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Background: Ear cleaning is often recommended for management of canine otitis externa (OE). Few in vivo studies evaluate how ear cleaning affects treatment outcome., Hypothesis/objectives: To determine the effect of ear cleaning on canine OE by comparing treatment outcome in cleaned versus noncleaned ears., Animals: Twenty-three client-owned dogs with ceruminous or purulent OE., Materials and Methods: Forty ears were randomised to one of two groups: 20 ears were wiped with dry or saline-moistened gauze (grp1), and 20 external ear canals received manual cleaning (grp2) with a commercial product. One millilitre of a commercial otic suspension containing hydrocortisone aceponate, miconazole nitrate and gentamicin sulfate was applied to affected ears every 24 h for five days. Cytological scores, modified otitis index score (OTIS3), pruritus Visual Analog Scale (PVAS), modified PVAS and client questionnaire were compared on Day (D)0 and D7., Results: There were no statistical differences between cytological scores, modified OTIS3, PVAS, modified PVAS or client assessments between groups at D7. Both groups had significant reduction in all treatment parameters from D0 to D7 with the exception of cytological rod scores that were only significantly decreased in cleaned ears. There was a significant difference in OTIS3 before and after cleaning on D0., Conclusions and Clinical Relevance: Both groups achieved successful outcome regardless of cleaning. Ear cleaning may be more important when rod-shaped bacteria are present., (© 2024 ESVD and ACVD.)

Introduction: Phimosis is defined as the inability to retract the foreskin, preventing partial or complete exposure of the glans., Objectives: To compare the efficacy of topical treatment with betamethasone alone and in combination with hyaluronidase, evaluate systemic absorption of cortisol, and identify factors that predispose the success of topical treatment of phimosis in children aged 3-10 years., Methods: This randomized double-blinded clinical trial involved 152 participants (3-10 years old) with phimosis. The children were divided into two groups: betamethasone associated with hyaluronidase (betamethasone valerate 2.5 mg + hyaluronidase 150 UTR; Group A) and betamethasone (betamethasone valerate 2.5 mg; Group B). Parents were instructed on how to use the ointment (twice a day, after hygiene, for 60 days) and on collecting salivary cortisol measurements at 11pm and 9am, before and after treatment. Participants were evaluated after 30 and 60 days. Fisher's exact test and paired t-test were used to analyze the data., Results: Ninety children (69.77%) were successfully treated with the proposed treatment, with the success rate for Group A being 75.38% versus 64.06% for Group B, p = 0.18). Systemic absorption evaluated by salivary cortisol did not show differences after the intervention (p > 0.05), indicating that there was no systemic absorption when using ointments with or without hyaluronidase. The factors of age (OR = 0.98 - CI: 0.97-1.00), adherence (OR = 1.49 - CI: 0.53-4.16), balanoposthitis (OR = 1.85 - CI: 0.47-7.19), and previous use of corticosteroids (OR = 1.21 - CI: 0.53-2.72) also did not show influenced results CONCLUSION: Topical therapy for true phimosis with betamethasone 0.2% + hyaluronidase, despite showing no differences when compared with betamethasone 0.2% alone, for a period of up to 60 days, proved to be safe, effective, and with good results. The variables analyzed could not predict the expected clinical response., Rebec: RBR-76bhgyb., Competing Interests: Conflict of interests None declared., (Copyright © 2024 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

Background: Diagnosis of primary aldosteronism (PA) is complicated by the need to withdraw antihypertensive medications that interfere with test results, particularly renin. This study examined whether machine learning-based steroid-probability scores offer a renin measurement-independent approach for testing less prone to interference than the aldosterone-to-renin ratio (ARR)., Methods: This prospective multicenter cohort study involved the use of plasma steroidomics and the ARR in 839 patients tested for PA, including 190 with and 578 without PA (71 indeterminate). Receiver operating characteristic curves for steroid-probability scores and the ARR were examined with and without interfering medications. Impacts of individual medications on plasma aldosterone, 18-oxocortisol, 18-hydroxycortisol, steroid-probability scores, renin, and ARRs were examined by multivariable and paired analyses in patients with and without PA., Results: Receiver operating characteristic curves indicated a significant impact of interfering antihypertensive medications on the diagnostic performance of the ARR and minimal impact on steroid-probability scores. Mineralocorticoid receptor antagonists increased plasma aldosterone, 18-oxocortisol, and 18-hydroxycortisol in patients without PA and resulted in false-positive test results for steroid-probability scores and false-negative results for the ARR. Diuretics increased aldosterone, 18-oxocortisol, and steroid-probability scores in patients without PA, whereas angiotensin-converting enzyme inhibitors decreased aldosterone, steroid-probability scores, and ARRs. Beta-adrenoceptor blockers, dihydropyridine calcium channel blockers, and angiotensin receptor blockers had negligible impact on mineralocorticoids and steroid-probability scores., Conclusions: Among antihypertensive drugs that impact plasma aldosterone, 18-oxocortisol, and 18-hydroxycortisol, mineralocorticoid receptor antagonists stood out as a cause of false-positive results for derived steroid-probability scores. Other antihypertensives have minimal or no impact, an advantage for use of steroid-probability scores over the ARR when those medications cannot be withdrawn., Registration: URL: https://drks.de/search/en/trial/DRKS00017084; Unique identifier: DRKS00017084., Competing Interests: None.

Compounding pharmacies commonly use ready-to-use vehicles such as TrichoSolTM to produce hair solutions for alopecia. However, chemical and microbiological compatibility are paramount to be determined so those can be safely implemented as the vehicle of choice. This study aimed to assess the physical-chemical and microbiological stabilities of selected active pharmaceutical ingredients in TrichoSolTM. For that, HPLC analyses and Antimicrobial Effectiveness Testing were conducted in bracketed studies. The beyond-use dates (BUDs) found were: 180 days for cetirizine hydrochloride 0.5%-2.0%, dutasteride 0.1%, hydrocortisone acetate 0.5%, nicotinamide 0.25%-0.50%, progesterone 1.0%, and pyridoxine hydrochloride 0.25%-5.0%. BUDs of 150 days were observed for hydrocortisone acetate 1.0%, 120 days for Dutasteride 0.25%, and 90 days for progesterone 2.5%., (Copyright© by International Journal of Pharmaceutical Compounding, Inc.)

Hydrocortisone succinate (1) is a synthetic anti-inflammatory drug and key intermediate in the synthesis of other steroidal drugs. This work is based on the fungal biotransformation of 1, using Monascus purpureus and Cunninghamella echinulata strains. Comopound 1 was transformed into four metabolites, identified as hydrocortisone (2), 11β-hydroxyandrost-4-en-3,17-dione (3), Δ 1 -cortienic acid (4), and hydrocortisone-17-succinate (5), obtained through side chain cleavage, hydrolysis, dehydrogenation, and oxidation reactions. These compounds have previously been synthesized either chemically or enzymatically from different precursors. Though this is not the first report on the biotransformation of 1, but it obviously is a first, where the biotransformed products of compound 1 have been characterized structurally with the help of modern spectroscopic techniques. It is noteworthy that these products have already shown biological potential, however a more thorough investigation of the anti-inflammatory properties of these metabolites would be of high value. These results not only emphasize upon the immense potential of biotransformation in catalysis of reactions, otherwise not-achievable chemically, but also holds promise for the development of novel anti-inflammatory compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Budesonide and tixocortol pivalate as markers of contact allergy to corticosteroids have been questioned, as they are not able to detect a significant percentage of allergic patients., Objectives: To investigate the potential role of clobetasol propionate in enhancing corticosteroid sensitisation detection., Methods: Between January 2022 and December 2023, patients who attended centres involved in the Spanish Registry of Research in Contact Dermatitis and Cutaneous Allergy were tested with an extended baseline series that included budesonide, tixocortol pivalate, clobetasol propionate 0.1% in ethanol and 1% in petrolatum., Results: A total of 4338 patients were tested. Twenty-four patients were allergic to budesonide (0.55%, 95% CI: 0.37-0.82); nine patients were allergic to tixocortol pivalate (0.21%, 95% CI: 0.11-0.39); and 23 patients were allergic to clobetasol (0.53%, 95% CI: 0.35-0.79). Only four of those patients allergic to clobetasol were detected by budesonide and one by tixocortol pivalate. No significant differences in the number of positive tests were found between clobetasol in petrolatum or ethanol., Conclusions: In Spain budesonide remains the main corticosteroid allergy marker whereas the role of tixocortol pivalate is questionable. The addition of clobetasol propionate to the Spanish baseline series would improve the ability to detect patients allergic to corticosteroids., (© 2024 The Author(s). Contact Dermatitis published by John Wiley & Sons Ltd.)

