The role of cortisone acetate in liver RNA synthesis of gamma-irradiated rats.

The possible role of induced endogenous glucocorticoid levels stimulated by ionizing radiation and their subsequent effect on rat-liver RNA was investigated in adrenalectomized rats using an intraperitoneal injection of cortisone acetate immediately prior to exposure to gamma-radiation (1800 rad). Adrenalectomized rats were divided into four groups: (1) control rats, (2) irradiated rats, (3) nonirradiated rats treated with cortisone acetate (10 mg/rat), and (4) irradiated rats which received cortisone acetate immediately before irradiation. Newly synthesized RNA was measured by an intraperitoneal injection of 8 muCi (6-14C) orotic acid either immediately after irradiation or 40 min prior to killing. All the rats were killed 6 hours post-irradiation. The results of these investigations indicate that (1) newly synthesized (40 min labelling) RNA increased in irradiated adrenal-intact rats, but not in adrenalectomized animals; (2) cortisone acetate by itself stimulated RNA synthesis whether the radioactive precursor was administered 6 hours of 40 min prior to killing; and (3) irradiation of adrenalectomized rats pre-treated with cortisone acetate induced more RNA synthesis than did cortisone alone. The results of this study support the suggestion that ionizing radiation may induce rat-liver RNA synthesis, at least in part, via stimulated adrenal secretion of glucocorticoids.     

Isotope ratio mass spectrometry analysis of the oxidation products of the main and minor metabolites of hydrocortisone and cortisone for antidoping controls

Metabolites of hydrocortisone (HC) and cortisone (C), namely tetrahydrocortisol (THF), tetrahydrocortisone (THE), allo-THF, allo-THE for the main metabolites and 11-hydroxyandrosterone, 11-hydoxyetiocholanolone, 11-ketoandrosterone, and 11-ketoetiocholanolone for the minor metabolites, as well as the two main metabolites of testosterone, androsterone and etiocholanolone, were separated from each other using HPLC fractionation of urine extracts. An isotopic ratio mass spectrometry (IRMS) analysis determined the absolute δ¹³C values of 5α-androstanetrione (5α-AT) and 5β-androstanetrione (5β-AT) as the oxidation products (ox-products) of the HC and C metabolites and as target compounds (TCs). We also performed IRMS analysis of 5α-androstanedione (5α-AD) and 5β-androstanedione (5β-AD) as the ox-products of etiocholanolone and androsterone and as endogenous reference compounds (ERCs). Urine samples came from two male volunteers treated with a single 10-mg oral dose and a single 100-mg intramuscular dose of HC hemisuccinate, a male volunteer treated with a single 25-mg oral dose of C acetate, and a control group of 30 drug-free athletes. The mean −3SD of δ¹³C depletion values from the controls were −1.46, −1.98, −1.78 and −2.42 for 5β-AT-5β-AD, 5α-AT-5β-AD, 5β-AT-5α-AD and 5α-AT-5α-AD, respectively, indicating −3‰ as a safe cut-off value for differentiating the pharmaceutical from the natural form. In the main metabolite fraction, δ¹³C depletion values peaked around −5‰ and −9‰ after oral and intramuscular administration of HC, respectively, and around −6‰ after oral administration of C. In comparison, less impressive results were obtained when IRMS analysis focused on the ox-products of the minor metabolites.     

Inhibitory effect of cortisone acetate on the stimulation of rat liver cytosol l-serine Pyruvate aminotransferase by dibutyryl adenosine 3′,5′-monophosphate

An injection of cortisone acetate at a dose of 5 mg/100 g body weight concomitant with dibutryl cyclic AMP prevents the increase in the activity of rat liver cytosol serine aminotransferase (L-serine: pyruvate aminotransferase, EC 2.6.1.51) elicited by the nucleotide with a lag of about 2 h. If the glucocorticoid is given 2 h prior to the nucleotide inducer, the lag disappears. The inhibitory effect of cortisone acetate gradually decays and is no longer detectable 12 h following its administration. Theophylline, insulin and glucose at doses which affect significantly the level of tyrosine aminotransferase, have no effect on the level of serine aminotransferase and on the cortisone inhibition. The inhibitory effect of the glucocorticoid on the dibutyryl cyclic AMP-mediated increase in serine aminotransferase diminishes with the age of animals. Increase in the enzyme activity by a single dose of glucagon can also be inhibited by cortisone acetate and actinomycin D as in the case with dibutyrl cyclic AMP as an inducer. The possibility of the existence of a specific inhibitory factor which is formed in response to cortisone acetate is discussed.     

