Supplementary Materials

Urinary metabolomic profiling to identify biomarkers of a flavonoid-rich and flavonoid-poor fruits and vegetables diet in adults: the FLAVURS trial

Maria M. Ulaszewska1*, Kajetan Trost1,2, Jan Stanstrup1, Kieran M. Tuohy1*, Pietro Franceschi1, Mary Foong-Fong Chong3,4 , Trevor George3,5 , Anne Marie Minihane3,6, Julie A. Lovegrove3, Fulvio Mattivi1*. For the FLAVURS study group

1Department of Food Quality and Nutrition, Research and Innovation Centre, Fondazione Edmund Mach (FEM), Via Mach 1, 38010 San Michele all'Adige (TN) - Italy;

2 Wine Research Centre, University of Nova Gorica, Vipavska 13, 5000 Nova Gorica, Slovenia

3 Hugh Sinclair Unit of Human Nutrition and the Institute for Cardiovascular and Metabolic Research (ICMR), Department of Food and Nutritional Sciences, University of Reading, Whiteknights, PO Box 226, Reading RG6 6AP, United Kingdom

4 Now Singapore Institute for Clinical Sciences, Brenner Centre, 30 Medical Drive, 117609 Singapore

5 Now Department of Applied Sciences, Faculty of Health and Life Sciences, Northumbria University, Ellison Place, Newcastle upon Tyne, NE1 8ST, United Kingdom

6 Now Department of Nutrition, Norwich Medical School, James Watson Road, University of East Anglia, Norwich, NR4 7UQ, United Kingdom

*corresponding authors

Dr Maria Ulaszewska: , Tel. +39 0461 615 540

Dr Kieran Tuohy: , Tel. +39 0461 615 613

Dr Fulvio Mattivi: , Tel. +39 0461 615 259

Table 1S. Statistically significant ions (adducts, and in-source fragments) and adjusted p values of biomarkers of HF and LF diets.

Abscisic acid glucuronide

m/z / 6 wk / 6 wk / 12 wk
POS mode / LF vs CT / LF vs HF / LF vs CT
441.1747 / 1.9E-02 / 5.0E-02 / -
442.1782 / 1.0E-02 / - / 4.0E-02
247.1327 / 1.0E-03 / 2.0E-02 / 2.0E-02
265.1433 / 1.0E-03 / - / -
463.1567 / 3.0E-03 / - / -

Coumaroyl malic acid

m/z / 6 wk / 6 wk / 12 wk / 12 wk / HF
NEG mode / HF vs CT / LF vs HF / HF vs CT / LF vs HF / 12wk vs 18wk
279.0512 / 3.00E-03 / 2.31E-03 / 9.61E-04 / 2.28E-04 / 3.49E-02
133.0148 / 5.53E-03 / 7.32E-03 / 6.39E-04 / 8.91E-05 / 2.22E-02
163.0404 / 2.20E-02 / 1.54E-02 / 1.59E-03 / 7.45E-04 / 8.73E-02

Cyclohexadiene carboxylic acid glycine

m/z / 6 wk / 6 wk / 18 wk
NEG mode / LF vs CT / LF vs HF / LF vs CT
180.06696 / - / 4.63E-02 / -
181.07017 / 5.70E-03 / 2.34E-03 / 5.70E-03

Cyclohexane carboxylic acid glycine

m/z / 6 wk / 18 wk
NEG mode / LF vs CT / LF vs CT
184.0985 / 2.0E-04 / 7.0E-02
185.1015 / 1.0E-04 / 5.0E-02
220.0749 / 6.0E-04 / 5.0E-02
221.0784 / 4.0E-03 / -
222.0717 / 4.0E-04 / -

Dihydroxyphenyl-γ-valerolactone sulfate

m/z / 12 wk / 12 wk / HF
POS mode / HF vs CT / LF vs HF / 6wk vs 12 wk
306.0641 / 2.8E-04 / 4.6E-04 / 1.0E-02
289.0380 / 6.0E-03 / 2.0E-03 / 3.0E-02
NEG mode
287.0230 / 5.0E-04 / 6.0E-04 / -
288.0270 / 8.0E-04 / 1.0E-03 / -
289.0196 / 7.0E-04 / 5.0E-04 / -

Fatty Acid Hydroxy-C13:1-GLC; Hydroxy-tridecenoic acid glucuronide (Isomer I)

m/z / 12 wk / 12 wk / 18 wk / 18 wk / HF
NEG mode / HF vs CT / LF vs HF / HF vs CT / LF vs HF / 6wk vs 18wk
403.1987 / 1.36E-03 / 6.76E-03 / 9.70E-06 / 1.01E-03 / 4.34E-02

