1. Introduction
Cardiovascular diseases (CVD) is the greatest contributor to global morbidity and mortality, according to the World Health Organization (
http://www.who.int). The causes of cardiovascular diseases are diverse but the most common is atherosclerosis, a low-grade inflammatory disease that starts with the recruitment of circulating leukocytes and their adhesion to the vascular endothelium [
1]. During these cell adhesion processes, endothelial cells are activated by inflammatory cytokines, enhancing the expression of adhesion molecules and synthesizing chemokines and lipid chemoattractants that are presented on their luminal surface [
2]. This process, known as endothelial activation, is a key event in the onset of atherosclerosis [
3]. Compelling experimental data support the protective role of tomato ingredients against the processes involved in atherogenesis such as endothelial activation [
4,
5].
Tomato (
Lycopersicon esculentum L.) is one of the most popular and extensively consumed vegetable crops worldwide [
6], and may be consumed either fresh or as tomato processed products (sauce, juice, paste, puree, ketchup and soup). Tomato products are usually cooked with the addition of oil, and both cooking and the addition of a fatty matrix increase the bioavailability of their bioactive compounds [
7]. Thus, food matrixes may play a key role in determining the absorption, distribution and final biological action of tomato compounds in the human body [
8].
Tomato and its byproducts are rich in phytochemicals such as carotenoids (mainly lycopene and β-carotene), phenolic compounds (mainly flavonoids, such as naringenin), vitamins C and E, potassium and folates [
9,
10]. Previous epidemiological studies have suggested that tomato intake may decrease the risk of CVD and several cancers [
11] due to some of these compounds, mainly lycopene [
12]. In this way, tomato consumption could reduce or delay the development of CVD by inhibiting cholesterol synthesis, improving immune function and reducing inflammation [
13].
Previous studies have observed that tomato processing may influence the metabolism of tomato phenolics and, consequently, their plasma bioavailability and urinary excretion [
14,
15,
16,
17]. However, the effect of acute intake of raw tomato and tomato sauces on plasma and cellular inflammation biomarkers related to the atherosclerotic process remains unknown. We embarked, therefore, on a randomized, controlled feeding intervention trial in order to evaluate the postprandial effects of a single dose of raw tomato (RT), tomato sauce cooked without oil (TS) and tomato sauce cooked with refined olive oil (TSOO) on markers of inflammation and cardiovascular disease.
2. Experimental Section
2.1. Participants
Forty healthy volunteers were included in the study. None reported any history of CVD or other medical conditions. All subjects were non-smokers and were not receiving medication or vitamin supplements. The Ethics Committee of Clinical Investigation of the University of Barcelona (Spain) and the Institutional Review Board of the Hospital Clinic of Barcelona approved the study protocol. All the participants gave written consent before entering into the trial. The International Standard Randomized Controlled Trial Number of the trial is ISRCTN20409295 (
www.controlled-trials.com).
2.2. Study Design
The study was an open, prospective, randomized, crossover, controlled feeding trial (
Figure S1, Supplementary Material). All subjects underwent a three-day washout period in which they were asked to consume their regular diet, but avoiding the intake of any tomato or tomato-based products and, during the 24 h immediately preceding the dietary intervention as well as the day of the study they were asked to follow a polyphenol-free diet. Subjects received a list showing permitted and forbidden foods and two menus in order to help them to follow the diet correctly.
Each volunteer randomly received: 7.0 g of RT/kg of body weight (BW), 3.5 g of TS/kg BW or 3.5 g of TSOO/kg BW and 0.25 g of sugar solved in water/kg BW on four different days with a month interval between each. For the elaboration of 250 g of tomato sauces, an amount of 500 g of tomato was used; therefore, the intervention with raw tomatoes was 500 g per 70 kg BW. The amount of sugar used in the control group was assessed in order to give the same amount of kilocalories from carbohydrates in all four intervention groups, around 17.5 g each. All subjects fasted for more than 8 h before each intervention. The day of the intervention, blood samples were collected into plasma ethylenediaminetetraacetic acid (EDTA) tubes at baseline (0 h) and 6 h following the test meal intake. Blood samples were immediately centrifuged after collection (1500 g for 20 min at 4 °C), and were then aliquoted and stored at −80 °C until analysis. Urine was collected in plastic containers 10–20 min before consuming the intervention (baseline) and after the ingestion (0–6 h). Before the 6th hour post-intervention, participants were instructed to empty their bladders. The urine was aliquoted and store at −80 °C prior to analysis.
