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Cyclodextrins (CDs) can improve the productivity of steroid biotransformation by enhancing substrate solubility. CDs can be recycled by grafting them with appropriate carriers. Loofah fiber is an excellent grafting material for CDs, and can be applied to the biotransformation and recycling of β-cyclodextrin (β-CD). In this work, a technique for recycling β-CD in cortisone acetate (CA) biotransformation by Arthrobacter simplex CPCC 140451 was studied. Loofah fiber-grafted β-CD (LF-β-CD) was prepared using epichlorohydrin, which is a cross-linking agent. The grafting yield of β-CD was 74.8 mg g -1 dried fibers. LF-β-CD could increase the solubility of CA and enhance biotransformation. The initial conversion rate of CA was 1.5-fold higher than that of the blank group. LF-β-CD was also used in biocatalytic reactions for eight cycles, and it maintained the conversion ratio of CA at approximately 90%. Given the above positive results, LF-β-CD can be utilized in biotechnological recycling applications. This method can also be applied to CD derivatives and hydrophobic compounds.

A specific and sensitive method based on high-performance liquid chromatography with ultraviolet absorbance detection (HPLC-UV) was developed for the simultaneous determination of urinary cortisol (F), cortisone (E), 6β-hydroxycortisol (6β-OHF) and 6β-hydroxycortisone (6β-OHE) using dexamethasone as the internal standard. The method involved solid-phase extraction of the five compounds from urine using Oasis HLB Waters cartridges with an elution solvent of ethyl acetate-diethyl ether (5 mL; 4:1, v/v), followed by 1 mol/L of NaOH (1 mL) and 1.0% acetic acid (1 mL). Separation of the five analytes was achieved within 31 min by using a reversed-phase C18 analytical column (200 × 4.6 mm, 5 µm, Agilent). A UV detector operated at 245 nm was used. According to the method validation, inter-run and intra-run precision was below 9.45% and accuracy ranged from 98.16 to 115.50%. The lower limits of quantitation were 5 ng/mL for four analytes. This is the first HPLC method that can simultaneously determine F, E, 6β-OHF and 6β-OHE in human urine. The assay was applied to research the ratio of (6β-OHF + 6β-OHE)/(F + E) as a non-invasive biomarker for the metabolism of tacrolimus., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Glucocorticoids (GC) are implicated in the development of metabolic syndrome, and patients with GC excess share many clinical features, such as central obesity and glucose intolerance. In patients with obesity or type 2 diabetes, systemic GC concentrations seem to be invariably normal. Tissue GC concentrations determined by the hypothalamic-pituitary-adrenal (HPA) axis and local cortisol (corticosterone in mice) regeneration from cortisone (11-dehydrocorticosterone in mice) by the 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme, principally expressed in the liver. Transgenic mice have demonstrated the importance of 11β-HSD1 in mediating aspects of the metabolic syndrome, as well as HPA axis control. In order to address the primacy of hepatic 11β-HSD1 in regulating metabolism and the HPA axis, we have generated liver-specific 11β-HSD1 knockout (LKO) mice, assessed biomarkers of GC metabolism, and examined responses to high-fat feeding. LKO mice were able to regenerate cortisol from cortisone to 40% of control and had no discernible difference in a urinary metabolite marker of 11β-HSD1 activity. Although circulating corticosterone was unaltered, adrenal size was increased, indicative of chronic HPA stimulation. There was a mild improvement in glucose tolerance but with insulin sensitivity largely unaffected. Adiposity and body weight were unaffected as were aspects of hepatic lipid homeostasis, triglyceride accumulation, and serum lipids. Additionally, no changes in the expression of genes involved in glucose or lipid homeostasis were observed. Liver-specific deletion of 11β-HSD1 reduces corticosterone regeneration and may be important for setting aspects of HPA axis tone, without impacting upon urinary steroid metabolite profile. These discordant data have significant implications for the use of these biomarkers of 11β-HSD1 activity in clinical studies. The paucity of metabolic abnormalities in LKO points to important compensatory effects by HPA activation and to a crucial role of extrahepatic 11β-HSD1 expression, highlighting the contribution of cross talk between GC target tissues in determining metabolic phenotype.

Aspergillus terreus is emerging as a causative agent of life-threatening invasive aspergillosis. Prognosis for affected patients is often worse than for A. fumigatus infections. To study A. terreus-mediated disease, we developed 3 infection models. In embryonated hen's eggs and leucopenic mice, the outcome of invasive aspergillosis was similar to that described for A. fumigatus. However, 10(2)- and 10(3)-fold higher conidia concentrations were required for 100% lethality. In corticosteroid-treated mice, only 50% mortality was observed, although bioluminescence imaging revealed transient disease in all infected animals. In surviving animals, we observed persistence of ungerminated but viable conidia. Cytokine levels in these mice were comparable to uninfected controls. In contrast to A. fumigatus infections, all mice infected with A. terreus developed fatty liver degeneration, suggesting the production of toxic secondary metabolites. Thus, at least in mice, persistence and subclinical liver damage are unique features of A. terreus infections.

