Metabolomics is a powerful technique which focuses in highthroughput

1. Diet absent of fruit-vegetables and food derived products Day 1 Day 2 Day 3 Day 4 200 mL 8:00 a.m 200 mL 8:00 a.m 200 mL 8:00 a.m 200 mL 8:00 a.m Wash out 2 days 200 mL 18:00 p.m 200 mL 18:00 p.m 200 mL 18:00 p.m 200 mL 18:00 p.m 24 h urine collection and frozen at -80º C Databases of compounds and MS/MS pattern Future identification of metabolites Significative biomarker changes after aronia juice intake by human volunteers using metabolomics tools Medina, S.1; García-Viguera, C.1; Ferreres, F.1; Savirón, M. 2; Orduna, J. 2; Gil-Izquierdo, A.1* 1Research Group on Quality, Safety and Bioactivity of Plant Foods, CEBAS-CSIC, Murcia, Spain 2Instituto de Ciencias de Materiales de Aragón. CSIC- Universidad de Zaragoza, Zaragoza, Spain *corresponding author: angelgil@cebas.csic.es Introduction Results Data LC-MS from treatment samples (after aronia juice intake: ) and control samples (before aronia consumption: O) were analyzed with multivariate statistical analysis, PCA (Principal Component Analysis) and Student´t t-test (Figure 1). In this analysis six hundred masses were aproximately generated of which six were selected as the most significative ones (three in positive mode and three in negative mode) (P-value <0.05) (Table 1 and 2). We have chosen the ion at m/z 581.2308 as an example to show the score and loading plot. In these graphs, the separation between treatment and control groups can be displayed showing an interesting dispersion in the loading plot. It was observed that aronia juice had an effect on the intensity of six metabolites. Some ions increased due to aronia juice consumption (e.g. 470.1343 m/z), and others reduced their intensity (e.g. 373.2671 m/z) (Figure 2). After statistical analysis, the signicative ions were processed by SmartFormula (Figure 3). These molecular formula of the target features were generated based on the MS data. Metabolomics is a powerful technique which focuses in highthroughput characterization of metabolome (the complete set of low-MW metabolites in biological samples) in order to obtain a molecular profile. This approach has shown a considerable potential in the field of nutrition, so, there is an increasing interest in the use of metabolomics as a tool for the understanding of the interaction between diet and metabolome and to be a promising technique for biomarker discovery related to food intake [1]. Our study was conducted with the aronia fruit (Aronia melanocarpa) common name black chokeberry which belongs to the Rosaceae family. This berry stands out in high amount of flavanols, quercetin (71.13 mg Kg-1; 93.07%) and kaempferol (5.3 mg Kg-1; 6.9%). Aronia berries are one of richest plant sources of anthocyanins, mainly containing cyanidin glycosides [2]. In a recent study, anthocyanins showed significant potency of antiobesity and ameliorate adipocyte function in vitro and in vivo systems, as well as, antioxidant, hypoglycemic and hypolipidomic activities and important implications for preventing metabolic syndrome of a chokeberry juice, indicate their contribution to the potential health benefits [3,4]. The aim of this study was to observe the single and five days changes in the metabolic profile of healthy volunteers following the consumption of aronia juice and to identify the key metabolites. Table 1. Significative features in positive mode. Table 2. Significative features in negative mode. Figure 1. PCA (Pareto scaling algorithm) score and loading plot. a) b) Assay design Number of patients: 8 (4 men and 4 women) Age: 28-47 Race: Caucasian BMI means: 24.5 Kg/m2 Figure 2. Intensity of metabolites in positive mode (a) and in negative mode (b). • Workflow for metabomic analysis: Figure 3. SmartFornula result of formula for bucket 11.5 min; 581.50 m/z. Urine samples (treatment and control) Conclusions Multivariate statistical analysis PCA (Principal Component Analysis)with Pareto scaling algorith and Student´s t-test (P-value <0.05 was considered significant) Data adquisition LC-MS-q-TOF (Bruker Daltonics, Bremen, Germany) Column ACE 3 C18: 150 x 0.075 mm Fase A: H2O/0.1% HCOOH Fase B: ACN/0.1% HCOOH Flow rate: 312.50 nL/min Injection volume: 6.25 nL Positive and negative mode Full scan: 50-900 m/z Data preprocessing Profileanalysis 2.0. (Bruker Daltonics, Bremen, Germany) Software for profiling and statistical evaluation of LC/MS data set. • Metabolomics approach with HPLC-q-TOF analysis is a powerfull tool for the discovery of metabolic changes in nutritional interventions with humans. • Four days of aronia juice intake alters the urinary metabolic profile. • New markers of aronia juice consumption were isolated. • Metabolomics can be used to distinguish the group consuming the aronia juice, moreover it can be able to separate of control group. Biomarker discovery References Acknowledgement [1].Lodge, K. 2010. Proceeding of the Nutrition Society. 69, 95-102. [2]. Jakobek, L.; Seruga, M.; Medvidovic-Kosanovic, M.; Novak, I. 2007. Agriculturae Conspectus Scientificus. Vol 72, No 4: 301-306. [3]. Tsuda, T. 2008. J. Agric. Food Chem. 56, 642-646. [4]. Valcheva- Kuzmanova, S.; Kuzmanov, K.; Tancheva, S.; Belcheva, A. 2007. Methods and Findings in Experimental and Clinical Pharmacology, 29, 101-105. This work has been fulfilled by financial support of the Spanish CICYT (AGL2007-61694) and CONSOLIDER-INGENIO 2010 (CSD2007-00063) projects. SM wish to thank also Spanish CSIC for PhD fellowship grant, partially funded by the European Social Fund. Authors are grateful to Hero España, S.A for supplying the aronia juice.