Precisionsnäring för att förbättra kardiometabolisk hälsa med dietära (poly)fenoler (PRE-CARE-DIET)

Subjects belonging to a specific aggregate phenolic metabotype (X) to which a diet with an increase of 50% in the actual (poly)phenol intake will be given, through personalized dietary plans.

Prescription of dietary plans increasing the intake of (poly)phenols through consumption of foods rich in these compounds, in the context of a balanced, personalised diet

Nutritional challenge with standardized (poly)phenol-rich tablets

Subjects belonging to a specific aggregate phenolic metabotype (X) to which general dietary recommendations for a healthy diet will be given.

Nutritional challenge with standardized (poly)phenol-rich tablets

Prescription of balanced dietary plans

Subjects belonging to a specific aggregate phenolic metabotype (Y) to which a diet with an increase of 50% in the actual (poly)phenol intake will be given, through personalized dietary plans.

Prescription of dietary plans increasing the intake of (poly)phenols through consumption of foods rich in these compounds, in the context of a balanced, personalised diet

Nutritional challenge with standardized (poly)phenol-rich tablets

Subjects belonging to a specific aggregate phenolic metabotype (X) to which general dietary recommendations for a healthy diet will be given.

Nutritional challenge with standardized (poly)phenol-rich tablets

Prescription of balanced dietary plans

Assessing the effect of a (poly)phenol-rich diet on 10-year risk of cardiovascular disease using the Systematic Coronary Risk Estimation (SCORE2) for volunteers aged between 40 and 69 years, considering different aggregate phenolic metabotypes. Interpretation of the result depends on the patient's age as the cut-off risk levels are numerically different for various age groups: low-moderate Cardiovascular Disease (CVD) risk (< 2.5% for < 50 years; < 5% for 50-69 years), high CVD risk (2.5% to < 7.5% for < 50 years; 5% to < 10% for 50-69 years), very high CVD risk (≥ 7.5% for < 50 years; ≥ 10% for 50-69 years).

Assessing the effect of a (poly)phenol-rich diet on 10-year risk of cardiovascular disease using the Systematic Coronary Risk Estimation-Older Persons (SCORE2-OP) for volunteers aged between 70 and 89 years, considering different aggregate phenolic metabotypes. Interpretation of the result depends on the patient's age as the cut-off risk levels are numerically different for various age groups: low-moderate CVD risk (< 7.5% for ≥ 70 years), high CVD risk (7.5% to < 15% for ≥ 70 years ), very high CVD risk (≥ 15% for ≥ 70 years).

Weight (measured in kilograms) and height (measured in meters) will be assessed using a balance and a stadiometer, respectively. These measures will be combined to report the Body Mass Index (BMI) using the following formula: kg/m^2.

Waist circumference (measured in centimeters) and hip circumference (measured in centimeters) will be assessed using an inelastic meter. These measures will be combined to report waist-to-height ratio and waist-to-hip ratio using the following formulas: waist circumference (cm)/height (cm) and waist circumference (cm)/hip circumference (cm), respectively.

Body composition estimation will be assessed using a bioelectrical impedance analysis (BIA). It is a tool that provides an estimate of qualitative and quantitative analysis of body composition and distribution.

Systolic and diastolic blood pressure measured in millimeters of mercury (mmHg) of each volunteer will be obtained after a 5-min rest in a seated position.

Heart rate, measured in beats per minute (bpm) of each volunteer will be obtained after a 5-min rest in a seated position.

Blood samples will be collected for the assessment, measured in milligrams per deciliter, of total cholesterol (mg/dL), HDL-cholesterol (mg/dL), triglycerides (mg/dL), glucose (mg/dL), lipoprotein a (mg/dL), apolipoprotein A1 (mg/dL), apolipoprotein B (mg/dL) and uric acid (mg/dL). Non-HDL cholesterol, triglyceride/glucose index, triglyceride/waist circumference index, and ApoB/ApoA ratio will be estimated using the reference formula.

Blood samples will be collected for the assessment, measured in enzymatic unit activity per liter, of alanine aminotransferase (U/L), aspartate aminotransferase (U/L) and gamma glutamyl transferase (U/L).

Homocysteine, measured in milligrams per liter, will be assessed from serum samples.