Fatty Acid Hydroxy-C13:1-GLC; Hydroxy-tridecenoic acid glucuronide (Isomer II)

m/z / 12 wk / 12 wk / 18 wk / 18 wk / HF / HF
POS mode / HF vs CT / LF vs HF / HF vs CT / LF vs HF / 6wk vs 12 wk / 6wk vs 18wk
427.1943 / 7.0E-04 / 3.0E-02 / 8.0E-03 / 3.0E-02 / 9.0E-04 / 8.0E-04
428.2006 / 6.0E-04 / 3.0E-02 / 8.0E-03 / 2.0E-02 / 1.0E-03 / 1.0E-03
463.1732 / 3.0E-03 / - / 9.0E-03 / 5.0E-02 / 3.0E-03 / 1.0E-03
464.1766 / 4.0E-03 / - / 3.0E-02 / - / 2.0E-03 / 2.0E-03
NEG mode
403.1969 / 1.0E-03 / - / 1.0E-05 / 3.0E-04 / - / -
404.2001 / 1.0E-03 / - / 1.0E-05 / 1.0E-04 / - / -
439.1734 / 5.0E-02 / - / 5.0E-05 / 2.0E-03 / - / -
440.1770 / 5.0E-02 / - / 2.0E-05 / 2.0E-03 / - / -

Fatty Acid Hydroxy-C13:1-GLC; Hydroxy-tridecenoic acid glucuronide (Isomer III)

12 wk / 12 wk / 18 wk / 18 wk
m/z / HF vs CT / LF vs HF / LF vs CT / HF vs CT
POS mode
422.23884 / 4.30E-05 / 4.51E-02 / 5.48E-02 / 1.10E-02
NEG mode
403.19878 / 6.97E-04 / - / - / -
404.20134 / 7.56E-04 / 8.33E-02 / - / -

Fatty Acid Dicarboxylic C14:1-GLC; Dicarboxylic Tetradecenoic Acid Glucuronide

m/z / 18 wk / 18 wk / HF
NEG mode / HF vs CT / LF vs HF / 6wk vs 18wk
431.2298 / 2.0E-02 / - / -
432.2331 / 6.9E-05 / 1.0E-02 / 1.0E-02

Fatty Acid C15:2-GLC; Pentadienoic acid glucuronide

m/z / 12 wk / 18 wk / 18 wk / HF
POS mode / HF vs CT / HF vs CT / LF vs HF / 6wk vs 18wk
415.2323 / 3.0E-02 / 8.0E-02 / 3.0E-02 / 2.0E-02
432.2605 / 4.0E-02 / 4.0E-02 / 5.0E-03 / 5.0E-02
437.2157 / 5.0E-02 / 4.0E-02 / 3.0E-02 / -
203.1797 / 9.0E-02 / 4.0E-03 / 5.0E-03 / 7.0E-03
221.1905 / 4.0E-02 / 2.0E-02 / 2.0E-02 / 5.0E-02
NEG mode
413.2177 / 1.0E-02 / 2.0E-02 / 2.0E-02 / -
414.2223 / 1.0E-02 / 2.0E-02 / 1.0E-02 / -

Fatty Acid Hydroxy-C15:3-GLC; Pentatrienoic acid glucuronide

m/z / 12 wk / 12 wk / 18 wk / 18 wk / HF / HF
POS mode / HF vs CT / LF vs HF / HF vs CT / LF vs HF / 6wk vs 12 wk / 6wk vs 18wk
429.2126 / 2.0E-04 / 4.0E-03 / 3.0E-03 / 1.0E-02 / 6.0E-04 / 7.0E-04
253.1798 / 1.0E-03 / 2.0E-02 / 1.0E-02 / 1.0E-01 / 5.0E-04 / 2.0E-03
235.1694 / 6.0E-04 / 3.0E-02 / 7.0E-03 / 3.0E-02 / 4.0E-04 / 4.0E-04
236.1731 / 1.0E-03 / 4.0E-02 / 7.0E-03 / 4.0E-02 / 1.0E-03 / 1.0E-03
451.1937 / 1.0E-04 / 6.0E-03 / 1.0E-02 / 3.0E-02 / 1.0E-03 / 1.0E-02
NEG mode
427.1978 / 1.0E-03 / 4.0E-02 / 8.0E-03 / 3.0E-02 / 1.0E-03 / 1.0E-03
428.2006 / 7.0E-04 / 3.0E-02 / 9.0E-03 / 2.0E-02 / 1.0E-03 / 1.0E-03
463.1732 / 3.0E-03 / - / 1.0E-02 / 5.0E-02 / 4.0E-03 / 1.0E-03

Fatty Acid Dicarboxylic C17:0-GLC; Dicarboxylic heptadecanoic Acid Glucuronide

m/z / 12 wk / 12 wk / 18 wk / 18 wk / HF
NEG mode / HF vs CT / LF vs HF / HF vs CT / LF vs HF / 6wk vs 18wk
475.2543 / 5.0E-03 / 3.0E-02 / 3.0E-04 / 1.0E-03 / 7.0E-03

Ferulic Acid

m/z / 12 wk / 12 wk / 18 wk / 18 wk / HF / HF
NEG mode / HF vs CT / LF vs HF / HF vs CT / LF vs HF / 6wk vs 12wk / 6wk vs 18wk
193.051 / 1.0E-03 / 3.0E-04 / 1.0E-06 / 5.0E-05 / 5.0E-02 / 2.0E-03

