Metabolism and bioavailability of flavanones after administration of normal and functional citrus juices to healthy human volunteers. Medina, S.1; García-Viguera, C.1; Ferreres, F.1; Pineda, J.L.2; García-Mora, P.1; Gil-Izquierdo, A.1* 1Research Group on Quality, Safety and Bioactivity of Plant Foods, CEBAS-CSIC, Murcia, Spain. 2Laboratorio Químico-Microbiológico, S.A., San Ginés, Murcia, Spain *corresponding author: angelgil@cebas.csic.es Results Introduction The bioavailability and metabolites of flavanones were compared in urine from human volunteers consuming citrus juices and citrus juices functionalised with aronia juice. Several changes in the qualitative and quantitative profile were detected between two treatments. Volunteers consuming aronia-citrus juice showed more variety of flavanones metabolites in urine than those ingested citrus juice and the rate “number of volunteers/flavanone metabolites” was higher in those that ingested aronia-citrus juice than in the ones that consumed the citrus juice (Figure 1). Citrus juices exhibit a high level of consumption per capita. These juices can be highlighted by their high content of flavonoids, especially flavanone and flavone glycosides. Extensive in vivo and in vitro esperiments of flavanones showed beneficial health activities as protective agents against cancer and cardiovascular, inflammatory and allergic disorders [1]. From a nutritional point of view, citrus juices intake implies the cleavage of the rahmnoglycosides bonds of the flavanones to be absorbed in the colon. Part of these deglycosylated flavonoids are glucuronidated at the liver or at the intestinal lumen. In the gut, they urdago the ring fision by the colonic bacteria and are catabolised up to a wide variety of phenolic acids [3,4]. In general, the bioavailability of dietary phytochemicals may be influeced by the food matrix, however, in particular, in case of citrus flavanones the hydrolysis by the gut microbiota probably constitures the limiting step of their absorption. Another source of variation in the absorption can be the differences in the composition of enzymes and transporters in the intestinal epithelial cells, equally the age is also a possible effect on urinary excretion of flavanones, all them are inter-individual variations on the bioavailability of citrus flavanones [2]. The aim of this study was to observe the changes in the qualitative profile and bioavailability of flavanones in urine of healthy volunteers following the consumption of citrus juices and citrus juices functionalised with aronia juice. Figure 1. Rate number of volunteers/flavanone metabolites in citrus juice and aronia-citrus juice. Figure 2. Aglycones 72% 46.5% The intake of aronia-citrus juice by human volunteers provided an increase of 30 % and 72 % of hesperetin-7-O-glucuronide and hesperetin in raw urine, respectively compared to the concentration detected in urine of human volunteers who consumed the citrus juice (μmol/24 h). Figure 2 and 3. Material. To develop the study a citrus juice and citrus-aronia functional juice were used. Both juices were kept in darkness at 4º C, before administration to the subjects. Volunteers. Number of patients: 8 (4 men and 4 women) Age: 28-47 Race: Caucasian BMI means: 24.5 Kg/m2 30 % Figure 3. Wash out 2 days Diet absent of fruit-vegetables and food derived products 24 h urine collection and frozen at -80º C 200 mL 8:00 a.m 200 mL 18:00 p.m Control urine collection under fasting conditions (7:30 a.m) Week 1 Week 2 Thirty seven compounds were detected with MRM transitions of each metabolites and a specific retention time (Table 1). Conclusions Volunteers consuming aronia-citrus juice showed more variety of flavanones metabolites in urine than those ingested citrus juice. The intake of aronia-citrus juice by human volunteers provided an increase of 46.5% and 72% of total aglycones and hesperetin in raw urine respectively. Hesperetin-7-O-glucuronide has increased a 30% in volunteers after aronia-citrus juice consumption compared to the concentration detected in urine of human volunteers who consumed the citrus juice (μmol/24h). The rate “number of volunteers- flavanones metabolites” was higher in those who ingested aronia-citrus functional juice. Workflow for analysis: Urine samples Data adquisition. Agilent HPLC coupled an Agilent 6460 triple quadrupole mass spectrometer. HPLC column YMC (250 x 2.1 mm, 3 μm particle size). Flow rate: 200 μL/min. MRM (Multiple reaction monitoring) Full scan: 50-900 m/z in positive and negative mode. Data processing. MassHunter software (Agilent) The samples were centrifuged at 12000 rpm for 5 minutes. References [1]. Gil-Izquierdo, A.; Riquelme, M.T.; Porras, I.; Ferreres, F. 2004. J. Agric. Food Chem. 52, 324-331. [2]. Manach, C.; Morand, C.; Gil-Izquierdo, A.; Bouteloup-Demange, C.; Rémésy, C. 2003. Eur. J. Clin. Nutr. 57, 235-242. [3]. Lafay, S.; Gil-Izquierdo, A. 2008. Phytochem. Rev. 7, 301-311. [4]. Crozier, A.; Jaganath, I.B.; Clifford; M.N. 2009. Nat. Prod. Rep. 26, 1001-1043. Acknowledgement. Authors are grateful to CONSOLIDER-INGENIO 2010 “Fun-C-Food” and the Spanish CICYT Project number AGL2007-67694/ALI. S. Medina is holder of a JAE-CSIC fellowship.