*Article* **Urinary Tartaric Acid, a Biomarker of Wine Intake, Correlates with Lower Total and LDL Cholesterol**

**Inés Domínguez-López 1,2, Isabella Parilli-Moser 1,2, Camila Arancibia-Riveros 1, Anna Tresserra-Rimbau 1,2, Miguel Angel Martínez-González 2,3,4, Carolina Ortega-Azorín 2,5, Jordi Salas-Salvadó 2,6,7, Olga Castañer 2,8, José Lapetra 2,9, Fernando Arós 2,10, Miquel Fiol 2,11, Lluis Serra-Majem 2,12, Xavier Pintó 2,13, Enrique Gómez-Gracia 2,14, Emilio Ros 2,15, Rosa M. Lamuela-Raventós 1,2,\* and Ramon Estruch 2,16,\***


**Abstract:** Postmenopausal women are at higher risk of developing cardiovascular diseases due to changes in lipid profile and body fat, among others. The aim of this study was to evaluate the association of urinary tartaric acid, a biomarker of wine consumption, with anthropometric (weight, waist circumference, body mass index (BMI), and waist-to-height ratio), blood pressure, and biochemical variables (blood glucose and lipid profile) that may be affected during the menopausal transition. This sub-study of the PREDIMED (Prevención con Dieta Mediterránea) trial included a sample of 230 women aged 60–80 years with high cardiovascular risk at baseline. Urine samples were diluted and filtered, and tartaric acid was analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Correlations between tartaric acid and the study variables were adjusted for age, education level, smoking status, physical activity, BMI, cholesterol-lowering, antihypertensive, and insulin treatment, total energy intake, and consumption of fruits, vegetables, and raisins. A strong association was observed between wine consumption and urinary tartaric acid (0.01 μg/mg (95% confidence interval (CI): 0.01, 0.01), *p*-value < 0.001). Total and low-density lipoprotein (LDL) cholesterol were inversely correlated with urinary

**Citation:** Domínguez-López, I.; Parilli-Moser, I.; Arancibia-Riveros, C.; Tresserra-Rimbau, A.; Martínez-González, M.A.; Ortega-Azorín, C.; Salas-Salvadó, J.; Castañer, O.; Lapetra, J.; Arós, F.; et al. Urinary Tartaric Acid, a Biomarker of Wine Intake, Correlates with Lower Total and LDL Cholesterol. *Nutrients* **2021**, *13*, 2883. https://doi.org/ 10.3390/nu13082883

Academic Editors: J. Mark Brown, Paolo Magni, Andrea Baragetti and Andrea Poli

Received: 1 July 2021 Accepted: 19 August 2021 Published: 22 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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 (https:// creativecommons.org/licenses/by/ 4.0/).

tartaric acid (−3.13 μg/mg (−5.54, −0.71), *p*-value = 0.016 and −3.03 μg/mg (−5.62, −0.42), *p*-value = 0.027, respectively), whereas other biochemical and anthropometric variables were unrelated. The results suggest that wine consumption may have a positive effect on cardiovascular health in postmenopausal women, underpinning its nutraceutical properties.

**Keywords:** PREDIMED; Mediterranean diet; lipid profile; cardiovascular risk; polyphenols; menopause; body fat; biomarkers; tartaric acid

#### **1. Introduction**

Cardiovascular disease (CVD) is the leading cause of death worldwide in both sexes. Nevertheless, important sex-specific differences exist. According to the American Heart Association, menopause is listed as a female-specific cardiovascular risk factor (CVRF) [1]. During the menopause transition women experience adverse changes in their lipid profile, body fat distribution, metabolic syndrome risk, and vascular health [2–5]. Previous studies suggested that menopause is associated with increased total and low-density lipoprotein (LDL) cholesterol [6] and changes in body composition such as increased fat mass and loss of lean mass [7]. Changes in blood pressure (BP), waist circumference (WC), body mass index (BMI), and blood glucose and insulin have not been specifically associated with menopause and appear to reflect chronological aging [5,6,8]. Therefore, menopauseinduced increases in cholesterol, body fat, and possibly other CVRFs may accelerate the risk of developing CVD.

Diet and lifestyle can also affect the incidence of CVD. Modifiable factors, such as smoking cessation, healthy diet, and regular physical activity, play a crucial role in reducing cardiovascular risk [9]. The Mediterranean diet (MedDiet) has been associated with a better control of several CVRFs [10] through improvements in BP, lipid profile, glucose metabolism, arrhythmia risk, and gut microbiome [11,12]. One of the main characteristics of the MedDiet is the abundant consumption of olive oil, vegetables, fruits, nuts, legumes, fish, and cereals, and moderate wine consumption [13,14]. Epidemiologic studies and randomized clinical trials reported that moderate consumption of wine (1 or 2 glasses/day) during meals has been consistently associated with a lower risk of CVD [15–17]. In the context of a MedDiet, moderate alcohol consumption appears to be synergistic with other cardioprotective components of the MedDiet that increase high-density lipoprotein (HDL) cholesterol, decrease platelet aggregation, promote antioxidant effects, and reduce inflammation [13].

Wine consumption is mainly determined through dietary questionnaires. A biomarker of wine intake reflects its consumption more reliably than a questionnaire, since people may not accurately report the amount of alcohol consumed due to perceived social rejection of excessive consumption [18]. Tartaric acid, the main organic acid in wine and the molecule responsible for wine acidity, is present in high amounts in wine (1.5–4.0 g/L) but is rare in most common foods [19,20]. Urinary tartaric acid has been considered as a sensitive, selective, and robust biomarker of moderate wine intake [21,22]. Therefore, determining tartaric acid stands out as a useful tool to further study the impact of moderate wine drinking on health. The aim of this study was to determine the association between urinary tartaric acid as a biomarker of wine consumption and CVRFs in postmenopausal women at risk of developing CVD.

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

#### *2.1. Study Design*

This study is a cross-sectional analysis of baseline data from a subsample of participants in the PREDIMED (PREvención con DIeta MEDiterránea) study, a large, parallelgroup, multicenter, randomized, controlled, 5-year clinical trial conducted between 2003 and 2009. The objective was to assess the effect of a Mediterranean diet supplemented with

extra-virgin olive oil or mixed nuts as compared to a low-fat diet on the primary prevention of CVD in 7447 participants at high cardiovascular risk. Eligible participants were men (55–80 years old) and women (60–80 years old) who had type 2 diabetes mellitus or at least 3 of the following major CVRFs: smoking, hypertension, elevated LDL cholesterol, low HDL cholesterol, overweight or obesity, and/or family history of premature coronary heart disease [23]. All participants provided written informed consent, and the study protocol and procedures complied the ethical standards of the Declaration of Helsinki.

For the present sub-study of the PREDIMED trial, urinary tartaric acid was analyzed in a subsample of women equivalent to 5% of the total female population of the PREDIMED study. The 230 women that were randomly selected had undergone the menopausal transition and their urine samples were available at baseline. Participants who had no available data of total energy intake or reported extreme values (>3500 kcal/day) were excluded from the analysis (*n* = 8).

#### *2.2. Anthropometric, Dietary, and Physical Activity Assessments*

Trained personal performed the anthropometric and clinical measurements (height, weight, WC, and BP). BMI was obtained by dividing the body weight in kilograms by the square of height in cm. The waist-to-height ratio (WtHR) was calculated by dividing the WC in centimeters by height in meters. Systolic (SBP) and diastolic blood pressure (DBP) were measured in triplicate with a validated semi-automatic oscillometer (Omron HEM-705CP, Lake Forest, IL, USA). A validated semi-quantitative food frequency questionnaire (FFQ), which included 137 food items [24], and the Minnesota Leisure-Time Physical Activity Questionnaire [25] were used to assess dietary habits over the previous 12 months and physical activity (metabolic equivalent tasks per minute per day, METs min/day) of the participants.

#### *2.3. Clinical Measurements*

Medical conditions, family history of disease, and risk factors were collected though a questionnaire during the first screening visit. Biological samples (plasma and urine) were collected at baseline after 12 h overnight fast and stored at −80 ◦C until assay. Blood glucose, total cholesterol, triglycerides, and HDL cholesterol were determined by standard enzymatic methods, and LDL cholesterol was calculated by the Friedewald equation [26].

#### *2.4. Tartaric Acid Determination*

#### 2.4.1. Reagents and Standards

Formic acid (approximately 98%), picric acid (98%, moistened with approximately 33% water), and sodium hydroxide (≥98%) were obtained from Panreac. L-(+)-Tartaric acid and creatinine were purchased from Sigma. The labelled internal standard DL-(±)-tartaric-2,3-d2 acid was obtained from C/D/N Isotopes. Solvents were high-performance liquid chromatography grade, and all other chemicals were analytical reagent grade. Ultrapure water was obtained from a Milli-Q Gradient water purification system (Millipore, Bedford, MA, USA).

Stock solutions of tartaric acid were prepared in water. Working standard solutions that ranged from 0.01 to 5 μg/mL were made by appropriate dilution in 0.5% formic acid in water and then stored in amber glass vials at −20 ◦C.

#### 2.4.2. Sample Preparation

Determination of urinary tartaric acid was performed following a previously validated stable-isotope dilution LC-ESI-MS/MS method by our research group [27]. Briefly, urine samples (20 μL) were diluted 1:50 (*v*:*v*) with 0.5% formic acid in water, and 10 μL of a deuterated isotope standard solution in water (DL-(±)-tartaric-2,3-d2 acid, 200 μg/mL) were added. The sample dilution was passed through a 0.20 μm filter and analyzed by LC–ESI-MS/MS. Urinary tartaric acid data were corrected by urine creatinine, measured according to the adapted Jaffé alkaline picrate method for thermo microtiter 96-well plates, according to Medina-Remón et al. [28]. Finally, urinary tartaric acid was expressed as μg of tartaric acid per mg of creatinine. According to previous data, the cut-off of 8.84 μg/mg creatinine was used to discriminate daily consumers and non-consumers of wine [21].

#### 2.4.3. LC–ESI-MS/MS Analysis

After filtration, tartaric acid was analyzed using an Atlantis TE C18, 100 mm × 2.1 mm, 3 μm (Waters, Milford, MA, USA) reversed-phase column coupled for detection to the triple quadrupole mass spectrometer API 3000 (Applied Biosystems, Foster City, CA, USA). The mass spectrometer was operated in negative electrospray ionization mode. The column was maintained at 25 ◦C throughout the analysis. Mobile phases A and B were 0.5% formic acid in water and 0.5% formic acid in acetonitrile, respectively. The following linear gradient was used: holding at 100%A for 3.5 min, decrease to 10%A over 2 min and holding for 2 min, return to initial conditions for 1.5 min, and re-equilibration for 6 min. The flow rate was set at 350 μL/min and the injection volume was 10 μL. Post-column addition of acetonitrile (250 μL/min) was carried out to improve analyte ionization efficiency. The detection was accomplished in multiple reaction monitoring (MRM) mode, and the following MS/MS transitions were used for quantification and confirmation, respectively: m/z 149/87 and m/z 149/73 for tartaric acid, and m/z 151/88 and m/z 151/74 for the deuterated isotope.

#### *2.5. Statistical Analyses*

The baseline characteristics of participants are presented as means and standard deviations (SD) for continuous variables, and frequency (*n*) and percentage (%) for categorical variables.

The normality of continuous variables was assessed with the Shapiro–Wilk test. The variables without normal distribution were transformed into logarithms. Multiple adjusted linear regression models were used to assess the differences between urinary tartaric acid and wine consumption as well as anthropometric and biochemical measurements. Three different adjustment models were applied. Model 1 was minimally adjusted for age (continuous). Model 2 was additionally adjusted for educational level, smoking status, BMI (except for anthropometric criteria), physical activity, and cholesterol-lowering, antihypertensive, and insulin treatment. Model 3 was further adjusted for total energy intake and consumption of fruits, vegetables, and raisins. We used robust variance estimators to account for the recruitment center in all linear models. To illustrate the relationship between wine consumption and urinary tartaric acid, the mL per month of wine reported in the FFQ were transformed into glasses of wine (with 1 glass equivalent to 100 mL).

