowering effect of the Portfolio diet was found to be 21% in efficacy trials and 12% in effectiveness trials. Other CVD risk factors like systolic and diastolic blood pressure and C-reactive protein were also lowered [69].


*Nutrients* **2020**, *12*, 2671

et al. [51].

Following a MED or a Nordic dietary pattern is also associated with beneficial blood lipid effects. Based on a meta-analysis of RCTs, the Nordic diet lowered TC by −0.39 mmol/L (95% CI: −0.76 to −0.01) and LDL-C by −0.30 mmol/L (95% CI: −0.54 to −0.06) with no significant changes in HDL-C and TG [82,83]. Beneficial effects on reducing systolic and diastolic blood pressure were also seen [83]. The beneficial effects of the Nordic dietary pattern can be attributed to the high DF intake and the low intake of saturated fat. Likewise, the MED dietary pattern was found to significantly lower LDL-C by −0.07 mmol/L (95% CI: −0.13 to−0.01) and TG by −0.46 mmol/L (95%CI: −0.72 to −0.21) [83]. Small or no reductions in TC and/or LDL-C were reported in a recent review that compared a MED diet intervention vs. either no intervention or another dietary intervention in primary prevention of CVD [84]. Blood pressure is also modestly reduced by the MED diet, but effects are less than those observed with the Nordic diet [83].

Several meta-analyses addressed the effects of a vegetarian dietary pattern on blood lipids. Two meta-analyses of RCTs found that vegetarian diets significantly lowered TC by −0.32 to −0.36 mmol/L and LDL-C by −0.32 to −0.34 mmol/L [85,86]. HDL-C was also significantly lowered by −0.09 to −0.10 mmol/L while TG were not significantly altered [85,86]. Another recent meta-analysis of RCTs studying vegetarian diet patterns and lipid risk factors in T2DM found a reduction in LDL-C of −0.12 mmol/L (95% CI: −0.20 to 0.04) with no significant effects on HDL-C and TG [83]. Taken together, vegetarian diet patterns, particularly vegan diets, are associated with lower blood cholesterol concentrations. That vegetarian diets are low in total and especially saturated fat, low in dietary cholesterol, and particularly high in DF intake may explain their cholesterol-lowering effect. The higher intake of phytochemicals such as PS, phenolics compounds, and carotenoids with vegetarian/vegan diet patterns may further contribute to this effect.

#### *6.3. E*ff*ects on CVD Risk and Outcomes*

Evidence for beneficial effects of dietary patterns on CVD risk and related outcomes derives mostly from observational studies; RCTs that studied the effect of dietary patterns on CVD-related endpoints are rare or non-existent.

There is no direct evidence from observational studies for a CVD risk benefit, e.g., on CVD incidence or mortality of the Portfolio diet. Nevertheless, the combined effects of that dietary pattern on CVD risk factors such as LDL-C and blood pressure as observed in RCTs was assumed to decrease the estimated 10-year CHD risk by ~13% [69].

The DASH diet that is well-accepted for its blood pressure lowering effect was found to reduce CVD incidence by 20%, CHD incidence by 21%, and stroke incidence by 19% [79,83]. These CVD risk-related benefits are attributable to the substantial reductions in blood pressure and in other CVD risk factors, i.e., TC and LDL-C.

The effects of the MED diet on CVD risk have been assessed in several meta-analyses of observational studies. Rosato et al. [87] found in their meta-analysis of prospective studies a 19% lower risk for unspecified CVD, a 30% lower risk for CHD/acute myocardial infarction, a 27% lower risk for unspecified stroke, and 18% lower risk for ischemic stroke when comparing the highest vs. the lowest adherence to this dietary pattern based on a MED diet score [72,87]. Another meta-analysis of prospective studies reports a risk reduction (RR) in total CVD, CHD, and stroke mortality of 0.79 (95% CI: 0.77, 0.82), 0.83 (95% CI: 0.75, 0.92), and 0.87 (95% CI: 0.80, 0.96), respectively, next to a lower CHD incidence (RR: 0.73; 95% CI: 0.62, 0.86) and stroke incidence (RR: 0.80; 95% CI: 0.71, 0.90), comparing the highest vs. lowest categories of MED diet adherence [88]. A meta-analysis of three RCTs revealed that the MED diet is associated with a 38% lower risk of total CVD and a 35% lower risk of total myocardial infarction (MI) but a nonsignificant reduction in CVD mortality [83,88]. Especially the PREDIMED trial, a primary prevention RCT that investigated a modified MED diet supplemented with either extra virgin olive oil or nuts compared to a reduced-fat control diet, revealed an approximately 30% lower incidence of a composite of CVD endpoints (MI, stroke, and death from CV causes) with these MED diet patterns [20]. A re-analysis of the original outcome data assessing individual endpoints

revealed a reduction in stroke incidence but little or no effect on total and CVD mortality or MI [20,84]. In their review, Rees et al. [84] concluded that there is still some uncertainty regarding the effects of a MED-style diet on clinical endpoints and CVD risk factors for both primary and secondary prevention.

Less, though more conflicting, evidence for CVD risk-related effects are reported for the Nordic diet. Adhering to a healthy Nordic diet was associated with a lower risk of CVD in men and women from the Danish Diet, Cancer and Health cohort [89,90] but not in women of the Swedish Women's Lifestyle and Health cohort [91]. A recent systematic review and meta-analysis of prospective studies assessing the Nordic diet reports a modest reduction in CVD risk (RR: 0.93; 95% CI, 0.88, 0.99) and stroke risk (RR: 0.87 95% CI, 0.77, 0.97) but not in CVD mortality [83].

Meta-analyses of observational study evidence support that a vegetarian diet pattern is associated with a 22% lower CHD mortality while no association was found for CVD or stroke mortality. Furthermore, vegetarian diets are associated with a 28% reduced risk of CHD [80,83]. These results are comparable to a previous meta-analysis reporting a 25% risk reduction in CHD incidence and mortality but no significant effects for CVD and stroke and all-cause mortality [92].

#### **7. Dietary Patterns and Environmental Aspects**

Dietary choices clearly contribute to the risk for developing CVD. Evidence supports the intake of specific nutrients, food groups, or certain dietary patterns to positively influence dyslipidemia and to promote the prevention of CVD. Next to their impact on CV health, dietary patterns also impact the environment in various ways. Plant-based dietary patterns have been shown to have a smaller impact on climate change (greenhouse-gas emissions), fresh-water use, cropland use (deforestation), and biodiversity loss [8,93] than consumption of animal-based foods such as (red and processed) meat. A recent global modeling study combined analyses of nutrient level, diet-related, and weight-related chronic disease mortality, and environmental impact in different sets of diet scenarios for more than 150 countries [94]. Four different energy-balanced diets were found to reduce environmental impact, nutrient deficiencies, and diet-related mortality. All four dietary patterns were predominately plant-based diets with limited red and processed meat intake. These patterns were described as flexitarian, pescatarian, vegetarian, and vegan diets [94].

It must be noted that not all plant-based diets are necessarily beneficial in view of diet-related chronic diseases such as CVD risk [74]. Observational evidence from cohort studies found that plant-based diets containing higher amounts of refined grains, juices/sugar-sweetened beverages, sweets, potatoes/fries were associated with an increased CHD risk, while plant-based diets including higher amounts of whole grains, fruits, vegetables, legumes, nuts, and vegetable oils were associated with lower risk of CHD [74,95]. While predominately plant-based diets have beneficial effects on CV risk, their impact on body weight and prevention or treatment of obesity are less clear [78]. Hence, consuming an energy-balanced diet in view of avoiding weight gain and obesity seems crucial.

There are several possibilities for adopting a predominately plant-based dietary pattern based on personal food taste and preference as well as individual needs. Characteristics for choosing a predominantly plant-based healthy diet are summarized in Table 3.



Complied based on dietary recommendations described in Mach et al. [2], the UK Eat Well guide (https: //www.gov.uk/government/publications/the-eatwell-guide), the 2015–2020 dietary guidelines for Americans [96]. \* Plenty refers to 5 portions of fruits and vegetables per day.

#### **8. Current Recommendations for a Healthy Dietary Pattern**

The CV health benefits of certain dietary patterns have been recognized by numerous dietary and clinical practice guidelines. For instance, the DASH dietary pattern is recognized by the 2015–2020 dietary guidelines for Americans [96], the AHA/ACC guideline on lifestyle management to reduce cardiovascular risk [27], the Canadian Cardiovascular Society guidelines for the management of dyslipidemia [64], and by the ESC/EAS 2019 guidelines for the management of dyslipidaemias [2]. The Canadian Cardiovascular Society [64] also mentions the Portfolio diet as a healthy dietary pattern. Adopting a healthy MED diet pattern for the promotion of healthy eating and the prevention of CVD is acknowledged by the 2015–2020 dietary guidelines for Americans [96], the 2019 ACC/AHA guideline on the primary prevention of CVD [27], and by the ESC/EAS 2019 guidelines for the management of dyslipidaemias [2]. Eating a plant-based Nordic diet as a healthy alternative to the MED-dietary pattern is advocated by the Nordic nutrition recommendations [97]. A healthy vegetarian eating pattern is mentioned by the 2015–2020 dietary guidelines for Americans [96] and the Canadian Cardiovascular Society guidelines for the management of dyslipidemia [64].

Notably, the emphasis of eating a predominantly plant-based diet as a healthy eating pattern to prevent diet-related chronic diseases such as CVD is a common theme of many food-based dietary guidelines [9,96]. Another driving factor for recommending a more plant-based diet relates to

environmental sustainability [8,9]. Nevertheless, the environmental aspects of dietary patterns are not yet widely addressed in food-based dietary guidelines, only a few countries have so far included environmental sustainability in their guidelines [78].

#### **9. Conclusions**

This review describes the beneficial effects of specific macro- and micro-components of a plant-based diet (vegetable fats, DF, and PS) in the management of dyslipidemia and CVD prevention. Moreover, the role of common plant-based dietary patterns in CV health is discussed.