Objectives: Amphotericin B inhalation powder (ABIP) is a novel dry-powder amphotericin B formulation that is directly delivered to the lung, resulting in elevated lung tissue drug concentrations of this polyene. We evaluated the prophylactic efficacy of single dose administration of ABIP in a guinea pig model of invasive pulmonary aspergillosis., Methods: Guinea pigs were immunosuppressed with cyclophosphamide and cortisone acetate and challenged with Aspergillus fumigatus conidia in an aerosol chamber. Guinea pigs received prophylaxis with a single inhaled dose of ABIP at 0.05, 0.5, 4 or 10 mg/kg administered 24 h prior to infection. Treatment with oral voriconazole at doses of 5 or 10 mg/kg twice daily beginning 24 h post-challenge served as the positive control., Results: Improvements in survival were observed with ABIP prophylaxis. A single inhaled dose of 4 mg/kg ABIP and treatment with 5 mg/kg voriconazole both improved median and percentage survival compared with untreated controls. In addition, pulmonary fungal burden, as assessed by cfu, quantitative PCR and galactomannan, was also reduced in a dose-dependent fashion with ABIP prophylaxis as well as with both doses of voriconazole treatment., Conclusions: Single-dose prophylaxis with inhaled ABIP as prophylaxis demonstrated a significant survival advantage and reductions in pulmonary fungal burden in this model of invasive pulmonary aspergillosis. Optimization of the dose and dosing frequency of ABIP dose may help to further enhance the anti-Aspergillus activity of this novel amphotericin B formulation.

Objective: Failure to mirror the diurnal cortisol profile could contribute to the impaired subjective health status in Addison's disease (AD). Some patients report benefit from the use of various nutritional compounds. The objective of this study was to investigate the impact of licorice and grapefruit juice (GFJ) on the absorption and metabolism of cortisone acetate (CA)., Design: Patients (n=17) with AD on stable CA replacement therapy were recruited from the outpatient clinic at Haukeland University Hospital, Norway. They were assessed on their ordinary CA medication and following two 3-day periods of co-administration of licorice or GFJ., Methods: Time series of glucocorticoids (GCs) in serum and saliva were obtained, and GCs in 24 h urine samples were determined. The main outcome measure was the area under the curve (AUC) for serum cortisol in the first 2.6 h after orally administered CA., Results: Compared with the ordinary treatment, the median AUC for serum cortisol increased with licorice (53 783 vs 50 882, P<0.05) and GFJ (60 661 vs 50 882, P<0.05). Median cortisol levels in serum were also elevated 2.6 h after tablet ingestion (licorice 223 vs 186 nmol/l, P<0.05; GFJ 337 vs 186 nmol/l, P<0.01). Licorice increased the median urinary cortisol/cortisone ratio (0.43 vs 0.21, P<0.00001), whereas GFJ increased the (allo-tetrahydrocortisol+tetrahydrocortisol)/tetrahydrocortisone ratio (0.55 vs 0.43, P<0.05)., Conclusion: Licorice and in particular GFJ increased cortisol available to tissues in the hours following oral CA administration. Both patients and physicians should be aware of these interactions.

Production of the proinflammatory cytokines interleukin (IL)-1 alpha and tumor necrosis factor (TNF)-alpha and of the chemotactic chemokine macrophage inflammatory protein (MIP)-1 alpha by bronchoalveolar macrophages (BAMs) from mice in response to Aspergillus conidia was tested after in vivo administration of saline, dexamethasone, cortisone acetate, granulocyte-macrophage colony-stimulating factor (GM-CSF), or a combination. Dexamethasone suppressed production of IL-1 alpha, TNF-alpha, and MIP-1 alpha; GM-CSF reduced secretion slightly but antagonized dexamethasone suppression when the two were given in combination. Cortisone acetate gave results similar to dexamethasone, but cortisone acetate suppression of BAM responses lasted 7 days, > or = 4 days longer than dexamethasone suppression. The effect of GM-CSF on cortisone acetate suppression lasted at least 7 days. GM-CSF could promote resistance to conidia by maintaining proinflammatory responses.

Invasive pulmonary aspergillosis is an emerging devastating infection in the immunocompromised host that is treated with corticosteroids for neoplastic disease or for organ transplantation. By use of a model of invasive pulmonary aspergillosis in corticosteroid-treated CF-1 mice, prior infection and 2 Aspergillus fumigatus vaccine preparations (sonicate and filtrate) administered intranasally and subcutaneously were tested for efficacy in protecting against subsequent lethal A. fumigatus infection. The mortality rates were as follows: control subjects, 100%; prior infection, 12.5%; sonicate administered intranasally, 29%; sonicate given subcutaneously, 0%; filtrate given intranasally, 75%; and filtrate given subcutaneously, 50%. Prior infection and A. fumigatus sonicate vaccine administered by 2 routes protected corticosteroid-treated animals against subsequent lethal invasive pulmonary aspergillosis. The sonicate vaccine was more protective, but the subcutaneous route was more effective.

Classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency is characterized by a defect in cortisol and aldosterone secretion and adrenal hyperandrogenism. Current treatment is to provide adequate glucocorticoid and mineralocorticoid substitution to prevent adrenal crises and to suppress excess adrenal androgen secretion. Satisfactory adrenocortical suppression often requires supraphysiological doses of hydrocortisone, which may produce an unacceptable degree of hypercortisolism. A new four-drug treatment regimen of flutamide, testolactone, reduced hydrocortisone dose, and 9alpha-fludrocortisone has been shown to achieve normal growth and development after 2 yr of therapy and may, therefore, represent a potential alternative approach to the treatment of children with classic congenital adrenal hyperplasia. We investigated the effect of flutamide and testolactone, and flutamide alone, on cortisol clearance by performing clearance studies twice in 13 children (6 males and 7 females; age range, 7.0-14.5 yr) with classic 21-hydroxylase deficiency. All studies were conducted at least 3 months after institution of the four-drug treatment regimen. In eight patients (group 1), the first cortisol clearance study was performed on the four-drug regimen, and the second study was performed after a 48-h washout period off flutamide and testolactone. In five patients (group 2), the first study was conducted 1 wk after discontinuation of testolactone and while patients were receiving flutamide, hydrocortisone and 9alpha-fludrocortisone, and the second study was performed after a 48-h washout period off flutamide. Oral hydrocortisone was held on the day of the clearance studies, and all patients received a continuous infusion of hydrocortisone (0.6 mg/m(2).h) from 1800 h to 0200 h, with cortisol concentrations measured once hourly. In addition, an in vitro study was conducted to exclude the possibility of an analytical interference of flutamide, 2-hydroxyflutamide, and testolactone with the serum cortisol immunoassay. Total body cortisol clearance was significantly lower during treatment with the four-drug regimen than during treatment with hydrocortisone and 9alpha-fludrocortisone (153.5 +/- 26.8 vs.355.4 +/- 65.8 ml/min; P = 0.001). Similar results were obtained comparing flutamide, hydrocortisone, and 9alpha-fludrocortisone therapy to hydrocortisone and 9alpha-fludrocortisone therapy (155.8 +/- 26.5 vs. 281.8 +/- 96.2 ml/min; P = 0.037). The in vitro study indicated that an interference with the serum cortisol immunoassay was unlikely. These findings indicate that the addition of flutamide and testolactone to the treatment regimen of hydrocortisone and 9alpha-fludrocortisone decreases cortisol clearance in patients with classic 21-hydroxylase deficiency, and this effect seems to be due to flutamide. Glucocorticoid replacement doses should be reduced when flutamide is added to the treatment regimen of patients receiving hydrocortisone.

Cortisol is metabolized irreversibly by A-ring reductases (5alpha- and 5beta-reductases) and reversibly (to cortisone) by 11beta-hydroxysteroid dehydrogenases (11betaHSDs). In rats, estradiol down-regulates 11betaHSD1 expression. In humans, ratios of urinary cortisol/cortisone metabolites differ in men and women. In this study, urinary cortisol metabolites and hepatic 11betaHSD1 activity were measured in healthy young men and women at different phases of the menstrual cycle. Ten men and 10 women with regular menstrual cycles collected a 24-h urine sample, took 250 microg oral dexamethasone at 2300 h, took 25 mg oral cortisone at 0900 h (after fasting), and had blood sampled for plasma cortisol estimation over the subsequent 150 min. Women repeated the tests in random order in menstrual, follicular, and luteal phases. Women excreted disproportionately less A-ring-reduced metabolites of cortisol [median 5alpha-tetrahydrocortisol, 1811 (interquartile range, 1391-2300) microg/day in menstrual phase vs. 2723 (interquartile range, 2454-3154) in men (P = 0.01); 5beta-tetrahydrocortisol, 1600 (interquartile range, 1419-1968) vs. 2197 (interquartile range, 1748-2995; P = 0.03)] but similar amounts of cortisol, cortisone, and tetrahydrocortisone. Analogous differences were observed in urinary excretion of androgen metabolites. Conversion of cortisone to cortisol on hepatic first pass metabolism was not different (peak plasma cortisol, 733 +/- 60 nmol/L in women vs. 684 +/- 53 nmol/L in men; mean +/- SEM; P = 0.55). There were no differences in cortisol or androgen metabolism between phases of the menstrual cycle. We conclude that sexual dimorphism in cortisol metabolite excretion is attributable to less A-ring reduction of cortisol in women, rather than less reactivation of cortisone to cortisol by 11betaHSD1. This difference is not influenced acutely by gonadal steroids. 11BetaHSD1 has been suggested to modulate insulin sensitivity and body fat distribution, but caution must be exercised in extrapolating inferences about its regulation from rodents to man. A-Ring reductases may have an equally important influence on metabolic clearance of cortisol and intracellular cortisol concentrations.