Iberin N-acetyl Cysteine

m/z / 12 wk / 12 wk / HF / HF
NEG mode / HF vs CT / LF vs HF / 6wk vs 12 wk / 12wk vs 18wk
325.06618 / 2.52E-05 / 2.75E-05 / - / 1.72E-03
326.07083 / 3.95E-05 / 4.03E-05 / - / 2.23E-03
327.06218 / 5.28E-05 / 3.47E-05 / - / 2.64E-03
84.04544 / 5.76E-06 / 3.01E-06 / 3.84E-02 / 2.74E-04
162.0223 / 4.67E-06 / 2.08E-06 / 4.74E-02 / 2.25E-04
163.02604 / 4.71E-05 / 2.28E-05 / - / 1.06E-03
196.02489 / 1.87E-06 / 1.43E-06 / 2.20E-02 / 1.09E-04
219.98111 / 2.96E-05 / 1.38E-05 / 2.03E-02 / 8.54E-05
POS mode
327.0839 / 3.3E -02 / 4.0E-06 / 4.0E-06 / 1.0E-02
328.0879 / 3.3E -02 / 2.0E-06 / 2.0E-06 / 6.0E-03
329.0786 / 3.3E -02 / 2.0E-06 / 2.0E-06 / 6.0E-03
164.0374 / 1.6E -02 / 1.0E-04 / 1.0E-04 / -
349.0659 / 3.5E-02 / 7.0E-05 / 3.0E-05 / 7.0E-02
198.0403 / 2.0E -02 / 1.0E-04 / 1.0E-04 / -
122.0268 / 1.2E -02 / 4.0E-04 / 5.0E-04 / -
105.0699 / 1.1E -02 / 2.0E-05 / 5.0E-05 / 3.0E-02

Hydroxy phenylacetic acid

m/z / 18 wk / 18 wk
NEG mode / HF vs CT / LF vs HF
151.0405 / 3.0E-03 / 3.9E-04
152.0439 / 3.0E-03 / 2.7E-04

Phenylacetic Acid

m/z / 12 wk / 12 wk / 18 wk / 18 wk / HF / HF
NEG mode / HF vs CT / LF vs HF / HF vs CT / LF vs HF / 6wk vs 12 wk / 6wk vs 18wk
135.0455 / 5.71E-05 / 6.30E-05 / 2.01E-02 / 1.76E-03 / 1.15E-04 / 2.85E-03

Proline betaine

m/z / 12 wk / 12 wk / 18 wk / 18 wk / HF
POS mode / HF vs CT / LF vs HF / HF vs CT / LF vs HF / 6wk vs 12 wk
144.1019 / 2.0E-06 / 3.0E-05 / 5.0E-03 / 2.0E-03 / 4.0E-02
145.1053 / 5.0E-06 / 7.0E-05 / 7.0E-03 / 2.0E-03 / 6.0E-02
102.0551 / 1.0E-05 / 9.0E-05 / 1.0E-02 / 2.0E-03 / 8.0E-02
286.2123 / 1.0E-03 / 9.0E-04 / 5.0E-02 / 4.9E-02 / -
85.08408 / 1.0E-03 / 9.0E-03 / 6.0E-02 / 1.0E-02 / -
NEG mode
142.0508 / 3.0E-04 / 7.0E-04 / 2.5E-03 / 2.0E-02

Trihydroxybenzoic acid (Gallic Acid)

m/z / 6 wk / 6 wk / 18 wk / 18 wk / LF
NEG mode / LF vs CT / LF vs HF / LF vs CT / LF vs HF / 12wk vs 18wk
169 / 1.0E-02 / 5.0E-02 / 6.0E-03 / 5.0E-04 / 4.0E-02

Trihydroxyphenyl-γ-valerolactone

m/z / 12 wk / 18 wk / 18 wk / HF
NEG mode / HF vs CT / HF vs CT / LF vs HF / 6wk vs 12 wk
259.0286 / 2.0E-02 / 3.0E-04 / 3.0E-02 / -
261.0440 / 5.0E-03 / 2.0E-03 / 7.0E-02 / 4.0E-02

Vanillic Acid Sulfate

m/z / 12 wk / 12 wk / 18 wk / 18 wk / HF
NEG mode / HF vs CT / LF vs HF / HF vs CT / LF vs HF / 6wk vs 12 wk
246.9919 / 5.0E-02 / 2.0E-03 / 1.0E-02 / 3.0E-03 / -
248.9884 / 6.0E-02 / 2.0E-02 / 1.0E-03 / 2.0E-02 / -
247.9953 / - / 4.0E-02 / 2.0E-03 / 2.0E-02 / -
124.0490 / 3.0E-02 / 1.0E-03 / 2.0E-02 / 1.0E-02 / 2.0E-02
123.0459 / 8.0E-02 / 5.0E-03 / 1.0E-02 / 7.0E-03 / 6.0E-02