The volunteers refrained from consuming other foods during the 6 h after intervention, while remaining in the clinical ward to avoid the possibility of eating or drinking.
2.3. Tomato Sauce Analysis
A commercial tomato variety (
Lycopersicum esculentum L.) suitable for tomato sauce elaboration (“Smooth tomato”) was used for the study. Tomato sauces were processed at the Torribera campus at the University of Barcelona (Barcelona, Spain) following a standardized procedure. Five percent of refined olive oil was added to the TSOO and the same amount of water was added to the TS in order to standardize the concentration of tomato compounds ingested at each intervention. Both tomato sauces were kept in the refrigerator until served to the subjects in order to avoid any detrimental effect. Tomatoes and tomato sauces were served at room temperature. The phenolic and carotenoid profiles of RT and tomato sauces were determined using an ultra-high pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) and a high pressure liquid chromatography with ultraviolet detection (HPLC-UV), respectively, as previously reported by Vallverdú-Queralt
et al. [
18,
19].
2.4. Dietary Assessment
Before each intervention, the subjects were asked to fill out a 24-h food recall to assess their compliance with the prescribed diet. Total energy, macronutrient, and micronutrient intake were calculated using the Food Processor Nutrition and Fitness Software (esha Research, Salem, OR, USA). To evaluate any possible adverse effects from the interventions, the dietitian administered a checklist that included mouth symptoms, bloating, fullness or indigestion, altered bowel habit and any other diet-related symptom the day after each intervention.
2.5. Measure of Compliance
Tomato phenolic metabolites were determined at baseline in urine spot samples sauces using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) as previously reported by Martínez-Huélamo
et al. [
20].
2.6. Clinical Measurements
Routine analyses were performed at the CORE laboratory of our Institution that fulfills all required quality criteria. In brief, blood parameters analyzed were the following: blood glucose by glucose oxydase method, total cholesterol and triglycerides by enzymatic procedures; high density lipoprotein (HDL) cholesterol after precipitation with phosphotungstic acid and magnesium chloride all performed in a clinical chemistry analyzer Advia 2400 from Siemens Healthcare (Siemens, Tarrytown, NJ, USA), all reagents were provided by the instrument manufacturer; serum folic acid was measured by an automated electrochemiluminescence immunoassay system Advia-Centaur, Siemens (Siemens, Tarrytown, NJ, USA) with reagents provided by the instrument manufacturer; and insulin by a customized Human Multi Analyte Profiling assay (Human MAP, Rules Based Medicine Inc., Austin, TX, USA).
2.7. Peripheral Blood Mononuclear Cells (PBMC) Immunophenotyping
PBMC were isolated from whole blood by density gradient centrifugation with the Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden). The expression of adhesion molecules was analyzed on PBMC surface via double direct immunofluorescence using commercial monoclonal antibodies following the manufacturer’s instructions. The adhesion molecules analyzed were: lymphocyte function-associated antigen-1 (LFA-1) (CD11a; Bender MedSystems, Burlingame, CA, USA), macrophage-1 antigen (or integrin αMβ2) (Mac-1) (CD11b/CD18; Bender MedSystems, Burlingame, CA, USA), very late activation antigen-4 (VLA-4) (CD49d;Cytogmos, Barcelona, Spain), Sialyl-Lewis X (SLex) (CD15s; Beckman Coulter, Fullerton, CA, USA) and CD40 (Caltag Laboratories, Burlingame, CA, USA) for lymphocytes and monocytes, and CD36 (Beckman Coulter Inc., Fullerton, CA, USA) and CCR2 (R&D Systems, Minneapolis, MN, USA) were only measured in monocytes. T-lymphocytes and monocytes were identified separately using anti-CD2 and anti-CD14 (Caltag Laboratories, Burlingame, CA, USA) monoclonal antibodies, respectively. Cell counting (10,000 and 3500 events for the T-lymphocyte and the monocyte regions, respectively) and fluorescence analysis were performed in a FACSCalibur Flow Cytometer (Becton Dickinson, San Jose, CA, USA) using the CellQuestPro software (version 3.3, BD Biosciences). Results are expressed as median fluorescent intensity (MFI) in arbitrary units (AU).
