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1 , 1 , 2 , 3 , 2 , 4 , * 1
1  Laboratory of Analytical Chemistry, School of Chemistry, Aristotle University of Thessaloniki, University Campus 54124 Thessaloniki
2  Laboratory of Forensic Medicine and Toxicology, Medical School, Aristotle University of Thessaloniki, University Campus 54124 Thessaloniki
3  Laboratory of Animal Physiology, School of Veterinary Medicine, Aristotle University of Thessaloniki, University Campus 54124 Thessaloniki
4  Department of Chemistry, University of Cyprus, P.O.Box 20537, 1678 Nicosia, Cyprus


   Ceratonia siliqua L. Fabaceae, commonly known as the carob tree, is native to the eastern Mediterranean countries and its products are widely used in the diet of people living in Mediterranean Europe, Middle East and North Africa. Carobs are considered to be of high nutritional value, as they are virtually fat-free, rich in proteins, antioxidants, vitamins and contain several important minerals. Different types of carob products are available in the local market, such as carob syrup, powder, flour, snack, cream, etc. However, the potential positive health effects of carob-containing products are largely unknown and have not been extensively studied. The aim of this study was to determine significant urine and fecal metabolome alterations in 8 rats treated with carob powder for 15 days as compared to 8 non-treated ones (controls) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and to underly specific metabolites that changed according to the treatment.
   Urine and fecal samples were collected in five time points during a 15 day period of treatment with carob powder throughout water consumption (10 g powder / L). A targeted HILIC-UPLC-MS/MS method was applied for the determination of 101 polar metabolites (sugars, amino acids, organic acids, amines, etc) in a single run of 40 min in both rat urine and feces. Chromatographic separation was performed on an Aquity BEH amide column (2.1 x 100 mm, i.d. 1.7 μm); the mobile phase was consisted of A: Acetonitrile:H2O 95:5 v/v (+ 10 mM ammonium formate) and B: H2O:Acetonitrile 70:30 v/v (+10 mM ammonium formate). The solvents flow rate was set at 0.5 mL/min. Mass spectrometry parameters were optimized for each of the 101 pre-selected analytes.
   Approximately 55 urinary and fecal metabolites were identified in both specimens. Data were further processed with multivariate (SIMCA 13) and univariate statistics (ANOVA). The differentiation of treated rats and controls was highlighted using discriminant multivariate models. 

Acknowledgements: The authors would like to thank the “Black Gold” project financially supported by the University of Cyprus.

Keywords: targeted metabolomics, carob, rat, urine, feces, LC-MS/MS
Comments on this paper
Clare Daykin
Thank you for submitting your presentation to the conference. I have been involved in nutritional metabolomics myself for almost 20 years now, so understand some of the issues that can be faced in such studies, especially when inter-individual differences are large and biological effects of nutrition are very small. I wonder if you could explain how you standardised the amount of carob consumed by the carob treated rats?

Thank you.
Olga Begou
We preferred to follow a mild, non invative protocol to administrate carobs in rats and thus we selected to prepare drinking solution enriched with carob. Also we monitored food consumption and rat weight and evaluted each rat separately via multivariate statistical analysis in which each sample contribute to the differentiation. Furthermore, we added same volume of the drinking solution every day up to 750 ml. The aninals were allowed to drink ad libitum.

Thank you for the interesting question.
Clare Daykin
Thank you for your reply. Can you therefore be sure that any metabolic changes observed were a direct result of ingestion of carob, or is it possible that there was a difference in the amount of water consumed between the groups (or other unidentified factors) and this contributed to the metabolic changes observed?

I also wonder what precautions did you take to avoid over-fitting of your data? The number of rats per group is very small, whilst the number of metabolites profiled is very large, leading to a high risk of observing chance correlations. Will you perhaps be carrying out a larger scale study to see if your prelimiary results can be replicated?
Olga Begou
In order to standarize the exact amount of carob that rats consume, the only way is force-feeding. The use of gavage requires delicate animal handling and there is a great risk of animal's loss. Regarding the differentiation of treated rats versus controls, the only parameter changed between the two groups, was carob treatment. Fecal sample is an ideal specimen reflecting the alteration of the treated rat's metabolome. The presented results are preliminary and untargeted metabolic profiling will follow.

Another great issue is undoubtedly data processing. A larger sample balk could lead to more powerful models, but a larger number of rats enhance the cost and the requirements. The obtained data were subjected in both univariate and multivariate analysis and validated with the available tools.

Thank you.