Insulin, measured in micro Insulin Units per milliliter, will be assessed from plasma samples.

Blood samples will be collected for the assessment, measured in percentage of total hemoglobin, of glycated hemoglobin (HbA1c (%)) and glycated albumin (%).

Fasting insulin sensitivity will be estimated through the quantitative insulin-sensitivity check index (QUICKI) - using the following formula: 1/log(fasting insulin (uUI/mL)) + log(fasting glucose (mg/dL)) -, McAuley's index, and the metabolic score for insulin resistance (METS-IR).

Insulin secretion capacity will be estimated through the homeostasis model assessment beta-cell function (HOMA-β) index using the following formula: 360 * insulin (uUI/mL)/glucose (mg/dL) - 55.

Insulin resistance will be estimated through the homeostasis model assessment of insulin resistance (HOMA-IR) index using the following formula: glucose (mg/dL) * insulin (uUI/mL)/82 * 7.

Plasma samples will serve to determinate the inflammatory status monitoring specific cytokines (Tumor Necrosis Factor α, InterLeukin-1β, InterLeukin-6, InterLeukin-8, InterLeukin-10, and Monocyte Chemoattractant Protein-1), adhesion molecules (Vascular cell adhesion protein-1, Intercellular Adhesion Molecule-1, E-selectin), and high sensitivity C reactive protein (hsCRP), using a customized magnetic bead-based multiplex assays.

The Framingham General Cardiovascular Risk Score (FRS) - 10 years will be assessed to understand the relationship with the aggregate phenolic metabotypes observed. The FRS estimates the 10-years risk of manifesting clinical CVD (CAD, Stroke, cardiac death). Risk is considered low if the FRS is less than 10%, moderate if it is 10% to 19%, and high if it is 20% or higher.

The Framingham General Cardiovascular Risk Score (FRS) - 10 years will be assessed to understand the relationship with the aggregate phenolic metabotypes observed. The FRS estimates the 30-years risk of manifesting clinical CVD (CAD, Stroke, cardiac death). Risk is considered low if the FRS is less than 12%, moderate if it is 12% to 40%, and high if it is 40% or higher.

The Framingham Heart Study Primary Risk Functions for Hard Coronary Heart Disease (HCHD) 10-year risk will be assessed to understand the relationship with the aggregate phenolic metabotypes observed. The HCHD estimates the 10-years risk of manifesting myocardial infarction or coronary death. Risk is considered low if the HCHD is less than 1%, moderate if it is 1%, and high if it is 2% to 11% for female; risk is considered low if the HCHD is less than 1%, moderate if it is 1%, and high if it is 2% to 25% for male.

The Framingham Heart Study Primary Risk Functions for First Coronary Heart Disease (2-years risk) will be assessed to understand the relationship with the aggregate phenolic metabotypes observed. This risk score estimates the 2-years risk of manifesting a first event of myocardial infarction or coronary death. Risk is considered low if the score is less than 0%, moderate if it is 0% to 1%, and high if it is 1% or higher for female; risk is considered low if the score is less than 0%, moderate if it is 0% to 1%, and high if it is 1% to 6% for male.

The Framingham Heart Study Primary Risk Functions for Diabetes will be assessed to understand the relationship with the aggregate phenolic metabotypes observed. This risk score estimates the risk of manifesting type 2 diabetes mellitus (fasting blood glucose at or above 126 mg/dL) in 8 years. Risk is considered low if the score is less than 3% and high if it is 4% to 18%.

The Framingham Heart Study Primary Risk Functions for Hypertension will be assessed to understand the relationship with the aggregate phenolic metabotypes observed. This risk score estimates the risk of incidence of hypertension. Risk is considered low if the score is less than 5%, moderate if it is 5% to 10%, and high if it is higher than 10%.

The Framingham Heart Study Primary Risk Functions for Stroke will be assessed to understand the relationship with the aggregate phenolic metabotypes observed. This risk score estimates the 10-year probability of stroke. The risk score is given as a percentage and higher percentage values correspond to an increased risk of stroke.

The Framingham Heart Study Primary Risk Functions for Fatty Liver Disease will be assessed to understand the relationship with the aggregate phenolic metabotypes observed. This risk score estimates the 10-year risk of CVD events in individuals with non-alcoholic fatty liver disease (NAFLD) The risk score is given as a percentage and higher percentage values correspond to an increased risk of CVD events.