Values are shown as 95% confidence interval (CI) and significance for all statistical tests was based on bilateral contrast set at *p* < 0.05. All the statistical analyses were performed using Stata statistical software package version 16.0 (StataCorp LP, College Station, TX, USA).

#### **3. Results**

#### *3.1. Study Population*

The main characteristics of the PREDIMED participants who were included in this sub study are summarized in Table 1. The mean age of the women was 66.9 + 0.4 years. Their burden of CVRFs was high: 42.1% had been diagnosed with type 2 diabetes, 87.3% with hypertension, and 76.5% with hypercholesterolemia. Among the drug treatments, statins were the most common medication, with 40.72% of them under treatment. Furthermore, 9.1% of the participants were current smokers. Finally, 82% of the participants had a low educational level.

Up to 45.7% of the participants reported wine consumption in the FFQ. The mean concentration of tartaric acid in urine was 28.34 μg/mg creatinine, and 40.4% were considered daily consumers of wine.


**Table 1.** Baseline characteristics of the women in the study population (*n* = 222).

ACE: angiotensin-converting enzyme; MET: metabolic equivalent task; BMI: body mass index; WC: waist circumference; WtHR: waist-to-height ratio; LDL: low-density lipoprotein cholesterol; HDL: high-density lipoprotein cholesterol. Data are expressed as the mean ± standard deviations (SD) for continuous variables and frequency (*n*) and percentage (%) for categorical variables.

The mean values of anthropometric measurements revealed that most participants were obese, as defined by their BMI, WC, and WtHR data [29] according to the International Diabetes Federation and the American Heart Association [30]. Regarding biochemical measurements, triglycerides and HDL cholesterol were at desirables levels, while total cholesterol, LDL cholesterol and glucose were borderline high [31,32].

The mean energy intake was 2161 kcal/day, of which carbohydrates accounted for 42.0% of the energy consumed, protein intake 16.8%, and fat intake 39.8%.

#### *3.2. Tartaric Acid as a Biomarker of Wine Consumption*

After adjustments for several covariates (age, education level, smoking status, physical activity, BMI, cholesterol-lowering, antihypertensive, and insulin treatment, total energy intake, and consumption of fruits, vegetables, and raisins), women who consumed more wine presented higher concentrations of tartaric acid in urine (0.01 μg/mg (95% CI: 0.01, 0.01), *p*-value < 0.001). Figure 1 illustrates the linear relationship between urinary tartaric acid concentrations and wine consumption expressed as glasses of wine, excluding those who reported not consuming wine.

**Figure 1.** Relationship between urinary tartaric acid concentrations in urine and wine consumption expressed as glasses of wine.

#### *3.3. Anthropometric Measurements and Urinary Tartaric Acid*

After adjustment for several covariates, we observed no association between urinary tartaric acid and BMI, WC, weight, WtHR, and systolic or diastolic BP (Table 2).


**Table 2.** Association between anthropometric variables and urinary tartaric acid (μg/mg creatinine).

BMI: body mass index; WC: waist circumference; WtHR: waist-to-height ratio; CI: confidence interval. Regression coefficients (95%CI) were obtained from multivariable adjusted linear regression models. β: Non-standardized coefficient. Model 1: adjusted for age. Model 2: adjusted for age, educational level, smoking status, physical activity, and cholesterol-lowering, antihypertensive, and insulin treatment. Model 3: adjusted for age, educational level, smoking status, physical activity, cholesterol-lowering, antihypertensive, and insulin treatment, total energy intake, and consumption of fruits, vegetables, and raisins. We used robust standard errors to account for recruitment center. *p*-values < 0.05 were considered significant.

#### *3.4. Biochemical and Clinical Measurements and Urinary Tartaric Acid*

A negative association was observed between urine tartaric acid and total and LDL cholesterol after full adjustment (−3.13 μg/mg (−5.54, −0.71), *p*-value = 0.016 and −3.03 μg/mg (−5.62, −0.42), *p*-value = 0.027, respectively). By contrast, no differences were observed for HDL with different concentrations of tartaric acid. Finally, no association was found between triglycerides and glucose and tartaric acid concentrations (Table 3).

**Table 3.** Association between biochemical variables and blood pressure and urinary tartaric acid (μg/mg creatinine).


BP: blood pressure; LDL: low-density lipoprotein cholesterol; HDL: high-density lipoprotein cholesterol; CI: confidence interval. Regression coefficients (95%CI) were obtained from multivariable adjusted linear regression models. β: Non-standardized coefficient. Model 1: adjusted for age. Model 2: adjusted for age, educational level, smoking status, physical activity, BMI, and cholesterol-lowering, antihypertensive, and insulin treatment. Model 3: adjusted for age, educational level, smoking status, physical activity, cholesterol-lowering, antihypertensive, and insulin treatment, total energy intake, and consumption of fruits, vegetables, and raisins. We used robust standard errors to account for recruitment center. *p*-values < 0.05 were considered significant.

#### **4. Discussion**

In this sub-analysis of a subset of postmenopausal women participating in the PRED-IMED trial, urinary tartaric acid concentrations as an objective biomarker of wine intake were significantly associated with lower concentrations of total and LDL cholesterol. No associations with anthropometric variables or blood pressure were observed. To the best of our knowledge, the current study is the first to evaluate wine consumption based on a biomarker in a postmenopausal population at increased risk of developing CVD.

Wine consumption has been widely studied due to its beneficial effects on cardiovascular and metabolic health [10]. However, most studies have evaluated wine intake using FFQs or self-questionnaires instead of biological biomarkers, a more reliable and objective way of assessing dietary habits [33]. It has been previously demonstrated that urinary tartaric acid is a specific and sensitive biomarker, as its major sources in the diet are grapes and wine [21,34]. Accordingly, we observed a positive association between wine consumption reported in the FFQs and the concentrations of tartaric acid present in urine. Other phenolic compounds, such as resveratrol and its metabolites, have been proposed as wine biomarkers. However, the resveratrol content in wine is subject to a high

variability and its metabolism shows interindividual differences [35]. Thus, selectivity and high correlation with reported intakes make tartaric acid a reliable dietary biomarker of wine consumption.

Different studies have evaluated how alcohol intake affects different parameters of body composition. A cross-sectional study in French adults suggested an inverse association in women of wine intake 100 g/day with BMI and WtHR [36]. Tresserra-Rimbau et al. analyzed the effects of red wine consumption on the prevalence of metabolic syndrome and its components, and found a negative association between moderate red wine consumption and BMI [16]. Tolstrup et al. also described inverse associations between alcohol consumption and WC in women [37], while other studies found no relationship between alcohol consumption and body weight in women [38,39]. The mentioned literature indicates that moderate consumption of wine, an alcoholic beverage that contributes to energy intake, is not related to weight gain or detrimental changes in body composition. Our study supports this notion, as we did not observe any differences in BMI, weight, WC, and WtHR with increasing wine consumption.

Evaluating the effect of alcohol, and specifically wine, on the risk of developing CVD in women is important due to the increase in cardiovascular risk after menopause. Among CVRFs, a recent metanalysis reported that triglycerides, total cholesterol, LDL cholesterol, and the total cholesterol-to-HDL-cholesterol ratio were significantly higher in postmenopausal women compared to premenopausal women, and suggested that age was partly responsible for the differences in lipid levels [40]. We found that women with higher concentrations of tartaric acid presented lower total and LDL cholesterol. Similarly to our results, Rifler et al. reported that after 2 weeks of drinking 250 mL of red wine daily, patients post myocardial infarction presented a 5% decrease in total and LDL cholesterol [41]. Furthermore, Taborsky et al. evaluated the effect of 1 year of wine consumption, and observed a reduction in total and LDL cholesterol [42]. In another clinical trial, authors reported a similar beneficial effect on the lipid profile after consumption of red wine in asymptomatic hypercholesterolemic individuals [43]. The data are almost consistent in showing that wine consumption reduces LDL cholesterol while increasing HDL cholesterol [44,45]. Moderate consumption of alcohol has been associated with higher concentrations of HDL cholesterol and diminished lipid oxidation stress [46]. Resveratrol metabolites in urine, as biomarkers of wine consumption, were significantly associated with lower triglycerides and higher HDL-cholesterol [47]. However, we were unable to confirm that higher urinary tartaric acid as a biomarker of wine consumption was associated with raised HDL cholesterol levels. A probable reason is that the women studied had rather high baseline HDL cholesterol levels, making it more difficult to further increase these with interventions.

Many clinical studies support that light to moderate alcohol consumption, in particular of red wine, is associated with lower CVD rates and an improved lipid profile and inflammatory system [17,48]. However, it remains unknown whether this effect of wine is due to alcohol per se, the phytochemicals of wine, their combined effect, or even the time of drinking, since postprandial oxidative stress after a meal appears to be counteracted by the ingestion of red wine [49]. In this sense, it has been found that wine micro-constituents modulate inflammatory mediators and therefore may be responsible for attenuating postprandial inflammation [50]. In addition, they protect against the effect of ethanol on cytokine secretion, which are involved in inflammatory processes [51]. In support of this view, a randomized clinical trial reported that wine bioactive compounds, such as resveratrol, can decrease total cholesterol by reducing mRNA expression of hepatic 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, in addition to the increased activation of the sirtuin system in all tissues [52]. Experimental work in cell cultures and animal models has shown that the enhancement of Sirtuin 1 can lead to better metabolic profiles and anti-inflammatory activities, as well as increased reverse cholesterol transport [53]. Overall, evidence supports that wine micro-constituents play a crucial role in the protective effect of wine on cardiovascular health by exerting anti-inflammatory actions.

The main strength of this study is that it used a biological biomarker, tartaric acid, to evaluate wine consumption, instead of less reliable methods such as FFQs or self-reported questionnaires. Moreover, it involved baseline data of participants in the PREDIMED trial; therefore, the results reflect real-life conditions. The main limitations were the modest sample size and the impossibility of determining causality due to the cross-sectional design.

#### **5. Conclusions**

The findings from the current cross-sectional study support the notion that wine intake has beneficial nutraceutical effects on the cardiovascular health of postmenopausal women, as its biomarker tartaric acid was associated with lower total and LDL cholesterol concentrations. Randomized trials are needed to confirm these results and determine the impact of wine consumption on cardiovascular health in a sensitive population such as that of postmenopausal women.

**Author Contributions:** Conceptualization, R.E.; Formal analysis and data curation, I.D.-L.; writing, I.D.-L., I.P.-M., and C.A.-R.; review and editing, A.T.-R., M.A.M.-G., C.O.-A., J.S.-S., O.C., J.L., F.A., M.F., L.S.-M., X.P., E.G.-G., E.R., R.M.L.-R., and R.E. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Interprofesional del vino by PID2020-114022RB-I00, CICYT [AGL2016- 75329-R], CIBEROBN from the Instituto de Salud Carlos III (ISCIII) from the Ministerio de Ciencia, Innovación y Universidades (AEI/FEDER, UE), and the Generalitat de Catalunya (GC) [2017SGR 196].

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and was approved by the Institutional Review Board of the 11 participating centers. The study was registered with the International Standard Randomized Controlled Trial Number (ISRCTN) 35739639.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study. Written informed consent was obtained from the patients.

**Data Availability Statement:** There are restrictions on the availability of data for the PREDIMED trial due to the signed consent agreements around data sharing, which only allow access to external researchers for studies following project purposes. Requestors wishing to access the PREDIMED-Plus trial data used in this study can make a request to the PREDIMED trial Steering Committee chair: restruch@clinic.cat. The request will then be passed to members of the PREDIMED Steering Committee for deliberation.

