Full 1 H and 13 C NMR spectral assignment of conjugated valerolactone metabolites isolated from urine of black tea consumers by means of SPE-prepLC-MS-LC-MS-NMR.

The health benefits of black tea have been linked to polyphenol metabolites that target specific modes of action in the human body. A major bottleneck in unravelling the underlying mechanisms is the preparative isolation of these metabolites, which hampers their structural elucidation and assessment of in vitro bioactivity. A solid phase extraction (SPE)-preparative liquid chromatography (prepLC)-MS-LC-MS-NMR workflow was implemented for preparative isolation of conjugated valerolactone metabolites of catechin-based polyphenols from urine of black tea consumers. First, the urine was cleaned and preconcentrated using an SPE method. Subsequently, the clean urine concentrate was injected on a preparative LC column, and conjugated valerolactones were obtained by MS-guided collection. Reconstituted fractions were further separated on an analytical LC column, and valerolactone fractions were collected in an MS-guided manner. These were reconstituted in methanol-d4 and identified and quantified using 1D and 2D homo- and hetereonuclear NMR experiments (at a field strength of 14.1 T), in combination with mass spectrometry. This resulted in the full spectral 1 H and 13 C NMR assignments of five conjugated valerolactones. These metabolites were collected in quantities of 8-160 µg and purities of 70-91%. The SPE-prepLC-MS-LC-MS-NMR workflow is suitable for isolating metabolites that occur at sub-µM concentrations in a complex biofluid such as urine. The workflow also provides an alternative for cumbersome and expensive de novo synthesis of tea metabolites for testing in bioactivity assays or for use as authentic analytical standards for quantification by mass spectrometry.

The effect of plant sterols and different low doses of omega-3 fatty acids from fish oil on lipoprotein subclasses.

SCOPE: Consumption of a low-fat spread enriched with plant sterols (PS) and different low doses (<2 g/day) of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil reduces serum triglycerides (TGs) and low-density lipoprotein-cholesterol (LDL-Chol) and thus beneficially affects two blood lipid risk factors. Yet, their combined effects on TG and Chol in various lipoprotein subclasses have been investigated to a limited extent. METHODS AND RESULTS: In a randomized, double-blind, placebo-controlled, parallel study, we determined TG and Chol in 13 LP subclasses in fasting serum of 282 hypercholesterolemic subjects, who consumed either a placebo spread or one of the four spreads containing PS (2.5 g/day) and EPA+DHA (0.0, 0.9, 1.3, and 1.8 g/day) for 4 weeks. After PS treatment, total LDL-Chol was reduced, which was not further changed by EPA+DHA. No shift in the LDL-Chol particle distribution was observed. The addition of EPA+DHA to PS dose-dependently reduced VLDL-Chol and VLDL-TG mainly in larger particles. Furthermore, the two highest doses of EPA+DHA increased Chol and TG in the larger HDL particles, while these concentrations were decreased in the smallest HDL particles. CONCLUSION: The consumption of a low-fat spread enriched with both PS and EPA+DHA induced shifts in the lipoprotein distribution that may provide additional cardiovascular benefits over PS consumption alone.

Structural elucidation and quantification of phenolic conjugates present in human urine after tea intake.

In dietary polyphenol exposure studies, annotation and identification of urinary metabolites present at low (micromolar) concentrations are major obstacles. To determine the biological activity of specific components, it is necessary to have the correct structures and the quantification of the polyphenol-derived conjugates present in the human body. We present a procedure for identification and quantification of metabolites and conjugates excreted in human urine after single bolus intake of black or green tea. A combination of a solid-phase extraction (SPE) preparation step and two high pressure liquid chromatography (HPLC)-based analytical platforms was used, namely, accurate mass fragmentation (HPLC-FTMS(n)) and mass-guided SPE-trapping of selected compounds for nuclear magnetic resonance spectroscopy (NMR) measurements (HPLC-TOFMS-SPE-NMR). HPLC-FTMS(n) analysis led to the annotation of 138 urinary metabolites, including 48 valerolactone and valeric acid conjugates. By combining the results from MS(n) fragmentation with the one-dimensional (1D)-(1)H NMR spectra of HPLC-TOFMS-SPE-trapped compounds, we elucidated the structures of 36 phenolic conjugates, including the glucuronides of 3',4'-di- and 3',4',5'-trihydroxyphenyl-γ-valerolactone, three urolithin glucuronides, and indole-3-acetic acid glucuronide. We also obtained 26 h-quantitative excretion profiles for specific valerolactone conjugates. The combination of the HPLC-FTMS(n) and HPLC-TOFMS-SPE-NMR platforms results in the efficient identification and quantification of less abundant phenolic conjugates down to nanomoles of trapped amounts of metabolite corresponding to micromolar metabolite concentrations in urine.