Congenital adrenal hyperplasia in children is often treated with cortisone acetate and fludrocortisone. It is known that certain patients with congenital adrenal hyperplasia require very high substitution doses of cortisone acetate, and a few patients do not respond to this treatment at all. A patient with 21-hydroxylase deficiency, for whom elevated pregnanetriol (P3) levels in urine were not suppressed during treatment with cortisone acetate (65 mg/m2 x day), was examined. The activation of cortisone to cortisol was assessed by measuring urinary metabolites of cortisone and cortisol. The patient's inability to respond to treatment with cortisone acetate was found to be caused by a low conversion of cortisone to cortisol, assumed to be secondary to low 11beta-hydroxysteroid dehydrogenase activity (11-oxoreductase deficiency). All exons and exon/intron junctions of the 11beta-hydroxysteroid dehydrogenase type1 gene (HSD11L) were sequenced without finding any mutations, but a genetic lesion in the promoter or other regulatory regions cannot be ruled out. The deficient 11-oxoreductase activity seems to have been congenital, in this case, but can possibly be attributable to a down-regulation of the enzyme activity. The results support the use of hydrocortisone, rather than cortisone acetate, for substitution therapy in adrenal insufficiency.

Assessment of mineralocorticoid replacement therapy in Addison's disease relies on clinical features and laboratory measurements, including plasma renin and potassium. Previous studies have questioned the value of measuring the plasma renin concentration (PRC), particularly in the setting of fludrocortisone overreplacement. The aim of this study was to evaluate the usefulness of plasma atrial natriuretic peptide (ANP) measurements as a marker of sodium and volume status in Addison's disease. Fourteen patients with Addison's disease receiving their usual glucocorticoid doses were placed on various doses of fludrocortisone (FC; 0 mg, 0.05 mg, 0.1 mg and 0.2 mg) in random order for four 2-week periods. At the end of each period, blood pressure and clinical symptoms were assessed, and blood was drawn for measurement of PRC and ANP levels. PRC was significantly elevated in patients receiving placebo (54.2 +/- 57.9 ng/mL x h) compared with PRC in those receiving baseline FC (24.7 +/- 42.4 ng/mL x h), 0.1 mg FC (15.2 +/- 25.9 ng/mL x h), and 0.2 mg FC (5.5 +/- 5.7 ng/mL x h). ANP levels were measured by either an extraction method (ANP(ext)) or directly from plasma (ANP(dir)). ANP(dir) was significantly elevated at 0.2 mg FC (87.1 +/- 20.1 pg/mL) compared with baseline (63.3 +/- 8.1 pg/mL), placebo (56.1 +/- 5.5 pg/mL), 0.05 mg FC (60.5 +/- 16.0 pg/mL), and 0.1 mg FC (65.4 +/- 13.7 pg/mL) values. ANP(ext) was elevated in patients receiving 0.2 mg FC (42.7 +/- 41.8 pg/mL) compared with that in patients receiving placebo (7.9 +/- 5.4 pg/mL), 0.05 mg FC (16.2 +/- 11.2 pg/mL), or 0.1 mg FC (19.7 +/- 11.1 pg/mL). Our data suggest that PRC is of value in determining mineralocorticoid underreplacement, whereas ANP is a more sensitive index of FC overreplacement. ANP levels may, therefore, be complementary to PRC in adjustment of mineralocorticoid doses in the upper dose range, where clinical symptoms and signs appear to be of little value.

