Effect of licorice on the reduction of body fat mass in healthy subjects.

The history of licorice, as a medicinal plant, is very old and has been used in many societies throughout the millennia. The active principle, glycyrrhetinic acid, is responsible for sodium retention and hypertension, which is the most common side-effect. We show an effect of licorice in reducing body fat mass. We studied 15 normal-weight subjects (7 males, age 22-26 yr, and 8 females, age 21-26 yr), who consumed for 2 months 3.5 g a day of a commercial preparation of licorice. Body fat mass (BFM, expressed as percentage of total body weight, by skinfold thickness and by bioelectrical impedance analysis, BIA) and extracellular water (ECW, percentage of total body water, by BIA) were measured. Body mass index (BMI) did not change. ECW increased (males: 41.8+/-2.0 before vs 47.0+/-2.3 after, p<0.001; females: 48.2+/-1.4 before vs 49.4+/-2.1 after, p<0.05). BFM was reduced by licorice: (male: before 12.0+/-2.1 vs after 10.8+/-2.9%, p<0.02; female: before 24.9+/-5.1 vs after 22.1+/-5.4, p<0.02); plasma renin activity (PRA) and aldosterone were suppressed. Licorice was able to reduce body fat mass and to suppress aldosterone, without any change in BMI. Since the subjects were consuming the same amount of calories during the study, we suggest that licorice can reduce fat by inhibiting 11beta-hydroxysteroid dehydrogenase Type 1 at the level of fat cells.

Regulation of corticosteroid receptors in patients with anorexia nervosa and Cushing's syndrome.

We have studied 16 patients with anorexia nervosa (11 with a stabilised weight loss and 5 in the weight-losing phase), 11 healthy controls, and 10 patients with Cushing's syndrome, by measuring plasma cortisol (by enzyme-immunoassay), ACTH (by RIA), corticosteroid (Type I-mineralocorticoid and Type II-glucocorticoid) receptors in mononuclear leukocytes (by radio-receptor assay), and lymphocyte subpopulations (by cytofluorimetry). In anorexic patients with a stabilised weight loss and in Cushing's syndrome the mean value of both Type I and Type II corticosteroid receptors in mononuclear leukocytes was significantly lower than in controls. The correlation between Type II receptors and plasma cortisol was inverse in stabilised anorexia nervosa and in Cushing's syndrome, and direct in healthy controls. Anorexic patients in the weight-losing phase showed a significant increase in plasma cortisol levels and a normal number of Type II receptors. From these results we hypothesise that in anorexia nervosa there is a progression from an increase in plasma cortisol in the weight-losing phase, to a concomitant decrease in Type II receptors when the disease is stabilised.

Pseudohypoaldosteronism: evaluation of type I receptors by radioreceptor assay and by antireceptor antibodies.

We have previously demonstrated a deficiency of mineralocorticoid receptors in pseudohypoaldosteronism, by radioreceptorassay. We now report findings with an antireceptor antibody derived from the immunogenic region of the receptor. Lymphocytes from normal controls and from two cases of pseudohypoaldosteronism previously shown to lack receptor binding were tested. After the plasma membrane of lymphocytes was permeabilized with methanol the cells were incubated with a 1:200 dilution of antibody followed by fluorescent antirabbit immunoglobulin mouse serum. After washing fluorescence was detected by microscopy and cytofluorimetry in both controls and patients with pseudohypoaldosteronism. Recent studies on mineralocorticoid receptor cDNA in pseudohypoaldosteronism have not established a mutation in the sequence. We thus suggest that the pathogenesis of pseudohypoaldosteronism is not related to an abnormality of the receptor but rather due to intracellular factor(s) which can block the binding of aldosterone to its receptor.

Corticosteroid receptors and aging.

We have measured plasma aldosterone, plasma cortisol, and glucocorticoid and mineralocorticoid receptors in mononuclear leukocytes in 54 healthy aged subjects (60-97 years old) and in a group of 21 controls (21-50 years old). In addition all parameters and age were plotted for correlation. Plasma cortisol was significantly higher in the aged group (346 +/- 140 nmol/l) than in controls (260 +/- 120). Mean plasma aldosterone was not different in the two groups. Both Type I and II receptors in mononuclear leukocytes were lower in the aged group than in controls (Type I 198 +/- 96 and 272 +/- 97 receptors per cell; Type II 1738 +/- 801 and 3339 +/- 918 receptors per cell). A direct correlation was found between cortisol and age and between Type I and II receptors in aged subjects, and between cortisol and Type I receptors, and cortisol and Type II receptors in controls. When all subjects are plotted together, a direct correlation was observed between cortisol and age and between Type I and II receptors, and an inverse correlation between age and Type I and age and Type II receptors. We conclude that the parallel reduction of both Type I and II receptors with age is not due to prior cortisol increase, but the increase of plasma cortisol can be considered the result of age-dependent involution of these receptors.

