Mediterranean diet effects on vascular health and serum levels of adipokines and ceramides

Background A randomized clinical trial to evaluate the effect of a Mediterranean-style diet on vascular health indices such as endothelial function indices, serum lipid and ceramide plasma and some adipokine serum levels. We recruited all consecutive patients at high risk of cardiovascular diseases admitted to the Internal Medicine and Stroke Care ward at the University Hospital of Palermo between September 2017 and December 2020. Materials and methods The enrolled subjects, after the evaluation of the degree of adherence to a dietary regimen of the Mediterranean-style diet, were randomised to a Mediterranean Diet (group A) assessing the adherence to a Mediterranean-style diet at each follow up visit (every three months) for the entire duration of the study (twelve months) and to a Low-fat diet (group B) with a dietary "counselling" starting every three months for the entire duration of the study (twelve months).The aims of the study were to evaluate: the effects of adherence to Mediterranean Diet on some surrogate markers of vascular damage, such as endothelial function measured by means of the reactive hyperaemia index (RHI) and augmentation index (AIX), at the 6-(T1) and 12-month (T2) follow-ups; the effects of adherence to Mediterranean Diet on the lipidaemic profile and on serum levels of ceramides at T1 and T2 follow-ups; the effects of adherence to Mediterranean Diet on serum levels of visfatin, adiponectin and resistin at the 6- and 12-month follow-ups. Results A total of 101 patients were randomised to a Mediterranean Diet style and 52 control subjects were randomised to a low-fat diet with a dietary "counselling". At the six-month follow-up (T1), subjects in the Mediterranean Diet group showed significantly lower mean serum total cholesterol levels, and significantly higher increase in reactive hyperaemia index (RHI) values compared to the low-fat diet group. Patients in the Mediterranean Diet group also showed lower serum levels of resistin and visfatin at the six-month follow-up compared to the control group, as well as higher values of adiponectin, lower values of C24:0, higher values of C22:0 and higher values of the C24:0/C16:0 ratio. At the twelve-month follow-up (T2), subjects in the Mediterranean Diet group showed lower serum total cholesterol levels and lower serum LDL cholesterol levels than those in the control group. At the twelve-month follow-up, we also observed a further significant increase in the mean RHI in the Mediterranean Diet group, lower serum levels of resistin and visfatin, lower values of C24:0 and of C:18:0,and higher values of the C24:0/C16:0 ratio. Discussion The findings of our current study offer a further possible explanation with regard to the beneficial effects of a higher degree of adherence to a Mediterranean-style diet on multiple cardiovascular risk factors and the underlying mechanisms of atherosclerosis. Moreover, these findings provide an additional plausible interpretation of the results from observational and cohort studies linking high adherence to a Mediterranean-style diet with lower total mortality and a decrease in cardiovascular events and cardiovascular mortality. Trial registration ClinicalTrials.gov Identifier: NCT04873167. https://classic.clinicaltrials.gov/ct2/show/NCT04873167.

However, the need for additional predictive biomarkers is underlined by the very substantial residual risk that exists above the standard clinical and biochemical risk predictors.Ceramides are members of the sphingolipid family and precursors of complex sphingolipids.Some studies in cultured cells and animal models repprted that the accumulation of ceramides may lead to the activation of pathologifc pathways altering normal cellular function, including the action of insulin Thus the excess de novo ceramide biosynthesis may be due to cellular stress stimuli, suche as high serum levels of saturated free fatty acids.Ceramide and its metabolites represent an intermediate link between over-nutrition and the pathogenetic basis of the cardiometabolic disease risk, including insulin resistance and low-grade inflammation.Thus it appears worthy of interest the evaluation of the role of serum ceramide leves as a expression of the interplay between lipidaemic pathways and vascular health status.
Adipocytes produce some hormones that directly interplay with the local microenvironment as well as distant tissues.Thus adipocytes have a possible pathogenetic role in the development of cardiometabolic diseases.Some adipokines produced by adipose tissue have been reported to be linked to inflammation and atherosclerosis and its related cardiovascular complications .
Few studies have prospectively analyzed the association between ceramides and the incidence of cardiovascular and cerebrovascular events.Laaksonen et al. reported a not well clear relationship of plasma ceramides with death from cardiovascular disease (CVD).It has been also suggested a possible role of the ratio of two ceramides as a possible predictive factor cardiovascular risk .The PREDIMED study suggested that dietary interventions according a Mediterranean-style diet positively modified the association between a blood ceramide score (comprised of individual ceramides and ceramide ratios) and risk of incident CVD.Furthermore, The CORDIOPREV in patients with established coronary heart disease randomly to receive a Mediterranean diet or a low-fat diet intervention, with a follow-up of 7 years analyzed the primary outcome of a composite of major cardiovascular events, including myocardial infarction, revascularisation, ischaemic stroke, peripheral artery disease, and cardiovascular death.Authors concluded that in secondary prevention, the Mediterranean diet was superior to the low-fat diet in preventing major cardiovascular events.
Nevertheless no study have evaluated in the same study the effects of Mediterranean Diet on endothelial function in parallel to its effects on serum lipid change including serum ceramide changes and on some adipokine serum pathway.
On this basis we designed a randomized trial to evaluate the effects of Mediterranean diet on endothelial function measured by the evaluation of reactive hyperaemia index (RHI) and the diet effects on serum ceramide and adipokines levels.

