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EXCESSIVE ingestion of licorice may result in sodium and water retention, hypertension, hypokalemia, and suppression of the renin-aldosterone system.1, 2 It was thought for years that licorice produced these effects through the binding of its active components, glycyrrhizic acid and its hydrolytic metabolite glycyrrhetinic acid, to mineralocorticoid receptors. Two findings, however, argue against this proposed mechanism. First, the affinity of glycyrrhetinic acid, the most active component of licorice, for mineralocorticoid receptors is 0.01 percent of that of aldosterone.3 Second, licorice, or glycyrrhetinic acid, does not have mineralocorticoid effects in patients with Addison's disease2 or adrenalectomized rats1 unless cortisone or hydrocortisone is administered concomitantly.
Stewart et al.4 have proposed that licorice acts by inhibiting Cortisol oxidase, a component of the widely distributed 11β-hydroxysteroid dehydrogenase system that converts Cortisol to cortisone, producing a state of apparent mineralocorticoid excess similar to that in children with 11/3-hydroxysteroid dehydrogenase deficiency5 (Fig. 1). In vitro, cortisol has the same binding affinity for mineralocorticoid receptors as aldosterone,6 whereas that of cortisone is much less. Licorice, by inhibiting 11β-hydroxysteroid dehydrogenase in aldosterone-responsive tissues such as the kidney, where it is found in high concentrations,7, 8 produces high renal levels of cortisol, which then binds to and activates mineralocorticoid receptors.4 The degree to which licorice inhibits 11β-hydroxysteroid dehydrogenase activity can be measured by examining the ratio of the metabolites of cortisone to those of cortisol in urine.
Epstein et al.9 studied several subjects with a history of chronic licorice ingestion and found that the renin-aldosterone axis was suppressed while the subjects were taking licorice, but normal function resumed within two to four months after licorice was discontinued. The pattern of clinical and hormonal recovery of suppressed 11β-hydroxysteroid dehydrogenase activity has not been determined, however. In the present study we examined the duration of the suppressive effects of licorice on 11β-hydroxysteroid dehydrogenase activity and the renin-aldosterone system before and after licorice withdrawal in a patient with chronic licorice intoxication.
Case Report
A 70-year old man was referred to us for an evaluation of hypertension and hypokalemia. Hypertension had been diagnosed four years earlier and had been severe during the previous year. Nine months before referral, therapy with a thiazide diuretic agent had produced severe hypokalemia (plasma potassium level, 1.9 mmol per liter) and rhabdomyolysis. The patient subsequently required potassium supplements and spironolactone to maintain normal potassium levels and blood pressure. His symptoms during the year before our evaluation included weakness, mental slowness, and the loss of approximately 15 kg in weight. An investigation of possible mineralocorticoid excess while he was taking medications revealed normal plasma levels of renin activity, aldosterone, and 18-hydroxycorticosterone.
He was admitted to our clinic to be evaluated for possible mineralocorticoid excess due to factors other than aldosterone. Further history taking, however, revealed that he had been eating 60 to 100 g of licorice candies, each weighing 2.5 g (Panda, Vaajakoski, Finland), daily for the past four to five years. The licorice contained approximately 0.3 percent glycyrrhizic acid, which is converted to glycyrrhetinic acid after ingestion. The patient's medications, which he continued to take until the day of admission, included potassium chloride (20 mmol three times daily), spironolactone (25 mg four times daily), and verapamil (240 mg daily). He stopped eating licorice one week before admission. Physical examination revealed the patient to be a thin man with a blood pressure of 124/63 mm Hg; the examination was normal in other respects. The plasma sodium level was 137 mmol per liter, and the plasma potassium level was 5.7 mmol per liter.