The Atherosclerotic Cardiovascular Disease (ASCVD) risk will be assessed to understand the relationship with the aggregate phenolic metabotypes observed. It is an index of the 10-year risk of having a cardiovascular problem, such as a heart attack or stroke. The ASCVD risk score is given as a percentage and there are four groupings: a 0 to 4.9 percent risk is considered low; a 5 to 7.4 percent risk is considered borderline; a 7.5 to 20 percent risk is considered intermediate; a greater than 20 percent risk is considered high.

The QRESEARCH risk estimator version 3 (QRISK3®) will be assessed to understand their relationship with the aggregate phenolic metabotypes observed. It is an index of the 10-year risk of having a cardiovascular problem, such as a heart attack or stroke. The QRISK3® risk score is given as a percentage and there are three groupings: low risk if the QRISK3 score of less than 10%; moderate risk if the QRISK3 of 10-20%; high risk if the QRISK3 score of more than 20%.

The QDiabetes Score® will be assessed to understand the relationship with the aggregate phenolic metabotypes observed. QDiabetes® is a validated tool which predicts the absolute risk of developing type 2 diabetes in the next 10 years. The QDiabetes Score® risk score is given as a percentage: high risk is defined for QDiabetes of 5.6% or greater, while it is low for lower values.

The Finnish Diabetes Risk Score (FINDRISC) will be assessed to understand the relationship with the aggregate phenolic metabotypes observed. FINDRISC is a prediction tool to identify patients at risk of developing diabetes. A risk score of 0-14 points indicates a low to moderate risk of diabetes (1-17% chance of diabetes over 10 years). A risk score of 15-20 points indicates a high risk of diabetes (33% chance of diabetes over 10 years). A risk score of >20 points indicates a very high risk of diabetes (50% chance of diabetes over 10 years).

The Calculating the individual risk score (Italian CVD risk score) will be assessed to understand the relationship with the aggregate phenolic metabotypes observed. Six cardiovascular risk categories were constructed, called MCV (from I to VI): the MCV risk category indicates how many persons out of 100 with the same characteristics will fall ill over next 10 years. Risk is considered: below 5% for MCV I; 5% to 10% for MCV II; 10% to 15% for MCV II; 15% to 20% for MCV IV; 20% to 30% for MCV V; over 30% for MCV VI

Adherence to healthy dietary patterns will be assessed by the Healthy Eating Index (HEI), which is a measure of diet quality used to assess how well a set of foods aligns with key recommendations and dietary patterns. The HEI uses a scoring system from 0 to 100. There are two groupings: 1) "Adequacy components" represent the food groups, subgroups, and dietary elements that are encouraged and for these components, higher scores reflect higher intakes, because higher intakes are desirable; 2) "Moderation components" represent the food groups and dietary elements for which there are recommended limits to consumption and for moderation components, higher scores reflect lower intakes, because lower intakes are more desirable.

Adherence to Italian Mediterranean diet will be assessed by the Italian Mediterranean Index. The score is calculated from intake of 11 items: high intakes of six typical Mediterranean foods (pasta, typical Mediterranean vegetables, fruit, legumes, olive oil and fish; low intakes of four "non-Mediterranean" foods (soft drinks, butter, red meat and potatoes); and alcohol. If consumption of typical Mediterranean foods is in the 3rd tertile of the distribution, the person receives 1 point; all other intakes receive 0 points. If consumption of non-Mediterranean foods is in the first tertile of the distribution the person receives 1 point. Alcohol receives 1 point for intake up to 12 g/day; abstainers and persons who consume >12 g/day receive 0. Possible scores range from 0 to 11.

To evaluate the adherence to and healthy and sustainable dietary, the Greek Mediterranean Index will be assessed with a nine-point score for the consumption of vegetables, legumes, fruit and nuts, cereal, fish, meat,dairy, alcohol, and lipids. Using the sex-specific median, a value of 0 will be given to participants whose consumption of vegetables, legumes, fruits and nuts, cereals, and fish is below the median consumption, while a value of 1 will be assigned in the opposite case. Study participants with a below-median consumption of meat and dairy products will be assigned a value of 1, whereas a value of 0 will be given to those with an above-median consumption. For ethanol, men consuming 10-50 g/day and women consuming 5-25 g/day will be given a value of 1. The score is categorized into three categories: low adherence, including individuals with a total score of 0 to 3 points; intermediate adherence, with a score of 4 to 5 points; and high adherence,with a score of 6 to 9 points.