Patients and recruitment consecutive patients at high risk of cardiovascular diseases admitted to the Internal Medicine and
Stroke Care ward at the University Hospital of Palermo between September 2017 and December 2020 will be enrolled The enrolled patients will be male subjects aged between 55 and 80 and female subjects between the ages of 60 and 80 who tested positive for at least two of the following eligibility criteria thus to be classified as subjects at high cardiovascular risk 1) Type 2 diabetes mellitus 2) Arterial hypertension 3) Body mass index (BMI) ≥ 25 4) Active smoking 5) Family history of early cardiovascular disease 6) Previous cardiovascular or cerebrovascular events (> 6 months)

Exclusion criteria
All patients with recent (<6 months) cardiovascular or cerebrovascular events were excluded

Randomization
The enrolled subjects, after the evaluation of the degree of adherence to a dietary regimen of the Mediterranean-style diet, were randomised to two different types of dietary schemes: 1. Group A (experimental arm) -Mediterranean Diet : adherence to a Mediterranean-style diet was assessed through dietary screening at each follow up visit (every three months) for the entire duration of the study (twelve months).
2. Group B (control arm) -Low-fat diet: for which the enrolled subjects received dietary "counselling" starting from their first visit at the time of enrolment and, subsequently, every three months for the entire duration of the study (twelve months).
The randomization code was based on computer-generated random numbers and a 2:1 randomization ratio for experimental/control arm was performed both to contain the high cost of clinical investigations to be performed, and to avoid the effects of a high drop out rate by allowing more power for a per-protocol analysis.As a consequence of the type of randomization chosen, in order to maintain the power of the study, an increase in sample size of 12% was planned.
The investigators involved in clinical data collection and the measurement of outcome variables were not directly involved in the patients' treatment and were masked to the randomization process.The randomization code was maintained only at the central data facility and was not broken until all data analysis was complete

Dietary Intervention Trial
We will adopt the dietary intervention protocol with regard of Mediterranean Diet and low-fat diet validated by the PREDIMED trial (5).

A. Mediterranean Diet :
The general guidelines to follow the Mediterranean diet that researchers will provide to participants included the following positive recommendations: a) abundant use of olive oil for cooking and dressing dishes; b) consumption of ≥ 2 daily servings of vegetables (at least one of them as fresh vegetables in a salad), discounting side dishes; c) ≥ 2-3 daily servings of fresh fruits (including natural juices); d) ≥ 3 weekly servings of legumes; e) ≥ 3 weekly servings of fish or seafood (at least one serving of fatty fish); f) ≥ 1 weekly serving of nuts or seeds; g) select white meats (poultry without skin or rabbit) instead of red meats or processed meats (burgers, sausages); h) cook regularly (at least twice a week) with tomato, garlic and onion adding or not other aromatic herbs, and dress vegetables, pasta, rice and other dishes with tomato, garlic and onion adding or not aromatic herbs.This sauce is made by slowly simmering the minced ingredients with abundant olive oil.Negative recommendations are also given to eliminate or limit the consumption of cream, butter, margarine, cold meat, pate, duck, carbonated and/or sugared beverages, pastries, industrial bakery products (such as cakes, donuts, or cookies), industrial desserts (puddings, custard), French fries or potato chips, and out-of-home pre-cooked cakes and sweets.The researchers insisted that two main meals per day should be eaten (seated at a table, lasting more than 20 minutes).For usual drinkers, the advice was to use wine as the main source of alcohol (maximum 300 ml, 1-3 glasses of wine per day).If wine intake was customary, a recommendation to drink a glass of wine per day (bigger for men, 150 ml, than for women, 100 ml) during meals was given.Ad libitum consumption was allowed for the following food items: nuts (raw and unsalted), eggs, fish (recommended for daily intake), seafood, low-fat cheese, chocolate (only dark chocolate, with more than 50% cocoa), and whole-grain cereals.