Methods
The patient was admitted to the General Clinical Research Center of San Francisco General Hospital Medical Center. Because he had hyperkalemia, licorice was again incorporated into his diet (approximately 100 g per day, or 300 mg of glycyrrhetinic acid per day, an amount similar to that used by Stewart et al.4) to determine sodium retention, potassium excretion, and 11β-hydroxysteroid dehydrogenase activity. The potassium supplements and spironolactone were stopped; verapamil treatment was continued throughout the two-week study period.
During the study, the patient ate a constant diet, which included 135 mmol of sodium and 85 mmol of potassium per day. His blood pressure, weight, and plasma sodium and potassium levels were measured daily, as was urinary sodium and potassium excretion. Plasma renin activity, levels of aldosterone, cortisol, and deoxycorticosterone, and urinary excretion of aldosterone, cortisol, glycyrrhetinic acid, and steroids were measured intermittently.
The licorice was discontinued after one week. We continued to measure urinary electrolytes, plasma renin activity, plasma aldosterone, and urinary glycyrrhetinic acid and steroids during a second week of hospitalization. After discharge, the patient was seen at intervals of one to two months for continued hormonal measurements, and plasma samples were collected under random conditions for the measurement of renin activity and aldosterone. The patient was asked to continue eating the same amounts of sodium and potassium after discharge.
The study was approved by our institutional review committee, and the patient gave informed written consent.
Assay of Plasma Steroids, Urinary Aldosterone, and Urinary Cortisol
Plasma concentrations of aldosterone, cortisol, and deoxycorticosterone were measured by radioimmunoassay after isolation by high-performance liquid chromatography, as previously described.10 Urinary aldosterone was measured as the 18-glucuronide metabolite by radioimmunoassay (normal range, 11 to 55.6 nmol per day), and urinary cortisol by radioimmunoassay after isolation by high-performance liquid chromatography (normal range, 3 to 119 nmol per day). Plasma renin activity was determined by radioimmunoassay of angiotensin I in plasma incubated in vitro.11
Gas Chromatography and Mass Spectrometry of Urinary Steroid Metabolites
Urinary steroids were measured by gas chromatography and mass spectrometry according to an automated procedure.12 The steroid conjugates were extracted with a C18 cartridge, hydrolyzed, and the hydrolysis mixtures extracted again. After formation of methyloxime and trimethylsilyl derivatives, the steroids were separated and quantified with a gas chromatograph-mass spectrometer (model 5970, Hewlett-Packard, Waltham, Mass.). Thirty-five urinary steroids, including metabolites of cortisol and androgens, were measured by a specific technique of selected ion monitoring to monitor changes in metabolism after licorice withdrawal.
Although numerous steroids were measured in each urine sample, only selected cortisol metabolites were considered to be relevant to this study. These were steroids that provided information concerning the relative activity of enzymes involved in cortisone reduction and cortisol oxidation (i.e., reactions catalyzed by 11β-hydroxysteroid dehydrogenase), the relative production of 5α- and 5β-reduced products of steroids, and the overall excretion of cortisol metabolites retaining the hormonal 3-oxo-4-ene structure (an intact A ring). These categories were selected on the basis of the following findings: in the syndrome of apparent mineralocorticoid excess in children, which licorice ingestion mimics, the conversion of cortisol to cortisone is impaired; 5α-reduction of cortisol is relatively more important than 5β-reduction; and there is an overall decrease in A-ring reduction. To evaluate 11β-hydroxysteroid dehydrogenase activity, we measured the ratio of tetrahydrocortisone to 5β-tetrahydrocortisol plus 5α-tetrahydrocortisol and the ratio of α-cortolone plus β-cortolone to α-cortol plus β-cortol. To evaluate the potential attenuation of 5β-reduction, we determined the ratio of 5β-tetrahydrocortisol to 5α-tetrahydrocortisol.