Lenvatinib (LEN), a multitarget tyrosine kinase inhibitor used in various cancer treatments, is mainly metabolized by cytochrome P450 3A (CYP3A) enzymes. The importance of therapeutic drug monitoring (TDM) in patients administered LEN has been proposed. Although some biomarkers of endogenous CYP3A activity have been reported, their utility in dosage adjustments has not been well evaluated. This study investigated the correlation between plasma LEN concentrations and endogenous urinary CYP3A biomarkers in clinical practice. Concentrations of plasma LEN (N = 225) and CYP3A biomarkers (cortisol, 6β-hydroxycortisol, deoxycholic acid, and 1β-hydroxydeoxycholic acid) in urine (N = 214) from 20 patients (hepatocellular carcinoma, N = 6; thyroid cancer, N = 3; endometrial cancer, N = 8; and renal cell carcinoma, N = 3) collected for consultation for up to 1 year were evaluated using liquid chromatography-tandem mass spectrometry. Moreover, plasma trough LEN concentrations were predicted using a three-compartment model with linear elimination for outpatients administered LEN before sample collection. Moderate correlations were observed between the quantified actual concentrations and the predicted trough concentrations of LEN, whereas there was no correlation with endogenous urinary CYP3A biomarkers. The utility of endogenous urinary CYP3A biomarkers could not be determined. However, TDM for outpatients administered orally available medicines may be predicted using a nonlinear mixed effect model (NONMEM). This study investigated the utility of endogenous urinary CYP3A biomarkers for personalized medicine and NONMEM for predicting plasma trough drug concentrations. These findings will provide important information for further clinical investigation and detailed TDM., (© 2024 The Author(s). Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

Introduction: Urine free cortisol measurements are routinely performed to evaluate hypercortisolism. Despite their analytical inaccuracy, immunoassay-based methods are frequently used. Advances in liquid chromatography-high-resolution mass spectrometry (LC-HRMS) facilitate the incorporation of powerful diagnostic tools into clinical laboratories. In addition to its high analytical specificity and simultaneous analysis of different metabolites, accurate mass measurement allows for untargeted compound identification, which may help to identify clinically relevant metabolites or drugs., Methods: The present study aimed to validate a simple routine LC-HRMS method to quantify cortisol, cortisone, 6β-hydroxycortisol, and 18-hydroxycortisol simultaneously in human urine. Additionally, the study also validated a GC-MS method for the same steroids, evaluated their cross-reactivity with commercial cortisol immunoassays, and quantified the 24 h urine excretion in patients under clinical suspicion or follow-up for hypercortisolism., Results: The LC-HRMS method involved liquid-liquid extraction using dichloromethane, micro-LC for chromatographic separation and detection using the accurate masses of the steroids, and simultaneous high-resolution full scan acquisition. The method presented acceptable linearity, precision, and accuracy. Significant interference from 6β-hydroxycortisol and cortisone was demonstrated in the cortisol immunoassays, which impacted their reliability in the follow-up of patients with hypercortisolism and significant changes in these cortisol metabolites (i.e., due to drug-induced changes in CYP3A4 activity)., Conclusion: A rapid and accurate routine LC-HRMS method was validated, which is useful for the evaluation of hypercortisolism and other disorders of glucocorticoid and mineralocorticoid metabolism.

Super-selective adrenal venous sampling (ssAVS) can collect the adrenal tributary venous blood in the aldosterone (ALD)-hypersecreting segments in primary aldosteronism. The concentrations of the C18-oxygenated steroids, especially 18-oxocortisol (18-oxoF), in the lesion segments might be more useful indices than those in the peripheral or adrenal central veins (current candidate indexes) for the differential diagnosis of unilateral ALD-producing adenoma (APA) and bilateral adrenal hyperplasia (BAH). To verify this hypothesis, we developed a liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) method for simultaneously quantifying ALD, 18-oxoF and 18-hydroxycortisol in the adrenal tributary venous serum sample collected by ssAVS (ssAVS serum) and compared their concentrations between APA and BAH patients. Only deproteinization was required for a 10 μl sample prior to the LC/ESI-MS/MS analysis. Endogenous corticoids did not interfere with the quantifications, and the intra-assay and interassay precisions (≤ 8.3%) and accuracies (94.2-102.7%) were acceptable. The clinical study revealed that the 18-oxoF concentration was significantly higher in the ALD-producing tumor tissues (from APA patients) than in the hyperplastic tissues (from BAH patients). However, in conclusion, the 18-oxoF concentration in the ssAVS serum sample can be a rough indication but cannot be decisive for the differential diagnosis between APA and BAH owing to the significant individual difference., (© 2024 John Wiley & Sons Ltd.)

Aim: A less invasive evaluation method of cytochrome P450 3A (CYP3A) activity provides an important tool for personalized medicine. We aimed to clarify the usefulness of the plasma 6β-hydroxycortisol to cortisol concentration (6β-OHF/F) ratio as a minimally invasive CYP3A phenotyping method., Methods: Plasma 6β-OHF and cortisol concentrations were measured via liquid chromatography/tandem mass spectrometry. The plasma 6β-OHF/F ratio was compared with 6β-hydroxylation clearance of endogenous cortisol (CL m(6β) ; which we previously developed as an index of CYP3A activity) before, during and after oral contraceptive administration in 3 healthy women. The plasma 6β-OHF/F ratio was observed during oral clarithromycin administration. The plasma 6β-OHF/F ratio was also measured in 39 healthy participants., Results: The plasma 6β-OHF/F ratio in 3 healthy women on Day 21 of starting oral contraceptive administration decreased by 39, 49 and 61% compared with Day 0. These values were similar to CL m(6β) values (43, 54 and 59%, respectively). Plasma 6β-OHF/F ratio and CL m(6β) exhibited a good correlation (r = .9053). The 6β-OHF/F ratio decreased from 0.00921 to 0.00577 only 3 h following clarithromycin administration. The plasma 6β-OHF/F ratio ranged 0.00565-0.01556 in 39 healthy participants., Conclusion: Based on its close relationship with CL m(6β) and its decrease upon inhibition by clarithromycin, the plasma 6β-OHF/F ratio serves as an index of CYP3A activity. Using this minimally invasive index, we can identify patients with extremely low CYP3A activity before treatment initiation and optimize the initial drug dose, thereby mitigating the risk of severe adverse reactions., (© 2023 British Pharmacological Society.)

Background: Erythemato-ceruminous otitis externa (ECOE) is frequently seen in dogs affected with an allergic skin disease, with recurrent secondary bacteria and yeast overgrowths (detected on cytological examination)., Objectives: The objective of the study was to compare the efficacy and safety of an ear spray containing only hydrocortisone aceponate glucocorticoid diester (HCA) to a control product (CTRL), an approved otic formulation containing prednisolone-miconazole-polymyxin combination, in dogs with ECOE., Animals: In total, 97 and 104 dogs with ECOE were respectively randomly assigned to the tested ear treatment product group (HCA) or the commercially available ear treatment control product group (CTRL)., Materials and Methods: Dogs were treated for 7-14 days, as needed. At Day (D)0, D7, D14, D28 and D42, Otitis Index Score-3, hearing test, pruritus and pain visual analogue scales, and cytological scores were graded. The overall response to treatment also was assessed., Results: All clinical parameters decreased rapidly and in a similar way without any significant difference at any time between treatment groups. A good-to-excellent response to treatment was seen in >90% of dogs of both groups as early as D14. The treatment was considered safe in all dogs., Conclusions and Clinical Relevance: A 7- to 14-day ear topical application of HCA alone to dogs with ECOE accompanied with bacterial and/or fungal (yeast) overgrowth was safe and led to no statistical difference in improvement of clinical scores relative to the CTRL combination. Based on these results, it may be necessary to reconsider the routine use of antimicrobial drugs such as antibiotics and antifungals as a first-line treatment for ECOE that is likely to have been caused by an allergic reaction., (© 2023 Virbac. Veterinary Dermatology published by John Wiley & Sons Ltd on behalf of ESVD and ACVD.)

