Identification of new biomarkers for fruit and vegetable intake by NMR and LC-microTOF-Q based metabolomics

Fruit and vegetables are believed to be health protective against several diseases such as coronary hearth disease and cancer. Epidemiological studies can help establish the association between fruit and vegetable intake and development of such diseases, but an accurate estimation of fruit and vegetable intake is critical to gain more knowledge about this relationship. Food diaries, food frequency questionnaires, and dietary recalls represent traditional methods for dietary assessment, but all of these methods are associated with both random and systematic errors. Serum β-carotene, vitamin C and dietary polyphenols excreted in urine have shown to be valid biomarkers for fruit and vegetable intake, but more is needed. NMR and LC/MS based metabolomics are excellent tools to help detect differences in metabolites excreted in urine after high and no intake of fruit and vegetables. These metabolites could serve as new and better biomarkers for fruit and vegetable intake. The purpose with this project is to search for new biomarkers for fruit and vegetable intake in urine obtained from the ‘6-a-day’ study by the use of NMR and LC-micrOTOF-Q-MS based metabolomics. The ‘6-a-day’ study, conducted in 2000, was a 25-day intervention study with complete control of dietary intake. The study included 43 subjects assigned to 1 of 3 groups. All groups were on the same basic diet devoid of fruit and vegetables, but the fruit and vegetable group received 600 g fruit and vegetables; the placebo group received a placebo pill, and the supplement group received a vitamin pill designed to contain vitamins and minerals corresponding to those in the 600 g fruit and vegetables. Twenty-four-hour urine samples were collected the day before intervention (day 0), at 4 occasions during the intervention period, and at follow-up 4 weeks after the end of intervention. Promising preliminary results have already been obtained from NMR analysis of a subset of urine samples from the ‘6-a-day’ study. PCA plots from these results show that several, yet unidentified, metabolites could become future biomarkers for fruit and vegetable intake. Further compound identification and metabolomic analysis of the urine samples have been performed on a LC-micrOTOF-Q-MS system and data analysis has been conducted using PCA analysis in combination with specific sorting algorithms. Utilizing both NMR and LC/MS will provide more information than using either technique alone and strengthen the results. Details about the markers identified by NMR and LCmicrOTOF-Q-MS will be presented.