**Acknowledgments:** The PREDIMED trial was supported by the official funding agency for biomedical research of the Spanish government (Instituto de Salud Carlos III) through grants provided to research networks specifically developed for the trial: RTIC G03/140 (Coordinator: R.E.) and RTIC RD 06/0045 (Coordinator: M.A.M.-G.). All investigators of the PREDIMED trial belong to Centro de Investigación Biomédica en Red (CIBER), an initiative of Instituto de Salud Carlos III. This sub-study of the PREDIMED trial was specifically funded by the Interprofesional del Vino de España. I.D.-L. thanks the FI-AGAUR Research Fellowship Program, Generalitat de Catalunya [FI\_B 00256]. I.P.-M. is thankful for the FI-SDUR (EMC/2703/2019) fellowship from the Generalitat de Catalunya.

**Conflicts of Interest:** E.R. reports grants, personal fees, non-financial support, and other from the California Walnut Commission during the conduct of the study; grants, personal fees, non-financial support and other from Alexion; and personal fees and other from Amarin, outside the submitted work. R.M.L.-R. reports personal fees from Cerveceros de España, personal fees, and other from Adventia, Wine in Moderation, Ecoveritas S.A., outside the submitted work. R.E. reports grants from the Fundación Dieta Mediterránea (Spain), and Cerveza y Salud (Spain), and personal fees for given lectures from Brewers of Europe (Belgium), the Fundación Cerveza y Salud (Spain), Pernaud-Ricard (Mexico), Instituto Cervantes (Alburquerque, USA), Instituto Cervantes (Milan, Italy), Instituto Cervantes (Tokyo, Japan), Lilly Laboratories (Spain), and the Wine and Culinary International Forum (Spain), as well as non-financial support for the organization of a National Congress on Nutrition and feeding trials with products from Grand Fountain and Uriach Laboratories (Spain).

#### **Abbreviations**

Angiotensin-converting enzyme (ACE); blood pressure (BP); body mass index (BMI); confidence interval (CI); cardiovascular disease (CVD); cardiovascular risk factors (CVRFs); food frequency questionnaire (FFQ); high-density lipoprotein (HDL); liquid chromatography with electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS); low-density lipoprotein (LDL); Mediterranean diet (MedDiet); Prevención con Dieta Mediterránea (PREDIMED); standard deviation (SD); waist circumference (WC); waist to height ratio (WtHR).

#### **References**


### *Review* **Nuts: Natural Pleiotropic Nutraceuticals**

**Emilio Ros 1,2,\*, Annapoorna Singh <sup>3</sup> and James H. O'Keefe <sup>3</sup>**


**Abstract:** Common nuts (tree nuts and peanuts) are energy-dense foods that nature has gifted with a complex matrix of beneficial nutrients and bioactives, including monounsaturated and polyunsaturated fatty acids, high-quality protein, fiber, non-sodium minerals, tocopherols, phytosterols, and antioxidant phenolics. These nut components synergize to favorably influence metabolic and vascular physiology pathways, ameliorate cardiovascular risk factors and improve cardiovascular prognosis. There is increasing evidence that nuts positively impact myriad other health outcomes as well. Nut consumption is correlated with lower cancer incidence and cancer mortality, and decreased all-cause mortality. Favorable effects on cognitive function and depression have also been reported. Randomized controlled trials consistently show nuts have a cholesterol-lowering effect. Nut consumption also confers modest improvements on glycemic control, blood pressure (BP), endothelial function, and inflammation. Although nuts are energy-dense foods, they do not predispose to obesity, and in fact may even help in weight loss. Tree nuts and peanuts, but not peanut butter, generally produce similar positive effects on outcomes. First level evidence from the PREDIMED trial shows that, in the context of a Mediterranean diet, consumption of 30 g/d of nuts (walnuts, almonds, and hazelnuts) significantly lowered the risk of a composite endpoint of major adverse cardiovascular events (myocardial infarction, stroke, and death from cardiovascular disease) by ≈30% after intervention for 5 y. Impressively, the nut-supplemented diet reduced stroke risk by 45%. As they are rich in salutary bioactive compounds and beneficially impact various health outcomes, nuts can be considered natural pleiotropic nutraceuticals.

**Keywords:** tree nuts; peanuts; fatty acids; prospective studies; randomized clinical trials; cardiovascular risk; type-2 diabetes; cancer; hypertension; cognitive function; mortality; body weight; blood lipids; inflammation; PREDIMED

#### **1. Introduction**

Common tree nuts include almonds, Brazil nuts, cashews, hazelnuts, macadamias, pecans, pine nuts, pistachios and walnuts. Botanically, the peanut (*Arachis Hypogaea*) is a legume, but it has a nutrient profile that is similar to the tree nuts listed above, which qualifies peanuts to be included in the nut food group [1]. The impact of nut consumption on health outcomes has been extensively investigated since the publication in 1992 of the pioneering Adventist Health Study, in which nut consumption was associated for the first time with a lower risk of coronary heart disease (CHD) [2]. Soon after, a landmark randomized clinical trial (RCT) demonstrated that walnut consumption significantly lowered blood cholesterol [3].

Nuts are nutrient-rich foods that have been a staple of humankind's diet throughout our long evolutionary history [4]. However, during the last century, most people in industrialized nations have markedly reduced their consumption of nuts, so that now nuts

**Citation:** Ros, E.; Singh, A.; O'Keefe, J.H. Nuts: Natural Pleiotropic Nutraceuticals. *Nutrients* **2021**, *13*, 3269. https://doi.org/10.3390/ nu13093269

Academic Editors: Paolo Magni, Andrea Baragetti and Andrea Poli

Received: 19 August 2021 Accepted: 14 September 2021 Published: 19 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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 (https:// creativecommons.org/licenses/by/ 4.0/).

comprise only a marginal source of dietary energy, except for vegetarians, health-conscious groups such as Seventh Day Adventists, and individuals following diets based on whole natural foods [5]. Tellingly, in the last two decades nut consumption has increased in Western countries in parallel with the United States (US) Food and Drug Administration's issue of a health claim that nut consumption is associated with a reduced risk of CHD [6], inclusion of nuts in many guidelines for health promotion [7], and wide media advertising of their beneficial health effects.

The scientific evidence behind nuts as health-promoting foods stems from both abundant epidemiological observations suggesting that their regular consumption relates inversely to incidence of and mortality from major non-communicable diseases [8] and from RCTs disclosing a consistent cholesterol-lowering effect of nut diets [9]. The mechanisms for these salutary effects include the optimal nutrient composition of nuts, their satiating effects, and their tendency to displace other less healthy foods.

Contrary to the popular belief that, due to the high energy content of nuts, their consumption has a fattening effect, evidence from both epidemiological studies and RCTs suggests that their regular consumption does not lead to increased body weight and may even promote weight loss [10]. This review summarizes current knowledge on the increasingly important topic of nuts as health-promoting foods and their sizable contribution to the nutritional quality of the diet, while laying out the scientific basis to consider them as natural pleiotropic nutraceuticals.

#### **2. Data Sources and Selection of Studies on Nuts and Human Health**

For this narrative review we conducted a comprehensive search of the PubMed®/MED LINE® (https://www.ncbi.nlm.nih.gov/pubmed/ (accessed up to 31 July 2021)) database through July 2021 for English language articles of epidemiological and clinical studies illustrating the effects of exposure to nuts (tree nuts and peanuts) and their components (mainly peanut butter) on health outcomes, and the latest reviews and meta-analyses of these studies. Meta-analyses pooling data from nuts and seeds were excluded. We also searched the references from original research studies and reviews, as well as articles citing clinical studies, reviews, and meta-analyses, as listed by the publishers of individual articles in their websites. Given that the information of the various meta-analyses on the same outcome tends to be redundant, each successive one synthesizing the results of the same studies plus newly published ones, for each outcome, only the information from the most recent meta-analysis is discussed. However, older systematic reviews may be cited if they contain relevant information (i.e., dose–response analyses) not covered in the subsequent meta-analyses. For completeness, the data from well-designed cohort studies published after each specific meta-analysis are also reviewed.

Data were examined for relevance, quality, consistency and independently extracted by the two senior authors (ER, JHO), who reached an agreement when in doubt about a specific citation. Given that few RCTs on the effects of nut consumption on clinical end points are available, we obtain the core of scientific evidence from epidemiologic studies relating frequency of nut consumption to disease outcomes and RCTs of nut-enriched versus control diets for effects on intermediate end points, with particular attention to meta-analyses of such studies.

#### **3. Historical Aspects**

Archeological sites throughout the world have produced proof of consumption of hard-shelled nuts by ancient humans going back to the mid-Pleistocene, one million years ago. The oldest evidence of cultivation of the common tree nuts almonds (*Prunus amigdalis*), hazelnuts (*Corylus avellana*), walnuts (English walnuts, *Juglans regia*), and pistachios (*Pistachia vera*) is from Asia, spanning from China to the Middle-East and the Anatolian peninsula (modern Turkey). These trees were subsequently cultivated in Greece, then in the territories of the Roman Empire and the Iberian peninsula, and were extended to all of Europe during the Middle Ages. In the 16th century, in one of the first and boldest

food globalizations, European colonizers introduced these tree nuts to the Americas and brought those native to the Americas back to Europe [11]. In North America, there were native hazelnuts and walnuts—the so-called black walnut (*Juglans nigra*), as well as other indigenous nuts such as pecans (*Carya illinoinensis*), while cashews (*Anacardium occidentale*) and Brazil nuts (*Bertholettia excelsa*) are native to South America. Another popular tree nut, macadamias (*Macadamia integrifolia*), is native to Australia. The common pine nuts (*Pinus pinea*) are often obtained from natural forests, mostly in the Mediterranean region, but they are also native to China and North America.

Peanuts (also called groundnuts in some areas) were first cultivated from wild varieties by the ancient Incas from Peru. European explorers during the 16th century first discovered peanut plants, which were being cultivated in Brazil and Mexico, and transported peanuts back to Spain. From there, traders and explorers exported peanuts to Asia and Africa, and eventually to North America in the 1700s. Peanut butter was developed more than a century ago as a soft protein meal for people with poor dentition [12]. Today, peanuts and peanut butter are popular; Americans per capita eat about 6 pounds per year.

In Europe, nut supply is highest in Mediterranean countries [13]. Indeed, nuts are an integral component and a defining feature of the traditional Mediterranean diet, a dietary pattern characterized by high consumption of vegetables, fruits, nuts, olive oil, cereal grains; moderate consumption of fish and alcohol—mostly wine; and a low consumption of dairy products, red meat and meat products, and sweets [14]. Nuts can be are incorporated into the usual diet in different ways, as snacks, mixed in meals or desserts and may be eaten whole (fresh or roasted), in spreads (i.e., almond paste, peanut butter), as oils or hidden in commercial products, sauces, pastries, cakes, ice creams, and baked goods. Due to their high energy content and tasty nature, nuts have been widely introduced into sports snacks and supplements.

#### **4. Nutrient Content**

Nuts are nutrient dense foods, coming only after vegetable oils as the natural plant food richest in fat. Their total fat content as percent of weight ranges from 44% in cashews to 76% in macadamias, and they provide 23 to 30 kJ/g (Table 1) [15,16]. However, the fatty acid composition of nuts is salutary because they have a low saturated fatty acid (SFA) content (range, 4% to 16%) and nearly one-half of their total fat content is formed by unsaturated fat—specifically, monounsaturated fatty acids (MUFA) in most nuts, polyunsaturated fatty acids (PUFA) predominating over MUFA in pine nuts, similar amounts of MUFA and PUFA in Brazil nuts, and mostly PUFA in walnuts (Figure 1). Of note, with around 10 g per 100 g, walnuts are particularly rich in α-linolenic acid (ALA), the plant-derived essential omega-3 fatty acid [1]. The favorable lipid content of nuts is an important contributor to the beneficial health effects conferred by their frequent consumption.