Corticosteroid receptors and lymphocyte subsets in mononuclear leukocytes in aging.

Plasma cortisol and aldosterone levels and number of related receptors in mononuclear leukocytes were measured in 49 healthy aged subjects (62-97 yr) and in 21 adult controls (21-50 yr). In all subjects, in addition, lymphocyte subsets were determined as an index of corticosteroid action. The mean number of type I and type II receptors was significantly lower in aged subjects than in controls (respectively, 198 +/- 96 and 272 +/- 97 receptors/cell for type I, and 1,794 +/- 803 and 3,339 +/- 918 for type II receptors). Plasma aldosterone and cortisol and lymphocyte subsets were not different in the two groups. All of the parameters were also tested for correlation, and a significant inverse correlation was found between age and type I and type II receptors when all subjects were plotted and between aged and CD4 and age and CD4/CD8 in the aged group. These data show that aged subjects have reductions of corticosteroid receptors that are not associated with increase of related steroids and that this situation probably represents a concomitant of the normal aging process.

The pathogenesis of pseudohyperaldosteronism from carbenoxolone.

Carbenoxolone is a derivative of glycyrrhetinic acid used for the treatment of peptic ulcer and gastritis, with salt and water retention a very common side-effect. To investigate this drug-induced pseudohyperaldosteronism we have studied 6 male volunteers before, during and after treatment with carbenoxolone for 7 days. Serum, urinary and sweat electrolytes values were consistent with a mineralocorticoid-like effect of drug administration. PRA was suppressed, and plasma cortisol and aldosterone progressively decreased over treatment. We have also determined by radioreceptor assay the plasma levels of factors which bind to mineralocorticoid receptors in rat kidney cytosol. The levels of these factors were decreased significantly at day 3 of treatment, suggesting a local renal effect of carbenoxolone to amplify endogenous steroid action. At day 7 the radioreceptor assay values were still decreased but significantly higher than at day 3, suggesting in addition a direct mineralocorticoid effect of the drug. We conclude that the drug is initially effective by amplifying the effect of endogenous steroids, and then when the plasma concentrations of the drug or its metabolites reach a higher plasma concentration, there may also be in addition a direct mineralocorticoid-like effect.

Canrenone and androgen receptor-active materials in plasma of cirrhotic patients during long-term K-canrenoate or spironolactone therapy.

Plasma levels of canrenone and androgen receptor-active materials (ARM) were determined during long-term oral K-canrenoate or spironolactone therapy in cirrhotics with chronic recurrent ascites. Mean plasma canrenone level was approximately 3 times higher under K-canrenoate than under spironolactone treatment; moreover, the levels were not dose related. Either type of treatment did not affect plasma aldosterone and testosterone concentrations. Plasma ARM during K-canrenoate treatment did not change, whereas in the spironolactone group a 3-fold increase of ARM occurred (p less than 0.05). No dose-related effect was evident with the latter treatment. The lower incidence of gynecomastia in the K-canrenoate group was not correlated with values of plasma canrenone or ARM (p greater than 0.05). Our study questions the traditional view that the mode of action of spironolactone is via its metabolite canrenone. The two antialdosterone drugs, although equally effective in clearing ascites from cirrhotics, appear to act through partially different metabolites. The lower incidence of antiandrogenic or estrogen-like side effects during K-canrenoate seems to be related to metabolites other than canrenone itself.

Determination of lymphocyte subpopulations by enzyme immunoassay. Comparison with conventional fluorescence microscopy.

An enzyme immunoassay for measuring lymphocyte subpopulations was evaluated and the results compared with those obtained with conventional fluorescence microscopy, using two different panels of antibodies. The enzyme immunoassay is a photometric method which expresses the results using a standard curve with known amounts of cells. The method was reproducible and accurate. The intra-assay variation for the standard curve ranged from 3.2 to 5.7% and the interassay variation from 9.5 to 13.8%. The intra-assay variation for clinical samples ranged from 3.3 to 8.2%. Results obtained with the enzyme immunoassay and with conventional fluorescence microscopy showed a significant correlation (P less than 0.05) for all the subclasses of lymphocytes tested using two different panels of monoclonal antibodies. We conclude that the choice of method should be related to the particular needs and manpower in the laboratory.