18-Oxocortisol is the product of the metabolism of 11-deoxycortisol by the mitochondrial enzyme aldosterone synthase (CYP11B2). The traditional concept is that the CYP11B2 is exclusively expressed in zona glomerulosa cells and the 17α-hydroxylase (CYP17A1) enzyme, required to synthesize 11-deoxycortisol, is in the zona fasciculata of the human adrenal. It has been postulated that the substrate for 18-oxocortisol is either cortisol from the circulation or from zona fasciculata cells adjacent to the zona glomerulosa. P-glycoprotein, which is highly expressed in steroidogenic cells of the adrenal gland, efficiently expels cortisol from the cell. Double immunofluorescence staining for the CYP11B2 and CYP17A1 enzymes in 7 human adrenals demonstrated that a highly variable number of cells in different areas of the zona glomerulosa co-expressed both enzymes. In addition, there were a variable number of cells that exclusively expressed the CYP17A1 embedded within the zona glomerulosa surrounded by CYP11B2-expressing cells. 18-Oxocortisol in the media of human adrenocortical HAC15 cells was measured by ELISA after incubation with and without 10 nM of angiotensin II to stimulate CYP11B2 activity, with and without the 3β-hydroxysteroid dehydrogenase (HSD3B) inhibitor trilostane, and with variable amounts of cortisol or 11-deoxycortisol. Cortisol was a poor substrate, while 11-deoxycortisol was a significant substrate for the synthesis of 18-oxocortisol. These data suggest that the biosynthesis of 18-oxocortisol in the human adrenal is likely catalyzed by co-expression of the two crucial enzymes CYP17A1 and CYP11B2 in a small proportion of cells within the zona glomerulosa. It is also possible that 11-deoxycortisol diffusing from cells expressing only CYP17A1 interspersed with cells expressing the CYP11B2 enzyme may be a paracrine substrate in the synthesis of 18-oxocortisol., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Eighteen-hydroxycortisol (18-OHF) is a potential biomarker for differential diagnosis of the two major primary aldosteronism subtypes, aldosterone-producing adenoma, and idiopathic hyperaldosteronism., Methods: Urine samples were processed, and the 18-OHF in urine samples were successfully quantified by in-house established dilute-and-shoot liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Separation was accomplished on a Sigma Ascentis Express C18 column with a gradient mixture of phase (A) 0.2% formic acid in water and phase (B) 0.2% formic acid in methanol at a flow rate of 0.4 ml/min. Mass spectrometric detection was performed in positive electrospray ionization mode via a mass spectrometer., Results: The linearity of urinary 18-OHF ranged from 4.28 to 8.77 × 10 3  nmol/L, with a lower limit of quantification at 4.28 nmol/L. The intra- and inter-precision were both below 3%. The range of analytical recovery was 97.8%-109.2%. The validated dilute-and-shoot LC-MS/MS method was compared with the SPE LC-MS/MS method modified from the one reported in 2013. The results by Passing-Bablok regression analysis and Bland-Altman plotting demonstrated a good agreement between the two methods. The presented method was then applied to establish sex-specific reference intervals from 62 males and 62 females, respectively. The calculated 2.5%-97.5% reference intervals for 24-h urinary 18-OHF were 113-703 nmol/day for males and 71.2-450 nmol/day for females., Conclusion: The presented dilute-and-shoot LC-MS/MS method for 18-OHF quantification showed a good performance in the clinical application. Furthermore, the sex-specific reference intervals for 24-h urinary 18-OHF were first established and quite important for its application in primary aldosteronism subtyping., (© 2022 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Controlled-release tablets and rectal suppositories of sulfasalazine (SLF) and hydrocortisone 21-acetate (HA) were prepared as recommended dosage forms for the treatment of acute episodes of ulcerative colitis, in patients who do not respond to monotherapy. A High-Performance Liquid Chromatography (HPLC) Diode-array method with a gradient elution mobile phase was developed to evaluate the production quality of both formulations (assay and dissolution profiles in gastric and intestinal fluids). Method's validation was carried out providing good linearity (r ≥ 0.9995), precision (RSD < 1.53%), recovery (96.9% - 103.7%) and limits of detection (LOD SLF  = 12 ng/mL, LOD HA  = 15 ng/mL). Experimental design and Plackett-Burman methodology was constructed to study the robustness of the analysis. In all composite substrates, a freezing lipid precipitation approach was used as purification step. The method was optimized by applying Central Composite design mode. The in-vitro/ex-vivo permeability studies of both formulations were evaluated by a Liquid Chromatography-Electron Spray Ionization Mass Spectrometry (LC-ESI/MS) +/- mode. The analysis of sulfamethazine (internal standard, SLM, m/z 279), HA (m/z 449, [M + HCOO] - ), SLF (m/z 399) and its active metabolite mesalazine (MSL, m/z 154) was performed using a C 18 column and gradient elution. The validation of the method met the requirements of the International Council for Harmonization (ICH) (r ≥ 0.9997, RSD ≤ 4.62%, Recovery > 95%, LOD SLF  = 1.28 ng/mL, LOD HA  = 1.07 ng/mL, LOD MSL  = 3.16 ng/mL). Based on the results, important conclusions were drawn concerning the role of excipients and SLF metabolism., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Several studies demonstrated the diagnostic accuracy of hair glucocorticoid measurement in patients with overt Cushing syndrome, but few data are available for patients with adrenal incidentaloma (AI) and cortisol autonomy. The aim of our study was to assess whether measurement of 5 corticosteroid hormones with the ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method in the keratin matrix is useful to stratify patients with AI by the presence of autonomous cortisol secretion [ACS] (defined as serum cortisol after 1 mg dexamethasone suppression test (DST) > 138 nmol/l) or possible ACS [PACS] (defined as serum cortisol after 1 mg DST > 50 nmol/l but ≤138 nmol/l). We analysed data of 67 AI patients (32 with cortisol autonomy) and 81 healthy subjects. We did not find any significant statistical difference comparing hair cortisol, cortisone, and 20β-dihydrocortisol concentrations between healthy controls and AI patients, while 6β-hydroxycortisol and 11-deoxycortisol were undetectable. Moreover, no significant difference was found in hair cortisol, cortisone, and 20β-dihydrocortisol levels of AI patients with or without cortisol autonomy. Finally, we did not find any correlation in patients with AI between hormonal concentrations in the keratin matrix and serum, salivary, and urinary cortisol levels, or with body mass index. In conclusion, our findings suggest that hair glucocorticoid measurement is not suitable as a diagnostic test for cortisol autonomy (ACS and PACS)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Puglisi, Leporati, Amante, Parisi, Pia, Berchialla, Terzolo, Vincenti and Reimondo.)

18-hydroxycorticosterone (18-OHB), 18-hydroxycortisol (18-OHF) and 18-oxocortisol (18-OXOF) are important biomarkers for the diagnosis of subtypes of primary aldosteronism. The detection of these three analytes by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is free from structurally similar compounds. The aim of this study was to develop and validate a new LC-MS/MS assay for the simultaneous quantification of 18-OHB, 18-OHF and 18-OXOF in plasma and to establish a reference intervals for apparently healthy population. Plasma samples were prepared by solid phase extraction and separated in an ultra-high performance reversed phase column. MS detection was achieved using a triple quadrupole mass spectrometer in both positive and negative ionization modes. The developed assay was then validated against standard guidelines. We collected 691 plasma samples from apparently healthy individuals (M:398, F:293) to establish the reference intervals. The analytes were separated and quantified within 5 min. The newly developed method demonstrated linearity for the detected steroid concentration in range of 5 to 3000 pg/ml for 18-OXOF (r 2  = 0.999) and 20 to 3000 pg/ml for 18-OHB (r 2  = 0.997) and 18-OHF (r 2  = 0.997). The lower limit of quantification (LLOQ) was 2.5 pg/ml, 20 pg/ml and 20 pg/m for 18-OXOF, 18-OHB and 18-OHF respectively. Specificity, precision, accuracy and stability were tested, and met the requirements of the guidelines. 18-OHB was higher in females than in males, but 18-OHF were higher in males than females. The reference intervals of 18-OHB, 18-OHF and 18-OXOF for both genders together were 90.5-1040.6 pg/ml, 224.4-1685.2 pg/ml, 4.0-70.5 pg/ml, respectively. Age was also an important factor influencing the levels of these three hormones. We have developed a sensitive and reliable method for the simultaneous quantification of 18-OHB, 18-OHF, and 18-OXOF. Our work provides a reference interval for the clinical application of these three steroid hormones., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Aim of the Study: Hydrocortisone is a soft steroid with low anti-inflammatory properties and a short duration of action, used to manage several ocular conditions. The clinical benefits and side effects associated with hydrocortisone are well documented, but its basic pharmacokinetic in the eye is yet to be fully elucidated. The purpose of this study is to investigate the anterior chamber penetration capabilities of hydrocortisone when used in different concentrations as eye drops treatment., Materials and Methods: This is a double-blind, single-centre, randomised clinical trial performed at the Department of Medicine and Surgery of the University of Perugia (Italy) on consecutive patients who undergone phacoemulsification with intraocular lens implantation. Patients were randomly assigned on the morning of surgery to receive a single instillation of 0.33% (group A) or 0.001% (group B) hydrocortisone sodium phosphate solution. Group of patients C did not receive any treatment and was used to measure the hydrocortisone endogenous levels. Before surgery, one aliquot of aqueous humor for each patient was aspirated. The time of collection for each sample was recorded. Hydrocortisone concentrations were then stratified into six interval classes of 30 minutes each., Results: The mean concentration of hydrocortisone was significantly higher in group A (25.2 ± 12.4 ng/mL) compared with group B (7.11 ± 1.51 ng/mL) and compared with the mean hydrocortisone endogenous levels (3.92 ± 1.18 ng/mL) (P < .0001). No statistically significant differences of hydrocortisone mean concentrations between group B and the mean endogenous levels were found., Conclusions: Considering the frequent need for prolonged topical steroid therapies and the possible consequent undesirable side effects, ophthalmologists should consider the lowest clinically effective dose of hydrocortisone useful to obtain the desired therapeutic effect and in an adequate time, to minimise the amount of steroids into the anterior chamber and to avoid side effects like intra-ocular pressure increase or cataract development., (© 2021 The Authors. International Journal of Clinical Practice published by John Wiley & Sons Ltd.)