Nuts are also contain other macronutrients and bioactives reputed as beneficial for health outcomes. They are a good source of vegetable protein (between 8% and 25% of energy) and are known to have a sizable content of the amino acid L-arginine, which is the substrate for the synthesis of endothelium-derived nitric oxide (NO), the main endogenous vasodilator and blood pressure (BP) regulator [17]. This explains in part why nut consumption helps improve endothelial function and may lower BP. Additionally, nuts are a good source of dietary fiber, ranging from 3 to 12.5 g per 100 g (Table 1) [15,16]. Indeed, a standard 1-oz (28-g) serving of nuts provides 5–10% of daily fiber requirements [1].

Among other nut components, there are several micronutrients that have salutary effects when taken in at doses beyond those necessary to prevent deficiency states. Nuts contain considerable amounts of the B-vitamin folate, peanuts being richest [15,18]. Almonds and hazelnuts are good sources of the antioxidant vitamins including tocopherols (e.g., vitamin E), while all nuts contain polyphenols, which are powerful antioxidants required to protect the germ from oxidative stress and preserve the reproductive potential of the seed [19]. Due to their protective characteristics, most polyphenols reside in the outer

peel of nuts (between the shell and the nut), a good reason to eat raw, unpeeled nuts when possible. Walnuts, pistachios, and pecans have the highest polyphenol content (Table 2).


**Table 1.** Average nutrient composition of tree nuts and oeanuts (per 100 g) [15,16].

Data for raw nuts, except when specified. ALA, α–linolenic acid; LA, linoleic acid; MUFA, monounsaturated fatty acids; PUFA; polyunsaturated fatty acids; SFA, saturated fatty acids.

**Figure 1.** Percent fatty acid profile of common nuts.


**Table 2.** Average composition of selected micronutrients in tree nuts and peanuts (per 100 g) [15,18].

Nuts are devoid of cholesterol, but their fat fraction contains chemically related non-cholesterol sterols, which belong to a heterogeneous group of molecules known as plant sterols or phytosterols. These compounds are non-nutritive plant components that play a structural role in their cell membranes just as cholesterol does in animal cell membranes [20]. Phytosterols interfere with cholesterol absorption, thus helping lower blood cholesterol concentration when present in doses of 1 g or higher in the intestinal lumen. The mechanism of action of phytosterols depends on their hydrophobic nature, as they have a large hydrocarbon molecule with a higher affinity for micelles than has cholesterol. As phytosterols displace cholesterol from micelles, the amount of sterol available for absorption is reduced. Pistachios and almonds are highest in phytosterols (Table 1). Not unexpectedly, evidence has been provided that phytosterols contribute to the cholesterol-lowering effect of nut consumption [21].

Nuts are also a rich source of beneficial minerals, such as calcium, magnesium, and potassium (Table 2). As in most plant foods, the sodium content of nuts is very low. Low sodium intake coupled with high intake of calcium, magnesium and potassium is associated with protection against hypertension, insulin resistance, and cardiovascular (CV) disease (CVD) [22], besides counteracting bone demineralization. Even lightly salted nuts retain a relatively low sodium content.

In summary, the macronutrients, micronutrients, and phytochemicals of nuts have all been documented to contribute to beneficial health outcomes, particularly a reduced risk of CVD and related metabolic alterations. As shown in Figure 2, bioactive nut components synergize to affect multiple metabolic and vascular physiology pathways leading to decreased cardiometabolic risk. For these reasons, whole unprocessed nuts may be considered as natural pleiotropic nutraceuticals. As such, daily consumption of nuts should be considered an essential feature of a health-promoting diet.

**Figure 2.** Schematic representation of the effects of nuts on risk of cardiometabolic diseases mediated by their main bioactive nutrients and phytochemicals (yellow boxes), which synergize to positively influence metabolic and vascular physiology pathways (thin arrows and orange boxes). The net effects on intermediate markers of CV risk are lowering of blood cholesterol, improved glycemic control, decreased blood pressure, improved vascular reactivity, and anti-inflammatory actions. Crucially, clinical trials of nuts have demonstrated all such effects. The overall result is reduced cardiometabolic risk (thick arrow connections), as observed in many prospective cohort studies and proven in the PREDIMED trial. Abbreviations: Ca, calcium; K, potassium; LDL-C, LDL-cholesterol; Mg, magnesium; NO, nitric oxide; TG, triglycerides. ↑: increase, ↓: decrease.

#### **5. Nut Consumption and Health Outcomes**

The bulk of evidence concerning the effects of nuts on health outcomes stems from prospective studies, the results of which have been summarized in numerous systematic reviews and meta-analyses conducted over the last two decades. Many RCTs have also been conducted examining the effects of nuts on intermediate risk factors, and corresponding meta-analyses have been published. As only one seminal RCT, the PREDIMED study [23], has assessed the effects of a long-term nut-enriched diet on hard CVD outcomes, it will be discussed separately from other RCTs.

#### *5.1. CVD Incidence and Mortality*

CVD, mainly CHD and stroke, are the leading causes of death globally. Most CVD could be avoided by addressing and modifying behavioral risk factors, such as incorporating healthy dietary habits [24]. In the last three decades, considerable evidence has accumulated on the effects of frequent nut consumption on CVD outcomes. The most recent systematic review and meta-analysis of 19 prospective cohort studies by Becerra-Tomás et al. [25] found an inverse association between total nut consumption (comparing highest vs. lowest categories) and CVD incidence (Relative Risk [RR] = 0.85; 95% Confidence Interval [CI], 0.80, 0.91; 3 studies), CVD mortality (RR = 0.77; 95%CI, 0.72, 0.82; 14 studies), CHD incidence (RR = 0.82; 95% CI, 0.69, 0.96; 7 studies), CHD mortality (RR = 0.76; 95% CI, 0.67, 0.86; 11 studies), and stroke mortality (RR = 0.83; 95% CI, 0.75, 0.93; 11 studies). No association was ascertained with incident stroke, either ischemic or hemorrhagic, in seven

and five studies, respectively. Regarding specific nut types, the reduced risk was noted with tree nuts and peanuts for most CVD outcomes, but not with peanut butter. However, concerning stroke mortality, reduced risk was found for high versus low consumption of peanuts (RR = 0.85; 95% CI, 0.79, 0.92), but not tree nuts. In dose–response analyses, total nut consumption and CVD outcomes showed non-linear inverse associations, with risk reductions up to a consumption of 5 g/day (stroke mortality), 10 g/day (CVD incidence), and 15–20 g/day (CVD and CHD mortality), namely, no further significant reductions were observed above these amounts. The findings of this meta-analysis concur with those of an earlier systematic review by Aune et al. [8].

In proof of the interest of the topic, data from additional large prospective cohort studies relating nut consumption to CVD outcomes, principally CVD mortality, have been released after that meta-analysis [25]. In an analysis of 16,217 men and women with diabetes from the prospective Nurses' Health Study (NHS)-I and -II and the Health Professionals Follow-Up Study (HPFS), highest versus lowest total nut consumption was associated with a lower risk of CVD and CHD incidence and all-cause mortality, with RRs similar to those ascertained in the mentioned meta-analysis, but not with stroke incidence and mortality [26]. In these cohorts, only tree nuts, not peanuts, were associated with reduced CVD outcomes. In a very large (*n* = 566,398) population-based prospective study in the US with a median follow-up of 15.5 y, data on cause-specific mortality confirmed the inverse association between higher total nut consumption and CVD deaths (Hazard Ratio [HR], 0.70; 95% CI, 0.66, 0.74), while no association for peanut butter consumption was found [27]. The Prospective Urban and Rural Epidemiology (PURE) study, conducted in 16 countries from 5 continents, examined nut consumption in relation to CVD outcomes in 124,329 participants followed for a median of 9.5 y [28]. Overall, CV mortality was lower (RR = 0.72, 95% CI, 0.56, 0.92) in high nut consumers, but no significant effects were detected for CHD or stroke. In the Women's Health Study (*n* = 39,167) with a mean followup of 19 y, higher versus lower nut consumption was associated with lower CVD mortality (HR = 0.73; 95% CI, 0.61, 0.87) [29]. In a recent Iranian population-based prospective cohort study comprised of 6504 participants, those in the highest quartile of nut consumption had a markedly decreased CVD risk (HR = 0.57, 95% CI, 0.47, 0.70) [30]. In a populationbased prospective study from Japan (*n* = 31,552), even though participants consumed very low amounts of nuts (1.6 g/d on average), mostly peanuts, higher versus lower peanut consumption was associated with reduced CVD mortality in women, while only trends towards inverse associations were found in men for peanuts and in both sexes for total nuts [31].

Another report from the large NHS and HPFS prospective cohorts relates nut consumption to CVD risk in a unique way by estimating risk associated with changes in nut consumption, either increases or decreases, during 4-year periods [32]. The researchers found that increasing consumption of total nuts, tree nuts, walnuts and peanuts, but not peanut butter, is associated with reduced risk of total CVD, CHD and stroke, while the converse (increase in risk of CVD and stroke) occurred when participants decreased nut consumption. This is one of few epidemiologic studies supporting a positive effect of nuts on stroke risk, though the PREDIMED RCT showed a marked reduction in stroke in those assigned to the Mediterranean diet with nuts arm [23].

Finally, two very large population surveys in Europe [33] and Latin America [34] analyzed the contribution of dietary factors to CVD mortality and found that one of the most important factors, accounting for the largest number of cardiometabolic deaths, was low nut and seed consumption.

Few prospective studies have examined the relationship of nut consumption with two additional CVD outcomes: atrial fibrillation and heart failure. The cited meta-analysis [25] synthetized data from two cohorts that examined the association of highest vs. lowest nut consumption categories with atrial fibrillation and found a RR of 0.85 (95% CI, 0.73, 0.99). On the other hand, no effect of nuts on heart failure (two studies) was observed [25].

In summary, consistent data from numerous large, well-conducted prospective studies and meta-analyses suggest that nuts are potent cardioprotective foods. The effect of higher nut consumption is strongest on CVD and CHD mortality, with reductions of 25–30%, followed by CVD and CHD incidence and stroke mortality (15–18% reduction), while effects on stroke incidence are less consistent. Total nuts, tree nuts and peanuts, but not peanut butter, generally share the same positive effects on CVD risk. These effects are likely ascribable to nuts' high content of healthy nutrients, such as PUFA, MUFA, non-sodium minerals, vitamins, and polyphenols and their potential to improve intermediate risk factors of CVD, as discussed in the corresponding section.

#### *5.2. Hypertension Incidence and Mortality*

The most recent review of epidemiological studies concerning total nut consumption in relation to cardiometabolic outcomes analyzes data from three meta-analyses of prospective studies with outcomes on incident hypertension [35]. An average 15% risk reduction, which was fairly constant in the three meta-analyses, was apparent when comparing high vs. low categories of total nut consumption. Based on data from four prospective studies with 11,962 incident hypertension cases, a 2017 dose–response meta-analysis (included in the review) estimated a 30% attenuation of hypertension risk for each daily serving (1-oz or 28 g) of nuts (RR = 0.70; 95% CI, 0.45, 1.08), with a linear dose–response [36]. No further prospective studies analyzing exposure to nuts in relation to hypertension risk have been published since that review.

In conclusion, in prospective studies nut consumption is associated with a consistent reduction of incident hypertension. As hypertension is the principal risk factor for stroke, this evidence clashes with the generally null epidemiological association of nut consumption with incident stroke, albeit, as discussed, increasing nut consumption is associated with lower stroke mortality. There is, however, sound RCT evidence that nut consumption lowers BP, as discussed in the sections on intermediate markers and health effects of nuts in the PREDIMED trial.

#### *5.3. Diabetes Incidence and Mortality*

The effects of nut consumption on risk of type-2 diabetes mellitus (T2D) in epidemiological studies have mostly been inconclusive and controversial [35,37]. A recent metaanalysis of nine studies (six prospective, three cross-sectional) published between 2002 and 2018 reported no association between extremes of consumption of total nuts, tree nuts or peanuts and the risk of T2D [38]. Walnuts, however, appeared to behave differently, as one large prospective study from the NHS and HPFS cohorts included in the meta-analysis found that walnut consumption related inversely to T2D risk (RR = 0.76; 95% CI, 0.62, 0.94) [39], while another large cross-sectional study found an even more beneficial effect of walnuts on T2D (RR = 0.47; 95% CI, 0.31, 0.71) [40].