The protective effect of predominately plant-based dietary patterns such as the MED and Nordic diet or the DASH and Portfolio diet on CVD risk and related risk factors, e.g., LDL-C, is associated with specific plant-based foods which are known for their CV health benefits [72,74–76,79]. These foods include fruits, vegetables, legumes, whole grains, nuts, and seeds [73,74]. Plant-based foods are typically rich in unsaturated fatty acids, DF, plant proteins and various micronutrients like vitamins and phytonutrients such as PS and polyphenols; they are low in saturated fats and often low in energy density compared to foods from animal sources [74]. The beneficial effects of these plant-based food components are explained by different underlying mechanisms which influence the development of CVD either directly or indirectly by influencing risk factors such as dyslipidaemia. For instance, replacing saturated fats in the diet with unsaturated fatty acids like MUFA and PUFA is known to lower LDL-C [13,16]. Observational studies and RCTs with clinical endpoints have further shown that replacing SAFA with especially vegetable oil PUFA lowers the risk of CVD, mainly the risk of CHD [13,17] whereas evidence for effects on CVD or CHD risk when SAFA is replaced by MUFA, carbohydrates, or proteins is insufficient or limited [13]. DF, especially viscous SF like beta-glucan, reduce the intestinal absorption of cholesterol and re-absorption of bile acids and produce short-chain fatty acids in the colon which may affect hepatic cholesterol synthesis, all contributing to a LDL-C-lowering effect [43]. Observational studies have shown a significant association between DF intake and lower risk of all-cause mortality [44] and mortality from CVD and CHD [43,44]. PS also inhibit intestinal cholesterol absorption resulting in lower circulating concentrations of LDL-C [61]. Although there are no RCTs showing the effects of long-term intake of specific DF like beta-glucan or PS on CVD outcomes, e.g., CV events, it seems reasonable that their intake may lower CVD risk based on the established LDL-C lowering effect. Replacing animal protein with plant protein has also been shown to lower LDL-C [98].

Noteworthy, not all plant-based dietary patterns are equally effective in lowering CVD risk as not all plant-based foods have beneficial CV effects [74,95]. Hence, the quality of plant-based foods and food components plays an important role. For instance, a dietary pattern with more refined grains than whole grains or more sugars-sweetened foods and beverages have been linked with a higher CVD risk. The same dietary patterns for which a positive impact on dyslipidemia and the prevention of CVD has been shown can also help reduce the impact of food choices on environmental degradation [8]. Hence, shifting to a more plant-based dietary pattern will not only improve CV health but will also be more environmentally sustainable. While plant-based, e.g., vegetarian, dietary patterns are generally perceived positively because of their benefits on health (CVD prevention) and the environment, there are also several barriers that hinder the switch to, and maintenance of a plant-based diet. Common barriers include health concerns that a plant-based diet may lack specific nutrients, reluctance to change dietary behavior, and enjoyment of eating meat and animal-based foods [99,100].

A limitation of this narrative review is that only selected macro- and micro components of a plant-based diet are described while other components such as vitamins and other phytonutrients like polyphenols with proposed CV benefits were not considered. Nevertheless, dietary fatty acids, DF, and PS were chosen because of their proven evidence for LDL-C-lowering and CVD risk benefits and because they are specifically referred to in dietary recommendations for CVD prevention [2]. While health benefits of these individual dietary components have been addressed before, this review addresses them in the context of a plant-based dietary pattern. As mostly observational study evidence

is available on CVD risk and clinical endpoints, more evidence from RCTs demonstrating such benefits for these dietary components in the context of a plant-based diet would be desirable, despite the challenges of carrying out long-term, controlled dietary intervention studies.

**Author Contributions:** E.A.T. was responsible for the conception of the review. E.A.T and S.M. contributed to the literature search, E.A.T. wrote the manuscript, and S.M provided critical input; both authors approved the final version for submission. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work received no external funding except that the publication costs were funded by Upfield.

**Conflicts of Interest:** S.M. is employed by Upfield. Upfield markets plant-based consumer goods such as margarines and spreads including foods products with added phytosterols. E.A.T. works as a consultant for Upfield. The authors declare no further conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Review*

### **Beyond Fish Oil Supplementation: The E**ff**ects of Alternative Plant Sources of Omega-3 Polyunsaturated Fatty Acids upon Lipid Indexes and Cardiometabolic Biomarkers—An Overview**

#### **Heitor O. Santos 1,\*, James C. Price <sup>2</sup> and Allain A. Bueno <sup>2</sup>**


Received: 27 September 2020; Accepted: 14 October 2020; Published: 16 October 2020

**Abstract:** Cardiovascular diseases remain a global challenge, and lipid-associated biomarkers can predict cardiovascular events. Extensive research on cardiovascular benefits of omega-3 polyunsaturated fatty acids (n3-PUFAs) is geared towards fish oil supplementation and fish-rich diets. Nevertheless, vegetarianism and veganism are becoming more popular across all segments of society, due to reasons as varied as personal, ethical and religious values, individual preferences and environment-related principles, amongst others. Due to the essentiality of PUFAs, plant sources of n3-PUFAs warrant further consideration. In this review, we have critically appraised the efficacy of plant-derived n3-PUFAs from foodstuffs and supplements upon lipid profile and selected cardiometabolic markers. Walnuts and flaxseed are the most common plant sources of n3-PUFAs, mainly alpha-linolenic acid (ALA), and feature the strongest scientific rationale for applicability into clinical practice. Furthermore, walnuts and flaxseed are sources of fibre, potassium, magnesium, and non-essential substances, including polyphenols and sterols, which in conjunction are known to ameliorate cardiovascular metabolism. ALA levels in rapeseed and soybean oils are only slight when compared to flaxseed oil. *Spirulina* and *Chlorella*, biomasses of cyanobacteria and green algae, are important sources of n3-PUFAs; however, their benefits upon cardiometabolic markers are plausibly driven by their antioxidant potential combined with their n3-PUFA content. In humans, ALA is not sufficiently bioconverted into eicosapentaenoic and docosahexaenoic acids. However, evidence suggests that plant sources of ALA are associated with favourable cardiometabolic status. ALA supplementation, or increased consumption of ALA-rich foodstuffs, combined with reduced omega-6 (n6) PUFAs intake, could improve the n3/n6 ratio and improve cardiometabolic and lipid profile.

**Keywords:** alpha-linolenic acid; flaxseed; lipids; omega-3; walnuts

#### **1. Introduction**

Cardiovascular diseases remain a major Public Health concern, with lipid-associated biomarkers being trusted predictors of major cardiovascular events [1,2]. Added to the pharmacological strategies and conscious effort to improve the patients' lifestyle by adequate nutrition and physical activity, nutraceutical strategies which include certain supplements, herbal extracts and functional foods, may be a helpful complementary approach for individuals with cardiovascular disease and dyslipidaemia [3–9].

The dietary replacement of saturated fats for polyunsaturated fatty acids (PUFAs) has shown beneficial effects upon lipid profile [10], as well as long-term protective benefits against major cardiovascular events and associated clinical complications [11,12]. For decades, omega-3 polyunsaturated fatty acids (n3-PUFAs) from either marine sources or fish oil (FO) supplementation were broadly referred to in cardiology guidelines [13–15]. For example, amongst several clinical investigations, Sagara et al. in 2011 showed that 2 g of DHA daily for five weeks improved blood pressure and lipid profile in a sample population of 38 middle-age men with hypertension and hypercholesterolaemia [16].

On the other hand, however, Manger et al. in 2010 did not find a significant trend in reduced risk of coronary events with increased consumption of n3-PUFAs from fish and fish supplements. Nonetheless, Manger argues that their sample population had a high intake of n3-PUA to begin with, and possibly the lack of association could have been attributed to a ceiling effect of n3-PUFAs [17]. Interestingly, a robust meta-analysis published in 2018, which included 79 Randomized Control Trials (RCTs) and a total of 112,059 participants, showed that n3-PUFAs supplementation actually did not show significantly greater efficacy on reducing the occurrence of cardiovascular events [18]. Such results must be interpreted carefully due to factors such as methodology employed, varied sample population investigated, other factors beyond the scope of the study, and a potential risk for bias. At the same time, it has also been demonstrated that plant sources of n3-PUFAs have shown some potential against cardiovascular events and dyslipidaemia [18].

The findings on fish consumption and FO supplementation upon cardiovascular health do not disregard the merit of investigating the usefulness of plant-derived n3-PUFAs in clinical practice. Studies on the potential efficacy of plant-derived n3-PUFAs become further justified as dramatic reductions of fish stocks have been reported in the North Atlantic Ocean and Mediterranean Sea [19,20]. In the present review, we have critically appraised the efficacy of plant-derived n3-PUFAs from foodstuffs, as well as its supplementation, upon the modulation of lipid profile and selected cardiometabolic markers. More specifically, we searched for the effects of chia seeds, flaxseeds, walnuts, as well as *Spirulina* and *Chlorella*. As those foodstuffs are gaining more popularity, a phenomenon possibly attributed to the increased awareness of environmental issues related to overfishing, combined with the increasing trend towards veganism, vegetarianism and flexitarianism across all segments of society, it is of greatest interest to clarify the clinical potential of plant sources of n3-PUFAs.

#### **2. Methods**

We employed the electronic databases Pubmed/Medline and Google Scholar to identify relevant publications. Randomised Controlled Trials (RCTs) written in English were the chosen sources of results as a means of translating current research findings into clinical practice. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [21] were used to evaluate and select the RCTs.

Limited to RCTs, we searched for plant sources of ALA by using the following combinations of Medical Subject Heading (MeSH) keywords: ("Chia Seeds" OR "Flaxseeds" OR "Hemp Seeds" OR "Walnuts" OR "Seaweeds" OR "*Spirulina*" OR "*Chlorella*") AND ("LDL-C" OR "Low-Density Lipoprotein Cholesterol" OR "Total Cholesterol" OR "Triglycerides" OR "HDL-C" OR "Low-Density Lipoprotein Cholesterol" OR "Blood Pressure" OR "Cardiovascular Disease" OR "Cardiometabolic Risk" OR "Inflammatory Biomarkers" OR "Proinflammatory Cytokines"). The period covered in the search included inception to August 2020. Summary findings from the selected papers are presented in Supplementary Tables S1–S3. After perusal of such findings, we discussed key aspects and manually expanded the review by selecting further articles that have investigated nutrition facts, mechanisms of action and clinical applications.

As the number and robustness of clinical studies that have investigated the metabolic effects of plant-derived n3-PUFAs are only a fraction of those that employed FO, we have also summarized in Supplementary Table S4 the outcomes of 38 selected FO supplementation studies identified using the key terms listed above and published in the last five years, with a combined sample population of 4136 individuals. The evidence gathered confirms that whilst some studies did show improvements in metabolic biomarkers after FO supplementation, some others did not.

