Scope: Biomarkers for intake of green leafy vegetables such as spinach can help investigate their health effects. However, only few potential intake markers have been reported in the literature so far.

Methods and results: Based on a cross-over study on whole leaf and minced spinach, we investigate changes in metabolites before and after spinach intake and differences between the two treatments and health status. Nineteen volunteers (12 healthy subjects and 7 short bowel patients) completed the study within 48 days. Urine samples (24-h intervals before and after spinach intake) and serum samples (baseline, post 8 d, and post 15 d) are collected and analyzed by ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS). The acquired data is analyzed by multivariate and univariate analyses. Three candidate biomarkers are observed in urine only after the spinach intake, including des-amino arginine pentenol ester, D/L-malic acid ester of cis-p-coumarate, D/L-malic acid ester of trans-p-coumarate, and 69 metabolites are present before spinach intake but showing an altered level after treatment. These metabolites are related to dietary habits or meal structure, and some changes are possibly affected by spinach intake. The candidate biomarkers are independent of spinach pre-processing and healthy status. No markers are discovered in serum samples.

Conclusion: We propose structures for three candidate spinach intake biomarkers; these markers will need further validation in independent studies. This article is protected by copyright. All rights reserved.