Inhibitory effect of cortisone acetate on the stimulation of rat liver cytosol L-serrine. Pyruvate aminotransferase by dibutyryl adenosine 3,5-monophosphate.

An injection of cortisone acetate at a dose of 5 mg/100 g body weight concomitant with dibutyryl cyclic AMP prevents the increase in the activity of rat liver cytosol serine aminotransferase (L-serine:pyruvate aminotransferase, EC 2.6.1.51) elicited by the nucleotide with a lag of about 2 h. If the glucocorticoid is given 2 h prior to the nucleotide inducer, the lag disappears. The inhibitory effect of cortisone acetate gradually decays and is no longer detectable 12 h following its administration. Theophylline, insulin and glucose at doses which affect significantly the level of tyrosine aminotransferase, have not effect on the level of serine aminotransferase and on the cortisone inhibition. The inhibitory effect of the glucocorticoid on the dibutyryl cyclic AMP-mediated increase in serin aminotransferase diminishes with the age of animall. Increases in the enzyme activity by a single dose of glucagon can also be inhibited by cortisone acetate and actinomycin D as in the case with dibutyryl cyclic AMP as an inducer. The possibility of the existence of a specific inhibitory factor which is formed in response to cortisone acetate is discussed.     

Evaluation of hepatic 11 beta-hydroxysteroid dehydrogenase activity by cortisone acetate test in young adults with diabetes mellitus type 1

Cortisone acetate test was performed in twelve young adult patients with diabetes mellitus type 1, after dexamethasone administration to suppress endogenous cortisol production. Previous screening revealed that all of the subjects had peak cortisol responses in the range from subnormal to normal, as determined by a low-dose Synacthen test. The aim was to find out whether these patients would exhibit different conversion of cortisone to cortisol by 11beta-hydroxysteroid dehydrogenase. Using multifactorial ANOVA the following significant relationships were obtained between cortisol or cortisol/cortisone ratio measured during the test and other parameters examined a) before dexamethasone suppression and b) during the test: a) Cortisol at 120(th) minute negatively correlated with daily insulin dose and positively with basal aldosterone. Cortisol/cortisone ratio at 60(th), 120(th), 180(th), and 240(th) minute negatively correlated with basal aldosterone/plasma renin activity ratio, urinary free cortisol/24 hours and positively with basal dehydroepindrosterone sulphate. b) Cortisol at 120(th) minute negatively correlated with suppressed basal serum glycemia; cortisol/cortisone ratio during the whole test negatively correlated with supressed basal ACTH. The examination of peripheral metabolism of cortisol using cortisone acetate test in patients with diabetes mellitus type 1 showed adaptive changes of 11beta-hydroxysteroid dehydrogenace activity associated with altered cortisol tissue supply.     

Uptake and metabolism of cortisone and cortisol by the fetal rabbit lung

Lungs of rabbit fetuses at 28 days of gestation were incubated with tritium-labeled cortisone (17α,21-dihydroxy-4-pregnene-3,11,20-trione) or Cortisol (11β,17α,21-trihydroxy-4-pregnene-3,20-dione). The fetal lungs metabolized efficiently cortisone yielding cortisol as the major product (64–71% conversion). Cortisol was poorly metabolized, only 10–14% being converted to cortisone and 68–75% of the substrate being recovered unchanged. A small amount of cortisone (5–7% of tissue radioactivity) was also found in the lungs twenty minutes after injection of labeled cortisol to the fetus $\text{in utero}$. Incubation of fetal lungs with labeled cortisone at 37° resulted in specific uptake and binding of radioactivity (predominantly cortisol) to nuclear macromolecules. The amount of cortisol bound to nuclear macromolecules was similar whether the tissue was incubated with cortisol or cortisone. These results demonstrate that the lungs of the rabbit fetus have the capacity to convert the biologically inactive cortisone to the biologically active cortisol, the reverse reaction occurring only to a limited extent.     

The enrichment of thymocytes bearing C3 receptors following cortisone involution.