Adherence to healthy dietary patterns will be assessed by the Dietary Approaches to Stop Hypertension (DASH) Index.

The DASH Index is comprised of 8 components (7 food groups and one nutrient). The scoring system is based on quintile rankings; for intakes of fruit (includes fruit juice), vegetables (excludes potatoes), low-fat dairy products, whole grains, and nuts, seeds, and legumes, individuals receive a score from 1 (lowest quintile) to 5 (highest quintile). In contrast, individuals receive a score from 1 (highest quintile) to 5 (lowest quintile) for intakes of sodium, sugar-sweetened beverages, and red and processed meat. Men and women are classified into quintiles separately. Component scores are summed to a total DASH score that ranges from a minimum of 8 to a maximum of 40 points.

Adherence to Mediterranean Diet will be assessed through the MEDI-Lite questionnaire.

Nutritional and energy requirements will be assessed considering the "Reference Intake Levels of Nutrients and Energy for the Italian Population for the Italian population" (LARN) recommendation using dietary records.

Nutritional and energy requirements will be assessed through the food frequency questionnaire European Prospective Investigation into Cancer and Nutrition (EPIC).

(Poly)phenol intake will be estimated using a food database. The same methodology will serve to calculate the intake of other plant bioactive compounds and dietary components associated with the gut microbiome, by matching food frequency questionnaires, and the dietary records data with databases.

General information, such as age, sex, smoking habits, childbirth, breastfeeding, will be assessed through an enrollment questionnaire.

Assessing the contribution of sleep quality through the questionnaire Pittsburgh Sleep Quality Index (PSQI). Scoring of answers is based on a 0 to 3 scale, whereby 3 reflects the negative extreme. A global sum of "5" or greater indicates a "poor" sleeper, while an overall sum of less than "5" indicates a "good" sleeper.

Assessing the contribution of sleep quality through the questionnaire Epworth Sleepiness Scale (ESS). The test is a list of eight situations in which it is rate the tendency to become sleepy on a scale of 0 to 3: 0 (no chance of dozing), 1 (slight chance of dozing), 2 (moderate chance of dozing), and 3 (high chance of dozing). The total ESS score is based on a scale of 0 to 24 and can be interpreted as follows: 0-5 Lower Normal Daytime Sleepiness; 6-10 Higher Normal Daytime Sleepiness; 11-12 Mild Excessive Daytime Sleepiness; 13-15 Moderate Excessive Daytime Sleepiness; 16-24 Severe Excessive Daytime Sleepiness

Assessing the contribution of sleep quality through the questionnaire Insomnia Severity Index (ISI).

Assessing the contribution of physical activity through the questionnaire International Physical Activity Questionnaire (IPAQ).

Assessing the contribution of state of health through the questionnaire Beck's Depression Inventory (BDI).

Assessing the contribution of state of health through the questionnaire Short Form Health Survey 36 (SF-36).

True internal exposure to dietary (poly)phenols will be assessed by analysing the circulating metabolites of (poly)phenols in the plasma, urine, and faecal samples collected. Validated targeted ultra performance liquid chromatography method coupled with triple quadrupole mass spectrometry (UPLC-QqQ-MS/MS) and untargeted Ultra Performance Liquid Chromatography-Ion Mobility Spectrometry-High-Resolution Mass Spectrometry (UPLC-IMS-HRMS) methods will be used.

True internal exposure to plant bioactive compounds will be assessed by analysing the circulating plant bioactive compounds (carotenoids, phytosterols, alkaloids (mainly purines and pyridines), thiosulfinates, and alkylresorcinols), in the plasma, urine, and faecal samples collected. Validated targeted ultra performance liquid chromatography method coupled with triple quadrupole mass spectrometry (UPLC-QqQ-MS/MS) and untargeted Ultra Performance Liquid Chromatography-Ion Mobility Spectrometry-High-Resolution Mass Spectrometry (UPLC-IMS-HRMS) methods will be used.