Surprisingly, peanut butter, assessed in two early prospective studies, was inversely associated with T2D risk in the pooled estimate (RR = 0.87; 95% CI, 0.77, 0.98). Yet, only the results of one of the two analyzed studies favored peanut butter for T2D risk. Notably, risk estimates for total nuts changed substantially from nonsignificant to significant for lower T2D risk when time-updated measurement of body mass index (BMI) obtained during follow-up was excluded from the model (RR = 0.85; 95% CI, 0.75, 0.95), which supports body weight changes as a mediator of the reduction in T2D risk [38]. In fact, there is increasing evidence from prospective studies that regular nut consumption is associated with less long-term weight gain and a lower risk of obesity, while short-term RCTs confirm the lack of fattening effect of nuts [35]. Hence, given that long-term nut consumption is associated with a lower BMI, adjustment for BMI may conceal the true relationship between nuts and T2D, as also highlighted in the review of meta-analyses of nut studies by Kim et al. [35].

The main cause of death in T2D is CVD. Regarding nut consumption in relation to T2D mortality, the analysis of data from participants with T2D in the prospective cohorts

of the NHS-I, NHS-II and HPFS showed that highest vs. lowest total nut consumption was associated with a 25% lower reduced CVD mortality and a 27% lower all-cause mortality [26]. In regard to T2D mortality, these data concur with the results of the earlier meta-analysis of Aune et al. [8].

In summary, epidemiological evidence suggests that consumption of total nuts is associated with a reduced T2D risk, which is mediated by nut-associated favorable weight changes that obscure the relationship when BMI is entered as covariate in adjustment models. RCTs also indicate favorable effects of nuts on glycemic control, as discussed in the section of intermediate markers below. Among the tree nuts, to date only the walnut has been associated with lower T2D risk. The unique nutrient composition of walnuts, rich in ALA, highly bioactive polyphenols and melatonin [41], may explain their differential effects on health outcomes, including T2D. A note of caution is necessary when considering the positive effect of peanut butter on T2D risk, as it was only ascertained in one prospective study. Additional large prospective studies are warranted to further elucidate the effects of nuts on T2D.

#### *5.4. Cancer Incidence and Mortality*

Cancer is a major cause of death and constitutes a huge public health hazard worldwide, as the global burden of cancer is expected to increase to 29.5 million new cancer cases and 16.4 million cancer-related deaths by 2040 [42]. At least 40% of cancers could be prevented by addressing modifiable risk factors such as tobacco use, dietary carcinogens, sedentary lifestyle, obesity and infectious agents [43]. In particular, adherence to a wholesome eating pattern such as a traditional Mediterranean diet has been shown to reduce the risk of some cancers by 4% to 57% [44]. There is substantial epidemiological evidence suggesting that consumption of nuts, a staple in the Mediterranean diet, is associated with reduced risks for cancer development and cancer-related deaths.

Based on the pooled results of eight prospective studies, the meta-analysis by Aune et al. showed that highest vs. lowest nut consumption was associated with a significant 15% reduction of total cancer incidence [8]. The most recent and comprehensive meta-analysis by Naghshi et al. comprised 51 epidemiological studies and reported that the summary effect size for risk of cancer, comparing extreme categories of total nut consumption, was similar to that described by Aune et al. [8], with an RR of 0.86 (95% CI, 0.81, 0.92) [45]. In the dose–response analysis, each 5-g/d increase of total nut consumption was associated with 3%, 6%, and 25% lower risks of overall, pancreatic, and colon cancers, respectively. Of note, this inverse dose–response relationship between nuts and incident cancer was not significant for peanuts and peanut butter consumption [45]. This could be due to different nutrient composition in peanuts vs. tree nuts [1]. Peanut butter, though comprised predominantly of ground peanuts that have been roasted, generally also contains additives such as sugar, salt and hydrogenated oils that may hamper its health benefits [46], as shown for CVD. Moreover, when peanuts or other nuts are improperly stored, they can be contaminated with aflatoxins, which are potent carcinogens produced by certain molds.

Nuts may also mitigate the increased breast cancer risk associated with alcohol use, as noted in a cohort study that showed reduced risk of benign breast disease, a precursor for breast cancer, with nut consumption, especially among individuals with substantial alcohol use [47].

The Naghshi et al. meta-analysis [45] also evaluated the effects of nuts on deaths due to malignancy and found statistically significant 18%, 8%, and 13% risk reductions in the risk of cancer mortality with the higher intake of tree nuts, peanuts, and total nuts, respectively. Like for the case of incident cancer, no significant association between peanut butter consumption and cancer mortality was ascertained [45]. Many other studies have evaluated the relationship between nut consumption and risk of cancer mortality. An earlier meta-analysis by Zhang et al. in 2020 showed that total nut consumption was associated with a reduced odds ratio (OR) of cancer-related mortality (OR = 0.90; 95% CI, 0.88, 0.92) [48]. In this meta-analysis, statistically significant inverse associations

between nut consumption and cancer site occurrence were present for colorectum, stomach, pancreas, and lung.

In summary, a consistent body of evidence, albeit based exclusively on observational studies, suggests that consumption of nuts may modestly reduce cancer incidence and cancer-related deaths. These benefits appear to be most significant for tree nuts, less so for peanuts, and nonexistent for peanut butter. More large prospective studies and RCTs are needed to clarify this important issue.

#### *5.5. Brain Health*

An unwanted consequence of increased lifespan and associated population aging in recent decades is a growing number of elderly individuals at risk of neurodegenerative disorders, particularly Alzheimer's disease—the most common type of dementia. Given that no effective disease-modifying pharmacologic treatments for mild cognitive impairment, a common harbinger of dementia, or dementia itself are available [49], there is an increasing interest in preventive strategies to implement in preclinical and early stages. Among them, lifestyle modifications, including dietary changes, are being actively investigated and there is incipient evidence that they may forestall cognitive decline and even prevent dementia, particularly in individuals at higher risk [50,51]. Brain oxidative stress and inflammation are currently considered to be causal factors leading to age-related neurodegeneration, a reason why dietary patterns and foods with anti-inflammatory properties are the most promising for improving brain health [52]. Additionally, a close link exists between Alzheimer's disease and vascular pathology, and there is evidence that treatment of CV risk factors contributes to maintain neuronal integrity and prevent cognitive dysfunction [53].

Nuts are rich in neuroprotective nutrients such as PUFAs and polyphenols and their increased consumption benefits vascular function and is consistently associated with reduced rates of CVD, therefore it can be predicted that they might also beneficially influence cognition and overall brain health. Although data from cohort studies relating nut consumption to dementia outcomes are lacking, evidence is accumulating on the potential of nuts to improve cognitive function. A recent systematic review synthetized data from 14 epidemiological studies and eight RCTs assessing effects of nut-enriched diets on cognitive function [54]. While some epidemiological studies showed a positive association, the quality of the evidence was low because nine studies were cross-sectional or case–control and only five were prospective. Nevertheless, studies targeting populations at higher risk of cognitive decline tended to have favorable outcomes. Notably, studies that specifically addressed the association between walnut consumption and cognitive performance had more homogeneous results, as out of six walnut studies, including two RCTs, only one failed to find a positive association. This may be due to the highly bioactive nutrients of walnuts previously mentioned in reference to T2D risk [41]. Indeed, many studies using walnuts in experimental models of brain aging and neurodegeneration have consistently uncovered beneficial effects [55]. A large population-based prospective study published after that systematic review, the Singapore Chinese Health Study, supports the cognitive benefit of nuts, as nut consumption at midlife was associated with a dosedependent reduction in risk of cognitive impairment 20 years later [56]. Interestingly, unsaturated fatty acid intake mediated close to 50% of the beneficial effect, pointing to the fatty acid composition of nuts as relevant in improving cognition.

Late-life depression is a common psychiatric disorder that compromises the quality of life of affected individuals. Depression is also a risk factor for cognitive decline, where chronic inflammation contributes to its pathophysiology, as is the case with neurodegenerative disorders [57]. For a similar reason, depression is also a risk factor for CHD, although the association is bidirectional [58]. Consequently, nuts can be postulated to have a salutary effect on depression. The epidemiologic evidence, however, is scanty and of suboptimal quality. Nut consumption was reported to benefit depressive symptoms in a large crosssectional study of Chinese adults [59]. In another cross-sectional report from the US, nut

consumers, and particularly walnut consumers, disclosed lower depression scores than subjects who were not consuming nuts, and this beneficial effect was more pronounced in women [60]. In that study, food consumption was assessed only via 24 h diet recalls, which can provide strong evidence for frequently consumed foods but, unless repeated, are much weaker for sporadically consumed foods such as nuts. In the Invecchiare in Chianti study, an Italian prospective investigation of 1058 adults followed for up to 9 years with repeated measurements of diet and depression scores, no association between consumption of nuts and depressive symptoms was observed [61].

Brain-beneficial nutrients contained in nuts, such as PUFAs and polyphenols, support their potential to delay cognitive decline, as evidenced in a few prospective studies and RCTs. Furthermore, other nut components such as phytomelatonin, phytosterols, antioxidant tocopherols, and folic acid may also support neurological health and cognitive wellness. Clearly, more well-designed prospective studies and RCTs, preferably conducted in individuals at high risk or with early dementia stages, are warranted to uncover the full potential of nuts to counteract cognitive decline.

#### *5.6. All-Cause Mortality*

While the major focus of epidemiological research with nuts has been CVD, many large population-based prospective cohort studies conducted globally have examined associations of exposure to nuts or nut components such as peanut butter with all-cause mortality. The latest meta-analysis by Chen et al. [62] synthetized data from 18 prospective studies and obtained a summary RR for high compared with low nut consumption of 0.81 (95% CI, 0.78, 0.84) for all-cause mortality. When data for total nuts and tree nuts and peanuts were analyzed separately, the RR estimates were similar for the three nut categories. Only two studies examined peanut butter separately from peanuts and their combined RR for all-cause mortality was 0.89 (95% CI, 0.80, 0.99). In dose–response analysis, the RR for all-cause mortality per one additional serving of total nuts per week was 0.96 (95% CI, 0.94, 0.97). Interestingly, dose–response analyses revealed nonlinear inverse associations between nut consumption and mortality, with risk reduction leveling off at consumption of approximately 3 servings/week (equivalent to 12 g/d), which suggests that maximum benefit on survival may be achieved with relatively low doses of nuts.

That low nut doses relate to lower overall mortality is underlined in a recent report from a large (*n* = 31,552) population-based prospective Japanese study, whereby higher compared with lower nut consumption was associated with reduced all-cause mortality (HR = 0.85, 95% CI, 0.75, 0.96) in men (not in women), in spite of an average consumption of only 1.8 g/d, peanuts accounting for 80% of total nuts [31]. Additionally, in spite of a similarly low average nut consumption in Korea, a recent large cross-sectional population survey relating dietary factors to all-cause and cause-specific mortality using a comparative risk assessment analysis found that a sizable proportion of deaths was related to low consumption of nuts [63]. The results of a recent very large (*n* = 566,398) population-based prospective study in the US with a median follow-up of 15.5 years support the inverse association between higher nut consumption and total mortality (HR = 0.78; 95% CI, 0.76, 0.81); in contrast consumption of peanut butter was not associated with lower risk of mortality [27]. Finally, the recent report from the PURE study described a significant reduction in total mortality (HR = 0.77; 95% CI, 0.69, 0.87) for highest (≥120 g/week) versus lowest (<30 g/month) nut consumption [28]. In PURE, tree nut consumption was associated with a decreased risk of mortality, whereas peanut consumption disclosed a nonsignificant trend towards a lower mortality risk.