Young CBA mice were injected with 2.5 mg of cortisone acetate, following which their spleen cells, thymocytes, and lymph node cells were tested for receptors for the third component of complement (C3) over a 20-day period. An erythrocyte-antibody-complement (EAC) rosette assay was used to detect C3 receptors. Cells bearing C3 receptors in the thymus emerged as early as 2 days after cortisone injection and peaked to a level of 18% at Day 7. This was followed by a decline to control levels by Day 14. There was no significant change in the percentage of C3 receptor-bearing cells in lymph node and spleen of the cortisone injected animals compared to appropriately matched uninjected animals. Evidence has been presented that cortisone-resistant thymocytes may bind EAC, and that these cells are surface Ig negative, contain no or very few macrophages, and bear thy-1 antigen. Complement receptor lymphocytes (CRLs) were separated from the nonrosetting thymus cells by sedimentation in an Isopaque-Ficoll gradient. These enriched C3 receptor-bearing cells were found to possess thy-1 antigens by indirect immunofluorescence. Specificity controls using antisera with thy-1.1 and thy-1.2 specificity and donor animals with thy-1.1 or thy-1.2 phenotypic expression indicated that C3 receptor-bearing cells appearing in the thymi of these respective donors following cortisone involution possessed the appropriate thy-1 phenotype.     

Effects of cortisone and thyroxine on suckling rat liver N-acetyl-ß-glucosaminidase isozymes: Comparison with adult reactivity

Suckling rat liver N-acetyl-β-glucosaminidase (hexosaminidase) activity undergoes considerable fluctuation during the first two weeks of life. As two major forms of hexosaminidase (A, heat-labile, and B, heat-stable) are known to exist in both human and adult rat liver, we choose to examine the effect of the maturative hormones, thyroxine and cortisone, upon these isozymes during the suckling period. Between days 7 and 15, the observed developmental change is attributable solely to an increase in the ‘A-like’ (heat-labile) form of the enzyme; an enhanced response is seen in thyroxine-injected 11–15-day old animals. The response may be considered ‘age-independent’, as adult animals react in the same manner. In contrast, cortisone-injected sucklings show a decrease in both A and B isozymes, while in adults no changes in total activity or isozyme distribution are evoked. The ratio of hexosaminidase A to hexosaminidase B in suckling rat liver appears to shift in favor of the labile (A) isozyme early in development.     

The effects of exogenous cortisone acetate on development (especially skeletal development) and on circulating levels of corticosteroids in chick embryos.

Chick embryos were treated with cortisone acetate on day 8 of incubation and the subsequent growth of the whole embryo and the development of the tibia studied to day 18 of incubation. Cortisone, at 10 ng to 2 mg/embryo decreased general body growth; above 0.5 mg/embryo it also retarded morphogenesis by as much as 3 Hamilton-Hamburger stages; and above 1 mg/embryo gross abnormalities were produced. The growth and differentiation of the tibia were affected to a greater extent than was the whole body. The reductions in tibial and total body weight were not linearly related to dose of cortisone injected. The exogenous cortisone acetate resulted in drastic alterations in the circulating levels of cortisone, cortisol, corticosterone, and progesterone, but plasma progesterone level was most readily correlated with the growth retardation. This study emphasizes that avian embryos can readily compensate for exogenous corticosteroids and that caution must be exercised when attributing causality to the substance administered.     

The response of lymphocytes to H-2D and H-2K region antigens: I. Differential effect of cortisone acetate

Cell surface antigens controlled by separate portions of the H-2 region differentially stimulate lymphocytes. Cells responding to antigens controlled by loci in or near the H-2D region transform later (5 days) than cells responding to antigens controlled by loci in or near the H-2K region (3 days). Treatment of lymphocyte donors with cortisone acetate shows that lymphocytes responding to some H-2K-associated antigens are cortisone resistant and that lymphocytes responding to H-2D-associated antigens are cortisone sensitive. Parallels are drawn between these characteristics and the lymphocytes responsible for cellular and humoral immunity.     