Background: Atopic dermatitis (AD) has a severe impact on quality of life (QoL)., Objectives: To analyze the impact of AD on QoL of small children with moderate-to-severe AD in a tertiary health care hospital in Helsinki, Finland., Materials & Methods: Based on interim analysis of this longitudinal follow-up study, we investigated treatment response (topical corticosteroids vs. tacrolimus) and QoL of 152 small children with moderate-to-severe AD., Results: The tacrolimus group had a significantly better treatment response at 12 months visit, but thereafter no differences were observed (p = 0.029; Mann-Whitney U test). The odds ratio for group comparisons was 2.258 (CI: 1.151-4.431). There was a significant improvement in QoL during follow-up in both treatment groups. Our study showed substantial improvements in disease severity and QoL based on active management and effective treatments in small children with AD. The main improvement was seen during the first year in both treatment groups with a lasting response., Conclusion: Effective treatment has a significant positive impact on the QoL of small children with AD and their families.

Arthrofibrosis is a joint pathology during which excess collagen is produced resulting in movement limitation, inflammation and pain. Itis caused by repeated injuries or surgical interventions, which is frequently managed by using certain medications such as glucocorticosteroids.The goal of our research was to study the effect of therapeutic exercise and hydrocortisone acetate (HA) phonophoresis combination therapy on the management of the rehabilitation process of knee joint functional limitations caused by arthrofibrosis and to establish the optimal parameters of ultrasound needed to achieve therapeutic effect.25 male patients between the ages of 18 and 50 (mean age 39±3.4 years) with moderate contracture of the knee and limitation of knee flexion, which varied between 70° and 90° participated in the randomized controlled experimental research. In addition, modified Cincinnati questionnaire scale (MCSc) was used. The following 3-week rehabilitation programs were developed: : I - the control group was assigned an individual home exercise program (HEP) 5 times a week; the experimental group II was assigned a treatment program which included the same exercise in addition to phonophoresis with 10% hydrocortisone gel (mixed ultrasound gel), duration - 10 minutes, frequency - 1.0 MHz, duty cycle - 50% (Pulsed), 1.0 W/cm2 high intensity ultrasound (HEP+PWHi); the experimental group III was assigned an almost identical program to the group II with the difference being 100% (Constant) duty cycle of the ultrasound (HEP+CWHi). As for groups IV and V - these groups were assigned the same home exercise programs alongside 10 minutes of low intensity 0.5 W/cm2, 1.0MHz, 50% (HEP+PWLi) and 100% (HEP+CWLi) ultrasound accordingly with 10% hydrocortisone gel, 15 procedures total.The results of the study revealed that using phonophoresis with high concentration hydrocortisone acetate that's mixed with ultrasound gel alongside therapeutic exercise in the treatment and rehabilitation of moderate post-traumatic or post-operative extension contracture of the knee caused by arthrofibrosis can be successful, which could enable us to achieve 40-50% of progress regarding knee flexion and functioning in 3 weeks in case 1.0 Mhz, 1 W/cm2, 50% pulsed ultrasound is utilized during 10 minutes, 5 times per week.

Dried blood spot (DBS) sampling is a minimally invasive method used to collect blood samples of any population for personalized medicine. We aimed to develop a sensitive and reliable analytical method for measuring 6β-hydroxycortisol (6β-OHF) and cortisol concentrations in DBS by liquid chromatography/tandem mass spectrometry so as to utilize DBS as a less invasive blood sampling method for calculating the ratio of 6β-OHF/cortisol. The lower limits of quantification obtained using four DBS were 1.08 pg/50 μl for 6β-OHF and 1.01 pg/50 μl for cortisol. The 6β-OHF and cortisol in DBS were stable for 28 days at room temperature. The intraday and interday accuracy and precision of the method was <12%. Additionally, the 6β-OHF and cortisol in DBS were measured before, during, and after 3 days of clarithromycin administration to two of the subjects. Then, their concentration was compared in the plasma and whole blood collected simultaneously. The concentrations of 6β-OHF and cortisol in four DBS ranged from 0.007 to 0.079 ng/50 μl and from 1.15 to 6.66 ng/50 μl, respectively. The 6β-OHF/cortisol ratio in DBS decreased by approximately 50% on administering clarithromycin compared with that before the administration of clarithromycin. The 6β-OHF/cortisol ratio in DBS also showed a strong correlation with that in whole blood (r = 0.9694) and plasma (r = 0.9383). This method provides high accuracy and precision for measuring 6β-OHF and cortisol in DBS. It also allows the use of DBS instead of plasma for calculating the 6β-OHF/cortisol ratio. The 6β-OHF/cortisol ratio could be an index of CYP3A activity in clinical setting., (© 2021 John Wiley & Sons, Ltd.)

The 6β-OH-cortisol/cortisol ratio (6β-OHC/C) in urine is an endogenous marker of drug-metabolizing enzyme cytochrome P450 3A (CYP3A). The primary aim of this single center, prospective, non-interventional cohort study, was to investigate the variability of 6β-OHC/C during the menstrual cycle. In addition, possible associations between the CYP3A activity and sex hormones, gut microbiota metabolite trimethylamine-N-Oxide (TMAO) and microRNA-27b, respectively, were investigated. Serum and urinary samples from healthy, regularly menstruating women followed for two menstrual cycles were analyzed. Twenty-six complete menstrual cycles including follicular, ovulatory, and luteal phase were defined based on hormone analyses in serum. 6β-OHC/C were analyzed in urine and sex hormones, TMAO and miRNA-27b were analyzed in serum at the same time points. 6β-OHC/C did not vary between the follicular, ovulatory, or luteal phases. There was a difference in the relative miRNA-27b expression between the follicular and ovulatory phase (p = .03). A significant association was found between 6β-OHC/C and progesterone during the follicular (p = .005) and ovulatory (p = .01) phases (n = 26 for each phase). In addition, a significant association was found between the ratio and TMAO during the ovulatory (p = .02) and luteal (p = .002) phases. 6β-OHC/C and gut microbiota TMAO were significantly associated (p = .003) when evaluating all values, for all phases (n = 78). Interestingly, the finding of an association between 6β-OHC/C in urine and levels of TMAO in serum suggest that gut microbiota may affect CYP3A activity., (© 2021 The Authors. Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