#### **3. Metabolic Pathways**

#### *3.1. Conversion of ALA in Humans*

Not only the insufficient intake of dietary n3-PUFAs, but also the inefficient elongation and desaturation of ALA to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in humans, result in low n3 long chain-PUFA content in blood and other peripheral tissues [22,23]. Accordingly, North America, Central and South America, and Africa, are geographical examples where EPA and DHA concentrations are endemically low [24]. More specifically, the intake of n3-PUFAs in the average USA population is low or very low [14].

Approximately only 5% of ALA is converted to EPA whilst less than 0.5% is converted to DHA in humans, although mammals have the essential enzymes used in this pathway [25–27]. A proof-of-concept study published in 2010 showed in newborn infants that only approximately 0.04% of ALA administered was elongated and desaturated to circulating EPA, whilst the subsequent conversion of EPA to DHA was comparatively more efficient [28]. The very low ALA to EPA bioconversion ratio identified in that study does not account for the EPA that was actually incorporated into solid tissues, but it does confirm the suggestion that even large amounts of dietary ALA will probably have negligible effects on plasma DHA levels [27].

Moreover, the conversion of ALA to EPA and subsequently DHA appears to be more efficient in women than in men, a phenomenon that could probably be explained by a possibly advantageous action of oestrogens in protecting the mother and the lactating child. Gender difference is a factor to be considered before making dietary recommendations for n3-PUFAs intake [25].

#### *3.2. n3-PUFAs Versus n6-PUFAs*

The inefficient bioconversion of ALA to EPA and DHA becomes more evident when considering that n3 and omega-6 (n6) PUFAs, although in much different concentrations in the typical westernized diet, compete almost equally for the same enzymatic pathway that elongates and desaturates the precursors ALA and linoleic acid (LA) to EPA and arachidonic acid (AA), respectively [29]. On the other hand however, potential roles for ALA in human health that may be independent of its bioconversion onto DHA have been proposed [27].

Experimental evidence alludes to a slower enzymatic metabolism of n3-PUFAs in relation to n6-PUFAs [30]. Therefore, focus on ALA intake is paramount for human health, but similarly important is the adequate intake of LA, as not to imbalance the n6/n3 ratio. Typical westernized diets show abundance of meats and poultry alongside deep-fried foodstuffs, an important dietary characteristic that favours high LA intake.

As seen in Figure 1, dietary sources of n6-PUFAs lead to raised levels of AA, culminating in increased synthesis of pro-inflammatory eicosanoids. Dietary sources of n3-PUFAs, in turn, allow the synthesis of anti-inflammatory eicosanoids. Increased n3-PUFA concomitant to decreased n6-PUFA decreases the AA content in platelets, vascular endothelial cells and vascular wall macrophages, thus reducing AA-derived pro-inflammatory mediators [31]. Cyclooxygenases (COX) and lipoxygenases (LOX) convert AA to prostaglandin E2, thromboxane A2, prostaglandin I2 and leukotriene B4, amongst other pro-inflammatory eicosanoids, whilst the same enzymatic pathways convert EPA to prostaglandin E3, thromboxane A3, prostaglandin I3 and leukotriene B5, amongst other anti-inflammatory eicosanoids [32–34].

**Figure 1.** Whilst the main dietary sources of the n3-PUFAs EPA and DHA are fatty fish (e.g., salmon and sardines) and algae, the n3 precursor ALA is found mainly in walnuts, chia seeds, flaxseeds, and rapeseed oil. Likewise, meat, poultry and eggs are important sources of the n6-PUFAs AA, and its n6 precursor LA is found mainly in safflower, sunflower, soybean and corn oils [35–37]. ALA and LA are converted by desaturases and elongases to EPA and AA, respectively, and subsequently converted in a series of complex enzymatic reactions to the longer forms DHA and n6-docosapentaenoic (n6-DPA), respectively [38,39].

#### *3.3. Cardiometabolic Pathways*

It has been observed in vitro that ALA is associated with decreased expression levels of vascular cell adhesion molecule-1 (VCAM-1), interleukin 6 (IL-6), proliferating cell nuclear antigen (PCNA), macrophage marker M3/84 (mac-3) and stearoyl-CoA desaturase-1 (SCD-1) [40]. Indirect effects of ALA upon cardiometabolic pathways have also been observed; ALA bioconversion to DPA is associated with decreased expression levels of COX-1, COX-2 and tumour necrosis factor-alpha (TNF-α), whilst its bioconversion to EPA and DHA is associated with decreased expression levels of peroxisome proliferator-activated receptor gamma (PPAR-γ) [40].

Given that such results have also been identified in aortic tissue [41], it is reasonable to speculate that the positive effects of ALA are driven by attenuation of inflammation, cell proliferation and oxidation [40]. In a more optimistic, long-term speculative scenario, such ALA-induced effects could retard the progression, or even promote an amelioration, of the atherosclerotic state.

ALA administration to primary cultured endothelial cells induced inhibitions of NAD-dependent deacetylase sirtuin-3 (SIRT3) reduction, superoxide dismutase 2 (SOD2) hyperacetylation, and mitochondrial reactive oxygen species (ROS) overproduction, alongside restoration of autophagic flux under damage induced by treatment with angiotensin II plus TNFα [42,43]. Such effects, apparently attributed to ALA, are in line with mitigation of endothelial dysfunction and experimental hypertension [42].

It is known that n3-PUFAs have the capacity to decrease liver triglyceride (TG) synthesis by competitive inhibition of 1,2 diglyceride acyltransferase (DAT), at the same time suppressing the activity of sterol regulatory element-binding protein 1c (SREBP-1c)—A protein that regulates the expression of genes involved in fatty acid and TG synthesis—and also to increase β-oxidation in adipose tissue [44,45]. Regarding the latter, the high affinity of n3-PUFAs for peroxisome proliferator-activated receptor alpha (PPAR-α) leads to a greater synthesis of enzymes involved in lipid catabolism, thus favoring not only fatty acid β-oxidation in peripheral tissues but also catabolism of circulating TG in chylomicrons and

very low-density lipoprotein cholesterol (VLDL-C) [44,46,47]. Moreover, substrates for TG synthesis are also decreased by reduced transport of non-esterified fatty acids to hepatocytes, consequently reducing VLDL-C synthesis [44,48]. Nonetheless, as the majority of the cardiometabolic pathways so far elucidated are based on experimental data [40–43], a more thorough, critical and applied appraisal of the effects of ALA is needed before any general conclusions can be made.

#### **4. Alternative Plant Sources of n3-PUFAs**

#### *4.1. Nuts and Seeds*

#### 4.1.1. Nutrition Facts

Nuts and seeds are important sources of ALA and also micronutrients, polyphenolic compounds, sterols and fibres, which are protective elements against the exacerbation of chronic diseases [49–51]. For instance, nuts and seeds that are sources of ALA also contain a considerable amount of calcium, magnesium, and potassium (Table 1), which are fundamental macrominerals for cardiovascular health, mainly in the management of hypertension [52–56]. Furthermore, nuts and seeds are a source of protein. Although at lower levels as compared to animal-derived protein in terms of needs for muscle hypertrophy, the consumption of nuts and seeds is inversely correlated with cardiovascular events and mortality, as demonstrated by recent studies [57,58].


**Table 1.** Nutrition facts of ALA-containing seeds.

Adapted from the United States Department of Agriculture (USDA) database [59]. ALA, alpha-linolenic acid; CA, calcium; CHO, carbohydrate by difference; FDC, FoodData Central; K, potassium; LA, linoleic acid; Mg, Magnesium.

Regarding the ALA content of nuts and seeds, one ounce (28 g) of flaxseed, chia seeds, hemp seed or walnuts exceeds the Adequate Intake (AIs) for ALA, which is 1.1 g/day for women and 1.6 g/day for men, and 1.4 g/day and 1.3 g/day during pregnancy and lactation, respectively [59,60]. In one ounce, there are 6.38 g (398% and 580% of AIs for men and women) of ALA in flaxseed, 4.99 g (175% and 254% of AIs for men and women) in chia seeds, 2.80 g (312% and 453% of AIs for men and women) in hemp seed, 2.54 g (159% and 230% of AIs for men and women) in walnuts (Table 1).

#### 4.1.2. Walnuts

Walnuts are an important source of ALA [61,62]. A 2-years follow-up study recruited 236 elderly subjects, segregated into two groups: a control group without nut consumption, and an intervention group in which 15% of the approximate daily energy intake consisted of walnuts, at approximately 30–60 g/day of walnuts [63]. The researchers found a reduction of 8.5 mmHg in the systolic blood pressure, whose baseline levels were >125 mmHg; however, no changes were observed in diastolic blood pressure. In the same study, the participants who consumed walnuts required lower dosages of antihypertensive drugs as compared to the control participants. The blood pressure of all participants in that study was monitored by the 24-h ambulatory blood pressure monitoring, which is considered the gold standard in the diagnosis of hypertension [64]. In contrast, a recent meta-analysis [65]

did not support walnut consumption per se as a blood pressure-lowering strategy. Despite being a meta-analysis, that study [65] may have had a few limitations due to heterogeneity. The results of Domènech et al. [63] provide what appears to be reliable evidence, based mainly on its long-term duration and sample size.

Le et al. [66] recruited 213 overweight and obese women to a weight loss study, and offered one of the three following dietary regimens: a walnut-rich diet which consisted of 35% energy from fat and 45% energy from carbohydrates, or a low-fat (20%) high-carbohydrate (65%) diet, or a high-fat (35%) low-carbohydrate (45%) diet. After six months of intervention, high-density lipoprotein cholesterol (HDL-C) levels were significantly increased (*p* < 0.05) in the walnut-rich group (from ≈60 to ≈63 mg/dL), whilst a small decrease was observed in the low-fat (from ≈60 to ≈57 mg/dL) and low-carbohydrate (from ≈58 to ≈57 mg/dL) groups.

Interestingly, Fatahi et al. [67] randomised 99 overweight and obese women into three low energy-diet groups: the first group consisted of 300 g/week of fatty fish such as salmon, avoiding the intake of plant sources of n3-PUFAs; the second group consisted of 18 walnuts/week, avoiding the intake of fish and other plant sources of n3-PUFAs; the third group consisted of 150 g fatty fish and nine walnuts/week, avoiding the intake of other sources of n3-PUFAs. After 12 weeks of dietary intervention, as compared to the fish-only group and walnut-only group, the fish + walnut group showed better metabolic profile overall, evidenced by greater increase in HDL-C (+3.6 mg/dL) levels, followed with greater decrease in systolic blood pressure (−5 mmHg), fasting blood glucose (−12 mg/dL), low-density lipoprotein cholesterol (LDL-C) (−6 mg/dL), high-sensitivity C-reactive protein (hs-CRP) (−0.51 mg/L), D-dimer (−0.45 mg/dL), fibrinogen (−22 mg/dL), alanine aminotransferase (ALT) (−6 IU/L), aspartate aminotransferase (AST) (−6 IU/L), TNF-α (−0.08 ng/mL) and IL-6 (−1.6 ng/mL).

