Identification of New Biomarkers of Banana and Tomato Intake

The intake of fruits and vegetables has been associated to a lower risk of developing metabolic diseases and cancer. The intake of tomato has been proposed to decrease the risk of prostate cancer while the high content of pro-vitamine A carotenes in banana have shown to alleviate Vitamin A deficiency in different countries. Interestingly in spite of their popularity, there are no biomarkers of banana intake reported in the literature while lycopene is the most frequently used metabolite to indicate tomato consumption however, its limited specificity and between-subjects variation sets doubt of its accuracy. Therefore, the identification of novel biomarkers for both banana and tomato is of great value. Untargeted metabolomics, allows a holistic analysis of the food metabolome allowing a deeper inquiry in the metabolism of different compounds and the recognition of patterns and individual differences that may lead to new hypothesis and further research. Therefore, the aim of the present study is to identify biomarkers of acute intake of banana and tomato using an untargeted approach on urine serum of 12 volunteers that participated in a crossover, randomized, controlled study. Volunteers consumed three different test foods: 1) 240g of banana, 2) 300g of tomato and 3) Fresubin 2kcal as control. Serum and urine samples were collected in kinetics over 24h and processed to be analyzed using LC-QTof analysis. The metabolomics profiles are compared using univariate (ANOVA) and multivariate statistical methods (PCA, PLSDA). The identification of discriminant compounds was performed by tandem mass fragmentation with a high-resolution LTQ-Orbitrab Mass spectrometer and by an extensive inquiry of different online databases.

The rise of metabolomics along with different platforms such as liquid chromatography mass spectrometers (LC-MS) have allowed the assessment of thousands of metabolites simultaneously in biological samples and the recognition of patterns that may constitute a fingerprint of the intake of different foods. Recent studies demonstrated the great potential of metabolomics to discover new biomarkers of intake in intervention and cohort studies.The diversity of compounds found in food metabolomics represents a major challenge and so in an international effort to improve dietary biomarkers identification and validation, the Food Biomarkers Alliance (FOODBALL) has been created. In this project, 22 institutions from 11 different countries will collaborate in three main tasks: 1) Discovery of new dietary biomarker using a metabolomic approach, 2) systemic validation of existing and newly discovered biomarker to achieve a good coverage of food intake in different European populations and 3) exploring biological effects using biomarkers of intake (http://foodmetabolome.org/). With the latter, the necessity of building a chemical library that allows the use of standards for further identification arises. Along with FOODBALL, The Food Compound Exchange (FoodComEx) aims to improve the availability of analytical standards of biological compounds to achieve a better and easier biomarker identification (http://foodcomex.org/).

As part of INRA collaboration to FOODBALL and FoodComEx, the present project attempts to identify biomarkers of banana and tomato intake, through the exploration of the serum and urine metabolome of 12 subjects who consumed these foods following a randomized, controlled, crossover design. The present study was comprised of 3 different intervention periods and a minimum of 3 days washout between interventions. The intervention periods were comprised of 2 run in days, 1 intervention day and 1 post intervention day. In the first day of the run in period, subjects were instructed to avoid the intake of banana or tomato or any of their products; the day prior to the intervention volunteers were asked to avoid the intake of phytochemical rich foods and beverages such as wine, coffee, chocolate, tea, and other plant based foods including banana and tomato.In the morning of the intervention, day subjects arrived in fasting state to the research center at 7.30 am. Volunteers were randomly assigned to one of the three interventions, Fresubin ® 2kcal fiber, 240g of banana plus control drink, or 300g of tomato plus control drink plus 12g of refined sunflower oil. Throughout the intervention, subjects had free access to water, maximum 250ml of water per hour until 6 hours after the intake of the test food.

A trained phlebotomist placed a catheter on the subject's arm before the intake of the test foods to collect the baseline sample. Then four other samples were collected postprandially after 1h, 2h, 4h, and 6h. A total of 7 urine samples were collected. The first void of urine was collected by the subjects at home upon the morning of Day 3 and the rest of the samples after the intake of the test foods as follows: 0-1h, 1h-2h, 2h-4h, 4h-6h. The urine samples corresponding to 6h-12h and 12-24h interval were collected by volunteers at home until the morning after the intake of the food.

After the 6h collection of blood, the peripheral catheter was removed and subjects had lunch composed of white bread and cooked pasta, then subjects were allowed to go home. Before leaving the Investigation center, participants were instructed to prepare a standardized dinner based on pan fried chicken with butter and boiled rice with salt. Volunteers were not allowed to eat or drink anything except water and the standardized dinner.

On the morning of the post intervention day, subjects arrived in fasting state to the research center to give the 24h blood sample and deliver the 06-12h and 12-24h urine collection. Afterward, subjects were served breakfast at the research center and resumed their normal diet until the next run in days of the next intervention period.

Urine samples and serum samples were aliquoted and stored at -80° C until analysis.

Eligibility Criteria:
Inclusion Criteria:

* Healthy males and females
* Aged 18- 40 years
* BMI \>18.5 and \< 30 kg/m2
* Willing/able to consume all test foods (tomato, banana, Fresubin drink) and the standardized meals (rice and chicken)

Exclusion Criteria:

* Smokers
* Diagnosed health condition (chronic or infectious disease)
* Taking nutritional supplements (e.g. vitamins, minerals) several times a week.
* Taking medication (oral contraceptive pill is allowed).
* Pregnant, lactating.
* Antibiotics treatment within 3 months prior to intervention.
* Vegetarians, as standardized meals will contain meat.
* Not willing to follow nutritional restrictions, including drinking alcohol during study days
* Not willing/able to give informed consent or to sign informed consent.
* Not affiliated to National Health Insurance.
* Being in exclusion on the National Volunteers Data file or refusing to be registered on the National Volunteers Data file.
* Currently participating or who having got 4500€ in this year to have participated in another clinical trial.