Context: Adrenocorticotropic hormone (ACTH) can contribute to aldosterone excess in primary aldosteronism (PA) via increased melanocortin type 2 receptor expression. Dynamic manipulation of the hypothalamic-pituitary-adrenal (HPA) axis could assist PA subtyping, but a direct comparison of dynamic tests is lacking., Objective: To investigate plasma steroid differences between aldosterone-producing adenoma (APA) and bilateral PA (BPA) relative to ACTH variations., Methods: We conducted comprehensive dynamic testing in 80 patients: 40 with APA and 40 with BPA. Peripheral plasma was collected from each patient at 6 time points: morning; midnight; after 1 mg dexamethasone suppression; and 15, 30, and 60 minutes after ACTH stimulation. We quantified 17 steroids by mass spectrometry in response to ACTH variations in all patients and compared their discriminative power between the 2 PA subtypes., Results: Patients with APA had higher morning and midnight concentrations of 18-hydroxycortisol, 18-oxocortisol, aldosterone, and 18-hydroxycorticosterone than those with BPA (P < 0.001 for all). In response to cosyntropin stimulation, the APA group had larger increments of aldosterone, 18-oxocortisol, 11-deoxycorticosterone, corticosterone, and 11-deoxycortisol (P < 0.05 for all). Following dexamethasone suppression, the APA group had larger decrements of aldosterone, 18-hydroxycortisol, and 18-oxocortisol (P < 0.05 for all), but their concentrations remained higher than in the BPA group (P < 0.01 for all). The highest discriminatory performance between the PA subtypes was achieved using steroids measured 15 minutes post-ACTH stimulation (area under receiver operating characteristic curve 0.957)., Conclusion: Steroid differences between APA and BPA are enhanced by dynamic HPA testing; such noninvasive tests could circumvent the need for adrenal vein sampling in a subset of patients with PA., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: Glucocorticoids (GCs) are steroids that play an essential role in physiological processes and are valuable therapeutic agents against various diseases. The aim of our study was to evaluate the antioxidant effects of piperine (PIP) on steroid-induced oxidative stress in liver tissue., Materials and Methods: We used 36 fertilized specific-pathogen-free (SPF) chicken eggs that were divided into the following 6 groups: group 1 (n=6), phosphate buffered saline (PBS) (pH 7.4 saline solution [0.9%] isotonic); group 2 (n=6), 0.50 µmol hydrocortisone succinate sodium (HC); group 3 (n=6), 0.50 µmol HC and 100 mg/kg piperine (PIP); group 4 (n=6), 0.50 µmol HC and 50 mg/kg PIP; group 5 (n=6), 0.50 µmol HC and 25 mg/kg PIP; and group 6 (n=6), 0.50 µmol HC and 10 mg/kg PIP. Chick embryos were removed from the eggs and the livers dissected from the embryos. The total antioxidant status (TAS), total oxidant status (TOS), reduced glutathione (GSH), and lipid peroxidation (malondialdehyde [MDA]) levels were measured., Results: The highest levels of GSH and TAS in the liver tissues were observed in group 3, with a significant difference from those in group 2 (p <0.001 and p =0.006, respectively). The lowest levels of MDA and TOS in the liver tissues were observed in group 3, with a significant difference from those in group 2 (p <0.001 and p =0.021, respectively)., Conclusions: The antioxidant and hepatoprotective properties of PIP were observed only at high doses.

Background and Objectives: Metabolomics facilitates the discovery of biomarkers and potential therapeutic targets for CKD progression., Design, Setting, Participants, & Measurements: We evaluated an untargeted metabolomics quantification of stored plasma samples from 645 Chronic Kidney Disease in Children (CKiD) participants. Metabolites were standardized and logarithmically transformed. Cox proportional hazards regression examined the association between 825 nondrug metabolites and progression to the composite outcome of KRT or 50% reduction of eGFR, adjusting for age, sex, race, body mass index, hypertension, glomerular versus nonglomerular diagnosis, proteinuria, and baseline eGFR. Stratified analyses were performed within subgroups of glomerular/nonglomerular diagnosis and baseline eGFR., Results: Baseline characteristics were 391 (61%) male; median age 12 years; median eGFR 54 ml/min per 1.73 m 2 ; 448 (69%) nonglomerular diagnosis. Over a median follow-up of 4.8 years, 209 (32%) participants developed the composite outcome. Unique association signals were identified in subgroups of baseline eGFR. Among participants with baseline eGFR ≥60 ml/min per 1.73 m 2 , two-fold higher levels of seven metabolites were significantly associated with higher hazards of KRT/halving of eGFR events: three involved in purine and pyrimidine metabolism (N6-carbamoylthreonyladenosine, hazard ratio, 16; 95% confidence interval, 4 to 60; 5,6-dihydrouridine, hazard ratio, 17; 95% confidence interval, 5 to 55; pseudouridine, hazard ratio, 39; 95% confidence interval, 8 to 200); two amino acids, C-glycosyltryptophan, hazard ratio, 24; 95% confidence interval 6 to 95 and lanthionine, hazard ratio, 3; 95% confidence interval, 2 to 5; the tricarboxylic acid cycle intermediate 2-methylcitrate/homocitrate, hazard ratio, 4; 95% confidence interval, 2 to 7; and gulonate, hazard ratio, 10; 95% confidence interval, 3 to 29. Among those with baseline eGFR <60 ml/min per 1.73 m 2 , a higher level of tetrahydrocortisol sulfate was associated with lower risk of progression (hazard ratio, 0.8; 95% confidence interval, 0.7 to 0.9)., Conclusions: Untargeted plasma metabolomic profiling facilitated discovery of novel metabolite associations with CKD progression in children that were independent of established clinical predictors and highlight the role of select biologic pathways., (Copyright © 2021 by the American Society of Nephrology.)

Ethnopharmacological Relevance: Because of the growing incidence of AD, psychosocial and economic burden of AD patients are often considerable. Steroid treatments are widely used, but long term use of this treatment can cause side effects. To reduce the burden of AD patients and find new efficient treatment, this study chose Soshiho-tang, a traditional medicine used in eastern Asia., Aim of the Study: Soshiho-tang (SSHT) is a traditional herbal medicine that has anti-inflammatory effects and improves immune function. This clinical trial evaluated the efficacy and safety of SSHT in atopic dermatitis (AD) patients with gastrointestinal disorders in comparison with placebo., Materials and Methods: This study was a single-center, randomized, double-blinded, placebo-controlled, and investigator-initiated clinical trial. A total of 60 patients aged 3-18 years with gastrointestinal disorders and diagnosed with AD by Hanifin & Rajka criteria with a Scoring Atopic Dermatitis (SCORAD) index between 15 and 49 were enrolled. Participants were randomly assigned to the SSHT or placebo groups in a ratio of 1:1 and efficacy evaluation was conducted at week 4 and 8. The participants orally administered SSHT or placebo three times a day for 4 weeks. The primary outcome was measured based on a change of SCORAD index. The secondary outcome measurements included the following: survey questionnaires of gastrointestinal disorder, amount and frequency of ointment application for AD, dermatology quality of life index, and safety evaluation (diagnostic test, adverse reaction, and vital sign monitoring)., Results: During efficacy evaluation, the SCORAD score and digestive symptoms in the experimental and placebo groups were not statistically significant. However, the amount and frequency of ointment application in the experimental group were reduced compared to those in the placebo group at week 8. Also, In the Children's Dermatology Life Quality Index (CDLQI), statistically significant Quality of Life (QOL) improvement was observed in the SSHT experimental group compared to the placebo group. In safety evaluation, all participants were within the normal range during the study period. Blood sample testing indicated that the lymphocytes ratio decreased, and neutrophils ratio increased in the experimental group, whereas the placebo group showed the opposite immune response pattern., Conclusion: We concluded that SSHT administration can reduce steroid ointment dependence and improve the QOL in AD patients by regulating neutrophil-lymphocyte ratio., (Copyright © 2021 Elsevier B.V. All rights reserved.)

To evaluate the diagnostic value of serum levels of adrenal steroids for diagnosing and subtyping Cushing's syndrome. Patients diagnosed with endogenous Cushing's syndrome (34 and 19 patients with adrenal and pituitary Cushing's syndrome, respectively) and healthy controls (n = 34) were consecutively enrolled at Seoul National University from 2016 to 2020. Morning serum samples were collected before and 3 months after treatment. Serum steroids were profiled using liquid chromatography-mass spectrometry. The diagnostic value of each and the combination of steroids were assessed using the area under the curve of receiver operating characteristic (AUROC) and decision tree analysis. Tetrahydrocortisone and 6β-hydroxycortisol showed the highest AUROC (0.893 and 0.890, respectively) for the diagnosis of endogenous Cushing's syndrome. The decision tree composed of tetrahydrocortisone and 6β-hydroxycortisol correctly classified 79/87 (90.8 %) subjects. For subtyping into adrenal or pituitary Cushing's syndrome, dehydroepiandrosterone sulfate (DHEA-S) showed the highest AUROC (0.988), which was similar to that of plasma ACTH (0.994, P = 0.458). The decision tree composed of only DHEA-S correctly classified 51/53 (96.2 %) of the Cushing's syndrome subtype. DHEA-S showed a significant linear correlation with the plasma ACTH level, but not with the 24 -h urine free cortisol or dexamethasone suppression test results. All steroids, except allo-tetrahydrocortisol and tetrahydrocortisone, decreased significantly at 3 months post-treatment with similar patterns in both adrenal and pituitary Cushing's syndrome. Serum steroid profiling using a single morning serum sample provides valuable information for diagnosing and subtyping Cushing's syndrome., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Oclacitinib is an effective systemic therapy for dogs with atopic dermatitis (AD). Few studies have evaluated concurrent topical treatment with oclacitinib in dogs., Objectives: To evaluate the efficacy and safety of combination therapy of oclacitinib and 0.0584% hydrocortisone aceponate (HCA) spray in dogs with AD., Animals: Eighteen dogs with AD., Methods and Materials: This study was a randomized, double-blinded, placebo-controlled trial. All dogs were treated with oclacitinib (0.4-0.6 mg/kg twice daily for 14 days, then once daily for 14 days) and randomized to receive either HCA spray or placebo spray, applied once daily for seven days then every other day through to Day (D)28. Clinical assessments included the Canine Atopic Dermatitis Extent and Severity Index, 4 th iteration (CADESI-4) and the pruritus Visual Analog Scale (PVAS) every seven days, and blood and urine tests every 14 days., Results: The mean CADESI-4 and PVAS scores were significantly reduced on D7 and D14 compared to D0 in both groups (P < 0.05). From D14 to D21, CADESI-4 and PVAS scores were significantly increased in the placebo group (P < 0.005), and not in the HCA-treated group. The mean reduction from baseline of the HCA-treated group was significantly higher than that of the placebo group for the PVAS and CADESI-4 on D21 (59.9% versus 27.6%, P = 0.0216) and D28 (56.0% versus 30.5%, P = 0.0109), respectively. One dog in the HCA-treated group was withdrawn as a consequence of developing diarrhoea., Conclusion: Topical application of 0.0584% HCA spray may be useful for preventing exacerbation of pruritus and clinical lesions when tapering oclacitinib therapy in dogs with AD., (© 2020 the European Society of Veterinary Dermatology and the American College of Veterinary Dermatology.)