Gas Chromatography and Mass Spectrometry of Urinary Glycyrrhetinic Acid
Urinary glycyrrhetinic acid was measured by gas chromatography and mass spectrometry (Hewlett-Packard) after silylation of a hydroxyl group and methylation of a carboxyl group. Internal standards, stigmasterol and cholesteryl butyrate, were added to a portion of the urine extracts. The urine extracts were dissolved in 0.5 ml of ethereal diazomethane (prepared in a Wheaton generator13), and methylation was completed within five minutes at room temperature. After the ether was evaporated under nitrogen, silylation was carried out with trimethylsilyl-imidazole.12 The carbonyl group at position 11 in glycyrrhetinic acid is not reactive under the conditions used for the formation of methyloxime; the methyloxime reaction was thus not necessary.
Derivatives of authentic glycyrrhetinic acid were found in the presence of the internal standards, and the mass spectrum was analyzed by gas chromatography and mass spectrometry. The molecular ion was observed at m/z 556, and prominent ions were seen at m/z 427 (M-129) and m/z 317 (M-129-90). These ions were considered suitable for the assay of urinary glycyrrhetinic acid derivatives by selected ion monitoring. The instrument was set up to monitor these ions and the ion m/z 368 from the internal standard cholesterol butyrate. We measured urinary glycyrrhetinic acid with a standard curve generated by using known amounts of glycyrrhetinic acid in the assay.
Results
Electrolyte Balance and Plasma Steroid Levels
At the time of admission the patient had hyperkalemia because he had continued to take potassium supplements and spironolactone for at least one week after ceasing to eat licorice (Fig. 2). After he began ingesting licorice again, the net sodium balance became positive and the net potassium balance negative, with a decline in plasma potassium to subnormal levels by the end of the two-week inpatient period. These changes in electrolyte balance were accompanied by an increase in body weight of 4.9 kg and a moderate increase in blood pressure (from 124/63 to 154/72 mm Hg). The effects of licorice on electrolyte metabolism persisted for a week after the patient discontinued licorice. During the ensuing months, his blood pressure was normal (verapamil therapy was continued), as was his plasma potassium concentration (without supplementation). Plasma levels of deoxycorticosterone (mean [±SD] level, 303±64 pmol per liter) and cortisol (309±22 nmol per liter) were consistently normal throughout the study period.
Renin-Aldosterone System
The levels of plasma renin activity, plasma aldosterone, and urinary aldosterone excretion were elevated at the time of admission, when the patient had hyperkalemia as a result of potassium supplementation and spironolactone therapy. During the week in which he received licorice in the hospital, these hormones all fell to low levels. Sixty-four days after licorice withdrawal, basal plasma renin activity and plasma and urinary aldosterone concentrations were slightly low or normal (Fig. 3), and they rose further by day 130.
Urinary Steroid and Glycyrrhetinic Acid Excretion
Urinary glycyrrhetinic acid excretion and selected urinary steroid ratios are shown in Figure 4 and Table 1. At the end of the one-week period of licorice treatment, urinary cortisol excretion was elevated and the ratio of urinary tetrahydrocortisone to 5β-tetrahydrocortisol plus 5α-tetrahydrocortisol was low, indicating the inhibition of 11β-hydroxysteroid dehydrogenase activity. Urinary glycyrrhetinic acid, which was present in the first three urine samples after the ingestion of licorice ended, was undetectable 18 days after licorice withdrawal. Concomitant with the decrease in urinary excretion of glycyrrhetinic acid, urinary cortisol excretion normalized. The ratio of tetrahydrocortisone to 5β-tetrahydrocortisol plus 5α-tetrahydrocortisol, which was much lower than normal when licorice was ingested, gradually increased to a normal level by day 27. The pattern of results for the ratios of cortolones to cortols was similar.
The measurements of 5α- and 5β-reduced cortisol metabolites (Table 1) revealed several unexpected findings. The ratio of 5β-tetrahydrocortisol to 5α-tetrahydrocortisol was initially elevated but decreased over a one-month period, indicating a relative increase in the 5α-reduced product. The ratio of unconjugated cortisol metabolites to total cortisol metabolites was elevated, but gradually declined to normal levels.