Nut consumption in relation to mortality has also been examined in a large prospective cohort of individuals with T2D. In the NHS and HPFS report on nuts and mortality among 16,217 men and women with diabetes, higher vs. lower nut consumption was associated with a significant 31% reduction in all-cause mortality [26]. When assessed separately, consumption of tree nuts and peanuts related to 33% and 20% lower mortality risks, respectively. Overall, the findings from prospective studies consistently point to an inverse

association of nut consumption with all-cause mortality, with an average of 1 in 5 deaths prevented or delayed by nut consumption at moderate levels.

#### *5.7. Intermediate Markers: Adiposity, Lipids, Blood Pressure, Glycemic Control, Endothelial Function, and Inflammation*

#### 5.7.1. Adiposity

The steady increase in the prevalence of overweight/obesity worldwide is a major public health problem. Due to the high energy density of nuts, increased body weight with long-term consumption has been an underlying concern. Yet, to the contrary, a growing body of epidemiological evidence suggests that daily nut consumption is a potentially effective strategy in the primary prevention of obesity [64].

A 2014 review of epidemiological and RCT data concluded that evidence was lacking on the common assertion that regular consumption of nuts increased adiposity [10]. This was confirmed in a recent network meta-analysis of 105 RCTs comparing the effects of diets enriched in various tree nuts and peanuts vs. control diets on body weight, BMI, waist circumference (WC), and percent body fat [64]. No significant increase was observed in any adiposity measures with any of the nuts, except for hazelnut-rich diets, which raised WC. On the other hand, results of pairwise comparisons between different nuts indicated that almond diets reduced WC compared to control diets; walnuts also reduced WC compared to pistachio, hazelnut and mixed nuts-enriched diets. In subgroup analyses considering only RCTs specifically designed to assess the weight loss effects of nut consumption, almonds were associated with reduced BMI and walnuts with reduced percent body fat. Importantly, among overweight and obese study subjects, those who consumed nutenriched diets experienced greater weight loss, reduced BMI and lower WC compared with their counterparts who consumed a nut-free isocaloric control diet (Figure 3).

**Figure 3.** Percentage change for adiposity outcomes in the healthy/normal weight groups vs. the overweight/obesity group with regard to the length of time following the nut interventions in 105 RCTs. Reproduced from reference [64], with permission. BW: body weight; BMI: body mass index; WC: waist circumference; % BF: body fat percentage.

Another recent meta-analysis of nut-feeding trials examined whether providing or not dietary substitution instructions to participants (recommending foods to be replaced by the nuts or just advising to eat the nuts on top of the usual diet) influenced adiposity changes [65]. The results showed the same absence of weight, BMI or WC changes for the two categories of studies.

A very large prospective study involving the three Harvard cohorts of the NHS-I, NHS-II, and HPFS assessed the association between changes in consumption of total and specific nuts per 4 y intervals and weight changes over 20–24 y of follow-up [66]. Increases in nut consumption, per 0.5 servings/d (14 g), were significantly associated with less weight gain per 4 y interval: −0.19 kg (95% CI, −0.21, −0.17) for total nuts, −0.37 kg (95% CI. −0.45, −0.30) for walnuts, −0.36 kg (95% CI, −0.40, −0.31) for other tree nuts, and −0.15 kg (95% CI, −0.19, −0.11) for peanuts. An increase in consumption of total nuts, per 0.5 servings/d, was associated with a modest but significant 3% lower risk of becoming obese, while a similar increase in consumption of walnuts and other tree nuts was associated with a 15% and 11% lower risk of developing obesity, respectively. Increasing peanut consumption, however, was not associated with reduced obesity risk.

Thus, both epidemiological and RCT data point to a slightly beneficial effect of nut consumption on adiposity rather than a harmful effect. That regularly eating a highly energy-dense food does not promote a positive energy balance is of particular interest. Several mechanisms underly the associations between nut consumption and lower risk of weight gain [10]. Nuts require considerable effort at mastication and chewing, and their high fat and fiber content can delay gastric emptying, increase satiety, suppress hunger and promote fullness. The fiber in nuts also increases binding of fatty acids in the gut, leading to greater fecal fat excretion. Similarly, the efficiency of energy absorption from nuts is reduced due to incomplete mastication and encasement of fat within unbroken cell walls in nut particles, hampering the bioaccessibility of fat from nuts in the gastrointestinal tract, with ensuing increases in fecal energy (fat) loss. Finally, there is evidence that the high unsaturated fat levels in nuts enhance fatty acid oxidation and increase thermogenesis and resting energy expenditure, which may also mitigate weight gain.

#### 5.7.2. Blood Lipids

Since the landmark RCT of Sabaté et al. demonstrating the cholesterol-lowering effect of a walnut diet [3], the effects of diets enriched with different nuts on blood lipids and lipoproteins have been examined in many RCTs [35].

To date, the 2015 meta-analysis of Del Gobbo et al. [9] is the most comprehensive. It reviewed 61 intervention trials (42 randomized and 19 non-randomized) lasting from 3 to 26 weeks designed to assess the effects of tree nuts on the blood lipid profile. All trials provided the study nuts to participants rather than simply giving advice to procure the nuts by themselves. Nut consumption (per serving/d) significantly decreased total cholesterol (−4.7 mg/dL), LDL-cholesterol (−4.8 mg/dL), and triglycerides (−2.2 mg/dL), but had no effect on HDL-cholesterol. Walnuts, followed by almonds and pistachios, were the nuts most frequently studied. The LDL-cholesterol lowering effect was dose related in a non-linear fashion, with stronger effects at doses of 60 g/d (approximately 2 servings), while triglyceride lowering had a linear dose–response. There was no heterogeneity by nut type or quality of the control diet. These authors reanalyzed the data as a function of the phytosterol content of nuts in each study and demonstrated that the phytosterol dose was strongly related to the observed LDL-cholesterol reduction, although this association was driven by the total nut dose [21].

An earlier analysis with pooled individual data from 21 RCTs indicated that, for an average consumption of 67 g/d of tree nuts or peanuts (two servings, approximately 20% of energy), the mean estimated reduction of LDL-cholesterol was 10 mg/dL (7%) [67]. Nuts had no significant effect on serum triglycerides, except in participants with triglycerides >150 mg/dL, in whom a significant 10.2 mg/dL reduction was observed. Importantly, there was a clear dose–response in LDL-cholesterol lowering. The statistical power of this

pooled analysis allowed detection of differential responses by baseline LDL-cholesterol level (greater response with higher values) and BMI (greater response with lower BMI) (Figure 4). The mean 10% LDL-cholesterol reduction with 2 servings/d of nuts in hypercholesterolemic individuals is similar to that described for functional foods fortified with plant sterols/stanols [68], which epitomizes the nutraceutical properties of nuts as cholesterol-lowering foods. Recently, in a network meta-analysis of 66 RCTs comparing the effects of 10 common food groups (refined grains, whole grains, nuts, legumes, fruits and vegetables, eggs, dairy, fish, red meat, and sugar-sweetened beverages) on cardiometabolic outcomes, nuts were ranked as the best food group at reducing LDL-cholesterol [69].

**Figure 4.** LDL-cholesterol responses to nut diets by baseline LDL-cholesterol and BMI. Data obtained in a pooled study of 25 nut RCTs (adapted from ref. [67] with permission).

The lipid effects for individual nut types have been examined in meta-analyses of RCTs using walnuts (24 studies) [70], almonds (27 studies) [71], pistachios (11 studies) [72], hazelnuts (3 studies) [73], and cashews (3 studies) [74]. All individual nuts except cashews reduced LDL-cholesterol to a similar extent than reported for total nuts in the mentioned systematic reviews [9,67], but cashews had no effect, which may be due to the low number of RCTs analyzed. Finally, a recent network meta-analysis of 34 RCTs of these five nuts for lipid outcomes used analyses based on the surface under the cumulative ranking curves and concluded that diets enriched in pistachios and walnuts were best for lowering LDLcholesterol and triglycerides compared with the other nut-enriched diets included in the study [75].

#### 5.7.3. Blood Pressure

The effects of nuts on office BP have been reported in many RCTs [9,35]. BP changes were a secondary outcome in the 2015 meta-analysis of Del Gobbo et al. [9], and no effect of nut-enriched diets on either systolic BP (SBP) or diastolic BP (DBP) was found. A 2015 meta-analysis including 21 RCTs of nut diets by Mohammadifard et al. [76] focused on BP changes. Results showed that diets supplemented with nuts had no effect on BP overall, except in individuals without T2D, who disclosed a weighted mean difference (WMD) in SBP of −1.29 mm Hg; (95% CI, −2.35, −0.22). In sub-analyses stratified by nut types, only diets enriched in pistachios resulted in a significant BP reduction, with a WMD of −1.82 mm Hg (95% CI, −2.97, −0.67) for SBP and of −0.80 mm Hg (95% CI, −1.43, −0.17) for DBP, while mixed nuts reduced only DBP, with a WMD of −1.19 mm Hg (95% CI, −2.35, −0.03).

Data on BP changes for specific nut types have also been reported. Thus, the 2018 meta-analysis by Guasch-Ferré et al. [70] of 24 RCTs focused on CV risk factor changes with walnut-enriched diets reported no effect on BP. A recent meta-analysis of 16 RCTs examining the effects of almonds on BP showed no differences for SBP between almond and control diets, but pooled analyses revealed a significant reduction of DBP by almond diets (WMD = −1.30 mm Hg; 95 % CI, −2.31, −0.30) [77]. A meta-analysis of 13 RCTs using pistachios for outcomes of CV risk factors by Asbaghi et al. [78] indicated a significant reduction of SBP (WMD = −2.12 mm Hg; 95 % CI, −3.65, −0.59), which supports the findings of the Mohammadifard et al. meta-analysis [76], although no effect on DBP was found. The meta-analysis of 3 cashew RCTs by Jalali et al. [74] also reported a significant reduction of SBP (WMD = −3.39 mm Hg; 95% CI, −6.13, −0.65), without changes of DBP.

The evidence on the effects of nuts on BP outcomes is inconsistent and, in general, does not support a relevant lowering effect, which contrasts with the epidemiological findings of an association of nut diets with a lower risk of incident hypertension, consistent across different meta-analyses [35,36]. Reasons for the failure of RCTs to detect BP changes with nut-enriched diets may be low statistical power (most RCTs included less than 50 participants), short duration of the intervention, exclusive use of office BP measurements, and the fact that they were usually a secondary outcome of lipid-focused trials, hence were not powered to detect changes in BP. Recently, the 2-year effects of a walnut diet on both office BP and 24-h ambulatory BP (the gold standard of BP measurements) in the Walnuts and Healthy Aging (WAHA) RCT conducted in 236 older individuals were reported [79]. The results showed that, compared with a control diet, a diet supplemented with walnuts at ≈15% of energy resulted in lower office SBP (−4.61 mm Hg) in the whole cohort and reduced 24-h ambulatory SBP (−8.5 mm Hg) in hypertensive participants. No changes in diastolic BP were observed. During the trial, participants in the walnut group required less uptitration of antihypertensive medication and had better overall BP regulation than controls. The WAHA trial overcomes the limitations of prior RCTs concerning BP effects of nut diets and shows a beneficial effect of long-term walnut consumption on SBP.

#### 5.7.4. Glycemic Control

Acute feeding studies have shown that nuts consumed with carbohydrate-rich foods having a high glycemic index reduce postprandial glucose responses in comparison with consumption of the same foods alone in both normoglycemic individuals and those with T2D [80,81], which suggests that nuts may be useful in glycemic control. The evidence from RCTs, however, is mixed. A recent meta-analysis of 40 RCTs with a median duration of 3 months concluded that consumption of tree nuts or peanuts had modest favorable effects on the homeostasis model assessment of insulin resistance (HOMA-IR) (WMD = −0.23) and fasting insulin (WMD = −0.40 μIU/mL), but not on fasting blood glucose or hemoglobin A1c [82]. Subgroup analyses showed similar results whether the study subjects were healthy individuals or those with prediabetes or T2D.