Inter-conversion of 7alpha- and 7beta-hydroxy-dehydroepiandrosterone by the human 11beta-hydroxysteroid dehydrogenase type 1

The dehydroepiandrosterone (DHEA) 7alpha-hydroxylation in humans takes place in the liver, skin, and brain. These organs are targets for the glucocorticoid hormones where 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) activates cortisone through its reduction into cortisol. The putative interference of 7alpha-hydroxy-DHEA with the 11beta-HSD1-catalyzed reduction of cortisone into cortisol has been confirmed in preliminary works with human liver tissue preparations of the enzyme demonstrating the transformation of 7alpha-hydroxy-DHEA into 7-oxo-DHEA and 7beta-hydroxy-DHEA. However, the large production of 7beta-hydroxy-DHEA could not be explained satisfactorily. Therefore our objective was to study the role in the metabolism of oxygenated DHEA by recombinant human 11beta-HSD1 expressed in yeast. The 7alpha- and 7beta-hydroxy-DHEA were each oxidized into 7-oxo-DHEA with quite dissimilar K(M) (70 and 9.5 microM, respectively) but at equivalent V(max). In contrast, the 11beta-HSD1-mediated reduction of 7-oxo-DHEA led to the production of both 7alpha- and 7beta-hydroxy-DHEA with equivalent K(M) (1.1 microM) but with a 7beta-hydroxy-DHEA production characterized by a significantly greater V(max). The 7alpha-hydroxy-DHEA produced by the cytochrome CYP7B1 in tissues may exert anti-glucocorticoid effects through interference with the 11beta-HSD1-mediated cortisone reduction.     

Cyclophosphamide and cortisone acetate inhibit complement biosynthesis by guinea pig bronchoalveolar macrophages.

To explore mechanisms of drug-induced alterations in local host defenses in the lung, the capacity of guinea pig alveolar macrophages to synthesize the second (C2) and fourth (C4) components of complement was studied following treatment with cyclophosphamide and cortisone acetate. Administration of either drug significantly inhibited biosynthesis of C2 and C4 within 1 day. After 1 week of treatment, local complement synthesis was inhibited approximately 80%, although serum levels of the corresponding proteins were normal.     

Effect of glucocorticoid excess on the cortisol/cortisone ratio

OBJECTIVE: The conversion of cortisol, which binds avidly to the mineralocorticoid receptor, to cortisone, which no longer has mineralocorticoid function, is predominantly catalyzed by the 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD 2). It was the objective of the present study to examine the impact of different forms of glucocorticoid excess on the cortisol/cortisone ratio and to differentiate their role in the genesis of hypertension. DESIGN AND METHODS: Plasma cortisol and cortisone levels were determined in 12 adults with Cushing's disease, 12 adults with hypercortisolism due to an adrenal tumor, and 20 healthy volunteers before and after an intravenous ACTH test, using specific radioimmunoassays after automated Sephadex LH 20 chromatography. RESULTS: The cortisol/cortisone ratios were significantly higher in patients with Cushing's disease (13.9 +/- 1.1), adrenal tumors (11.5 +/- 2.3), and in healthy volunteers after ACTH stimulation (14.1 +/- 2.0) than in untreated controls (6.0 +/- 0.5) (P < 0.001, P < 0.05, and P < 0.001, respectively). Similar differences were seen for cortisol plasma concentrations, whereas cortisone concentrations did not differ among the groups. CONCLUSIONS: Our data suggest that the excessive mineralocorticoid effects in patients with hypercortisolism are inflicted by elevated cortisol/cortisone ratios possibly due to an insufficient conversion of cortisol to cortisone by 11beta-HSD 2. This may provide a possible explanation for the occurrence of hypertension. This effect seems to be independent of the role of ACTH in the mechanism of hypercortisolism.     

Increased ratio of serum cortisol to cortisone in acute-phase response

BACKGROUND/OBJECTIVES: 11beta-Hydroxysteroid dehydrogenase (11beta-HSD) enzymes convert cortisol into inactive cortisone and vice versa. While 11beta-HSD type 2 (mainly localized in the kidney) unidirectionally inactivates cortisol to cortisone, type I isoform (mainly localized in the liver) acts bidirectionally and can thus potentially restore cortisone to active cortisol. The aim of this pilot study was to investigate whether the serum cortisol:cortisone ratio is altered during the acute-phase response, possibly due to altered modulation of 11beta-hydroxysteroid dehydrogenase isoforms. METHODS: Using liquid chromatography electrospray tandem mass spectrometry, cortisol and cortisone were measured in the serum of hospitalized patients with normal and abnormal CRP concentrations, the latter indicating acute-phase response. Fifteen unselected samples were analyzed, all with a CRP concentration within one of the following ranges to cover a wide range of CRP concentrations evenly: <5, 5-20, 21-50, 51-100, 101-200, and >200 mg/l. RESULTS: In the heterogeneous study population, increased CRP concentrations significantly correlated with an increased cortisol:cortisone ratio (p < 0.001; r = 0.65, Spearman correlation coefficient). This correlation was independent of increased serum cortisol concentrations found by multivariate regression analysis. The median ratio was 6.4 (interquartile range 5.5-7.4; n = 30) in patients with a CRP concentration < or =20 mg/l, and 11.2 (interquartile range 8.8-13.9; n = 60) in patients with CRP >20 mg/l (p < 0.01). CONCLUSION: The balance between serum cortisol and cortisone is altered during acute-phase response with a shift towards active cortisol, suggesting that 11beta-HSD isoenzymes play a role in the modulation of systemically available cortisol during acute illness.     