#### 4.1.3. Flaxseed

Flaxseed is an important source of ALA, and a few trials have identified beneficial effects of flaxseed intake upon lipid indexes and cardiometabolic biomarkers. In a study recruiting 21 patients with coronary artery disease [68], a pivotal population to ascertain the magnitude of a cardiometabolic intervention, the daily consumption of 30 g flaxseed for 12 weeks promoted better outcomes as compared to the control group in increasing flow-mediated dilation (5.1 vs. −0.55% change from baseline for the flaxseed and control groups, respectively), whilst decreasing the inflammatory status by reducing the levels of CRP (−1.18 mg/L), IL-6 (−7.65 pg/mL), and TNF-α (−34.73 pg/mL). Importantly, no significant change in body weight was observed in either groups [68], which appears to be a very relevant result as it suggests that flaxseed may improve cardiovascular parameters independently of weight loss.

In a systematic review and meta-analysis of RCTs with 1502 patients across 32 studies, flaxseed or its derivatives (whole or ground flaxseed, flaxseed oil, or lignan supplements) reduced the concentrations of hs-CRP (weighted mean difference (WMD): −0.75; 95% CI −1.19, −0.31) and TNF-α (WMD: −0.38; 95% CI −0.75, −0.01) but did not change IL-6 levels. Flaxseed was tested in the form of whole flaxseed, golden flaxseed, flaxseed oil, and lignan supplements at dosages ranging from 360 mg to 60 g, for 2 to 12 weeks, with an averaged intervention period of approximately 10 weeks [69].

A recently published clinical trial recruited 41 women suffering with polycystic ovary syndrome, randomly segregated into two groups, group 1 subjected to lifestyle changes (American Heart Association recommendations + >30 min moderate to intense activity 3x/week) plus 30 g/day brown flaxseed flour in salad, yogurt or cold drinks, and group 2 subjected to the same lifestyle changes only, for 12 weeks [70]. The authors found that the flaxseed group showed significant improvements in insulin, homeostasis model assessment of insulin resistance (HOMA-IR), TG, hs-CRP, IL-6, leptin, HDL-C and adiponectin, as compared to the non-flaxseed group [70].

In a RCT recruiting 100 eligible patients suffering with non-alcoholic fatty liver disease (NAFLD), Yari et al. [71] found that 30 g flaxseed daily plus positive lifestyle interventions for 12 weeks decreased serum concentrations of total cholesterol (TC) (−31.71 mg/dL), TG (−61.33 mg/dL), LDL-C (−22.64 mg/dL), ALT (−11.12 U/L), AST (−5.37 U/L) and gamma-glutamyltransferase (−11.54 U/L), results that were not matched in the group submitted to positive lifestyle interventions only. It should be noted however that both groups showed reductions in BMI (30.37 ± 4.42 to 28.05 <sup>±</sup> 3.89 kg/m2 in the flaxseed plus lifestyle improvement group, and 33.37 <sup>±</sup> 5.56 to 32.42 <sup>±</sup> 5.98 in the lifestyle improvement only group) as well as the intensity of hepatic steatosis, a result most likely attributed to decreased energy intake in both groups (2379.41 ± 473.74 to 2117.47 ± 378.46, and 2424.45 ± 470.89 to 1966.39 ± 449.52 kcal, respectively). Such findings reinforce the hypothesis of beneficial effects of flaxseed independently of changes in energy intake and body composition.

In a mirrored RCT [72], this time recruiting 98 patients suffering with metabolic syndrome, the same researchers from the previously mentioned study [71] observed comparable results, in which 30 g flaxseed plus positive lifestyle interventions for 12 weeks reduced by 76% the prevalence of metabolic syndrome, whilst the lifestyle intervention only group had this parameter reduced by 36.4% (*p* = 0.013 for the difference between groups). Likewise, both groups reduced their calorie intake before versus after, but without differences between them (2423.04 ± 468.98 to 2198.76 ± 455.47 and 2410.26 ± 451.87 to 2079.89 ± 465.46 for flaxseed and control groups, respectively).

#### *4.2. Oils*

#### 4.2.1. Lipid Profile of Oils

Oils rich in ALA are a powerful tool to investigate the effects of ALA within a less complex matrix. Despite the presence of other fatty acids and fat-soluble compounds in the oil, the removal of fibre, vitamins, especially water-soluble ones, and water-soluble matter surely minimize the effects of confounding variables. Flaxseed, walnut, and rapeseed oils are, respectively, the main sources of ALA. Soybean oil is often considered a small to reasonable source of ALA, once research into its fatty acid composition has shown ALA concentrations ranging from 2.7% to 7.8% [73,74]. The n6-PUFAs content is nonetheless crucial when contemplating the n3 content of plant oils, as sunflower, corn, walnut, cottonseed, soybean, and peanut oils are important sources of n6-PUFAs (Table 2).


**Table 2.** ALA content and principal nutrition facts of selected edible oils.

Highest to lowest sources of ALA among common oils used for cooking. In addition, the main sources of LA can be noted, which are sunflower, corn, walnut, cottonseed, soybean, and peanut oils, respectively. Adapted from the United States Department of Agriculture (USDA) database [59].

#### 4.2.2. Flaxseed Oil

A RCT investigated the effects of either 25 mL/d flaxseed oil or 25 mL/d sunflower oil administered for seven weeks to 60 patients suffering with metabolic syndrome [75]. Serum IL-6 levels decreased significantly in both groups (9.37 to 7.90 pg/mL, *p* < 0.001 for flaxseed oil, and 9.22 to 8.48 pg/mL, *p* < 0.006 for sunflower oil), but the flaxseed oil group presented a greater reduction (*p* = 0.017). Given that that was a dosage considered high, it is worth mentioning that no side effects were reported in either group [75]. Interestingly, in a study recruiting 60 women with gestational diabetes [76], the daily supplementation for 6 weeks with 2 g/day flaxseed oil capsules, which contained 800 mg/day ALA, reduced the concentrations of TG (−40.5 mg/dL), TC (−22.7 mg/dL), insulin (−2.2 μIU/mL), and hs-CRP (−1.3 mg/L), as compared to a matched group that received sunflower oil capsules. The flaxseed oil-receiving group also showed upregulated LDL receptor, downregulated IL-1 and TNF-α gene expression, decreased malondialdehyde levels and increased total nitrite and total glutathione levels.

In a recent study [77] recruiting 59 overweight and obese adults with stage I hypertension without pharmacological treatment, 10 g of refined cold-pressed flaxseed oil (4.7 g ALA) for 12 weeks decreased fasting free fatty acid (−58 μmol/L) and TNF-α (−0.14 pg/mL) plasma concentrations. In contrast, no changes were found in other metabolic risk markers (e.g., serum glucose and TG levels) nor vascular function markers (e.g., brachial artery flow-mediated vasodilation, carotid-to-femoral pulse wave velocity, and retinal microvascular calibres) before versus after testing, both on fasting and postprandially. We consider this result to be extremely relevant for our critical discussion, as the ALA intake in that study was about three to five times higher than the recommended daily intake and, even so, it failed to improve cardiovascular markers. Furthermore, the volunteers of that study not only had obesity or overweight and stage I hypertension, their average age was 60 ± 8 years, a finding that is positively associated with vascular ageing, which by definition poses a greater risk for hypertension and atherosclerotic disease than the more traditional risk factors, including lipid and glucose levels, smoking and sedentary lifestyle [78].

The Omega-3 Index (O3I) reflects the relative percentage amount of EPA and DHA in erythrocyte membranes, and is considered a surrogate biomarker for cardiovascular events [79]. Cao et al. investigated in individuals with low baseline n3-PUFA levels the effects of supplementation with fish oil or flaxseed oil upon O3I [80]. Cao found that supplementing 2.1 g/day FO (1296 mg EPA + 864 mg DHA) for eight weeks increased the O3I from 4.3% to 7.8% (*p* < 0.001), followed by a gradual decline to 5.7% and to 3.8% at 4 and 16 weeks after the end of the supplementation period, respectively. On the other hand, supplementation with flaxseed oil (3510 mg ALA + 900 mg LA/d) for the same period, in turn, did not significantly change the O3I, but it did increase not only EPA but also n3-docosapentaenoic acid (DPA) [80], a fatty acid that sits in between EPA and DHA in the elongation desaturation pathway.

It can be argued that the study of Cao et al. [80] did not cover a period of supplementation that would allow maximum incorporation and saturation of supplemented PUFAs into erythrocyte membranes, therefore not raising the O3I to its maximum achievable level. A O3I higher than 8% has been proposed to be favourable against cardiovascular events, whilst ≤ 4% is interpreted as low [24,79,81]. Accordingly, the benefits of ALA as a mitigator of cardiometabolic events can be supported by its role as a substrate for EPA. The latter is a well-known element against pro-inflammatory pathways in the cardiovascular system.

#### 4.2.3. Soybean Oil

Despite having an obviously different fatty acid profile as compared to FO, soybean oil, unarguably, is a source of some ALA, and a few studies have already demonstrated a mild but relatively positive potential for soybean oil. For example, in treated hepatitis C patients (*n* = 52), 6000 mg/day FO or a soybean oil control for 12 weeks significantly decreased serum insulin levels (17.1 to 10.9 μIU/mL *p* = 0.001, 12.6 to 10.6 μIU/mL *p* = 0.011 for FO and soybean oil groups, respectively) and HOMA values (3.8 to 2.4 *p* = 0.002, 3.1 to 3.0 *p* = 0.046 for FO and soybean oil groups, respectively) when comparing baseline versus end-of-intervention. The FO group was clearly more efficient; however, differences between both groups (*p* = 0.016 for insulin levels and *p* = 0.015 for HOMA-IR values) have been observed [82].