Discussion
A syndrome of mineralocorticoid excess (characterized by hypertension, sodium and water retention, and hypokalemia) resulting from the ingestion of licorice has been well described.1, 2 Stewart et al.4 proposed that licorice acts by inhibiting renal 11β-hydroxysteroid dehydrogenase activity, thereby diminishing the conversion of cortisol to cortisone and resulting in high renal levels of cortisol, which is available for binding to mineralocorticoid receptors. During licorice ingestion, plasma cortisol levels and the pituitary-adrenal axis are normal, although the plasma half-life of cortisol is prolonged.14 Our findings of elevated urinary excretion of cortisol, increased amounts of unconjugated cortisol metabolites relative to total cortisol metabolites in urine, and decreased urinary ratios of cortisone to cortisol metabolites (the ratios of tetrahydrocortisone to 5β-tetrahydrocortisol plus 5α-tetrahydrocortisol and of 5α- plus 5β-cortolones to cortols) confirm that licorice impairs normal cortisol metabolism.
The effects of licorice on 11β-hydroxysteroid dehydrogenase activity are similar to those in children with the syndrome of apparent mineralocorticoid excess. The elevated urinary ratio of 5β-tetrahydrocortisol to 5α-tetrahydrocortisol that occurs with licorice ingestion is, however, unlike the findings in such children, in whom 5α-reductase activity is increased relative to 5β-reductase activity.5 In children with the syndrome of apparent mineralocorticoid excess, reduced metabolism of the A ring (3-oxo-4-ene) also occurs, resulting in a high relative level of excretion of unconjugated cortisol metabolites.15, 16 In our patient, the relative level of unconjugated steroid excretion was elevated during the ingestion of licorice and decreased somewhat during the recovery period. Although the effects of licorice on A-ring metabolism are different from those of the apparent mineralocorticoid excess syndrome of children, the effects on cortisol production should not be different.
Epstein et al.14 investigated the effects of short-term (one to four weeks) licorice ingestion in normal subjects and found that the urinary excretion of cortisol remained elevated one week after licorice was withdrawn, although the ratio of cortisone to cortisol metabolites had become normal. In our patient, who had consumed licorice regularly for several years, we found that the suppression of 11β-hydroxysteroid dehydrogenase activity, as well as many of the changes in electrolyte balance, persisted for almost two weeks after licorice intake was discontinued. His excretion of glycyrrhetinic acid gradually diminished during the same two-week period. Thus, the prolonged suppression of 11β-hydroxysteroid dehydrogenase activity appeared to be due to the continued action of glycyrrhetinic acid; as urinary glycyrrhetinic acid levels fell, the suppression of 11β-hydroxysteroid dehydrogenase activity reversed.
Limited data exist concerning the recovery of the renin-aldosterone axis after the suppression that occurs during chronic ingestion of licorice. Epstein et al.9 found that the function of the renin-aldosterone system became normal within two to four months in four subjects who had ingested 25 to 200 g per day of licorice for six months to five years. In our patient, four to five years of licorice ingestion had profound effects: the unstimulated renin-aldosterone system was suppressed for nearly four months after the cessation of licorice ingestion. These prolonged suppressive effects are much like those that occur after the removal of an aldosterone-producing adenoma, which creates a similar state of chronic mineralocorticoid excess.17 Before the study period the patient's potassium and spironolactone treatment was appropriate for the licorice-induced hypermineralocorticoid state that probably maintained the renin-aldosterone system at a low set point. When spironolactone and potassium treatment was continued without licorice, however, the effects of spironolactone on the renin-aldosterone system were unopposed and resulted in relative stimulation, indicating that the system was capable of responding to a powerful stimulus. Whether direct stimulation with angiotensin II would have produced such a response is uncertain. In our patient, however, ambulatory plasma renin activity after discharge remained below normal overnight values in recumbent subjects for four months, implying prolonged suppression. This conclusion is supported by the low level of urinary aldosterone excretion during this time.