9 alpha-Fluorocortisol (9 alpha FF) is about 200 times more potent as a mineralocorticoid than cortisol (F) in man, although it binds with the same affinity as F and aldosterone to the human mineralocorticoid receptor. The low mineralocorticoid activity of F has been shown to be due to its rapid conversion by the kidney to cortisone (E), which does not bind to the receptor. Therefore, we compared the conversion of F to E with that of 9 alpha FF to 9 alpha-fluorocortisone (9 alpha FE) by 11-hydroxysteroid dehydrogenases in man in vivo and in vitro. Single oral doses of 9 alpha FF, 9 alpha FE, and F were given to normal males, and the excretion of free 9 alpha FF, 9 alpha FE, F, and E was measured in urine. Human kidney and liver slices were incubated with unlabeled steroids, and the free 11-hydroxy- and 11-oxosteroids were quantitated after high performance liquid chromatography separation by UV absorption. Oral F (5 mg) is excreted 70% as free E and 30% as free F (percentage of free steroids only). Oral 9 alpha FF (5 mg) is excreted 90% as free 9 alpha FF and 10% as free 9 alpha FE. Free 9 alpha FF excretion is 14 times greater than that of F after ingesting an identical dose. Oral 9 alpha FE (4 mg) is also excreted 90% as 9 alpha FF and 10% as 9 alpha FE. Kidney slices convert F much faster to E than 9 alpha FF to 9 alpha FE. The conversion of 9 alpha FE to 9 alpha FF is, on the contrary, much faster than that of E to F. Thus, the equilibrium of the reaction is on the 11-oxo side for F/E and on the 11-hydroxy side for 9 alpha FF/9 alpha FE. The interconversion of both pairs of steroids is inhibited by glycyrrhetinic acid in a dose-dependent manner. Liver slices do not measurably convert 9 alpha FF to 9 alpha FE, but do rapidly convert 9 alpha FE into 9 alpha FF. Reflecting this negligible conversion of 9 alpha FF to 9 alpha FE and the low plasma-protein binding of 9 alpha FF, free urinary 9 alpha FF excretion is much higher than that of F after the same oral dose.(ABSTRACT TRUNCATED AT 400 WORDS)

We report the case of a lady with multiple presentations and admissions with fever. The eventual diagnosis was made of adrenocorticosteroid deficiency.

Glucocorticoids have been shown to inhibit GH secretion in normal man when acutely and chronically administered in pharmacological amounts. Pyridostigmine (PD), an acetylcholinesterase inhibitor, is able to elicit GH secretion when administered alone and to enhance the GH response to GHRH in normal subjects probably via a decrease in the hypothalamic release of somatostatin. The aim of the present study was to investigate the influence of glucocorticoids on the GH response to PD administered either alone or in combination with GHRH in normal adult subjects. Six healthy adult volunteers underwent six experimental protocols. They received 1) human (h) GHRH(1-29)NH2, 100 micrograms injected as an iv bolus; 2) cortisone acetate, 50 mg administered orally (po) 60 min before an hGHRH iv bolus injection; 3) PD, 120 mg administered po, 60 min before an hGHRH iv bolus injection; 4) PD and cortisone acetate, administered po 60 min before an hGHRH iv bolus injection; 5) PD, administered po 60 min before a saline iv bolus injection; 6) PD and cortisone acetate administered po 60 min before a saline iv bolus injection. Mean GH levels, peak GH levels, and GH area under the curves (AUCs) were significantly lower after GHRH + cortisone as compared to GHRH alone. However, these parameters were not significantly different after PD + GHRH + cortisone when compared to PD + GHRH and after PD + cortisone when compared to PD alone. We conclude that acute administration of pharmacological amounts of glucocorticoids cannot inhibit the GH response to PD alone or in combination with GHRH. Thus, we hypothesize that the inhibitory action of glucocorticoids on the GH response to GHRH in man may be mediated by an enhancement of either somatostatin release by the hypothalamus or somatostatin action on the pituitary.

A method of reproducibility measuring human leukocyte beta-adrenergic receptor density and affinity has been developed and applied to the study of receptor regulation in man. The method has the advantages of using a membrane preparation which binds highly specifically and employing techniques such as using low concentrations of [3H]dihydroalprenol, analyzing the data by computer modelling techniques, and providing data from both granulocytes and lymphocytes in the same individual to minimize measurement errors. Using this methodology, human beta-adrenergic receptor regulation is examined. Cortisone acetate was found to induce an acute rise in granulocyte beta-adrenergic receptor density and adenylate cyclase activity and an acute fall in lymphocyte beta-adrenergic receptor density. This potentially differential regulation of a single receptor subtype in two lines of leukocytes has important implications for the study of receptor regulation in man using leukocyte models.

Glucocorticoid excess is associated with alterations in the vitamin D endocrine system. The aim of this study was to assess change in serum bone Gla protein (BGP) after low and high dose cortisone acetate treatment and to assess whether these alterations are altered or attenuated by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] administration. Five groups of rats were studied and compared to a control group [cortisone acetate in doses of 0.2, 3.3, and 5.0 mg/100 g BW; 1,25-(OH)2D3 in a dose of 100 ng/100 g BW; and a combination of 1,25-(OH)2D3 (100 ng/100 g BW) plus cortisone acetate (3.3 mg/100 g BW)]. Each animal received daily sc injections for 27 days. BGP decreased significantly by day 7 in the two groups receiving high doses of cortisone acetate compared to control group values (65.20 +/- 4.38 vs. 150.18 +/- 6.13 ng/ml in the intermediate dose group and 91.57 +/- 5.30 vs. 150.18 +/- 6.13 ng/ml in the high dose group; P less than 0.01); this effect persisted until day 28. Histomorphometry revealed decreased formation and resorption in the two high dose cortisone acetate groups, whereas low dose cortisone acetate produced no histological change. The combination therapy lessens any change in BGP until day 28 when BGP was lower than the control value (P less than 0.01); histomorphometry showed that combination therapy prevents the effect of cortisone acetate by increasing bone formation and resorption. The data demonstrate that high doses of cortisone acetate suppress bone formation and that this is reflected in the low serum BGP values. Thus, BGP may be a marker of glucocorticoid-induced bone disease. 1,25-(OH)2D3 protects against glucocorticoid-induced bone disease and the normal BGP level reflects this.