A 45-year-old brown-skinned woman presented with a 5-year history of asymptomatic grayish brown lesions on the face, arms, and legs. She had no medical history of previous diseases or contact dermatitis. She revealed that she had used olive oil all over her body for the last 8 years every other day. Physical examination showed multiple, well-defined, oval-shaped, dark brown, smooth-surfaced macules with no elevated active borders (Figure 1). There were no associated lesions on the nails, scalp, or mucosae. Serologic tests for autoantibodies and hepatitis A, B, and C virus infections were non-reactive. A patch test for olive oil was also negative. A skin biopsy revealed epidermal atrophy, orthokeratosis, basal cell vacuolation, and a band-like lymphocytic infiltrate in the upper portion of the dermis with abundant colloid bodies and pigmentary incontinence in the papillary dermis (Figure 2). A diagnosis of lichen planus pigmentosus (LPP) was confirmed, and betamethasone butyrate propionate was applied for 2 months over the lesions, with a limited therapeutic effect. Clinical improvement was seen only after she discontinued the olive oil application (Figure 3).

In this study, an accurate, simple, economical, precise and reproducible reversed-phase HPLC method was developed for the estimation of fusidic acid and hydrocortisone acetate, according to the International Conference on Harmonization guidelines, in a cream formulation. Chromatographic separation was achieved by isocratic elution, on a Shimadzu reversed-phased high-pressure liquid chromatography instrument, equipped with a C 18 column (150 × 4.6 mm, 5 μm) and UV detector at 225 nm wavelength, using acetonitrile and 0.05% trifluoroacetic acid (60:40), as a mobile phase and diluent, at flow rate 2 ml/min and an injection volume of 20 μl. The calibration curves were acquired with concentration range 80-120% and mean percentage recoveries for hydrocortisone acetate and fusidic acid were 100.14 and 100.81%, respectively. The limits of detection was obtained as 6.0667 and 6.807 μm ml -1 and the limits of quantification were 20.204 and 20.628 μm ml -1 for hydrocortisone acetate and fusidic acid, respectively. All of the validation parameters were within the acceptance criteria, as per International Conference on Harmonization requirements, for hydrocortisone acetate and fusidic acid. This method was found to be validated, simple, rapid and applicable for the simultaneous estimation of hydrocortisone acetate and fusidic acid by reversed-phased high-pressure liquid chromatography, for routine analytical testing in quality control, with a run time of 8 min., (© 2020 John Wiley & Sons, Ltd.)

There is increasing interest in using nonblood measures of glucocorticoids to assess the physiological response to chronic stress conditions. In sheep, cortisol has been measured in various matrices including saliva, feces, and wool, but comprehensive studies of the relationship between plasma concentrations of cortisol and concentrations in these nonblood matrices are lacking. Therefore, we tested the hypothesis that administration of cortisol to sheep would result in elevated concentrations of cortisol in blood, saliva, feces, and wool. Merino ewes were administered with saline or 2 mg/kg BW/d hydrocortisone acetate (HCA) by intramuscular (i.m.) injection for 28 d. This treatment was imposed to mimic circulating cortisol concentrations experienced during periods of chronic stress. Cortisol and cortisone were directly measured in plasma, saliva, and wool before, during, and after treatment with saline or HCA. A 14-d pre-treatment and a 14-d post-treatment period were used to measure time taken for glucocorticoid concentrations in each of the matrices to return to baseline levels. Cortisol was also measured in feces before, during, and after treatment. Wool growth was also measured. Before treatment, there was no difference in the concentration of cortisol or cortisone in plasma, saliva, feces, or wool in animals treated with saline or HCA. In contrast, treatment with HCA increased (P < 0.05) concentrations of both cortisol and cortisone in plasma, saliva, and wool and of cortisol in feces. In plasma, cortisol concentrations were higher than cortisone (P < 0.05), whereas saliva cortisol and cortisone concentrations did not differ significantly. In wool, the concentration of cortisone was about 19-fold higher than that of cortisol during treatment and post-treatment periods. Treatment with HCA inhibited wool growth. These results demonstrate that an increase in glucocorticoids in the blood of sheep is reflected in increases in saliva (after 7 d of treatment), feces (21 d), and wool (14 d). Therefore, measures of glucocorticoids in these matrices may provide a measure of activation of the adrenal glands over time in sheep, thereby providing a retrospective indicator of chronic stress. With respect to wool, it appears that cortisol is predominantly metabolized to cortisone in the skin or wool follicle and is stored as cortisone. Therefore wool cortisone may also provide an important measure in quantifiying chronic stress in sheep., (Copyright © 2020 Elsevier Inc. All rights reserved.)

ACT-539313 is a potent and selective orexin-1 receptor antagonist. CYP3A is the major cytochrome P450 (CYP) enzyme involved in the metabolism and clearance of ACT-539313 in man. The main objective of this study was to investigate the effect of ACT-539313 on the pharmacokinetics of orally administered midazolam. Thereby, this single-center, open-label, fixed-sequence study investigated the CYP3A interaction potential of ACT-539313 following single- (on day 2) and repeated-dose (on day 11) twice-daily administration of 200 mg ACT-539313. Exposure to midazolam was higher during concomitant administration of single as well as after repeated doses of ACT-539313 over 10 days compared to midazolam alone (day 1). In the presence of ACT-539313, the geometric mean ratio of the maximum plasma concentration and the area under the plasma concentration-time curve from time 0 to 24 hours increased by 1.18- and 1.79-fold on day 2, and by 2.13- and 4.54-fold on day 11, respectively. A similar outcome was also shown in the additionally evaluated urinary 6β-hydroxycortisol/cortisol ratio (6β-CR), as the geometric mean ratio of the 6β-CR showed a decrease to 0.78 on day 2 and to 0.61 on day 11. The most commonly reported adverse events (AEs) included somnolence and headache. All AEs were transient and of mild intensity. No treatment-related effects on vital signs, clinical laboratory, and electrocardiogram were observed. In summary, the observed corresponding decrease of both the validated, exogenous (midazolam/1-hydroxymidazolam ratio) and a frequently used endogenous (6β-CR) marker of CYP3A activity is indicative of CYP3A inhibition occurring after ACT-539313 treatment., (© 2020, The American College of Clinical Pharmacology.)