B. Control diet group. A low-fat, high complexcarbohydrate diet, as recommended by the National
CholesterolEducation Program, with <30% of total calories from fat (12 -14%MUFAs, 6-8% PUFAs, < 10% SFAs), 55% from carbohydrates and 15% from protein.In both diets, the cholesterol content was adjusted to<300 mg/day (6).The focus in the control group was to reduce all types of fat, with particular emphasis in recommending the consumption of lean meats, low-fat dairy products, cereals, potatoes, pasta, rice, fruits and vegetables.

Evaluation of adherence to the Mediterranean and to the low-fat diet
For participants in the Mediterranean-diet group, researcher will undertake individual and group dietary-training sessions at the baseline visit and every three months thereafter.In each session, participants completed a 14-item dietary questionnaire to assess adherence to the Mediterranean diet so that personalized advice could be provided to the study participants in these groups.
Information relating to the dietary habits of the enrolled patients will be collected through a specific questionnaire (semiquantitative food frequency questionnaire (F.F.Q.s) (17) dedicated to the evaluation of the frequency of different food items in the diet and adapted to the Sicilian population.
The subjects enrolled were classified based on the levels of adherence to a Mediterranean-style diet according to the methods proposed by Trichopoulou et al. (18).
The patients enrolled in the study will be assess for their adherence to a dietary regimen of the Mediterranean-style diet using the Mediterranean Diet Score, and the quantity and frequency of consumption of the food items characterising a Mediterranean-style diet were evaluated.
The consumption of foods presumed to be far from this diet pattern (i.e., rare or monthly consumption, meat and meat products, poultry and complete fat products) will be assign scores on an inverse scale.
A value of 0 or 1 will be assign to each indicated food component.For the beneficial components, patients whose consumption was below the median were assigned a value of 0. For components presumed to be detrimental, patients whose consumption was below the median were assigned a value of 1.
We will apply a non-monotonous function for the alcohol, i.e., score 5 for the consumption of less than 300 ml of alcohol per day, score 0 for no consumption or for the consumption of > 700 ml per by Sandwich ELISA (visfatin Phoenix Pharmaceuticals Inc.); the minimum detectable concentration for visfatin was 1.8 ng/ml.

Endothelial function evaluation
The pulse amplitude tonometry (PAT) probe will be placed on one finger of each of the two hands.
After 5 minutes of control measurement, the pressure cuff was inflated to 200 mmHg for 5 minutes to induce reactive hyperaemia and then deflated.
The RH-PAT will be analysed at enrolment and at the 6-and 12-month follow-ups.Measurement was made digitally using the Endo-PAT2000 software version 3.0.4device.The RH-PAT index reflects the extent of reactive hyperaemia.It will be calculated as the ratio of the average of the PAT signal amplitudes above the first minute of initial measurement to 1.5 minutes of measurement following deflation of the cuff (A: Control Arm; C: Occluded Arm) divided by the average of the PAT signal amplitudes over 2.5 minutes before inflation of the pressure cuff (B: Control Arm; D: Occluded Arm).This RH-PAT index, called the reactive hyperaemia index (RHI), is expressed by the formula RHI = (C/D)/(A/B) x basal correction.

Study samples and metabolomics profiling
All analyses will be performed by fasting (fasting for ≥8 hours) plasma EDTA samples collected at baseline and year 1.All samples will be processed at each recruiting centre no later than 2 hours after collection and stored in −80 °C freezers.Samples from cases and controla were randomly distributed before being shipped to the laboratory for metabolomics assays.LC-MS techniques were used to quantitatively profile ceramides in plasma samples.Plasma ceramide metabolites were measured concurrently with other lipid metabolites on the same platform and were identified based on total acyl carbon.Plasma ceramide concentrations (µg/mL) were assayed using a validated LC-MS/MS protocol at enrolment and at the 6-and 12-month follow-ups.