The 21-hydroxylation of plasma progesterone (P) has been demonstrated in pregnant, nonpregnant, and adrenalectomized women and in men. The fractional conversion of plasma progesterone to deoxycorticosterone (DOC), [rho]P-DOC BU, among those subjects was 0.009 +/- 0.001 (mean +/- SEM, n = 32). The [rho]P-DOC BU in a woman with congenital adrenal hyperplasia due to apparent adrenal steroid 21-hydroxylase deficiency was 0.010 when she was taking cortisone acetate, and the [rho]P-DOC BU determined when she was not taking cortisone acetate was 0.012. Moreover, the value computed for the fractional conversion of 17 alpha-hydroxyprogesterone to 11-deoxycortisol in this woman (0.004) was similar to that observed in a woman with normal adrenal function (0.005). Therefore, extraadrenal 21-hydroxylase activity in a woman with nonsalt-losing congenital adrenal hyperplasia due to 21-hydroxylase deficiency was similar to that found in persons with normal adrenal function.

6 alpha-Hydroxy metabolites of cortisol were determined in the urine of pregnant (36-40 weeks of gestation) and nonpregnant women and in amniotic fluid from nearly fullterm pregnant women because relatively large amounts of these compounds are excreted in the urine of 2-day-old infants (> 200 micrograms/day). The corticosteroids analyzed by high pressure liquid chromatography and gas chromatography-mass spectrometry were 6 alpha-hydroxy derivatives of (allo)tetrahydrocortisone (3 alpha, 17 alpha, 21-trihydroxy-5 epsilon-pregnan-11,20-dione), (allo)tetrahydrocortisol (3 alpha, 11 beta, 17 alpha, 21-tetrahydroxy-5 epsilon-pregnan-20-one), and alpha- and beta-cortolone (3 alpha, 17 alpha, 20 epsilon, 21-tetrahydroxy-5 beta-pregnan-11-one). All of these compounds were found in the urine samples from both groups of women and in the amniotic fluid samples in contrast to those found in the urine samples from the neonates where 6 alpha-hydroxy compounds of (allo)tetrahydrocortisol and allotetrahydrocortisone were not positively identified because of insufficient yields. The pregnant women excreted significantly larger amounts of 6 alpha-hydroxy metabolites of cortisol (approximately 600 micrograms/day) than the control women (approximately 90 micrograms/day), and the rate of urinary excretion of these 6 alpha-hydroxy compounds was 7.82 and 1.30 micrograms/kg . day, respectively, for these groups of women compared to 54.3 micrograms/kg . day for the neonates. The precursors of these metabolites within the fetal body originated largely from the maternal circulation, and, therefore, the 6 alpha-hydroxy metabolites of cortisol excreted by the mother refer mainly to fetal metabolism and to a lesser extent, to the fetal secretion of cortisol.

Cortisone acetate (250 micrograms/kg X day) was given by im injections to 40 21-day-old diabetes-prone BB rats. The animals were followed longitudinally to determine islet cell surface antibodies (ICSA), as an expression of an abnormal immune reaction against the pancreatic islet cells and plasma glucose to estimate the degree of metabolic control. ICSA were detected 10-150 days before the diagnosis of diabetes. In the cortisone-treated group the diabetic rats showed significantly higher ICSA values compared to the nondiabetic ones, both in frequency of positive tests (P less than 0.05) and in mean binding values (P less than 0.02). In the control group, no difference in ICSA levels were seen between diabetic and nondiabetic rats. The cortisone regimen also failed to influence the degree of insulitis, commonly associated with diabetes in these rats. These experiments in well defined animals which spontaneously develop diabetes do not support the use of low dose cortisone treatment in attempts to improve or prevent insulin-dependent diabetes in human subjects.