In a small controlled clinical trial recruiting 16 hypercholesterolaemic women in a 10 weeks intervention, participants received an amount of soybean oil that consisted of 20% of their energy intake in a weight-maintaining diet with <300 mg/day of cholesterol [83]. The control group consisted of the same participants in a weight-maintaining diet with <300 mg/day of cholesterol for eight weeks but no soybean oil. As compared to the eight-week control period, TC, HDL-C, LDL-C and small dense low-density lipoprotein-cholesterol (sdLDL-C) levels were reduced at the end of the 10 week-soybean oil intervention period [83]. Furthermore, the sdLDL oxidation lag time was reduced after soybean oil consumption. In addition to the unfavourable cardiovascular conditions of maintaining low HDL-C levels, the former is linked to the pathophysiology of atherosclerotic disease due to the damage potential of sdLDL-C subclasses, especially in their oxidised form, on arterial structures [6].

#### 4.2.4. Rapeseed Oil

In a meta-analysis of 27 RCTs [84], consumption of rapeseed oil was associated with reductions of approximately 7.24 mg/dL in TC (95% CI, −12.1 to −2.7) and of approximately 6.4 mg/dL in LDL-C (95% CI, −10.8 to −2) serum levels, as compared to sunflower oil and saturated fat. No changes were observed in TG nor HDL-C. Overall, the daily dose of rapeseed oil ranged from 12 to 50 g for 21 to 180 days. Most trials in that meta-analysis addressed individuals with lipid disorders and patients with heart disease, type 2 diabetes mellitus, obesity, metabolic syndrome, or non-alcoholic fatty liver disease. Importantly, the trials were under isocaloric conditions and, thus, partially avoided the bias of weight loss and its relationship with amelioration of lipid indices [84]. A clinical trial study in well-controlled conditions, recruiting 10 healthy men aged 25.3 ± 1 years, found that 24 h lipid oxidation was more pronounced when the participants received rapeseed oil-enriched meals for the duration of the study, as compared to a matched meal enriched with palm oil, a source of saturated fat [85].

It is well known that replacing dietary saturated fats with monounsaturated fatty acids (MUFAs) and PUFAs can reduce overall mortality [86]. The United Kingdom Scientific Advisory Committee on Nutrition in 2019 concluded that the recommendation for the population average contribution of saturated fat to total calorie intake should remain at no more than 10% of total dietary intake, and that reducing intakes of saturated fats reduces the risk of cardiovascular and heart disease events [87].

The alleged cholesterol-lowering properties of rapeseed oil could be attributed not only to its ALA content, but also possibly combined with its MUFA composition. Approximately 56% of the total fatty acids in rapeseed oil are MUFAs, with oleic acid as the most abundant one, at 54.5% of the total fatty acids, approximately [88]. The abundance of oleic acid in rapeseed oil may support its beneficial properties, as it has been shown that oleic acid elicits improvement on lipids and lipoproteins, as well as reduced risk of cardiovascular disease in humans [89]. The ALA content in rapeseed oil, in turn, is estimated at approximately 6 to 10% of total fatty acids [59,90,91]. Interestingly however, a study found a much lower ALA content in rapeseed oil, ≈1.2% [92].

A critical interpretation of published studies will see that other plant sources of MUFAs and PUFAs may show positive results in comparison to rapeseed oil. For instance, the consumption of sesame oil was more favourable for glycaemic control markers when compared to rapeseed oil in a recent RCT recruiting individuals with type 2 diabetes mellitus [91]. In that study, rapeseed oil increased serum fasting blood glucose (+7.72 ± 3.15 mg/dL, *p* < 0.05), whilst sesame oil decreased serum insulin (−6.00 ± 1.72 mIU/mL, *p* < 0.05) levels in a nine-week intervention period. The dietary recommendation was based on 30–32% of the total energy intake from fats but, despite the predominance of oil intake in each intervention, we noticed that the authors did not present in their study the exact or estimated amount of oil consumed. Some of the strengths of the study include its design, a triple-blind, cross-over clinical trial with 92 subjects completing all treatment periods, which were composed of four-week run-in and four-week wash-out periods based on sunflower oil.

#### *4.3. Seaweed*

#### 4.3.1. Lipid Profile of Seaweed

The use of seaweed for cooking and as a food supplement is gaining more popularity worldwide [93,94]. As a functional food, seaweed is a vegetarian source of n3-PUFAs, protein, and micronutrients [95]. Spirulina and chlorella are commercially available biomass extracts of cyanobacteria and green algae respectively, designed to attend a growing demand [96]. Both spirulina and chlorella are often claimed to be valuable sources of n3-PUFAs; however, the accuracy of such statement needs to be clarified in context, as to obtain approximately 2 to 3 g of total lipids from these microalgae it is necessary to ingest approximately 28 g of it in its powdered form. The total lipid content can be practically zero in supplemental dosages (≈3 g/day) [59].

*Chlorella minutissima* UTEX 2219 and UTEX 2341 feature 3.3% and 31.3% EPA, respectively, of the total fatty acid content, but DHA was not detected in either strain [97]. In a fatty acid profile analysis of *Spirulina platensis* from seven commercially available products, EPA and DHA were detected in only two samples, contributing with 1.79% and 7.70%, and 2.28% and 2.88%, respectively, of the total fatty acids [98]. Similarly to flaxseed and nuts, whether the effects of dietary intervention with seaweed are attributed to its n3-PUFAs per se remain to be investigated, as the food matrix should be considered. *Spirulina* and *Chlorella* contain not only macro and micronutrients but also other compounds with antioxidant properties which may play a role in positive health outcomes [99–101].

#### 4.3.2. Clinical Findings

A meta-analysis published in 2016 [102] found that in general the daily consumption of 1 to 10 g *Spirulina* led to significant improvements in lipid profile by reducing TC in ≈47 mg/dL (95% CI: −67.31 to −26.22), LDL-C in ≈41 mg/dL (95% CI: −60.62 to −22.03), TG in ≈44 mg/dL (95% CI: −50.22 to −38.24), whilst increasing HDL-C in ≈6 mg/dL (95% CI: 2.37–9.76). Seven placebo-controlled clinical trials with duration of 2–4 months were included in that meta-analysis, and the population appraised consisted of patients with diabetes, cardiac diseases, nephrotic syndrome and HIV infection, illnesses whose pathophysiologies are related to dyslipidaemia [103–105]. Seemingly, an effective and practical dose was about 4 g/day of *Spirulina*, which can be administered in capsules or powder [102]. Nevertheless, as discussed above, we believe that the n3-PUFAs content in *Spirulina* was not the sole player in yielding such outcomes.

Supplementation with 300 mg/day of *Chlorella* for 8 weeks decreased TNF-α levels, in comparison with the placebo group, in a RCT of 70 patients with NAFLD [106]. An 8 week-long RCT investigating 44 women suffering with primary dysmenorrhea and supplemented with 1500 mg/day *Chlorella* found a significant reduction in hs-CRP levels (from 2590.00 ± 1801.66 to 974.21 ± 292.85 ng/mL), as compared to the control group [107].

In a Japanese sample population, 40 daily tablets of *Chlorella* were provided to 17 individuals with borderline high fasting blood glucose, TC, and TG levels, as well as to 17 healthy individuals [108]. After 16 weeks of supplementation, the researchers found reductions in body fat percentage, serum TC, and fasting blood glucose levels in both groups [108]. In our view however, that study appears to show a few limitations that should be considered in the context of a broader clinical scenario. The researchers did not measure food intake, there was no matched control group, and body fat percentage was obtained through bioelectrical impedance, which as a method of body composition analysis has some limitations [109]. Lastly, *Chlorella* supplementation was used in an impracticable dosage that, although it may be tested, cannot be translated into a broader clinical recommendation. As 40 tablets were ingested together with a considerable volume of fluid every day, probably this posology in itself with fluid may have resulted in lower food intake due to stomach filling.

#### **5. Population**

Type 2 diabetes, obesity, dyslipidaemia and hypertension, conditions that we have attempted to address in the present study, are related to a pro-inflammatory state [110–113]. The evidence so far available suggests that the consumption of ALA-rich foodstuffs may attenuate the levels of inflammation-associated biomarkers. More importantly however, the consumption of ALA food sources appears to be associated with reduced incidence of cardiovascular events. Further long-term RCTs are imperative to further elucidate such preliminary findings.

In the context of liver diseases, NAFLD particularly, lifestyle improvement is a cornerstone in ameliorating the disease. We have identified studies that showed beneficial effects of ALA interventions in patients with NAFLD [71,84]. Accordingly, a favourable nutritional support based on ALA food sources could be considered in therapies for NAFLD patients in cases of low frequency of fatty fish intake or absence of FO supplementation.

Food sources of ALA may also be relevant during pregnancy and lactation due not only to their rich nutritional composition but also due to a thoroughly justified need to avoid complex herbal supplement mixtures that may jeopardize the health of the mother and of the child. Additionally, plant sources of n3-PUFAs could be seen as an option for pregnant women who do not tolerate fatty fish and for those who suffer with nausea and with hyperemesis gravidarum, relatively common manifestations during the gestational period [114,115]. The amount and profile of n3-PUFAs ingested by the lactating mother is paramount for infant health, as the mother's diet directly reflects upon her milk fatty acid profile [116].

Exclusively vegan diets are to be examined carefully due to the risk of n3-PUFAs deficiency. Apart from a lower intake of total and saturated fats, another characteristic of exclusively vegan diets is a higher proportional intake of n6-PUFAs, when compared to omnivorous and vegetarian diets [110,117]. For those reasons, recommendations for vegan diets that include appropriate amounts of ALA, necessarily combined with a balanced n3/n6 ratio, are paramount for the maintenance of long-term health.

It is widely accepted that peanut and peanut butter have been speculated by the layperson and by some health practitioners as friendly components of the exclusively vegan diet. Careful consideration however should be exercised regarding the amounts of peanut and peanut butter consumed, as a pilot study observed in 14 exercise-trained healthy individuals with an average age of 30 years that the daily consumption of approximately 103 g of peanut butter for 4 weeks led to changes in body composition, markedly increased body fat content [118]. Such results only confirm that careful calorie counting is pivotal when adding a new source of lipids to the diet plan, regardless of whether those are considered healthy food items. Furthermore, peanut is particularly rich in LA and virtually absent of ALA (Table 2).

#### **6. Decision-Making Practice**

A diet poor in n3-PUFAs and rich in n6-PUFAs in the long term leads to inflammation and increased risk of diseases, including cardiovascular diseases [119]. If the patient does not comply with a diet that includes fatty fish or FO supplements, ALA-rich foods are important to at least partially supply some of the n3 requirement, at the same time reducing the LA bioavailability. Furthermore, some individuals in society may choose not to adhere to food supplementation, be it because of added costs, dietary choices such as veganism and vegetarianism, the wish to avoid polypharmacy if they are already taking medicine tablets, or due to personal values. Such lifestyle choices and circumstances further emphasize the need for a careful nutritional planning that includes sources of n3-PUFAs.

