Targeted Metabolomics Identifies Glucuronides of Dietary Phytoestrogens as a Major Class of MRP3 Substrates In Vivo

Background & Aims

The physiologic function of the efflux transporter Multidrug Resistance Protein 3 (MRP3) remains poorly defined. In vitro, MRP3 transports several glucuronidated compounds, but the compounds transported under physiologic conditions are unknown. Knowledge of the compounds transported by MRP3 in vivo would greatly contribute to the elucidation of the physiologic function of this transport protein.

Methods

We used targeted metabolomics to identify substrates of MRP3 in vivo. Liquid chromatography coupled to mass spectrometry was used to specifically screen in plasma and urine of mice for compounds containing a glucuronic acid moiety.

Results

We found that several highly abundant compounds containing a glucuronic acid moiety have a much lower abundance in plasma and urine of Mrp3^(−/−) than of wild-type mice. We identified these as phytoestrogen-glucuronides, and we show that MRP3 transports these compounds at high rates and with high affinity in vitro.

Conclusions

We have identified the efflux transporter MRP3 as a major factor in the disposition of phytoestrogens, a class of compounds to which mammals are exposed via food of plant origin. Our targeted metabolomics approach is not restricted to MRP3 but applicable to many other transport proteins for which knockout mouse models are available. Similar screens could be developed for sulpho- and glutathione-conjugates, further increasing the potential of identifying new physiologic transporter substrates.

Abbreviations used in this paper: ABC, ATP-binding cassette, ATP, adenosine triphosphate, BSA, bovine serum albumin, cDNA, complementary DNA, CID, collision-induced dissociation, HEK, Human Embryonic Kidney cells, LC, liquid chromatography, MRM, multiple reaction monitoring, MRP3, Multidrug Resistance Protein 3, MS, mass spectrometry, m/z ratio, mass-to-charge ratio, PXR, pregnane-X-receptor, SPE, solid phase extraction, WT, wild-type