Bioactives Lead to Enhanced Disease defence

Rationale:
People who eat sufficient amounts of fruits and vegetables as part of their daily diet have a reduced risk of a number of chronic diseases, such as cancer, cardiovascular disease and type II diabetes mellitus. There is ample scientific evidence suggesting that these health benefits are the consequence of the combined action of different phytochemicals present in fruits and vegetables. However, the number of studies in humans is limited. More research is needed to unravel the most optimal combination of phytochemicals in fruits and vegetables that can protect against disease risk. Furthermore, the underlying mechanisms by these phytochemicals exert their effect remain unclear. In addition, people might respond differently to dietary changes due to their genetic make-up. It was shown that subjects with specific genetic characteristics may benefit more from certain combinations of phytochemicals than others. More combinations of fruits and vegetables should be tested in humans with different genetic characteristics at the level of appropriate phenotypic markers of effect, including whole genome gene expression changes in order to provide insight into the underlying molecular mechanisms. Therefore, the aim of the present human dietary intervention study is to evaluate the effect of various combinations of vegetables and fruit containing different complex mixtures of phytochemicals in healthy volunteers on the level of phenotypic markers of disease risk in combination with whole genome gene expression analyses, and taking genetic variability, based on a selection of genotypes, between subjects into account.

Objective:
The main objective of the human dietary intervention study is to investigate the beneficial health effects of food products containing various combinations of an equivalent of 400 grams vegetables and fruits in healthy volunteers. This will be evaluated in different subgroups with specific genetic characteristics on the level of different phenotypical markers, combined with gene expression profiling.

Study design:
This human dietary intervention study has a randomized controlled cross-over – repeated measures design, including only healthy volunteers. After a two week run-in period, subjects will be randomized into one of the 9 study groups. Each group will follow two intervention periods of two weeks in which at each period one of nine different food product containing different combinations of vegetables and fruits will be consumed, separated by a one week washout period. At baseline and after each intervention period, blood and urine is collected, and a photograph is taken from the fundus of the right eye for analyses of markers of oxidative stress, DNA damage, biomarkers of cardiovascular disease risk and type II diabetes, gene expression analyses and determination of genetic polymorphisms.

Study population:
All subjects will be recruited at the University of Maastricht (UM) in the province of Limburg, the Netherlands, using advertisements, flyers as well as other (social) media. Healthy subjects of both sexes will be selected based on predefined inclusion criteria (BMI: 18-27; 18-60 years of age) and exclusion criteria, and randomly assigned to one of the different experimental groups.

Intervention:
Nine different food products containing different combination of fruits and vegetables will be evaluated in this human dietary intervention study. Seven of these 9 different food products consist of a smoothie, containing 400 grams of vegetables and fruits which will be consumed during the day: Smoothies 1-4 contain a specific selection of vegetables and fruit resulting in an overrepresentation of a specific class of phytochemicals. Smoothies 5-7 will consist of a combination of the four different classes overrepresented in smoothies 1-4, with increasing biodiversity. Food product 8 and 9 will consist of a pearl which is a crouton-like product, consisting of a core of oats, and will be either coated or non-coated with the most diverse mixture of vegetables and fruits as used in smoothie number 7. Each subject will be randomized into one of the nine study groups, which will start of a two week run-in period followed by two intervention periods of two weeks separated by a washout-period. At each intervention period, one of the nine different food products will be consumed. At baseline, and after each intervention period, blood and urine will be collected, as well as a photographic image of the fundus of the right eye will be taken.

Main study parameters/endpoints:
- To measure differences at the level of different phenotypic markers of disease risk: oxidative DNA damage in ex-vivo treated lymphocytes, antioxidant capacity of blood plasma, reactive oxygen species levels in blood, excretion of lipid peroxidation products in urine, plasma levels of different markers for cardiovascular disease and type II diabetes, and changes in the retinal microvasculature.
- To measure whole genome gene expression analyses in order to provide more insight in to the underlying molecular mechanisms. Genes and involved molecular processes associated with the measured phenotypic markers will demonstrate a causal relationship between the particular intervention and the markers of disease risk.
- To measure the occurrence of different polymorphisms related to disease risk in order to identify particular subgroups that will benefit more from a particular intervention.

Nature and extent of the burden and risks associated with participation, benefit and group relatedness:
Healthy volunteers will have to sign an informed consent and must follow a strict diet in consultation with a dietician. The food products contain different combination of vegetables and fruits, at the level of recommendations, which are freely available and are considered to be healthy. Participants will have to come to the university 8 times. The first visit only comprises signing of the informed consent and oral explanation of the study design by the principle investigator. At baseline, and after the first and second intervention period, subjects will come to the university after an overnight fast and donate urine and blood. Also a photograph of the fundus of the right eye is taken. In total, subjects will have to collect three times 24 hour urine, at 3 times 34 mL of blood divided over 4 vacutainers will be drawn, and 3 images of the fundus of the right eye will be taken. The other visits will be used for picking up of the food products. The risk of collection of these samples is considered to be minimal. During the whole study period, subjects have to keep track of their diet using a food diary.

We hypothesize that consumption of mixtures of 400 grams vegetables and/or fruits for 2 weeks will result in lower ex-vivo induced oxidative DNA damage, increased antioxidant capacity of blood plasma, reduced levels of reactive oxygen species in whole blood, reduced excretion of 8-iso-prostaglandin F2 in urine, changes in microcirculation I.e. wider retinal arterioles and narrower venules, and changes in the expression of genes in such a way that the involved molecular processes will be positively modulated thereby contributing to prevention of chronic disease risk.
We hypothesize that subgroups with different genetic characteristics based on a selection of genotypes, respond differently to the intervention by establishing the impact of different polymorphisms related to disease risk (cancer, diabetes type II, cardiovascular disease) and metabolisms of bioactive compounds.

There are three timepoints:
1) pre-test: after two week run-in period
2) post-test 1: after the first intervention period of two weeks
3) post-test 2: after the second intervention period of two weeks

A dietary intervention study will be performed to establish the beneficial health effects of 9 different food products, produced by MiFood (a company involved in food production industry (www.mifood.nl)), containing various combinations of phytochemicals from different combinations of vegetables and fruits. Seven of these 9 different food products will consist of a smoothie, containing different combinations of vegetables and fruits. Smoothies 1-4 will contain a specific selection of vegetables and fruit resulting in an overrepresentation of a specific class of phytochemicals. Smoothies 5-7 will consist of a combination of the four different classes overrepresented in smoothies 1-4, with increasing botanical diversity. The remaining two dietary interventions will test a crouton (also called pearl), consisting of a core of oats and rice flour, either coated or non-coated with the most diverse mixture of vegetables and fruits as used in dietary intervention number 7.