Hymenolepis nana: Protective immunity against mouse-derived cysticercoids induced by initial inoculation with eggs

Mice were almost completely resistant to a mouse-derived cysticercoid (cyst) challenge after 6 to 10 months following an initial immunizing inoculation with eggs of Hymenolepis nana. Previously uninfected control mice of the same age became infected with the cyst-derived tapeworms. There was no age resistance to H. nana in mice. Immunity against the cyst challenge was acquired by initial egg inoculation and blocked by injecting cortisone acetate just prior to the challenge. However, the number of worms recovered from mice given cortisone was significantly less than that from nonimmunized controls. Unexpected evidence was obtained that a few of the egg-derived tapeworms can survive for 6 or more months in some of the immunized mice, which are resistant to both egg and cyst challenges. The relative immunogenicity of oncospheres and cysts is discussed. It is strongly suggested that the cysts are different from the oncospheres in their immunogenicity, and, because of this, H. nana can complete its life cycle in the same immunized host. It is also suggested that the host possesses at least two separate immune responses: One is an early response directed exclusively against the oncosphere and/or the early postoncospheral stage (s) acquired within 2 days of egg inoculation, and the other is a late response against the cyst acquired after a time lag of unknown duration.     

Hymenolepis nana: Maturation in an immunosuppressed unnatural rat host

When eggs of the dwarf tapeworm Hymenolepis nana, cycled exclusively and directly through mice for more than 10 years, were inoculated into previously uninfected inbred Fischer (F344) strain rats, they failed to mature in the rat intestinal lumen. Eggs of H. nana inoculated into the rat developed normally into cysticercoids (cysts) in the intestinal tissue, but thereafter failed to mature in the lumen except when the host was treated with cortisone acetate from the day of cyst maturation. The Fischer rat initially given eggs of H. nana became completely immune to egg challenge within 2 days of egg inoculation; no cysts derived from challenge eggs were found in the immunized rat. Immunosuppression, assessed by the success of cyst recovery in the tissue 4 days after egg challenge, had no promotive effect on the recovery of adult worms derived from eggs initially inoculated. Rats initially given eggs and immunosuppressed by cyclophosphamide or antithymocyte serum did not harbor any adult worms. Cortisone acetate treatment which was sufficient for eggs inoculated to mature (a total of 75 or even 200 mg, from Day 5 of egg inoculation) had no effects of immunosuppression, whereas cortisone acetate treatment which was sufficient for immunosuppression (a total of 150 mg from Day -2, two days prior to the initial egg inoculation) induced some adult formation as well. In addition, when mouse-derived cysts were inoculated into the rat instead of eggs, they also failed completely to mature even when the rat was treated with cyclophosphamide or antithimocyte serum. However, when the rat was treated with cortisone acetate from the day of cyst inoculation, the cysts developed into adult worms. Therefore, these results indicate that the Fischer rat clearly differs in its susceptibility to the tissue phase of egg inoculation and to the lumen phase of cyst inoculation of H. nana, and strongly suggest that the failure of maturation of H. nana in the unnatural host Fischer rat is not attributed to innate and/ or acquired immunity of the rat but to other nonimmunological mechanisms.     

