Identification of an Alternative Glycyrrhizin Metabolite Causing Liquorice-Induced Pseudohyperaldosteronism and the Development of ELISA System to Detect the Predictive Biomarker

Abstract

Liquorice is usually used as crude drug in traditional Japanese Kampo medicine and traditional Chinese medicine. Liquorice-containing glycyrrhizin (GL) can cause pseudohyperaldosteronism as a side effect. Previously, we identified 18β-glycyrrhetyl-3-O-sulfate (3) as a GL metabolite in Eisai hyperbilirubinuria rats (EHBRs) with the dysfunction of multidrug resistance-related protein (Mrp2). We speculated that 3 was associated with the onset of liquorice-induced pseudohyperaldosteronism, because it was mainly detected in serum of patients with suspected to have this condition. However, it is predicted that other metabolites might exist in the urine of EHBRs orally treated with glycyrrhetinic acid (GA). We explored other metabolites in the urine of EHBRs, and investigated the pharmacokinetic profiles of the new metabolite in EHBRs and normal Sprague-Dawley rats. We further analyzed the serum concentrations of the new metabolite in the patients of pseudohyperaldosteronism. Finally, we developed the analyzing method of these metabolites as a preventive biomarker for the onset of pseudohyperaldosteronism using an enzyme-linked immunosorbent assay (ELISA). We isolated a new GL metabolite, 18β-glycyrrhetyl-3-O-sulfate-30-O-glucuronide (4). Compound 4 significantly inhibited rat type-2 11β-hydroxysteroid dehydrogenase (11β-HSD2) and was a substrate of both organic anion transporter (OAT) 1 and OAT3. Compound 4 was also detected in the serum of patients with suspected pseudohyperaldosteronism at an approximately 10-fold lower concentrations than 3, and these concentrations were positively correlated. Compound 4 showed a lower serum concentration and weaker inhibitory titer on 11β-HSD2 than 3. We developed an enzyme-linked immunosorbent assay system using an anti-18β-glycyrrhetyl-3-O-glucuronide (3MGA) monoclonal antibody to measure the serum concentration of 3 to facilitate the measurement of biomarkers to predict the onset of pseudohyperaldosteronism. Although we found 4 as the secondary candidate causative agent, 3 could be the main potent preventive biomarker of liquorice-induced pseudohyperaldosteronism. Compound 3 was detected in serum at a higher concentration than GA and 4, implying that 3 may be a pharmacologically active ingredient mediating not only the development of pseudohyperaldosteronism but anti-inflammatory effects in humans administered GL or other liquorice-containing preparations.

Clinical Risk Factors of Licorice-Induced Pseudoaldosteronism Based on Glycyrrhizin-Metabolite Concentrations: A Narrative Review

“…and 18β-glycyrrhetyl-3-O-sulfate-30-glucuronide (compound 4) as other GL metabolites in the urine of EHBR orally treated with GA (34)(35)(36). We also found that the blood and urinary concentrations of these new metabolites were much higher than those of 3MGA in EHBR orally treated with GA, and that their pharmacokinetic behavior was similar to that of 3MGA (34,35).…”

“…As it is difficult to diagnose PsA based only on physical examination, including peripheral edema and hypertension, laboratory testing of parameters such as potassium and aldosterone levels is needed. The grade or occurrence of peripheral edema and hypertension in patients who consumed licorice did not correlate well with laboratory abnormalities, such as hypokalemia, low renin activity, aldosterone concentration, and GL metabolites (33,36). Several studies have shown the expression of mineralocorticoid receptor and 11βHSD1/2 in the heart and arteries (37)(38)(39).…”

“…Therefore, diagnosing PsA is challenging, especially in mild cases. The serum concentrations of GA3S, GA, and compound 4 were found to be negatively correlated with serum potassium concentration, plasma renin activity, and aldosterone concentration (33,36). From these results, it is suggested that such GL-metabolites could be used as an objective laboratory marker of "licorice-induced" PsA, and could be applied to the early detection and prevention of licorice-induced PsA especially in high risk patients.…”

“…patients in a multicenter retrospective case series of milder PsA (33,36). Therefore, we considered GA3S to be the most likely causative agent of PsA.…”