Our findings demonstrate that the rates of recovery differ in two enzyme systems that are suppressed by the long-term ingestion of licorice. The activity of 11β-hydroxysteroid dehydrogenase was suppressed for two weeks after licorice was withdrawn, but it then increased as urinary glycyrrhetinic acid levels decreased. In contrast, the basal activity of the renin-aldosterone system remained low for several months after licorice withdrawal. This prolonged suppression of the renin-aldosterone axis demonstrates the potency of licorice toxicity and emphasizes the need to consider licorice, and possibly other factors or drugs that affect 11β-hydroxysteroid dehydrogenase activity, as a cause of low-renin hypertension.
Supported in part by grants (DK06415 and DK34400) from the National Institute of Diabetes and Digestive and Kidney Diseases, and carried out in part in the General Clinical Research Center at San Francisco General Hospital Medical Center (RR00083) with support from the National Center for Research Resources. The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the awarding agencies.
We are indebted to Ms. Esther Roitman and Ms. Cathy Kletke for expert technical assistance in carrying out the mass spectrometric analyses, to Ms. Debra Greenblat for assistance in the preparation of the manuscript, and to Ms. Barbara Chang and Ms. Joy Hirai for their expertise in steroid analysis.
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Figure 1:
Overview of Cortisol Metabolism.
Licorice inhibits 11β-hydroxysteroid dehydrogenase (11β-OHSD) activity, resulting in a relative increase in cortisol (F) metabolites, such as 5α- and 5β-tetrahydrocortisol (The) and cortols, and a relative decrease in cortisone (E) metabolites, such as 5β-tetrahydrocortisone (THE) and cortolones.
[Image Omitted: See PDF]
Figure 2:
Plasma Potassium Concentrations (Circles), Urinary Excretion of Sodium (Solid Bars) and Potassium (Hatched Bars) Electrolytes, and Measurements of the Renin-Aldosterone System before and during Licorice Ingestion and for the First Five Days after Licorice Withdrawal.
The initial measurements were obtained before the patient was admitted to the hospital. Plasma renin activity was measured as levels of angiotensin I (normal range, 0.14 to 1.8 ng per liter · second). Urinary aldosterone was measured as the 18-glucuronide metabolite (normal range, 11.8 to 55.6 nmol per day). The normal range for plasma aldosterone concentration is 111 to 334 pmol per liter. ND denotes not determined.
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Figure 3:
Recovery of Plasma Renin Activity, Plasma Aldosterone Levels, and Urinary Aldosterone Excretion after the Withdrawal of Licorice.
Measurements were made with the patient in a recumbent position. Day 0 was the final day of licorice ingestion. Values at right are normal ranges.
[Image Omitted: See PDF]
Figure 4:
Urinary Excretion of Glycyrrhetinic Acid (Stippled Circles) and Cortisol (Bars), and the Ratio of Tetrahydrocortisone to 5β-Tetrahydrocortisol plus 5α-Tetrahydrocortisol (Solid Circles) in Urine after Licorice Withdrawal.
Day 0 was the final day of licorice ingestion.
[Image Omitted: See PDF]
Table 1:
Ratios of Urinary Cortisol Metabolites during and after the Ingestion of Licorice.
[Image Omitted: See PDF]
From the General Clinical Research Center, San Francisco General Hospital Medical Center, San Francisco (R.V.F., E.G.B., I.I., R.G.-F.); and the Children's Hospital Oakland Research Institute, Oakland, Calif. (C.H.L.S.). Address reprint requests to Dr. Biglieri at the Clinical Study Center, Bldg. 100, Rm. 321, 1001 Potrero Ave., San Francisco, CA 94110.
Copyright Massachusetts Medical Society Oct 24, 1991