A combination of heparin and cortisone acetate significantly inhibited both embryonic angiogenesis and the tumor growth of Lewis lung carcinoma (3LL) transplanted into C57BL/6 mice, although each of these agents used alone affected neither angiogenesis nor tumor growth. On the other hand, this combination neither decreased the number of metastatic foci in the lung nor prolonged the survival time of mice with 3LL. All tumor-bearing mice died of hemothorax due to pulmonary metastases. Cortisone acetate by itself increased metastasis, and addition of heparin did not affect accelerated metastasis. Because an antiangiogenic activity appears independent of metastasis acceleration by cortisone acetate, the use of steroids other than cortisone acetate having no metastasis-promotion effect should be required for an antiangiogenic tumor therapy in the presence of heparin. Heparin plus cortisone acetate prevented the DNA synthesis of cultured vascular endothelial cells but not that of cultured 3LL cells. Additionally, oral administration of this combination decreased the [3H]thymidine labeling of endothelial cells of tumor blood vessels prior to the suppression of tumor growth. The specific inhibition of the growth of endothelial cells by heparin plus cortisone acetate was revealed in both the in vitro and the in vivo tests.

In four patients who required maintenance glucocorticoid therapy after bilateral adrenalectomy for Cushing's disease, we compared the effects of im injection and oral ingestion of cortisone acetate and hydrocortisone hemisuccinate. By the former route of administration, cortisone acetate was not effective in elevating plasma cortisol levels or in suppressing plasma adrenocorticotropin, although hydrocortisone was. When given by mouth, no significant difference was found between the two steroids. Therefore, in the treatment of acute adrenal insufficiency or in the maintenance of patients with chronic adrenal insufficiency and in their preparation for surgery or other stressful situations, we advise against im injection of cortisone acetate. Oral ingestion, however, is appropriate for maintenance.

Heparin and heparin fragments in combination with corticosteroids have been shown to markedly inhibit tumor angiogenesis. Experiments were performed to test the hypothesis that heparin, heparin fragments, and the combination of heparin and corticosteroids affect DNA synthesis and the proliferation of cerebral microvessel endothelium (ME). In vitro, methyl-3H-thymidine incorporation in the ME cells was measured after a 24 hour pulse. Our results show that heparin, hydrocortisone, and heparin in combination with hydrocortisone had a slight inhibitory effect on DNA synthesis of ME (p less than 0.05), and hydrocortisone in combination with heparin had a slight inhibitory effect on ME cell growth (p less than 0.05). The hexa-, octa-, and deca-saccharide fragments of heparin stimulated DNA synthesis in ME (p less than 0.01). In vivo, DNA synthesis in cerebral endothelial cells at the margin of a freeze lesion to mouse cerebral cortex was assayed by quantitation of labeling indexes from methyl-3H-thymidine autoradiographs in mice treated with heparin, cortisone, or a combination of heparin and cortisone. A mean endothelial cell labeling index (LI) of 6% in the cortisone-treated animals was significantly lower than controls (32%, p less than 0.01). The addition of heparin to cortisone did not significantly alter the endothelial cell LI compared to the cortisone-treated animals, and heparin alone did not significantly alter the LI compared to the controls. These results indicate that cortisone markedly reduces the endothelial proliferation around a cortical freeze lesion in vivo. This effect is independent of heparin.

To examine the effect of glucocorticoid deficiency on the glucocorticoid receptor, we examine the binding of [3H] dexamethasone to lymphocytes in normal subjects and patients with adrenal insufficiency before and after glucocorticoid replacement therapy. Using a whole cell competitive binding assay, normal human lymphocytes had 5977 +/- 1487 (mean +/- SD) binding sites/cell and a dissociation constant of 10 +/- 2 nM. Lymphocytes from patients with untreated adrenal insufficiency had fewer binding sites (3364 +/-322) and a 2-fold increase in binding affinity (5.4 +/- 0.9 mM). The administration of conventional replacement doses of cortisone acetate for 6 months caused no change in receptor number, but was associated with a decrease in binding affinity toward normal. After long term glucocorticoid replacement therapy, binding parameters were similar to those in patients before treatment. The physiological implications of the decreased receptor number and increased binding affinity in adrenal insufficiency remain to be elucidated.

The ability of bronchoalveolar macrophages from normal, diabetic, and cortisone-treated mice to inhibit spore germination and kill fungal spores in vivo was investigated. The data indicated that the normal host controls different fungal infections in the lungs by different mechanisms. Prevention of mucormycosis required inhibition of fungal spore germination by alveolar macrophages. In contrast, pulmonary defense against aspergillosis depended on early killing of conidia by alveolar macrophages and not on inhibition of germination by bronchoalveolar macrophages. Bronchoalveolar macrophages in diabetic and cortisone-treated animals allowed fungal spore germination, thereby permitting infection by Rhizopus oryzae. In the cortisone-treated mouse, bronchoalveolar macrophages did not kill fungal conidia and progressive infection by Aspergillus fumigatus occurred. Fungicidal activity of bronchoalveolar macrophages was measured with a new in vivo killing assay.

