Licorice-induced pseudoaldosteronism is a common adverse effect in traditional Japanese Kampo medicine, and 3-monoglucuronyl glycyrrhetinic acid (3MGA) was considered as a causative agent of it. Previously, we found 22α-hydroxy-18β-glycyrrhetyl-3-O-sulfate-30-glucuronide (1), one of the metabolites of glycyrrhizin (GL) in the urine of Eisai hyperbilirubinuria rats (EHBRs) treated with glycyrrhetinic acid (GA), and suggested that it is also a possible causative agent of pseudoaldosteronism. The discovery of 1 also suggested that there might be other metabolites of GA as causal candidates. In this study, we found 22α-hydroxy-18β-glycyrrhetyl-3-O-sulfate (2) and 18β-glycyrrhetyl-3-O-sulfate (3) in EHBRs’ urine. 2 and 3 more strongly inhibited rat type 2 11β-hydroxysteroid dehydrogenase than 1 did in vitro. When EHBRs were orally treated with GA, GA and 1–3 in plasma and 1–3 in urine were detected; the levels of 3MGA were quite low. 2 and 3 were shown to be the substrates of organic anion transporter (OAT) 1 and OAT3. In the plasma of a patient suffering from pseudoaldosteronism with rhabdomyolysis due to licorice, we found 8.6 µM of 3, 1.3 µM of GA, and 87 nM of 2, but 1, GL, and 3MGA were not detected. These findings suggest that 18β-glycyrrhetyl-3-O-sulfate (3) is an alternative causative agent of pseudoaldosteronism, rather than 3MGA and 1.

Pseudoaldosteronism is a common adverse effect associated with traditional Japanese Kampo medicines. The pathogenesis is mainly caused by 3-monoglucuronyl glycyrrhetinic acid (3MGA), one of the metabolites of glycyrrhizin (GL) contained in licorice. We developed an anti-3MGA monoclonal antibody (MAb) and an ELISA system to easily detect 3MGA in the plasma and urine of the patients. However, we found that some metabolites of GL cross-reacted with this MAb. Mrp2-deficient Eisai Hyperbilirubinemia rats (EHBRs) were administered glycyrrhetinic acid (GA), and we isolated 22α-hydroxy-18β-glycyrrhetyl-3-O-sulfate-30-glucuronide (1) from the pooled urine with the guidance of positive immunostaining of eastern blot as the new metabolite of GL. The IC50 of 1 for type 2 11β-hydroxysteroid dehydrogenase (11β-HSD2) was 2.0 µM. Similar plasma concentrations of 1 and GA were observed 12 h after oral administration of GA to EHBR. Compound 1 was eliminated via urine, whereas GA was not. In Sprague–Dawley (SD) rats orally treated with GA, compound 1 was absent from both the plasma and the urine. Compound 1 was actively transported into cells via OAT1 and OAT3, whereas GA was not. Compound 1, when produced in Mrp2-deficiency, represents a potential causative agent of pseudoaldosteronism, and might be used as a biomarker to prevent the adverse effect.

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