Along those lines, some period supplementing FO and followed by some period incrementing plant sources of n3-PUFAs may be an example of what would be a "periodized" loading of n3 status. Such hypothesis can be sustained due to the incorporation of n3-PUFAs into cell membranes, which occurs over a few months after supplementation, whilst the period appears to be longer for decreasing n3 status than the progress of storage. In the case of the study of Cao et al. [80] for example, two months of FO supplementation was sufficient to almost achieve a reasonable n3 state, which was maintained by one month afterwards but returning to the same baseline level after 16 weeks of interruption. We believe that a "periodized" FO supplementation regimen could be conceivable two to three times a year for one to four months, followed with nutritional counselling and observing the intake of foodstuffs rich in ALA in the period without the FO supplementation, hoping to provide a better n3-DPA level. Nutritional advice however will always be tailored to the individual's needs and preferences, such as frequency of consumption of fatty fish and food sources of ALA, patient's income, and any coexisting morbidity. As nutritional strategy for individuals who, for one reason or another, do not eat fish nor take FO supplementation, adding in ALA-rich foods, e.g., flaxseed and chia seeds, or adding their oils within a dietary plan, alongside a careful consideration to dietary sources of n6-PUFAs, in itself seems to be paramount for long term overall health.

#### **7. Perspectives**

In the present study we focused on the effects of ALA found naturally in foodstuffs, seeds, nuts and oils, instead of isolated ALA supplementation. Further clinical research is urgently required to broaden the current knowledge of the potential of ALA upon cardiometabolic dysregulations and cardiometabolic protection. Well-designed RCTs could certainly minimize the residual confounding variables caused by other nutrients.

The limitations of our study, as well as of other studies that have investigated ALA cardiometabolic effects specifically, are many. Ultimately however, although the current study provides some insights in a real-world prescription-based intervention, we also encourage the execution of meta-analyses to expand the current knowledge of specific nutrients in specific populations, in an attempt to find ideal dose-responses, as well as to continuously update guidelines in nutrition and cardiology.

#### **8. Conclusions**

In case of suspected insufficient n3 status, such as in individuals with low intake of fatty fish, those who do not take FO supplement, and in vegan individuals with very narrow dietary habits, alternative plant sources of n3-PUFAs may be candidates for partially attending the n3 metabolic demands. Although plant sources of n3-PUFAs are less impactful on EPA and DHA levels, evidence suggests that those are foodstuffs positively associated with favourable cardiometabolic outcomes, which could be triggered by other plant components, in synergy with ALA. Not only ALA in isolation, but its proposed effect in combination with other plant fatty acids and other plant components such as fibre, potassium, magnesium, and non-essential substances, e.g., polyphenols and sterols, may be the players in yielding benefits in cardiovascular metabolism.

Consumption of walnuts and flaxseed seems to be the main plant sources of n3-PUFAs with strong scientific basis for translation into clinical practice. Regarding oil intake, we believe that flaxseed oil is more advantageous than walnut oil, because the former's ALA content is five times greater than that of the latter, which in turn, can be considered the second principal source of ALA. Although several studies have alluded to rapeseed and soybean oils as ALA sources, their ALA amount is slight when compared to flaxseed oil, so that a usual oil serving must be considered in order not to exceed the daily energy requirement in an attempt to achieve an optimal level of ALA. Regarding seaweed, *Spirulina* and *Chlorella* have gained attention but there are no discernible studies corroborating a relevant amount of n3-PUFAs in usual doses of supplementation. Seemingly, the benefits of seaweed over cardiometabolic markers appear to be driven by their antioxidant content.

The introduction of ALA-rich foods is a cornerstone for individuals looking for n3 sources beyond fish and fish oil. It is nevertheless of greatest concern that the proposal to increase the consumption of ALA ought to be integrated with a controlled calorie intake and controlled n6-PUFAs intake, since both of them can be raised concomitantly, thus ensuing in untoward effects such as increased fat mass and cardiometabolic dysregulations.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6643/12/10/3159/s1, Table S1: Identification of RCTs that investigated the effects of nuts and seeds rich in ALA through the database search, Table S2: Identification of RCTs that investigated the effects of ALA-rich oils through the database search, Table S3: Identification of RCTs that investigated the effects of seaweed through the database search, Table S4: Identification of recent RCTs that investigated the effects of fish oil supplementation through the database search.

**Author Contributions:** Conceptualization, H.O.S.; methodology, H.O.S., J.C.P.; writing—original draft preparation, H.O.S.; funding acquisition, A.A.B.; supervision, A.A.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors are grateful to the University of Worcester for funding the publication fees.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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### *Article* **Association between Three Low-Carbohydrate Diet Scores and Lipid Metabolism among Chinese Adults**

#### **Li-Juan Tan, Seong-Ah Kim and Sangah Shin \***

Department of Food and Nutrition, Chung-Ang University, Gyeonggi-do 17546, Korea; tanlijuan88@cau.ac.kr (L.-J.T.); sakim8864@gmail.com (S.-A.K.)

**\*** Correspondence: ivory8320@cau.ac.kr; Tel.: +82-31-670-3259; Fax: +82-31-675-1381

Received: 23 April 2020; Accepted: 29 April 2020; Published: 3 May 2020

**Abstract:** This study investigated the blood lipid levels of 5921 Chinese adults aged >18 years using data from the China Health and Nutrition Survey 2009. Diet information was collected through 3 day, 24 h recalls by trained professionals. The low-carbohydrate diet (LCD) score was determined according to the percentage of energy obtained from carbohydrate, protein, and fat consumption. Dyslipidemia was defined when one or more of the following abnormal lipid levels were observed: high cholesterol levels, high triglyceride levels, and low high-density lipoprotein cholesterol levels. Multivariate adjusted odds ratios (ORs) and their 95% confidence intervals (95% CIs) were calculated using logistic regression models. After adjusting the confounding variables, in males, the OR of hypercholesterolemia was 1.87 (95% CI, 1.23–2.85; *p* for trend = 0.0017) and the OR of hypertriglyceridemia was 1.47 (95% CI, 1.04–2.06; *p* for trend = 0.0336), on comparing the highest and lowest quartiles of the LCD score. The animal-based LCD score showed a similar trend. The OR of hypercholesterolemia was 2.15 (95% CI, 1.41–3.29; *p* for trend = 0.0006) and the OR of hypertriglyceridemia was 1.51 (95% CI, 1.09–2.10; *p* for trend = 0.0156). However, there was no significant difference between plant-based LCD scores and dyslipidemia. In females, lipid profiles did not differ much among the quartiles of LCD scores—only the animal-based LCD score was statistically significant with hypercholesterolemia. The OR of hypercholesterolemia was 1.64 (95% CI, 1.06–2.55), on comparing the highest and lowest quartiles of the LCD score. In conclusion, a higher LCD score, indicating lower carbohydrate intake and higher fat intake, especially animal-based fat, was significantly associated with higher odds of hypercholesterolemia and hypertriglyceridemia in Chinese males. Future studies investigating the potential mechanisms by which macronutrient types and sex hormones affect lipid metabolism are required.

**Keywords:** LCD score; CHNS; dyslipidemia; dietary factor; plant based; animal based; Chinese adults

#### **1. Introduction**

With an increasing prevalence worldwide, dyslipidemia has become a public health problem. Dyslipidemia is a well-known important risk factor for cardiovascular disease (CVD) and metabolic syndrome [1]. In 2012, the mortality rate of chronic non-communicable diseases in China was 533 per 100,000, including 272 deaths caused by cardiovascular and cerebrovascular diseases [2]. In the past 30 years, with rapid economic development and improvement in the standard of living in China, the blood lipid levels in the Chinese population have gradually increased, and the prevalence of dyslipidemia has increased significantly. In 2012, the prevalence of hypercholesterolemia was 4.9%, which is 69% higher than in 2002 (2.9%); the overall prevalence of dyslipidemia in Chinese adults was 40.4% in 2012, which was significantly higher than that in 2002 (18.6%) [2]. This indicates that the health care burden of dyslipidemia and its related diseases has consistently increased in the past decade.

Dyslipidemia is a disease characterized by elevated total cholesterol (TC) and/or triglyceride (TG) levels or a low high-density lipoprotein cholesterol (HDL-C) level, which contributes to the development of coronary heart disease and stroke [1]. According to the data reported by the Chinese Center for Disease Control and Prevention in 2015, 26.4% of ischemic heart disease cases in China are attributed to hypercholesterolemia, which is one component of dyslipidemia. Secondary dyslipidemia accounts for most dyslipidemia cases and is associated with lifestyle factors or medical conditions that interfere with blood lipid levels over time. Among these influencing factors, obesity (specifically abdominal obesity), alcohol consumption, and high-fat diets (specifically, high saturated and trans-fatty acid intakes) are well-known risk factors of dyslipidemia, as confirmed by several previous studies [3–5].

Carbohydrates significantly contribute to the total energy intake, particularly in the Chinese population who use rice and wheat as their staple food. At present, several studies have assessed the effect of carbohydrate intake on dyslipidemia [6–10]. Moreover, most of these studies have been conducted in European and American populations, and a few Asian studies have been conducted in the Korean population [9,10]. A study assessing the effect of carbohydrate intake on dyslipidemia in the Chinese native population has not been conducted yet.

The concept of and calculations to determine the low-carbohydrate diet (LCD) score have been introduced in detail previously [9,11,12]. In summary, participants with the highest scores have the lowest carbohydrate intakes. In 1992, the percentage of energy intake from carbohydrates in urban and rural residents in China was 66.2%, which decreased to 55.0% in 2012 according to The Report on the Status of Nutrition and Chronic Diseases of Chinese residents (2015). Therefore, it is of epidemiological value to study the effect of carbohydrate intake on dyslipidemia in the Chinese population. The present study used the low-carbohydrate diet (LCD) score to examine the association between LCD and dyslipidemia and its components in Chinese adults. Further, we identified the association by source foods (plant based and animal based).

#### **2. Materials and Methods**

#### *2.1. Study Population*

Data were obtained from the China Health and Nutrition Survey (CHNS), which used a multistage, random cluster sampling process to select samples from 15 provinces in China [13]. The sample scheme is reported in detail elsewhere [13]. Data on fasting blood glucose levels were first collected in 2009 (N = 9549). In the CHNS, the information was collected by trained interviewers to assure compliance and quality of data. The CHNS dataset and the Institutional Review Board information are available on the official website of the CHNS (https://www.cpc.unc.edu/projects/china). Participants provided written informed consent for inclusion in this study.