Over half of a group of 373 inpatients with advanced malignant disease were treated with corticosteroids for a variety of reasons. They received either prednisolone or dexamethasone, or replacement therapy with cortisone acetate. Forty percent of those receiving corticosteroids benefited from them. A higher response rate was seen when corticosteroids were prescribed for nerve compression pain, for raised intracranial pressure, and when used in conjunction with chemotherapy. No significant difference in efficacy was noted between the 2 drugs. The results, however, suggest that with a larger sample, dexamethasone would have been shown to be significantly better than prednisolone in the management of nerve compression pain. The incidence of side effects was broadly similar with dexamethasone and prednisolone. The most common side effect was oral candidosis and there was a highly significant relationship between the use of corticosteroids and the prescription of nystatin suspension. Dexamethasone was more likely than prednisolone to cause oro-pharyngeal candidosis. Dexamethasone was also associated with significantly more cases of psychological disturbance and hyperactivity. On the other hand, dexamethasone seems less likely to cause oedema, weight gain and dyspepsia. Corticosteroids were withdrawn because of side effects in only 11 patients (5%)--6 were receiving dexamethasone and 5 prednisolone. Dexamethasone has been adopted as the standard corticosteroid for terminal cancer patients at Sir Michael Sobell House.

The possible role of THF and THE as intermediates in the biotransformation of cortisol to the cortoic acids was studied by giving 3H-THF + 14C-cortisol tracers and 3H-THE + 14C cortisol tracers to two subjects each, measuring the 3H/14C isotope ratios of the urinary cortoic acid metabolites and relating these ratios to the dose ratio. Isotope ratios substantially higher than the dose ratio indicate that the tetrahydro compound is a better precursor than cortisol, and isotope ratios that are essentially identical to that of urinary THF or THE, respectively, indicate that the tetrahydro compound may be an obligatory intermediate in the cortisol leads to cortoic acids pathway. The isotope ratio data in these studies clearly establish that THF was a preferential precurosor of the 11 beta-hydroxy cortoic acids (cortolic and beta-cortolic) and THE was a preferential precursor of the 11-ketone cortoic acids (cortolonic and beta-cortolonic). Furthermore, the data strongly suggest that THF and THE may be obligatory intermediates in cortoic acid formation.

Basal release of gonadotropin and the response to an infusion of 100 mug of synthetic luteinizing hormone releasing hormone (LRH) were studied in a teenage girl with congenital adrenal hyperplasia (CAH). The initial study was done during a period of poor adrenal suppression, and second study was done after adequate adrenal suppression was achieved. To assess adrenal function, circulating levels of adrenal steroid hormones were evaluated continuously over a 24 h period. During the period increased production of adrenal androgens, the pattern of gonadotropin release was that of a prepubertal child. After 3 months of adrenal suppression the pattern of gonadotropin secretion was similar to that of a normal girl in mid-puberty. This demonstrates the rapid change from prepubertal to pubertal gonadotropin dynamics in a teenage patient following adequate suppression of androgens from the adrenal.

An insulin-requiring diabetic patient who developed Addison's disease is described. At diagnosis, investigations revealed biochemical hypothyroidism and these abnormalities resolved after replacement therapy for the adrenal failure only. The possible mechanism of this change is discussed in relation to the presence of organ-specific antibodies.

Glucocorticoids (GC) have stimulatory effects on GH secretion in vitro and suppressive effects when administered in pharmacological amounts in vivo. We studied six patients with ACTH deficiency and severely impaired serum GH responses to insulin tolerance tests and arginine infusion tests. All patients underwent the same tests during GC replacement while receiving cortisone acetate in doses ranging from 12.5-25 mg/day. The three patients with idiopathic ACTH deficiency and no evidence of pituitary mass lesions had impaired GH secretion, which returned to normal during GC replacement. In contrast, the three patients with ACTH deficiency and hypothalamo-pituitary mass lesions detected by a computed tomography scan had impaired GH secretion during GC replacement therapy. Our data indicate that in patients with idiopathic ACTH deficiency, an impaired GH response to stimuli reversible during GC replacement therapy may be the functional consequence of the low levels of circulating cortisol. We conclude that physiological serum cortisol levels are necessary for normal serum GH responses to provocative stimuli in man.

To distinguish between the contributions of neutrophils and cell-mediated immunity to defense against Nocardia asteroides in the lung, we induced pneumonia in BALB/c mice by intranasal inoculation. We studied the course of pneumonia by histology, bronchoalveolar lavage, and quantification of colony-forming units in lungs. Mice with intact host defenses had an initial inflammatory response, consisting mostly of neutrophils, followed by a mononuclear cell infiltrate. Nocardiae were eradicated during the seven days after inoculation. Mice in which the early, predominantly neutrophil inflammatory response was prevented with mechlorethamine or antiserum to neutrophils had little inflammation, and nocardiae grew extensively. Mice in which cell-mediated immunity was impaired with cyclosporin A or cortisone acetate developed abscesses; neutrophils were abundant, but nocardiae proliferated. The occurrence of these three separate and distinct patterns suggested that both neutrophils and cell-mediated immunity are necessary for optimal control of N. asteroides infection.