2.8. Soluble Inflammatory Markers
The concentration of soluble adhesion molecules is determined using a Human Cytokine Plex (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The molecules determined are: Intercellular Adhesion Molecule-1 (ICAM-1), Vascular Cell Adhesion Molecule-1 (VCAM-1), Monocyte Chemoattractant Protein-1 (MCP-1), Interleukin-1α (IL-1α), IL-6, IL-10 and IL-18. Plates were analyzed on a Luminex 100™ instrument (Luminex, Austin, TX, USA) using Bio-Plex Manager TM Software (Bio-Rad, Hercules, CA, USA). Concentrations are obtained by standard calibration curves. Results are shown in pg/mL, except for VCAM-1 and ICAM-1 that are expressed in ng/mL. All measurements are performed in duplicate.
2.9. Statistical Analyses
Values are expressed as means ± standard deviation (SD), unless otherwise indicated. The dietary composition was analyzed through a one-factor analysis of variance (ANOVA) for mean comparison since all variables followed a normal distribution according to the results of the Kolmogorov–Smirnov test. To analyze the changes within each treatment a Student’s t test for paired samples was performed between the data obtained before and after each intervention. To compare the differences of the changes in outcome variables between the interventions an ANOVA for repeated measures with the Bonferroni post-hoc test was used. All analyses were repeated with adjustment for sex. Differences were considered significant at p < 0.05. Statistical analysis was performed using the SPSS Statistical Analysis System (version 18.0; SPSS Inc, Chicago, IL, USA).
4. Discussion
In this feeding trial performed in 40 healthy subjects, we found that food matrix modified the short term effects of a single tomato intake on lipid profile by decreasing plasma total and ratio total cholesterol/HDL-c. In addition, a single tomato intake decreased some inflammatory biomarker concentrations such as LFA-1, IL-6, IL-18, MCP-1 and VCAM-1, and increased plasma IL-10 concentrations. Moreover, the findings observed demonstrate that tomato sauce in addition to a lipid matrix (refined olive oil) enhanced the effects of tomato intake on the CV system, since the effects of TSOO on plasma inflammatory biomarker concentrations were greater than those of RT and TS.
Diet is a mixture of multiple nutrients that profoundly affects many CVD risk factors [
22] and has been recognized to play an important role in the prevention and treatment of atherosclerosis, nowadays considered a low-grade inflammatory disease [
23]. Several epidemiological studies have ascribed beneficial anti-inflammatory effects to tomato products. However, published data are unclear, probably due to the fact that the anti-inflammatory effects of tomato products may be affected by industry and/or cooking processing, as well as composition. To date, few human feeding trials have been conducted to study the effect of processed tomato intake in different matrixes on CVD risk factors [
4,
24,
25,
26,
27]. Besides, in most studies on tomato and tomato products, lycopene was assumed to be responsible for the positive health effects. However, it has been postulated that the decreased risk for developing CVD was more strongly associated with tomato intake with all its components than with isolated lycopene intake [
27], suggesting that other compounds may also be involved in the protective cardiovascular effects of tomato. In fact, it should be taken into account that tomato products contain a great variety of compounds including micronutrients such as folic acid, vitamins C, and E, fiber, carotenoids, potassium, magnesium and polyphenols. In addition, the beneficial effects derived from tomato intake could be due, at least in part, to the synergistic effects of these bioactive compounds.
Nutrient bioavailability from dietary sources depends on several factors including the breakup of the food matrix, cooking processes and the presence or addition of lipids or other substances. Moreover, nutrients may interact between them or with other dietary components during digestion, changing their bioavailability. Tomato products’ processing usually involves heat and/or homogenization, both of which can break up the matrix and release and degrade compounds. Several studies have found that heat treatment may decrease the concentration of some micronutrients such as ascorbic acid, total phenolics, lycopene and antioxidant capacity [
28] but, at same time, other studies have proved that tomato processing may also increase the bioavailability of certain other bioactive compounds such as lycopene or phenolics [
24,
29,
30]. Besides, during industrial or domestic tomato processing oil is usually added. Since carotenoids are fat-soluble, adding small quantities of fat or oil to the meal may improve their bioavailability [
31,
32] and even the composition of the oil used may affect antioxidant activity [
4,
25]. In this setting, we decided to use a homemade tomato sauce instead of commercial preparations in order to allow direct extrapolation of our findings to everyday culinary practices. Thus, we examined the effect of the traditional processing of tomato sauce with refined olive oil, a type of oil that does not contain carotenoids or polyphenols. Therefore, the changes observed may be attributed to differences in the bioavailability of tomato compounds (raw tomato, tomato sauce without oil and tomato sauce with refined olive oil) rather than to the addition of olive oil polyphenols. On the other hand, the dose of active compounds such as carotenoids and polyphenols administered was higher in RT than in the sauces because of processing losses.