Background: Vertebrate glucocorticoid receptor (GR) is an evolutionary-conserved cortisol-regulated nuclear receptor that controls key metabolic and developmental pathways. Upon binding to cortisol, GR acts as an immunosuppressive transcription factor. Drosophila melanogaster, a model organism to study innate immunity, can also be immunosuppressed by glucocorticoids. However, while the genome of fruit fly harbors 18 nuclear receptor genes, the functional homolog of vertebrate GR has not been identified., Results: In this study, we demonstrated that while D. melanogaster is susceptible to Saccharomyces cerevisiae oral infection, the oral exposure to cortisol analogs, cortisone acetate or estrogen, increases fly sensitivity to yeast challenge. To understand the mechanism of this steroid-induced immunosuppression, we identified the closest genetic GR homolog as D. melanogaster Estrogen Related Receptor (ERR) gene. We discovered that Drosophila ERR is necessary for cortisone acetate- and estrogen-mediated increase in sensitivity to fungal infection: while ERR mutant flies are as sensitive to the fungal challenge as the wildtype flies, the yeast-sensitivity of ERR mutants is not increased by these steroids. Interestingly, the fungal cortisone analog, ergosterol, did not increase the susceptibility of Drosophila to yeast infection. The immunosuppressive effect of steroids on the sensitivity of flies to fungi is evolutionary conserved in insects, as we show that estrogen significantly increases the yeast-sensitivity of Culex quinquefasciatus mosquitoes, whose genome contains a close ortholog of the fly ERR gene., Conclusions: This study identifies a D. melanogaster gene that structurally resembles vertebrate GR and is functionally necessary for the steroid-mediated immunosuppression to fungal infections.

Different methods have been used for CYP3A phenotyping, such as probe drugs or the urinary index 6β-hydroxycortisol/cortisol ratio (6β-OHF:C). This work describes a simple and affordable method for the simultaneous determination of the endogenous compounds cortisol and 6β-hydroxycortisol in urine using a background subtraction approach. The method was applied to investigate the CYP3A activity in HIV-infected pregnant women (n = 9) in the third trimester and postpartum periods. Also, the within-day variability in the 6β-OHF:C index was also evaluated. The sample preparation consists of a pre-cleanup with acetonitrile followed by liquid-liquid extraction with ethyl acetate. The analytes were resolved by employing an Acquity UPLC®BEH C18 column with a mobile phase that consisted of a mixture of acetonitrile containing 0.1% formic acid and 0.1% formic acid in gradient mode. The method presented linearities of 1-1.000 ng/mL and 2-1.000 ng/mL for C and 6β-OHF, respectively, and presented acceptable precision and accuracy. Qualitative and quantitative matrix effects tests were also performed. A high 6β-OHF:C within-day variability was observed in both phases. In the third trimester period, the 6β-OHF:C ranged from 2.57 to 51.69, with a mean ± standard deviation (SD) of 15.12 ± 5.41 (n = 9). Similar values were obtained in the postpartum period, with 6β-OHF:C ranging from 3.48 to 44.54 with a mean ± SD of 14.37 ± 5.73 (n = 7). Even though the 6β-OHF:C is a non-invasive index for CYP3A phenotyping, its use is susceptible to high within-day variability., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objectives: Dexamethasone is associated with adrenal insufficiency in adults and children with chronic disease. This association has not been studied after single-dose oral dexamethasone, the standard of care for children with croup. We hypothesized that single-dose oral dexamethasone in children with croup is associated with a transient decrease in endogenous glucocorticoids., Methods: We conducted a prospective, 2-arm, pharmacodynamic study of single-dose oral dexamethasone 0.6 mg/kg (maximum, 12 mg) in children older than 2 years with croup compared with controls (children with febrile upper respiratory tract infections who did not receive dexamethasone). Primary outcome was urinary 6β-hydroxycortisol-cortisol ratio., Results: Twenty-seven children were analyzed (22 with croup and 5 with upper respiratory tract infections). Median 6β-hydroxycortisol-cortisol ratios before dexamethasone, the following morning, and on days 1, 3, and 7 were 2.8, 2.2, 2.0, 2.8, and 2.6, respectively. Among controls, the median 6β-hydroxycortisol-cortisol ratios at the same time intervals was 1.9, 1.5, 1.8, 2.5, and 1.7, respectively. There were no significant differences in the change from time 0 between groups at any time point. There were no serious adverse events or infectious complications., Conclusions: Single-dose oral dexamethasone is not associated with decreased endogenous corticosteroid levels in children with croup. Future studies should use criterion standard tests to rule out suppression of the hypothalamic-pituitary-adrenal axis and be powered sufficiently to identify adverse clinical outcomes.

Adrenal insufficiency (AI) remains a significant cause of morbidity and mortality in children with 1 in 200 episodes of adrenal crisis resulting in death. The goal of this working group of the Pediatric Endocrine Society Drug and Therapeutics Committee was to raise awareness on the importance of early recognition of AI, to advocate for the availability of hydrocortisone sodium succinate (HSS) on emergency medical service (EMS) ambulances or allow EMS personnel to administer patient's HSS home supply to avoid delay in administration of life-saving stress dosing, and to provide guidance on the emergency management of children in adrenal crisis. Currently, hydrocortisone, or an equivalent synthetic glucocorticoid, is not available on most ambulances for emergency stress dose administration by EMS personnel to a child in adrenal crisis. At the same time, many States have regulations preventing the use of patient's home HSS supply to be used to treat acute adrenal crisis. In children with known AI, parents and care providers must be made familiar with the administration of maintenance and stress dose glucocorticoid therapy to prevent adrenal crises. Patients with known AI and their families should be provided an Adrenal Insufficiency Action Plan, including stress hydrocortisone dose (both oral and intramuscular/intravenous) to be provided immediately to EMS providers and triage personnel in urgent care and emergency departments. Advocacy efforts to increase the availability of stress dose HSS during EMS transport care and add HSS to weight-based dosing tapes are highly encouraged., Competing Interests: Competing interests: BSM is a consultant for AbbVie, Ascendis, Ferring, Novo Nordisk, Pfizer, Sandoz, Soleno and Tolmar and has received research support from Alexion, Ascendis, BioMarin, Endo Pharmaceuticals, Genentech, Genzyme, Novo Nordisk, Opko, Sandoz, Sangamo, Shire, Tolmar and Versartis. MK received grant support from T1D Exchange Quality Improvement Collaborative. MG is a consultant for Spruce Biosciences, Millendo, Pfizer, and BridgeBio. KS receives research support from the DHHS Federal Food and Drug Administration, NIH National Cancer Institute, March of Dimes, National Science Foundation, Spruce Biosciences, Alexion and Neurocrine., (© American Federation for Medical Research 2020. Re-use permitted under CC BY-NC. No commercial re-use. Published by BMJ.)

Background Cytochrome P450s (CYPs, EC 1.14.14.1) are the main enzymes of drug metabolism. The functional significance of CYPs also includes the metabolism of foreign chemicals and endogenic biologically active compounds. The CYP3A4 isoform contributes to the metabolism of about half of all marketed medicinal preparations. The aim of this study was to investigate the effects of two biologically active compounds: 2-aminoethane-sulfonic acid (taurine) and 3-hydroxy-4-trimethylaminobutyrate (L-carnitine) on urinary 6β-hydroxycortisol/cortisol (6β-OHC/cortisol) metabolic ratio as a biomarker of the CYP3A4 activity of healthy volunteers. Taurine is used for the treatment of chronic heart failure and liver disease. Cardiologists, nephrologists, neurologists, gerontologists in addition to the main etiopathogenetic therapies, use L-carnitine. The quantification of the 6β-OHC/cortisol metabolic ratio as a biomarker of CYP3A4 activity in human urine was used for the assessment of CYP3A4 catalytic activity as a non-invasive test. Methods The study included 18 healthy male volunteers (aged from 18 to 35 years old). The volunteers took taurine in a dose of 500 mg twice a day or L-carnitine in a dose of 2.5 mL 3 times a day for 14 consecutive days. The test drug was given 20 min before meals. The collection of urine samples was performed before and after 3, 7, 10, and 14 days after taurine intake. The metabolic ratio of 6β-OHC/cortisol in morning spot urine samples was studied by the liquid chromatography/mass spectroscopy (LC/MS) method. Results The ratio of 6-6β-OHC/cortisol was used as a biomarker to study the taurine and L-carnitine influence on CYP3A4 metabolism of cortisol. The ratio of urinary 6β-OCH/cortisol in the morning urine samples of volunteers before the beginning of taurine therapy (baseline ratio) was 2.71 ± 0.2. Seven days after the administration of taurine in a dose of 500 mg twice a day, the 6β-OCH/cortisol ratio was 3.3 ± 0.2, which indicated the increased catalytic activity of CYP3A4 towards cortisol. As for the L-carnitine supplementation, analysis of the 6β-OCH/cortisol ratio in the urine for 14 days did not show any significant changes in this baseline ratio, indicating the lack of L-carnitine influence on the catalytic activity of CYP3A4 to cortisol. Conclusions The results obtained demonstrated the influence of taurine on 6β-OCH/cortisol metabolic ratio as a biomarker of CYP3A4 catalytic activity to cortisol. L-carnitine did not affect the activity of CYP3A4. The lack of a clinically meaningful effect of L-carnitine was established.