This study used one round of survey data from 2009. Participants aged less than 18 years (*n* = 1160) were excluded. Subsequently, we excluded 54 participants with insufficient blood sample data (i.e., TC, HDL-C, low-density lipoprotein cholesterol [LDL-C], TG, and fasting plasma glucose levels) and 22 participants with implausible energy intake (<800 kcal/day or >6000 kcal/day for males; <600 kcal/day or >4000 kcal/day for females) [14]. Additionally, we excluded participants taking medications for hypertension, diabetes, and myocardial infarction and participants who reported a medical history of heart attack, stroke, or diabetes (*n* = 2392). Finally, a total of 5921 participants (2743 males and 3178 females) were included in this study. A flowchart of the selection process of the study population is shown in Figure 1.

**Figure 1.** Selection process for the study population in the 2009 China Health and Nutrition Survey (CHNS).

#### *2.2. Dietary Assessment and Calculation of the Low-Carbohydrate Diet Score*

The 3 day, 24 h dietary recalls were used to assess the dietary intake of each participant. The quantities and types of food and beverages consumed during the past 24 h were determined from the participants. The interviewers measured the participants' dietary intake with picture aids and food models during household interviews. The participants' energy and nutrient intakes were calculated using the China Food Composition Table (FCT) (FCT 2002 edition and FCT 2004 edition).

To evaluate LCD based on the method used by Halton et al. [11], we referred to the concept of the LCD score. According to the percentage of energy from carbohydrate, protein, and fat consumption, the participants were ranked separately and were subsequently divided into 11 groups. Using the assignment method by Halton et al., the highest score (LCD score = 30) group had the lowest carbohydrate intake and the highest fat and protein intakes, while the lowest score (LCD score = 0) group had the highest carbohydrate intake and the lowest fat and protein intakes. To facilitate the analysis, we further calculated the scores for animal-based food (the percentage of energy from carbohydrate, animal protein, and animal fat consumption) and plant-based food (the percentage of energy from carbohydrate, plant protein, and plant fat consumption) in a similar manner. The division of animal- and plant-based food is based on the FCT 2002 and FCT 2004 editions. Table 1 presents the percentages used to determine the LCD score, animal-based LCD score, and plant-based LCD score. According to the scores (from low to high), the participants were divided into four groups according to gender.


**Table 1.** Energy percent of macronutrients used in calculating the low-carbohydrate diet (LCD) scores, animal-based LCD scores, and plant-based LCD scoresadults,ChinaHealthandNutrition

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Simultaneously, based on the study by Kim et al. [9], when comparing the acceptable macronutrient distribution range (AMDR) for carbohydrates as recommended by the Chinese Dietary Reference Intakes (CDRIs) Handbook (2013), the participants in each quartile were divided into three groups according to the percentage of energy from carbohydrates: lower (<50%), standard (50–65%), and higher (>65%). Moreover, we adjusted the carbohydrate intake criteria according to previous research criteria [15,16]: low-carbohydrate diet (<40%), moderate-carbohydrate diet (40–65%), and high-carbohydrate diet (>65%). Finally, fat intake in each quartile was also determined in our study on comparing the AMDR using the fat intake criteria from the CDRIs Handbook: lower (<20%), standard (20–30%), and higher (>30%).

#### *2.3. Determination of Dyslipidemia*

Dyslipidemia is diagnosed when one or more of the following abnormal lipid levels are observed: high cholesterol levels (hypercholesterolemia), high triglyceride levels (hypertriglyceridemia), and low HDL-C levels based on the Guidelines for the Prevention and Treatment of Dyslipidemia in China (2007) [2]. Hypercholesterolemia is a disease characterized by increased TC levels in fasting serum (>240 mg/dL (6.22 mmol/L)) or increased LDL-C levels (>160 mg/dL (4.10 mmol/L). Hypertriglyceridemia is a disease characterized by increased TG levels in fasting serum (200 mg/dL (2.26 mmol/L)), and low HDL-C levels is a disease characterized by decreased HDL-C levels (<40 mg/dL (1.04 mmol/L)) [2].

#### *2.4. Assessment of Other Variables*

Body mass index (BMI) was obtained by dividing the body weight by the square of the height (kg/m2). Participants were divided into four groups based on their BMI values: underweight (BMI < 18.5 kg/m2), normal weight (≥18.5 kg/m<sup>2</sup> and <24 kg/m2), overweight (≥24 kg/m2 and <sup>&</sup>lt;28 kg/m2), and obese (BMI <sup>≥</sup> 28 kg/m2) according to the Guidelines for the Prevention and Control of Overweight and Obesity in Chinese Adults (2003). Sociodemographic variables such as age, gender, and income level and lifestyle variables such as alcohol consumption, smoking status, and physical activity level (PAL) were obtained using well-validated questionnaires distributed by trained interviewers. Information regarding alcohol consumption was divided into two groups according to frequency: "yes" (consumed alcohol more than once in the past year) or "no." Information regarding smoking status was divided into three groups: "current smoker" (still smoking), "past smoker" (previously smoking but currently not smoking), or "never smoker".

PAL included the following three domains: transportation activities, occupational activities, and sports. PAL was measured in terms of metabolic equivalent of task (MET) minutes in each week [17,18], which were converted using the time spent on each activity. The MET scores for each activity were based on the 2011 Compendium of Physical Activities. Physical activity was also divided into three categories: light PAL (<600 MET minutes/wk), moderate PAL (≥600 MET minutes/wk and <3000 MET minutes/wk), and vigorous PAL (≥3000 MET minutes/wk) [18,19].

The area of residence was categorized into "rural" and "urban". According to the results of the questionnaire, "urban neighborhood" (primary sampling units = 36) and "suburban neighborhoods" (primary sampling units = 36) were classified as "urban"; "towns" (primary sampling units) and "rural villages" (primary sampling units = 108) were classified as "rural". Other covariates that were defined in the model included age (years), educational level (less than primary school, middle school, technical school, college, or university or above), and ethnicity (Han, other ethnicities).

#### *2.5. Statistical Analyses*

Participants' general characteristics, such as age, income level, BMI, obesity, alcohol consumption, smoking status, PAL, and nutrient intake were analyzed using the generalized linear model and chi-square test for continuous and categorical variables, respectively, according to the quartiles of LCD scores based on gender. Moreover, all results for the continuous variables are presented

as the mean ± standard error, and the results for the categorical variables are presented as *n* (%). After adjusting for potential confounding variables (including age, educational level, body mass index, ethnicity, physical activity level, alcohol consumption, smoking status, and individual income), logistic regression models were used to calculate the odds ratios (ORs) of dyslipidemia and their 95% confidence intervals (95% CIs). All statistical analyses were performed using the Statistical Analysis System software version 9.4. A *p* value < 0.05 was considered statistically significant.

#### **3. Results**

Table 2 shows the general characteristics of the participants according to LCD score quartiles based on gender. The male and female LCD median scores both ranged from 5.00 in the lowest quartile (Q1) to 25.00 in the highest quartile (Q4). Moreover, participants with higher LCD scores had higher income levels, lived in urban areas, consumed alcohol, performed more physical activity, and had higher educational levels compared to participants in Q1 (all *p* < 0.05). Males in Q4 were more likely to have obesity (*p* < 0.05). Further, regarding lipid profiles, participants with higher LCD scores had higher levels of TC and LDL-C. Furthermore, males in Q4 had higher levels of TG than males in Q1 (all *p* < 0.05).

Macronutrient intake was assessed according to LCD score quartiles based on gender (Table 3). Carbohydrate intake was decreased and protein and fat intakes were increased according to the LCD score. Interestingly, intake of plant-based protein was decreasing according to the LCD score.

In Table 4, carbohydrate and fat intakes are presented according to the quartiles of the LCD score. Compared with the AMDR for carbohydrates as recommended by the CDRIs Handbook, only 34.85% of males and 36.25% of females consumed carbohydrates within the recommended level, whereas 58.95% of males and 58.34% of females consumed carbohydrates above the recommended level. None of the participants consumed carbohydrates below the recommended level in Q1. In total, 56.58% and 57.49% of males and females, respectively, had lower fat intake than the AMDR, and most of these participants were assigned to Q1 and Q2. According to previous research criteria, 39.7% of males and 40.59% of females consumed moderate-carbohydrate diets, whereas only 1.35% of males and 1.07% of females adhered to a LCD. All participants in Q1 consumed high-carbohydrate diets. Even in Q4, 94.88% of males and 95.87% of females consumed moderate-carbohydrate diets, whereas only 5.12% of males and 4.13% of females adhered to a LCD.

The multivariate adjusted ORs and their 95% CIs for the components of dyslipidemia according to the LCD score quartiles based on gender are summarized in Table 5. Male participants with higher LCD scores had a higher risk of hypercholesterolemia (OR, 1.87; 95% CI, 1.23–2.85) than those in Q1 (*p* for trend = 0.0017) after adjusting for age, educational level, BMI, ethnicity, PAL, alcohol consumption, smoking status, and income level. Meanwhile, a similar significant association between the LCD scores and hypertriglyceridemia was observed in males (OR, 1.47; 95% CI, 1.04–2.06) on comparison with participants in Q1 (*p* for trend = 0.0336) after adjusting for the mentioned confounding variables previously. However, in females, there were no significant associations between the ORs for dyslipidemia and LCD scores in comparison to the participants in Q1.

Table 6 illustrates the multivariate adjusted ORs and their 95% CIs for the components of dyslipidemia according to the animal- and plant-based LCD score quartiles based on gender. Comparing the fourth and the first animal-based LCD score quartiles, the multivariate ORs for hypercholesterolemia were 2.15 (95% CI, 1.41–3.29; *p* for trend = 0.0006) and the multivariate ORs for hypertriglyceridemia were 1.51 (95% CI, 1.09–2.10; *p* for trend = 0.0156) in males. In females, there was a borderline linear trend for hypercholesterolemia from the first quartile to the fourth quartile (*p* for trend = 0.0651). Regarding the plant-based LCD score, there was no significant linear trend for dyslipidemia from the first quartile to the fourth quartile both in males and females.