In the current study, we also included a control intervention in order to discard changes occurring due to postprandial physiological processes. In this way, changes observed in all interventions including the control cannot be attributed to tomato consumption. Thus, the decrease observed in systolic and diastolic BP after all interventions cannot be attributed to the tomato intake. Despite several mid- and long-term studies have reported beneficial effects on BP [
10,
27,
33] and glucose metabolism [
28,
31] after antioxidant-rich diets, we did not observe this effect in a postprandial state. In this way, we hypothesize that a single intake of tomato is not enough to obtain the beneficial effects of long term consumption.
Postprandial lipemia is strongly associated with the risk of development of atherosclerotic lesions. It is characterized by the raised levels of triglyceride-rich lipoproteins [
34] and is induced by fat meals. In our study, we observed a decrease in triglycerides after the three tomato interventions and, as expected, this change was mitigated after the consumption of TSOO. Moreover, after control intervention, postprandial triglycerides levels returned to baseline, confirming beneficial effect of tomato consumption on postprandial triglyceride plasma concentration. Some studies [
30], but not all also observed a reduction in triglycerides after tomato sauce intake, but only when the sauce was prepared with olive oil, and not when sunflower oil was used. Similar to triglycerides, total cholesterol and LDL-cholesterol showed significantly lower concentrations at six hours after the three interventions but not after the control. Regarding cholesterol fractions HDL was found to increase significantly only after TSOO intervention and LDL diminish significantly after TS intake. Since postprandial lipemia is considered a pro-inflammatory process these results are of great importance since systemic inflammation is the basis of the onset and development of atherosclerosis.
Along this line, the interaction of T-lymphocytes and monocytes with the endothelium through adhesion molecules is a crucial event in atheroma plaque formation [
23]. Soluble forms of endothelial adhesion molecules found in plasma are considered as an index of endothelial activation and even a biomarker of atherosclerosis [
35]. Accordingly, the reduction in IL-6 and VCAM-1 concentrations after a single intake of TSOO (16.9% and 10.4%, respectively) may be due to an inhibited endothelial activation through the inhibition of transcription factors, such as nuclear factor κB, via an activation of a family of inhibitors called IκBs (inhibitors of κB) [
23]. Similar results were obtained in another intervention study in which 500 mL a day of
n-3 PUFA-enriched tomato juice or plain tomato juice were administered to healthy women during 15 days; serum VCAM-1 and ICAM-1 concentrations decreased significantly by 24% and 47%, respectively [
36]. Further, Burton-Freeman
et al. [
37] observed that tomatoes reduced postprandial elevation of IL-6 concentrations and concluded that this response could be attributed, at least in part, to the antioxidant effects of tomatoes altering cellular redox status. We also observed a decrease in MCP-1 concentrations after the three interventions with tomato. However, there is only one study in healthy humans evaluating the effects of a tomato product (Mediterranean vegetable soup) on MCP-1. In this study, a decrease in MCP-1 concentrations was found after 14-day intervention [
38]. Likewise, we observed a decrease in IL-18 although it only attains statistical significance after the consumption of the two sauces. In contrast, concentrations of IL-10 in plasma increased but only achieved significance after the intake of TS and RT. Interleukin expression are also regulated by the genes involved in the inflammatory response via changes in expression of NF-κB [
23].
To our knowledge this is the first study regarding tomato consumption and IL-18 and IL-10 concentrations. Thus, the intake of tomato products may reduce the oxidant and pro-inflammatory effect of meals in the postprandial situation.
Our study has some limitations. Firstly, the study population was healthy and, therefore, the effects observed cannot be extrapolated to high cardiovascular risk populations. Secondly, the acute effects of tomato product consumption may not represent the long-term effects of its consumption. Third, we used a solution of sugar in the control group. Although the number of calories from carbohydrates was similar in the four groups, we cannot exclude a certain acute inflammatory effect of the sugar added [
39]. However, our study also has major strengths, such as the study design (cross-over trial including washout periods between interventions), the monitoring of the intake and the compliance, that was excellent according to the results of the analysis of polyphenols in urine samples.
Palmira Valderas-Martinez 1,2, 
, 