Metabolites playing a role in the diet-gut microbiome-host interactions will be evaluated in plasma/urine/faeces samples using targeted ultra performance liquid chromatography method coupled with triple quadrupole mass spectrometry (UPLC-QqQ-MS/MS) and untargeted Ultra Performance Liquid Chromatography-Ion Mobility Spectrometry-High-Resolution Mass Spectrometry (UPLC-IMS-HRMS) methods. Main compounds to be targeted are short-chain fatty acids, L-carnitine, choline, phosphatidylcholine, trimethylamine, trimethylamine N-oxide, tryptophan and indole derivatives, phenylalanine and phenylacetate, tyrosine derivatives, 4-ethylphenylsulfate, p-cresol-sulfate, and primary and secondary bile acids.

Multiple metabolites associated with the consumption of food groups and foodstuffs will be analysed in urine samples and, in case, in plasma, using ultra performance liquid chromatography method coupled with triple quadrupole mass spectrometry (UPLC-QqQ-MS/MS) methods, to provide objective information on participants' diet. Moreover, a comprehensive panel of biomarkers related to healthy dietary patterns will be analysed in plasma/urine samples by using Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) methods procedures reported in literature.

Urine/plasma/fecal samples will be subjected to untargeted Liquid Chromatography-Ion Mobility Spectrometry-Mass Spectrometry (LC-IMS-MS) metabolomics approach to assess potential differences among the metabolomes of individuals belonging to different aggregate phenolic metabotypes.

Whole Exome Sequencing (WES) will be conducted using an exome-wide sequencing approach, using short reads, able to detect gene variability across the exome. Genomic DNA will be prepared from blood samples.

Microbial profiling in faeces will be carried out by whole genome shotgun metagenomics analysis, for the sequencing of the entire bacterial DNA, aiming at greater accuracy and precision in taxonomic recognition and function inferences related to the microbiota.

Microbial profiling in saliva samples will be carried out by an internal transcribed spacer (ITS)-based method for cataloguing of bacterial communities at (sub)species level.

A panel of biomarkers related to the adipose tissue function (adiponectin, leptin, and resistin) will be evaluated in plasma samples.

A panel of biomarkers related to food intake regulation (ghrelin, glucagon-like peptide 1, cholecystokinin, gastric inhibitory peptide) will be analysed in plasma samples.

Insulin resistance corrected by adiponection (HOMA-AD)

HOMA2 will be used to determinate insulin sensitivity, insulin resistance, and beta cel function calculated by a computer model from C-peptide concentrations

C-reactive protein to triglyceride-glucose index (CRP-TyG); Systemic Immune-Inflammation Index (SII); Systemic Inflammatory Response Index (SIRI); INFLA-score; Neutrophil-to-Lymphocyte Ratio (NLR); Neutrophil-to-HDL Cholesterol Ratio (NHR); and Neutrophil-to-Albumin Ratio (NAR).

Adipose Tissue Insulin Resistance index (Adipo-IR)

Atherogenic Index of Plasma (AIP)

DNA methylation analysis of the samples will be performed after sodium bisulfite treatment of the extracted DNA and analysed using the Illumina Infinium methylation 450k bead chip arrays (Illumina) on the NextSeq550 analyser (Illumina). These arrays cover between up to 860,000 humans genomic CpG sites, including 99% of RefSeq genes and 96% of CpG islands. The resulting methylated and unmethylated signal intensity data will be normalized according to Illumina's background correction method using the minfi package in R. All the samples will be examined for genome-wide methylation density, and those deviating from a bimodal distribution will be excluded.

Telomerase analysis will be performed using qPCR to assess telomere length and telomerase activity strictly correlated with ageing.

Blood samples in tubes containing EDTA will be processed to obtain lymphomonocytes. Part of these will be stored at -80°C to allow subsequent transcriptomic or proteomic analysis, while the remainder will be stored in liquid nitrogen to allow in vitro use.

Taste system functionality is evaluated using the validated taste strips method. Participants identify the taste quality of filter paper strips impregnated with varying concentrations of taste solutions and rate the perceived taste intensity on a 10-point scale and the hedonic response on a 10-point hedonic scale.

Whole-gut transit time is assessed using the sunflower seed method: participants record the exact time elapsed between seed consumption and their first visual appearance in the stool.