 1


valuesof 30; a low LCD score indicates weaker adherence, whereas a high score indicates greater adherence and higher protein and fat intake. 4 Classified by "rural" and "urban" according to the questionnaire. 5 Classified into "low", "middle", "high" according the file from National Development and Reform Commission. 6 Determined by body mass index: underweight (<18.5 kg/m2), normal weight (≥18.5 kg/m2 and < 24 kg/m2), overweight (≥24 kg/m2 and <28 kg/m2), and obesity (≥28 kg/m2) according to the Guidelines for the Prevention and Control of Overweight and Obesity in Chinese Adults (2006). 7 "Yes" indicated drank alcohol over the past year. 8 "Past" indicated that the person has smoked over their lifetime but has not smoked recently, and "current" indicated that the person still smokes. 9 "Heavy" indicated the metabolic equivalent of task (MET) hours per week ≥ 3000, "moderate" indicated MET ≥ 600 and <3000, and "light" indicated MET < 600 according to the 2011 Compendium of Physical Activities. 10 Divided into four groups: "less than primary school", "middle school", "technical school", and "college or university".







203

40–65% energy;

high-carbohydrate

 diet, >65% of energy.


**Table 5.** Multivariate adjusted odds ratios (ORs) for dyslipidemia according to the quartiles (Q) of the low-carbohydrate diet (LCD) scores of Chinese adults, China Health and Nutrition Survey. 1


using generalized Hypercholesterolemia mmol/L (240 mg/dL) fasting glucose test, low-density lipoprotein cholesterol level ≥ 4.14 mmol/L (160 mg/dL). 4 Adjusted for age, educational level, body mass index, ethnicity, physical activity level, alcohol consumption, smoking status, and individual income. 5 Hypertriglyceridemia was defined as a triglyceride level ≥ 2.26 mmol/L (200 mg/dL). 6 A low high-density lipoprotein cholesterol (HDL-C) level was defined as a HDL-C level < 1.04 mmol/L (40 mg/dL).

*Nutrients* **2020**, *12*, 1307



1 *p* adherence,whereas a high score indicates greater adherence and higher protein and fat intakes. 3 Hypercholesterolemia was defined as a total cholesterol level ≥ 6.22 mmol/L (240 mg/dL) on a fasting blood glucose test, low-density lipoprotein cholesterol level ≥ 4.14 mmol/L (160 mg/dL). 4 Adjusted for age, educational level, body mass index, ethnicity, physical activity level, alcohol consumption, smoking status, and individual income. 5 Hypertriglyceridemia was defined as a triglyceride level ≥ 2.26 mmol/L (200 mg/dL). 6 A low high-density lipoprotein cholesterol (HDL-C) level was defined as a HDL-C level<1.04 mmol/L (40 mg/dL).

#### **4. Discussion**

Using the CHNS 2009, the present study examined the impact of dietary carbohydrate intake on dyslipidemia and its components using the LCD scores. It was found that higher LCD scores were significantly associated with higher intake of legumes, nuts, fish, and other non-carbohydrate food sources, consistent with the results of an improved carbohydrate quality index from an original study [20] (Table A1). Furthermore, in the present study, it was also found that participants with higher LCD scores lived in urban areas, had high income, consumed alcohol, performed light physical activity, and had higher educational levels. Among these, the PAL and drinking status have been widely studied and confirmed as important factors on lipid metabolism [4,21,22]. Due to their inescapable impact, in the present study, we adjusted PAL and drinking status as covariables in the multivariate model to minimize their impact, improve the validity of research results and focus on the analyses of the impact of diet on lipid metabolism. In the follow-up study, we will consider PAL and drinking status as exposure factors in order to study their impact on lipid metabolism.

Furthermore, it was found that a higher LCD score was significantly associated with a higher risk of hypercholesterolemia and hypertriglyceridemia in males after adjusting for eight confounding variables. These observations are consistent with the observations noted in a series of studies comprising Chinese adults [1,14]. Furthermore, higher animal-based LCD scores were also associated with a higher risk of hypercholesterolemia and hypertriglyceridemia in males and with a borderline increase in TC levels in females, while the plant-based LCD score was statistically insignificant in males and females.

We grouped the participants according to the quartiles of the LCD scores and there was a clear dose–response relationship between dietary macronutrients and the components of dyslipidemia, specifically for carbohydrates. In our study, the higher the LCD score, the lower the carbohydrate intake, the higher the fat intake, and the higher the risk of hypercholesterolemia and hypertriglyceridemia in males.

A previous study [14] reported that males with the highest carbohydrate intake (>68.2% of energy) had a significantly lower TC level after adjusting for several confounding variables. This result was consistent with the result of the present study, in which participants with high LCD scores had a higher risk of hypercholesterolemia than participants with lower LCD scores.

The effect of the LCD score on lipid metabolism can be analyzed from the perspective of a low-carbohydrate and high-fat diet using our results in Table 4. According to Volek et al. [16], carbohydrates are considered as the main source of energy and glucose in the general diet, and they directly or indirectly regulate the distribution of excessive dietary nutrients through insulin to regulate lipolysis and lipoprotein assembly and processing. Thus, carbohydrates affect the association between dietary intake of saturated fat and circulating lipid levels.

Furthermore, the group with high LCD scores obtained a higher percentage of energy from fat, as shown in Table 4. Moreover, 41% and 37.08% of male and female participants in Q4 had a high-fat diet, respectively, resulting in an increase in the serum TC and LDL-C levels [23,24]. Hence, the higher the LCD score, the higher the odds of hypercholesterolemia.

Recent studies have focused not only on the quantities of fat and carbohydrate intake but also on the quality of fat and carbohydrates. In our study, participants with higher LCD scores consumed less cereals and tubers, which are the main sources of dietary carbohydrates. Nuts contain high levels of unsaturated fatty acids [25], and various animal products have different saturated, polyunsaturated, and trans-fatty acid levels [16,26]. Limiting the intake of certain forms of carbohydrates is the preferred dietary strategy for improving cardiovascular health. Dietary fiber, which is a form of carbohydrate, reduces the risk of atherosclerosis and CVD [27]. Several types of fatty acids and their intake also have different effects on lipid metabolism [28,29]. Moreover, according to Acosta-Navarro et al. [30], higher consumption of an animal-based diet leads to higher TC levels. Hence, animal-based LCD scores were associated with differences in TC levels according to gender in this study. In both males and females, higher animal-based LCD scores were significantly associated with a higher risk of hypercholesterolemia, while the plant-based LCD score showed no statistical significance. This result

is inconsistent with the result of a previous study [31], mainly because the previous study, which assessed plant-based diets, emphasized not eating meat or eating less meat. However, in our study, the plant-based LCD score refers to the percentages of protein and fat that the participants consumed from plant-sourced foods during the 3 day diet survey, and the participants themselves were not prohibited from eating meat (Table 1).

The difference between males and females in statistical significance according to our results is possibly attributed to the different body fat distributions and hormonal differences between males and females. In normal-weight individuals, significant differences are observed in lipid and fat distributions between males and females [32]. Serum TG levels are relatively lower and HDL-C levels are higher in females than in males (Table 2), due to the differences in gender hormones and body fat distribution [33,34]. Additionally, the response to a high-fat diet in terms of lipoproteins has a gender dimorphism [35]. These findings indicate that the effect of a high-fat diet on serum HDL-C levels is more significant in females than in males, as confirmed in our study, and a multivariate adjusted OR for HDL-C levels was also observed in our study (Table 5, OR, 0.48; 95% CI, 0.28–0.82).

This study based on the CHNS indicates that higher LCD scores result in a higher risk of hypercholesterolemia and hypertriglyceridemia in males, but we cannot exactly determine the amount and type of carbohydrate or fat intake using LCD scores. Therefore, the comprehensive effect of several macronutrients in the general diet on lipid metabolism should be further considered.

Although the data collection of CHNS 2009 is a little dated, the CHNS is a study that constitutes a wide age range and large sample size after adjusting for a comprehensive range of potential confounding variables. The analysis results of the representative datasets are reliable and can be extended to the general Chinese population. To the best of our knowledge, this is the first study to use LCD scores to study the risk of dyslipidemia among Chinese adults. Compared to a food frequency questionnaire, a 24 h recall is more accurate and has a lower estimation bias [13].

However, this study has several limitations. First, since this is a cross-sectional study, we were not able to determine the causal association between dietary macronutrients and the risk of dyslipidemia. Second, the dataset assessed dietary intake using 3 day, 24 h recalls, which may have a relatively limited correction for the internal differences among the participants and may be affected by seasons. Third, as we assessed confounding variables, such as PAL, using a questionnaire, bias may exist. Finally, although we adjusted for confounding variables, there are still unknown variables that can influence the results. Hence, larger-scale prospective studies are required to determine the association between LCD and dyslipidemia.

#### **5. Conclusions**

In conclusion, among Chinese males, the group with higher LCD scores has a higher fat intake (especially animal-based fat), a diet pattern that is closer to a LCD, and greater odds of hypercholesterolemia and hypertriglyceridemia. We advise that people should pay more attention to the proportion of macronutrients in their diet, especially animal-based fat. The association between LCD score and dyslipidemia is significantly complex. In the future, careful attention should be paid to the quality of macronutrients for preventing dyslipidemia in the Chinese population. Further prospective studies and those involving detailed dietary nutrient intake evaluations are required to verify these findings in Chinese and other populations.

**Author Contributions:** Conceptualization, L.-J.T.; methodology, L.-J.T.; software, L.-J.T.; formal analysis, L.-J.T.; data curation, L.-J.T.; writing–original draft preparation, L.-J.T.; writing–review and editing, S.S.; visualization, S.-A.K.; supervision, S.S.; project administration, S.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No.2020R1C1C1014286). MSIT: Ministry of Science and ICT.

**Acknowledgments:** This study uses data from the CHNS. We thank the National Institute for Nutrition and Health, China Center for Disease Control and Prevention, Carolina Population Center (P2C HD050924, T32 HD007168), the University of North Carolina at Chapel Hill, the NIH (R01-HD30880, DK056350, R24 HD050924, and R01-HD38700), and the NIH Fogarty International Center (D43 TW009077, D43 TW007709) for their financial support for CHNS data collection and analysis from 1989 to 2015 and future surveys. We also thank the China–Japan Friendship Hospital and the Ministry of Health for support for the CHNS 2009, the Chinese National Human Genome Center at Shanghai since 2009, and Beijing Municipal Center for Disease Prevention and Control since 2011.

**Conflicts of Interest:** The authors declare no conflict of interest.


### **Appendix A**

**Table A1.** Food intake and energy percent according to the LCD score based on gender, China Health and Nutrition Survey. 1


#### **References**


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