A meta-analysis of 16 RCTs that assessed effects of walnut diets on biomarkers of glycemic control failed to find any benefit [83]. Likewise, a recent in-depth narrative review of almonds and health outcomes based on findings of 64 RCTs and 14 meta-analyses and/or systematic reviews concludes that almonds have inconsistent and/or insignificant beneficial effects on glycemic control [81].

#### 5.7.5. Endothelial Function

The endothelium plays a central role in arterial health and throughout all stages of atherosclerosis. Endothelial function can be viewed as an integrative biomarker of the overall harmful effects of CV risk factors on the arterial wall, a reason why endothelial dysfunction is an independent predictor of future CVD events [84]. Endothelial dysfunction is characterized by a decreased bioavailability of NO and increased expression of pro-inflammatory cytokines and cellular adhesion molecules and can be evaluated non-invasively by several methods; flow-mediated dilation (FMD) measured by brachial artery ultrasound is considered the most sensitive and accurate in assessing endothelial function [85].

Two meta-analyses have summarized results of RCTs testing nut diets for effects on FMD [86,87]. The meta-analysis of Neale et al. [86] examined RCTs of nut diets providing data on inflammatory molecules, but also regarding effects on endothelial function. FMD was explored in nine strata (five testing the effects of walnuts) from eight RCTs, resulting in significant improvements in FMD of the nut versus the control diets (WMD = 0.79%; 95% CI, 0.35, 1.23). When subgroup comparisons were made according to nut type, only the walnut interventions resulted in improved FMD. The meta-analysis of Xiao et al. [87] of 10 RCTs was focused exclusively on effects of nuts on FMD. The pooled estimates showed that nut consumption significantly improved FMD (WMD = 0.41%; 95% CI, 0.18, 0.63). Again, subgroup analyses indicated that only walnut interventions improved FMD. Walnuts are particularly rich in ALA, polyphenols, arginine (the precursor of NO, the endogenous vasodilator) and other bioactives, as reviewed [41], which may explain their differential effects on endothelial function. However, almonds may also improve endothelial function, as shown by a recent 6-wk RCT that tested almond snacks (about 2 servings/day) versus control snacks (muffins) in adults at above-average CV risk for effects on FMD, among other cardiometabolic risk variables [88]. The results showed a noticeable increase in FMD by almonds (WMD = 4.1%; 95% CI, 2.2, 5.9), much higher than that reported in the cited meta-analyses [87,88]. No effects on BP were observed despite the use of 24 h ambulatory BP monitoring.

A recent review analyzed 16 nut intervention trials using noninvasive techniques other than FMD to assess vascular function, such as pulse wave velocity, pulse wave analysis, digital volume pulse, impedance cardiography, and peripheral arterial tonometry [89]. The results were mixed, with only 6 out of 16 studies showing improved vascular function ensuing nut diets.

#### 5.7.6. Inflammation

Chronic non-communicable diseases, such as atherosclerosis with major CV events, obesity, T2D, neurodegenerative disorders, cancer, and auto-immune diseases are characterized by a state of low-grade inflammation, which plays a central role in disease progression and perpetuation. Changes in this inflammatory state can be identified by determination of circulating biomarkers of inflammation, including C-reactive protein (CRP), tumor-necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), E-selectin, and adhesion molecules intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, all of which have garnered much interest in CV risk prediction [90]. The critical role of chronic inflammation in CVD has been substantiated recently by landmark RCTs demonstrating that interventions selectively targeting inflammation can improve clinical outcomes in patients with atherosclerosis [91].

The effects of nut-enriched diets on soluble inflammatory biomarkers have been investigated in many RCTs, usually as secondary outcomes, therefore not powered to detect changes in these outcomes [35]. A 2017 meta-analysis of 32 RCTs by Neale et al. [86] concluded that nut interventions induced no significant changes in inflammatory markers, including CRP, TNF-α, IL-6, ICAM-1 and VCAM-1, or in the anti-inflammatory biomarker adiponectin. A 2018 meta-analysis of 23 RCTs by Xiao et al. [92] showed that nut consumption reduced ICAM-1 (WMD = −0.17; 95% CI, −0.32, −0.03), but had no consistent effects on CRP or other soluble inflammatory molecules. Other meta-analyses focused on the lipid effects of total nuts [9] and walnuts [70] and a recent review on the cardiometabolic effects of almonds [81] concur in reporting no significant changes in CRP levels.

While based on results of generally small and short-term RCTs, it appears that consumption of nuts has a negligible impact on inflammatory markers, a recent report from the large, long-term WAHA trial provides a different view. The walnut intervention at ≈15% of energy for 2 years in 634 older participants recruited in two sites, Barcelona, Spain and Loma Linda, California, resulted in significant mean reductions ranging from 3.5% to 11.5% in several inflammatory biomarkers, including granulocyte-monocyte colony stimulating factor, interferon-γ, IL-1-β, IL-6, TNF-α, and E-selectin, but had no effect on CRP, ICAM-1 or VCAM-1 [93]. Thus, high statistical power and a long duration of the interventions might be necessary to uncover the anti-inflammatory effects of nuts. Regardless, these data provide novel mechanistic insight for the benefit of nut (walnut) consumption on CVD risk beyond that of lipid lowering.

#### *5.8. Other Health Outcomes*

Few prospective studies or single RCTs have examined the effects of nuts on alternative health outcomes, such as gallbladder disease, metabolic syndrome (MetS), non-alcoholic fatty liver disease (NAFLD), physical function, healthy aging, bone health, and reproductive health. There is also incipient evidence that nut diets elicit changes in microbiota.

Two reports from the large prospective cohorts of the NHS in women and the HPFS in men examined the association between frequency of nut consumption and risk of gallstone disease. In the NHS, women consuming ≥5 servings of nuts per week had a significantly lower risk of cholecystectomy (RR = 0.75, 95% CI, 0.66, 0.85) than did those who rarely or never consumed nuts [94], while in the HPFS, men consuming ≥5 servings of nuts per week had a significantly lower risk of symptomatic gallstone disease (RR = 0.70, 95% CI, 0.60, 0.86) compared to those who rarely or never consumed nuts [95]. The results of the two studies suggest that regular nut consumption protects men and women equally against gallstone disease. This beneficial effect is attributable to the richness of nuts in bioactive components capable of influencing intestinal bile acid and cholesterol biology, particularly unsaturated fatty acids, fiber, and non-sodium minerals.

An individual meets diagnostic criteria for MetS when harboring at least three of the following risk factors: increased WC, high triglycerides, low HDL-C, elevated BP, and high fasting blood glucose, and this cluster of risk factors increases the risk of CVD and all-cause mortality beyond the risk imparted by each separate factor [96]. The pathophysiological basis of MetS is insulin resistance, generally linked to central fatness, and as such MetS is an epidemic condition worldwide. Lifestyle changes directed to weight loss and cardiometabolic risk factor control are critical for preventing and treating MetS, thus nut consumption might play a role [97]. A 2014 meta-analysis included 49 RCTs of ≥3 weeks duration reporting effects of nut consumption on at least one criterion of the MetS [98]. Pooled analyses showed a beneficial effect of nuts on MetS via modest decreases in triglycerides and fasting blood glucose. However, it should be noted that a recent meta-analysis found no evidence of benefit of nut diets on blood glucose levels [82], which underlines the limitations of present data on a putative beneficial effect of nuts on MetS. Likewise, the large WAHA trial found no effect of a 2-year walnut-enriched diet on MetS [99].

NAFLD, the accumulation of fat (triglycerides) in the liver in the absence of excessive alcohol intake is the hepatic manifestation of MetS, a prevalent condition globally and a public health concern. NAFLD not only increases risk of CVD, but also of liver cirrhosis and hepatocarcinoma [100]. Like in MetS, abdominal obesity and T2D are major drivers of NAFLD, and its primary treatment consists of lifestyle and dietary changes directed at weight loss. The favorable effects of nuts on body weight, glycemic control and CVD risk would predict a beneficial effect in NAFLD, and a few prospective studies have suggested that increased nut consumption is associated with a lower incidence of NAFLD, as recently reviewed [101]. Nuts may contain the carcinogenic agent aflatoxin, a fungal metabolite and mycotoxin that can contaminate improperly stored nuts and other seeds. This has been a reason of concern for patients with NAFLD due to their increased risk of liver cancer. However, in Western countries, where aflatoxin contamination of crops is rare due to strict

regulations, health benefits provided by increased nut consumption likely outweigh the risks associated with chronic increases in aflatoxin exposure. This may not be the case in countries known for high rates of aflatoxin contamination of peanuts, like Indonesia [101].

There is evidence from a single prospective study conducted in Spain, where nut consumption is rather high, that it may lower the risk of impaired agility/mobility and increase overall physical function in older individuals [102], while another report from the NHS cohort suggests that consumption of total nuts and, particularly, walnuts is associated with healthy aging, i.e., survival beyond 65 years with no chronic diseases, no memory impairment, no physical disabilities, and intact mental health [103].

Dietary components are important for providing crucial constituents for bone health and regulating cellular metabolism within bone [104]. Nuts might promote bone health because they are rich sources of antioxidant, anti-inflammatory flavonoids, and calcium. Resveratrol is a stilbene-type polyphenol present in some nuts that is a powerful activator of the longevity-linked sirtuin-1 molecule, which regulates processes related to longevity, including apoptosis, DNA repair and energy expenditure [105]. Nevertheless, there are no data from prospective studies relating nut consumption to bone health, while RCTs are limited. A single small RCT assessed the effects of an ALA diet sourced from walnuts and flaxseed oil in comparison with an average American diet and a linoleic acid-rich diet on bone turnover, assessed by serum concentrations of N-telopeptides and bone-specific alkaline phosphatase [106]. N-telopeptide levels were significantly lower following the ALA diet relative to the average American diet, suggesting that plant sources of dietary n-3 PUFA may have a protective effect on bone metabolism via decreased bone resorption. In summary, the evidence on the efficacy of nuts to promote bone health is very limited. Both well-powered prospective studies and RCTs are warranted to examine this important issue.

Concerning reproductive health, in the last decades there has been a steady increase in infertility worldwide, in great part related to declining semen quality. Exposure to environmental toxins, smoking, and unhealthy diets are believed to underlie impaired spermatogenesis [107,108]. Two RCTs have tested nut diets for outcomes of semen quality. Robbins et al. [109] randomized 117 healthy men to consume 75 g of walnuts/d for 12 weeks on top of their usual Western-style diet or usual diet alone and found improved sperm vitality and motility after the walnut diet, but no changes in total sperm count. The FERTINUTS trial [110] was a 14-wk RCT involving 119 healthy men 18–35 y-old that assessed the effects on various sperm parameters of 60 g/day of mixed nuts (30 g walnuts, 15 g almonds, and 15 g hazelnuts) in the context of a Western-style diet vs. the same diet without nuts. Compared to the control group, the nut group showed significant improvements in total sperm count and vitality, motility and morphology. Nuts appear to improve male fertility, but clearly more research is needed.

Another prevailing pathology related to men's sexual heath is erectile dysfunction, a condition in which endothelial dysfunction at the level of penile vasculature is causal in the failure to initiate and/or maintain an erection [111]. Erectile dysfunction is intimately linked to CV risk factors and associated with an increased incidence of CVD [112]. Given that nut consumption is associated with reduced CV risk and improved endothelial function, in part due to their content in arginine, the precursor of the endogenous vasodilator NO, it is plausible that nut diets would benefit erectile function. Indeed, in folk medicine nuts (particularly cashews and walnuts) are promoted as aphrodisiacs and a remedy for impotency. A single RCT, a secondary analysis of the FERTINUTS trial [110], tested the effect of mixed nuts on erectile function in healthy young men [113]. Compared to the control group, small but significant increases in self-reported orgasmic function and sexual desire, but not erectile function, were observed following the nut intervention. There were no between-group differences in changes of peripheral concentrations of NO and E-selectin. Well-powered RCTs conducted in individuals with an objective diagnosis of erectile dysfunction are necessary to reach definitive conclusions on the efficacy of nuts to help men with this prevalent and troublesome pathology.

