LC-HR-MS/MS based untargeted metabolomics for urinary biomarker discovery of chronic high and low flavonoid fruit and vegetables intake in humans

LC-HR-MS/MS BASED UNTARGETED METABOLOMICS FOR URINARY

BIOMARKER DISCOVERY OF CHRONIC HIGH AND LOW FLAVONOID

FRUIT AND VEGETABLES INTAKE IN HUMANS.

Flavonoid-poor F&V

Flavonoid-rich F&V

+2

+2

+ 4

+ 6

Habitual

diet

?

+ 4

+ 6

To determine the impact of diets enriched in fruit and vegetables (F&V), 2, 4 and 6 portion increases for consequative 6 week intervals, on urine metabolite profiles in people at risk of cardiovascular disease (CVD) (The FLAVURS study, University of Reading).

Colour scale: exponential 1e+08 5e+07 1e+07 5e+06 1e+06 5e+05 1e+05 50000 10000 5000 1000 500 100 50 10 5 1 0 200 400 600 800 1000 10 0 20 0 30 0 40 0 50 0 60 0 70 0 m/ z DATA ANALYSIS:

Data processing - XCMS using the

“matchedFilter” peak picking

method with Spectra Filter

Window Mower function.

For each mass feature two linear mixed models were fitted, diet-time interaction and diet-time alone.

METHODS:

•LTQ FT Orbitrap MS with Dionex HPLC system.

•Chromatographic separation: Kinetex C18 column (150 mm × 2.1 mm I.D., 3.5 um).

•Positive and negative ionization mode, full scan (Res.30000), 3 consecutive MS/MS scan events (res.7500)

STUDY DESIGN:

• Randomised, controlled, dose-dependent, parallel study

• Sequentially increasing doses of high flavonoid (HF) or low flavonoid (LF) F&V, compared to control habitual diet (CT).

•Male/female with a >1.5-fold increased risk of CVD (n = 174)

Both models were adjusted for baseline. p values for all features were corrected for multiple testing according to the two-stage Benjamini and Hochberg step-up false discovery rate (FDR).

CONCLUSIONS: Chronic, 16 week, intake of high/low-flavonoid F&Vs modulated the metabolic profiles of urine in people at risk of CVD. Biomarkers were principally end-products of microbial polyphenol metabolism like hippuric acid and hydroxy-hippuric acid isomers. Moreover phenylacetyl glutamine (correlated with bifidobacteria activity), tyrosine (related to amino acids), hydroxyphenyl-γ-valerolactone-O-methyl-glucuronides&sulfates, vanilloylglycine (catechin metabolites) and proline betaine were found to be significantly higher in diet HF compared to CT, what is in good agreement with previous studies and our microbiological analysis (data not shown). LC-HR-MS/MS based untargeted metabolomics succeeded in identifying novel biomarkers of chronic F&V intake. Further studies are needed to correlate metabolite profiles with the primary clinical outcome of the study, a significant improvement in vascular function and inflammatory status following enrichment with F&V.

Acknowledgments: The FLAVUR project was financially supported by Food Standards Agency, UK (clinical study), and Provincia Autonoma di Trento (IT) within ADP 2013 programme (metabolomics study)

Maria M. Ulaszewska

, Kajetan Trost

, Kieran Tuohy

, Jan Stanstrup

, Pietro Franceschi

,

Julie A. Lovegrove

, The FLAVURS Group, and Fulvio Mattivi

1

_{Department of Food Quality and Nutrition, Research and Innovation Centre, Fondazione Edmund Mach}

(FEM), Italy;

Hugh Sinclair Unit of Human Nutrition and Institute for Cardiovascular and Metabolic

Research, University of Reading, UK.

Rt Annotation; Elemental Composition, MW, adjusted p value 1.10 Proline Betaine; MMW: 143.0946, p 0.002 ↑Diet A;

3.80 Hydroxy Hippuric Acid (isomer); MMW: 195.0531, p 0.02 ↑Diet A; 4.40 Hydroxy Hippuric Acid (isomer); MMW: 195.0531, p 0.002 ↑Diet A, 4.82 Vanilloylglycine, MMW: 225.0637; p.0.03 ↑ Diet A, B;

5.70 Hippuric Acid, MMW: 179.0582; p 0.002 ↑Diet A

5.89 Phenylacetylglutamine, MMW: 264.1110, p 0.04 ↑Diet A; 6.15 Tyrosine , MMW: 181.0731, p 0.04; ↑Diet B

6.26 Dihydroxyphenyl-γ-valerolactone-O-sulphate MMW:288.0306 p 0.0003 ↑Diet A; 6.56 Dihydroxyphenyl-γ-valerolactone-O-methyl-O-GLC, p 0.01 ↑ Diet A;

7.27 Unknown cysteine derivative, p 0.02 ↓DietA

7.35 Hydroxy Hippuric Acid (isomer), MMW: 195.0531, p 0.01 ↑ Diet A; 7.76 Hydroxy-tridecenoic acid GLC, MMW: 404.2046, p 0.001 ↑ Diet A; 9.95 Unknown C19H28O4-GLC; MMW: 496.2308, p 0.03 ↑ DietA

12.30 Unknown menthol-glucuronide derivative; MMW: 316.1885, p 0.02 ↓ Diet A;

INTRODUCTION:

Human intervention trials are providing evidence for protective effects of various (poly)phenol-rich foods against chronic diseases like cardiovascular disease, neurodegeneration, osteoporosis and cancer. However, the overall impact of polyphenols on human metabolome is not fully understood.

0 500 1000 1500 2000

HYDROXY-HIPPURIC ACID (isomer)

0 500 1000 1500 2000 0 500 1000 1500 PROLINE BETAINE 0 6 12 18 weeks Sq rt( int)

Diet HF, Diet LF, Diet CT

Diet HF, Diet LF, Diet CT

Diet HF Diet LF Diet CT

HYDROXY-HIPPURIC ACID (isomer)

1 1 t h N u G O w e e k , N u t r i g e n o m i c s o f F o o d . 8 - 1 1 S e p t e m b e r 2 0 1 4 , T h e V e s u v i a n I n s t i t u t e , C a s t e l l a m m a r e d i S t a b i a , I T

Rest of biomarkers is still under construction ....

0 6 12 18 weeks

0 6 12 18 weeks