Apparent cortisone reductase deficiency: a rare cause of hyperandrogenemia and hypercortisolism

A 55-year-old woman presented with androgenetic alopecia which had started at age 40. Her clinical history revealed that, unlike her younger sister, she was unable to conceive and was diagnosed as being sterile at age 30. At age 45, 21-hydroxylase deficiency (late-onset CAH) was assumed and glucocorticoid treatment suggested, but not initiated. There was slight hirsutism, but no other sign of virilization. Retesting of plasma steroids revealed elevated 17-OH-progesterone and free testosterone. Treatment with prednisone, cyproterone acetate, and spironolactone was started with significant clinical success. Surprisingly, the analyses of urinary steroid metabolites revealed a pattern that did not support the diagnosis of 21-hydroxylase deficiency (pregnanetriolone absent, pregnanediol, 17-OH-pregnanolone and pregnanetriol not increased). Abdominal CT showed bilateral adrenal hyperplasia and masses in both ovaries. Bilateral adnexectomy was performed, and cystic teratomas were diagnosed. Postoperative urinary steroid analyses showed a decreased tetrahydrocortisol/tetrahydrocortisone ratio (values around 0.08 as compared to age- and sex-matched controls with a ratio of about 0.5-0.8). Plasma cortisol appeared to be repeatedly elevated with exogenous sources excluded. Mass spectrometry showed that, while the tetrahydro metabolites were mainly cortisone-derived, the metabolites not reduced in A ring were mostly cortisol derivatives. This constellation clearly indicates cortisone reductase deficiency, a defect of hepatic 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD1). This enzyme catalyzes the oxidation of cortisol to cortisone and the reduction of cortisone to cortisol. In contrast to the corresponding kidney enzyme (11 beta-HSD2), its primary activity is, however, reductive. Although this is only the fifth reported case of that defect, more attention should be paid to this condition in hyperandrogenic women, even if elevated 17-OH-progesterone and testosterone suggest a more frequent cause.     

Inhibition of cortisone action in mice by heparin.

A single dose of heparin applied in a depot-form (Freund's incomplete adjuvant or Ca-phosphate gel) inhibits the effects of intraperitoneally injected cortisone on the lymphoid organs (thymus and spleen), on the peritoneal and peripheral lymphoid cell count and serum gamma globulin level as well as on the liver glycogen deposition in mice. The same dose of heparin did not influence the action of hydrocortisone measured on the thymic and spleen involution and liver glycogen content. The route of cortisone administration seems to be critical, as heparin shows no or only minor effects when cortisone is applied subcutaneously; moreover, the action of cortisone per se is more marked by subcutaneous than by intraperitoneal administration. The results suggest the hypothesis that heparin inhibits cortisone-cortisol conversion and this inhibition is mediated by macrophages.     

Prenatal development of acid beta-glycosidases in the rat liver, effect of triiodothyronine or cortisone administered to pregnant rats.

We have found that acid beta-galactosidase, beta-glucuronidase and N-acetyl-beta-glucosaminidase in the rat fetal liver increase during the last week of pregnancy. These enzyme activities were influenced by treatment of pregnant rats (daily from day 16) with L-tri-iodothyronine (20 or 50 microgram/100 gm b.w.) or cortisone acetate (10 or 50 mg/100 gm b.w.) as studied in their fetuses obtained on day 22 by caesarian section.     

Urinary free cortisol and cortisone excretion in healthy individuals: influence of water loading

The influence of water loading on urinary excretion of free cortisol and cortisone was investigated in healthy men. The results were as follows: water loading tests (intake of 0.25-1.5 L) in a single individual showed that a water load of 1.5 L reliably increased the excretion of urine, free cortisol and cortisone (p < 0.01). Regression analyses gave significant correlations of urine volume with free cortisol and free cortisone, and of free cortisol and free cortisone. Corresponding results were obtained when water loading tests were performed in males who ingested 1.5 L of water (n = 8): the excretion of urine, free cortisol and free cortisone were significantly augmented; correlated was urine volume with free cortisol and free cortisone, and free cortisol with free cortisone. In a third set of tests, volunteers collected one 5 h urine (10:00-15:00 h) after the intake of 3 x 0.1 or 0.5 L at 11:00, 12:00 and 14:00 h. Excretion of urine, free cortisol and free cortisone in males of the low water loading group (3 x 0.1 L) was 0.59 mL/min, and 8.2 or 15.0 microg/5 h; corresponding values in individuals ingesting 3 x 0.5 L of water were 1.5 mL/min (p < 0.01), 12.3 microg/5 h (p > 0.05) and 26.3 microg/5 h (p < 0.02). In summary, urinary free cortisol and cortisone excretion in healthy men depends on urine volume, especially during water diuresis. Thus, interpretation of free cortisol and especially of free cortisone excretion is only possible if subjects strictly control their fluid intake and if urine volume is considered an important pre-analytical parameter-otherwise, interpretation of urinary free cortisol results is difficult and of urinary free cortisone data remains tenuous at best.