In-vivo metabolites of spironolactone and potassium canrenoate: determination of potential anti-androgenic activity by a mouse kidney cytosol receptor assay.

We have established a sensitive and specific radioreceptor assay for androgen receptor active materials in plasma, using tritiated methyltrienolone ([3H]R1881) as tracer, and spayed mouse kidney cytosol receptor as the binding species. On radioreceptor assay, plasma from mice chronically administered spironolactone contained approximately 10 times higher levels of androgen receptor active material than from mice administered potassium canrenoate. In parallel bioassays (antagonism of the effect of testosterone on seminal vesicle weight), spironolactone was greater than 4 times as potent an antiandrogen as potassium canrenoate. Administered potassium canrenoate circulates as canrenoic acid, in equilibrium with its lactonized congener canrenone. Since over 80% of administered spironolactone is irreversibly converted to canrenone/canrenoic acid, its much higher anti-androgen activity on radioreceptor assay and bioassay may point to the generation of unidentified, minor metabolites with very high affinity for androgen receptors and/or a very long plasma half-life.

Affinity of liquorice derivatives for mineralocorticoid and glucocorticoid receptors.

Liquorice abuse causes a syndrome of pseudohyperaldosteronism. Much less commonly, glucocorticoid-like effects have been reported. The electrolyte-active principle of liquorice is glycyrrhizic acid (GI), which can be hydrolyzed to glycyrrhetinic acid (GE). Previous studies have reported that GE, but not GI, may occupy mineralocorticoid and glucocorticoid receptors. We here report that both GE and GI can bind to both mineralocorticoid and glucocorticoid receptors. The affinity of GI for mineralocorticoid receptors is four orders of magnitude lower than aldosterone and for glucocorticoid receptors five orders of magnitude lower than dexamethasone. The affinity, though low, is sufficient to explain the mineralocorticoid-like side effects, given the large amount of liquorice required to produce such a syndrome.

The mechanism of mineralocorticoid action of carbenoxolone.

The principal side effects of the drug carbenoxolone (Biogastrone; 18 beta-glycyrrhetinic acid sodium hemisuccinate) are sodium retention, hypokalemic alkalosis, suppressed plasma renin, and hypertension. In previous animal studies, carbenoxolone appeared not to have intrinsic mineralocorticoid activity but, rather, to enhance aldosterone action by displacing it from nonspecific binding sites. We here report studies showing that carbenoxolone has demonstrable affinity for rat kidney mineralocorticoid receptors, intrinsic mineralocorticoid activity in the adrenalectomized rat at doses consistent with its receptor affinity, and, in addition, a powerful action of amplifying the electrolyte effects of near-maximal doses of aldosterone.

Free urinary cortisol radioimmunoassay (application in the diagnosis of adrenal diseases).

The availability of a specific antiserum has made it possible to develop in our laboratory a radioimmunoassay for free urinary cortisol which is quite simple and rapid to perform. No preliminary procedures of chromatographic purification are necessary, and no correction for losses is required. Precision and accuracy tests are satisfactory. Seventeen normal subjects, 10 obese subjects, 18 patients with Cushing's syndrome, 4 hypopituitaric patients and 4 with Addison's disease were studied. The values of free urinary cortisol were compared with those of urinary 17-OHCS, plasma cortisol and, in some cases, the cortisol secretion rate. In normal subjects, the mean free urinary cortisol was 77.22 +/- 7.74 microgram/24 h, in obese subjects it was 112.05 +/- 13.64 microgram/24 h, and in patients with Cushing's syndrome it was at significantly higher levels with a mean value of 488.06 +/- 64.39 microgram/24 h. There was no overlap between the values obtained in the first two groups and those of subjects with adrenal hyperfunction, and the results were similar to those shown by the cortisol secretion rate. The same was not true for urinary 17-OHCS and plasma cortisol. Thus, the assay of free urinary cortisol, that is the free biologically active circulating fraction, may provide a relatively simple alternative to measurement of the cortisol secretion rate, as a single differential test for the diagnosis of conditions with adrenal hyperfunction. Less satisfactory were the results obtained in patients with primary and secondary adrenal hypofunction. Another limitation of the method is its relative non-specificity, since the results of 16 urinary samples measured without preliminary chromatography were 29.6% higher than those after previous chromatographic purification. However, in our experience, this overestimation does not affect the value of the method as a screening test for the diagnosis of Cushing's syndrome.