British Journal of Nutrition

Molecular Nutrition

Proline betaine and its biotransformation products in fasting urine samples are potential biomarkers of habitual citrus fruit consumption

Amanda J. Lloyda1, Manfred Beckmanna1, Gaëlle Favéa2, John C. Mathersa2 and John Drapera1 c1

a1 Institute of Biological Environmental and Rural Sciences, Aberystwyth University, Aberystwyth SY23 3DA, UK

a2 Human Nutrition Research Centre, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne NE2 4HH, UK


The lack of robust measures of dietary exposure hinders a quantitative understanding of causal relationships between diet and health. Non-targeted metabolite fingerprinting was used to explore the relationships between citrus exposure in free-living human subjects, estimated by a FFQ, and the chemical content of urine. Volunteers (study 1, n 12; study 2, n 11) were classified into high-, medium- and low-frequency citrus consumption groups. Overnight and spot fasting urine samples were obtained after exposure to a standardised citrus-free evening meal. The urine samples were analysed by flow injection electrospray-ionisation MS followed by supervised multivariate data classification analysis to discover discriminatory features associated with the level of citrus exposure. Good separation of high and low citrus consumption classes was achieved. Deeper exploration of high-ranked explanatory mass signals revealed several correlated signals derived from proline betaine. Targeted analysis of the relative levels of proline betaine in both fasting and overnight urine samples demonstrated good correlation with FFQ exposure data. Acute exposure of volunteers to orange juice resulted in the appearance of proline betaine and several biotransformed products in postprandial urine samples. Biomarker validation showed sensitivities of 80·8–92·2 % and specificities of 74·2–94·1 % (false discovery rate-adjusted P values < 0·05) for elevated proline betaine in participants who reported high citrus consumption. Proline betaine biotransformation products displayed weaker quantitative relationships with habitual citrus exposure. Targeted screening for the presence of biotransformation products of hesperidin and narirutin, known to be abundant in oranges, revealed that they were relatively poor indicators of citrus exposure.

(Received October 14 2010)

(Revised January 10 2011)

(Accepted February 08 2011)

(Online publication May 09 2011)


c1 Corresponding author: J. Draper, fax +44 1970 621981, email


Abbreviations: AUC, area under the receiver-operating characteristic curve; CRF, Clinical Research Facility; FIE-MS, flow injection electrospray-ionisation MS; FT-ICR-MS, Fourier-transform ion cyclotron resonance ultra mass spectroscopy; MEDE, Metabolomics to characterise Dietary Exposure; PC, principal component; RF, random forest