An expanding area of clinical research is the intestinal microbiome, which is primarily controlled by the nutritional quality of the diet and is believed to play a major role in a vast array of biological functions [114]. Colonic microbiota can be modulated by different lifestyle and dietary factors and impact the risk of developing obesity, T2D and other cardiometabolic diseases, as well as infectious diseases. Nuts have been suggested to have a prebiotic effect (that conferred by a substrate selectively used by the host microorganisms translating into a health benefit) on the gut microbiome [115]. The non-bioaccessible components of nuts (fiber, polymerized polyphenols and fat contained within undigested cell walls in incompletely masticated nut particles) make up a rich supply of nutrients to the intestines for feeding the microbes residing there. The field of nuts and microbiota is still at an early stage, but a recent comprehensive meta-analysis synthesized data from nine RCTs investigating almonds (*n* = 5), walnuts (*n* = 3) and pistachios (*n* = 1) for effects on fecal bacterial diversity [116]. Nut consumption increased the relative abundances of the genera *Clostridium*, *Lachnospira* and *Roseburia*, which are considered beneficial because they produce butyrate, a short-chain fatty acid critical in nourishing the intestinal epithelium and maintaining its integrity. Nut consumption had little overall impact on bacterial diversity, a metric considered as positive for health, except for a marginal enhancement from almond consumption, which could be explained by the particular matrix of almonds and small cell walls limiting fat availability for digestion, but increasing fat delivery to the colon and thus feeding the microbiota [81]. The overall meta-analytical evidence of a modulatory effect of microbiota by nuts is weak because microbial determinations were a secondary outcome in most RCTs, which were not powered to detect changes of this outcome. Nut effects on microbiota is a relevant topic for future research.

#### *5.9. Health Effects of Nuts in the Predimed Trial*

The landmark PREDIMED trial targeted both the effects of nut consumption on intermediate cardiometabolic markers and clinical outcomes, such as CVD and T2D, among others. The PREDIMED study [23] was a multicentric, parallel group, nutrition intervention RCT for the primary prevention of CVD. It was conducted in Spain and enrolled 7447 men and women aged 55–80 years at high risk of CVD but no CVD at recruitment. Participants were allocated to three study arms: two Mediterranean diets, supplemented with either extra-virgin olive oil (50 mL or more/day) or mixed nuts (30 g/day: 15 g walnuts, 7.5 g almonds, and 7.5 g hazelnuts), or control diet (advice on a low-fat diet) and followed for 5 y. The supplemental foods (olive oil and raw, unpeeled nuts) were delivered periodically to participants in the corresponding groups. Registered dietitians delivered the interventions at quarterly individual visits and group sessions separate for each group. As PREDIMED intended to assess the effects of the nutrition intervention alone, the diets were energyunrestricted and increased physical activity was not promoted. The primary end point was a composite of major CVD events (non-fatal myocardial infarction, non-fatal stroke, and CV death). An event adjudication committee, whose members were blinded to group allocation, was responsible for event ascertainment. Attesting to the high CV risk of participants, the mean age was 67 years and the mean BMI was 30 kg/m2, almost one-half had T2D, two-thirds had dyslipidemia, and 4 out of 5 had hypertension. Since its inception in June 2003, the trial has generated a steady stream of data on the beneficial health effects of Mediterranean diets enriched with either of the supplemental foods, culminating with the publication of results on the primary CVD outcome, demonstrating a ≈30% reduction with the two Mediterranean diets compared with the control diet [23]. The incidence of myocardial infarction was reduced non significantly with the two Mediterranean diets. The main results concerning the Mediterranean diet enriched with nuts are summarized below.

The most striking result was that incident stroke, a component of the PREDIMED main outcome, was significantly reduced by 45% (HR = 0,55; 95% CI, 0.35, 0.86] in the group allocated the Mediterranean with nuts compared to the control group (Figure 5) [23]. Regarding other hard cardiometabolic outcomes, the Mediterranean diet with nuts resulted in a 49% reduction (HR = 0.51; 95% CI, 0.32, 0.83) in the incidence of peripheral artery

disease [117] and a non-significant 18% reduction (HR = 0.82; 95% CI, 0.61, 1.10) in incident T2D [118]. In participants with MetS at baseline (*n* = 3392), the nut-supplemented diet resulted in a 28% (HR = 1.28; CI 1.08, 1.51) higher probability of reversion of MetS compared with the control diet, and this beneficial effect was driven mainly by reduced WC [119]. The risk of heart failure, a secondary outcome of the trial, was unaffected by the Mediterranean diets [120]. A post hoc analysis revealed no effect of the nut-supplemented diet on incident atrial fibrillation (HR = 0.89; 95% CI, 0.65, 1.20) [119]. The trial was not powered to examine mortality risk; however, when considered as an observational cohort, nut consumption was associated with a significantly reduced risk of all-cause mortality: compared to nonconsumers, participants consuming nuts >3 servings/week (32% of the cohort) had a 39% lower mortality risk (HR = 0.61; 95% CI, 0.45, 0.83) [121]. A similar protective effect against CVD and cancer mortality was observed.

**Figure 5.** Cumulative incidence of stroke by intervention group in the PREDIMED trial [23]. Copyright © (2018) Massachusetts Medical Society. Reprinted with permission. Med diet, Mediterranean diet; EVOO, extra-virgin olive oil.

Concerning intermediate outcomes, data from the full PREDIMED cohort showed a stable body weight, but WC (which tends to increase with age in older populations) increased less in the Mediterranean diet with nuts group, with and adjusted difference in 5-y changes of –0.92 cm (95% CI, –1.60, –0.24) compared with the control group [122]. These results provide first-level evidence that an ad libitum Mediterranean diet high in fat because of supplementation with nuts does not promote weight gain or visceral adiposity. Data from PREDIMED sub-studies revealed beneficial changes of the Mediterranean diet with nuts on: blood lipids and fasting glucose [122,123]; office BP, insulin resistance and soluble inflammatory markers (except CRP) [122]; BP as assessed by 24 h ambulatory monitoring, with 1 y changes of nearly −4 mm Hg for SBP and −2 mm Hg for DBP compared with the control diet, remarkable given that most participants were hypertensive and received standard anti-hypertensive medications [123]; and carotid plaque regression compared with progression in the control group after intervention for 2.4 y [124]. In another sub-study with 334 participants, a comprehensive neuropsychological test battery was administered at baseline and after a mean follow-up of 4.1 y. The results showed that, compared with the control diet, both Mediterranean diets resulted in delayed age-related cognitive decline, while the nut-supplemented diet performed better in the memory domain [125]. This PREDIMED sub-study is the first RCT demonstrating that a dietary pattern enhances cognitive function.

It must be emphasized that the PREDIMED interventions were meant to improve the overall diet, but the major between-group differences in food consumption were for the provisioned supplemental foods. It follows that nut consumption was probably responsible for most of the observed benefits in the Mediterranean diet with nuts group. The PREDIMED results illustrate the remarkable potential of nuts and other healthy foods such as extra-virgin olive oil to beneficially impact health outcomes. Given the age of PREDIMED participants, an important lesson of the trial is that it is never too late to change dietary habits to improve CV health and brain function.

#### **6. Conclusions**

Nuts, by virtue of their beneficial nutrients and phytochemicals, appear to bestow favorable and wide-ranging health dividends. The PREDIMED RCT showed a protective effect of nuts against CVD. Other RCTs have demonstrated that nuts lower LDL-cholesterol concentration, reduce insulin resistance and improve vascular reactivity. Epidemiological studies report largely congruent findings indicating that nut consumption is associated with lower risks for CVD, total mortality, atrial fibrillation, hypertension, and cancer. Habitual nut consumption does not promote obesity and may even result in less weight gain over time, particularly among individuals with overweight/obesity. Table 3 summarizes the main beneficial effects of nuts on health outcomes.

**Table 3.** Associations of nut consumption with health outcomes and disease risk factors. Summary of scientific evidence.



**Table 3.** *Cont.*

Abbreviations: +/−, equivocal evidence; +, limited evidence from few studies; ++, evidence from many studies; HDL, high-density lipoprotein; LDL, low-density lipoprotein; PAD peripheral artery disease. \* Evidence collected in the PREDIMED trial, among others. \*\* Evidence collected only in the PREDIMED trial.

The cumulative scientific evidence indicates that nuts are one of the most wholesome and nutritious foods in the usual diet, but which nuts are best for health? The three nuts that were supplied and consumed by the participants in the Mediterranean diet plus nuts arm of the PREDIMED study were walnuts, almonds and hazelnuts. Thus, these are the only three nuts with first level evidence for improving CV outcomes in the context of a RCT.

However, the nutrient profiles of other nuts make them excellent dietary options as well. For example, Brazil nuts are especially rich in selenium, pecans and peanuts are great sources of polyphenol antioxidants, pistachios are particularly high in carotenoids, tocopherols and phytosterols, and macadamias are replete with monounsaturated fats and flavonoids. Consequently, consuming a mixture of nuts, aiming for a daily dose of at least 30 g/d, is ideal for optimizing health.

As nuts are naturally high in non-sodium minerals and virtually devoid of sodium, lightly salted nuts are still a healthy low-salt snack that many people find more palatable than unsalted nuts. The inner peel between the shell and the nut is rich in polyphenols. Given that the peel and its polyphenols are lost when nuts are roasted, raw, unpeeled nuts are generally the healthiest and those that can be rightly considered as the natural food with most pluripotential nutraceutical properties. It is noteworthy that with the choice of a single wholefood we can positively impact multiple cardiometabolic risk factors, promote healthy aging, and live longer [126]. Regular nut consumption is an indispensable component of any healthy, plant-based dietary pattern.

**Author Contributions:** E.R. conceptualized the research. E.R. and J.H.O. examined the relevance, quality, and consistency of the scientific literature on nuts and health, and independently extracted and reached a consensus when in doubt about a specific citation. A.S. assisted in retrieving articles and writing. All authors participated in writing of this manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** CIBEROBN is an initiative of Instituto de Salud Carlos III, Spain.

**Conflicts of Interest:** E.R. reports grants, personal fees, non-financial support and other from California Walnut Commission, during the conduct of the study; grants, personal fees, non-financial support and other from Alexion; personal fees and other from Amarin, outside the submitted work. J.H.O. reports a major ownership interest in CardioTabs, a nutraceutical company. A.S. has not conflicts of interest.

#### **Abbreviations**

ALA—α-Linolenic Acid BP—Blood Pressure BMI—Body Mass Index CHD—Coronary Heart Disease CI—Confidence Interval CRP—C-Reactive Protein CV—Cardiovascular CVD—Cardiovascular Disease DBP—Diastolic Blood Pressure FMD—Flow-Mediated Dilation HOMA-IR—Homeostasis Model Assessment of Insulin Resistance HPFS—Health Professionals Follow-Up Study HR—Hazard Ratio ICAM—Intercellular Adhesion Molecule IL—Interleukin MetS—Metabolic syndrome MUFA—Monounsaturated Fatty Acids NAFLD—Non-Alcoholic Fatty Liver Disease NHS—Nurses' Health Study NO—Nitric Oxide OR—Odds ratio PREDIMED—PREvención con DIeta MEDiterránea PUFA—Polyunsaturated Fatty Acids PURE—Prospective Urban and Rural Epidemiology RCT—Randomized Controlled Trial RR—Relative Risk SBP—Systolic Blood Pressure SFA—Saturated Fatty Acids T2D—Type-2 Diabetes Mellitus TNF-α—Tumor-necrosis Factor-α VCAM—Vascular Cell Adhesion Molecule WAHA—Walnuts and Healthy Aging WC—Waist Circumference WMD—Weighted Mean Difference

#### **References**