Recent genetic examinations and multisteroid profiles have provided the basis for subclassification of aldosterone-producing adenomas (APAs). The objective of the current study was to produce a comprehensive, high-resolution mass spectrometry imaging (MSI) map of APAs in relation to morphometry, immunohistochemical profiles, mutational status, and clinical outcome. The study cohort comprised 136 patients with unilateral primary aldosteronism. Matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance MSI was conducted, and metabolite profiles were analyzed with genotype/phenotype information, including digital image analysis from morphometry and IHC of steroidogenic enzymes. Distinct molecular signatures between KCNJ5- and CACNA1D-mutated APAs with significant differences of 137 metabolites, including metabolites of purine metabolism and steroidogenesis, were observed. Intratumor concentration of 18-oxocortisol and 18-hydroxycortisol were inversely correlated with the staining intensity of CYP11B1. Lower staining intensity of CYP11B1 and higher levels of 18-oxocortisol were associated with a higher probability of complete clinical success after surgery. The present study demonstrates distinct metabolomic profiles of APAs in relation to tumor genotype. In addition, we reveal an inverse correlation between cortisol derivatives and CYP11B1 and the impact of 18-oxocortisol and CYP11B1 on clinical outcome, which provides unprecedented insights into the pathophysiology, clinical features, and steroidogenesis of APAs.

Background: It is important to determine the vasoconstrictor potencies of topical corticosteroids used to treat psoriasis to ensure appropriate clinical use. Objective: To compare the vasoconstrictive potencies of fixed-dose combination calcipotriol (50 μg/g) and betamethasone dipropionate (0.5 mg/g) (Cal/BD) cutaneous foam with other topical corticosteroids. Methods: In this Phase I, single-center, healthy volunteer study, Cal/BD foam, clobetasol propionate 0.05% cream (CP; very potent), BD 0.05% ointment (potent), mometasone furoate 0.1% cream (MF; potent), hydrocortisone-17-butyrate 0.1% ointment (HB; moderately potent), and foam vehicle were applied, then removed after 16 h. Skin blanching was visually assessed 2 h later (scale of 0-4). Results: Thirty-six volunteers were randomized. Skin blanching with Cal/BD foam (median [range], 2.00 [0.75-3.00]) was significantly lower than CP cream (3.00 [1.75-4.00]; p  < .001), was not significantly different from BD ointment (1.75 [0.75-3.00]; p  = .30) and MF cream (2.00 [1.00-3.75]; p  = .22), and was significantly greater than HB ointment (1.25 [0.50-3.00]; p  < .001) and vehicle (0 [0-0.50]; p  < .001). There were no local tolerability reactions or adverse events. Conclusions: The corticosteroid potency of Cal/BD foam was not significantly different from BD ointment and MF cream, significantly stronger than HB ointment, but weaker than CP cream in healthy volunteers.

Introduction Due to the effects of hemorrhoids on physical and mental health, this study aimed to compare the effect of Myrtus communis herbal and anti-hemorrhoid ointments on symptoms of hemorrhoid and quality of life (primary outcomes) and satisfaction of the treatment and side effects (secondary outcomes). Methods This triple-blind randomized controlled trial was performed on women with grade I and II hemorrhoid referring to health centers in Tehran, Iran, in 2017. Individuals were randomly assigned to two groups of 67 people through block randomization method. The intervention group received the Myrtus communis herbal ointment and the control group received anti-hemorrhoid ointment twice a day, every 12 ± 2 h, an applicator of the drug through the rectum for 4 weeks. The Colorectal Evaluation of a Clinical Therapeutics Scale (CORECTS) was used to assess the severity of symptoms of hemorrhoid. To assess the quality of life, the World Health Organization Quality of Life questionnaire (WHOQOL-BREF) was used to measure the general quality of life of participants. This questionnaire was completed once before the start of the study, then on the fourth and the eighth week after the start of the intervention. Repeated measure ANOVA, Chi-square, Mann-Whitney U and independent t-test were used for data analysis. Results The severity of all symptoms of hemorrhoid decreased in both two group and there was no statistically significant difference between the two groups (p>0.05). However, the mean of anal itching at 4 and 8 weeks after the intervention was significantly lower in the Myrtus communis ointment group (p<0.05). There was no significant difference between groups in terms of quality of life at 4 and 8 weeks after the intervention (p>0.05). There was a significant difference between the two groups in terms of satisfaction with the drug (p=0.019) and the participants in the Myrtus communis ointment group were more satisfied with their drug use. Conclusions Myrtus communis herbal ointment was able to reduce the symptoms of hemorrhoid in the affected women. Therefore, it is likely that the use of this drug will promote the health of mothers with hemorrhoid.

Background: Phimosis is a common condition affecting most infant boys and generally resolves over time without symptoms. Severe cases of phimosis can lead to balanoposthitis, urinary tract infections, and urinary retention. Medical treatment for symptomatic phimosis includes topical corticosteroids with manual foreskin retraction., Objective: Over-the-counter hydrocortisone 1% cream was compared in a randomized controlled fashion with prescription triamcinolone 0.1% cream for the medical management of symptomatic phimosis., Methods: The study institution conducted a randomized open-label trial for the treatment of grades 4-5 phimosis (phimosis grade scale 0-5). Boys aged 3-13 years were randomized to hydrocortisone 1% cream or triamcinolone 0.1% cream dosed at least twice daily for a course of 12 weeks. Instructions were provided for appropriate application and manual retraction of the foreskin. Evaluations were performed at 4, 8, and 12 weeks. Successful completion of the study was determined by reaching phimosis grade 2 or less or after completing 12 weeks of treatment., Results: A total of 52 boys enrolled in the trial, with a total of 32 boys completing the 12-week duration. Of the 13 boys in the hydrocortisone arm, there was a 30.8% success rate at 4 weeks, 53.8% success rate at 8 weeks, and 61.5% success rate at 12 weeks. Of the 19 boys in the triamcinolone arm, there was a 31.6% success rate at 4 weeks, 52.6% success rate at 8 weeks, and 68.4% success rate at 12 weeks. There was no statistical difference between the two arms at each interval., Discussion: To the study authors' knowledge, this is the first open-label trial with direct comparison of hydrocortisone 1% cream with triamcinolone 0.1% cream. The study results support those reported in other studies when each topical steroid was compared with placebo. Limitations of the study include loss to follow-up, unblinded treatment allocation, and reduced power to detect differences by treatment frequency and duration., Conclusion: Over-the-counter hydrocortisone 1% cream is not inferior to triamcinolone 0.1% cream when paired with manual retraction for the treatment of grade 4-5 phimosis. Successful treatment response may be seen up to 12 weeks., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

Visualizing tissue distribution of steroid hormones is a promising application of MALDI mass spectrometry imaging (MSI). On-tissue chemical derivatization using Girard's T reagent has enhanced the ionization efficiency of steroids. However, discriminating between structural isomers with distinct bioactivities remains a challenge. Herein, we used ion trap MS/tandem MS (MS 3 ) to distinguish a mineralcorticoid aldosterone (Aldo) and a glucocorticoid cortisol (F), from their structural isomers. Our method is also useful to detect hybrid steroids (18-hydroxycortisol [18-OHF] and 18-oxocortisol) with sufficient signal-to-noise ratio. The clinical applicability of the tandem MS method was evaluated by analyzing F, Aldo, and 18-OHF distributions in human adrenal glands. In such clinical specimens, small Aldo-producing cell clusters (APCCs) were identified and were first found to produce a high level of Aldo and not to contain F. Moreover, a part of APCCs produced 18-OHF, presumably converted from F by APCC-specific CYP11B2 activity. Catecholamine species were also visualized with another derivatization reagent (TAHS), and those profiling successfully discriminated pheochromocytoma species. These tandem MSI-methods, coupled with on-tissue chemical derivatization has proven to be useful for detecting low-abundance steroids, including Aldo and hybrid steroids and thus identifying steroid hormone-producing lesions.

A new simple and robust HPLC-DAD method was developed for the concurrent analysis of hydroquinone (HQ), hydrocortisone acetate (HCA) and tretinoin (TRN) triple combination for the first time using an Inertsil ODS 3-C18 column (150 mm × 4.6 mm, 5 μm particle size) column with 0.05 M phosphate buffer (pH 5.0) and acetonitrile at a ratio of (10:90, v/v) as a mobile phase, eluted by an isocratic elution mode at a flow rate of 1.0 mL/min and detected at 265 nm. Mefenamic acid was used as an internal standard (I.S.). The method produced linear responses in the concentration range of 10-200, 5-100 and 1-40 μg/mL, with detection limits of 2.01, 1.13 and 0.28 × 10-3 and quantitation limits of 6.11, 3.41 and 0.87 × 10-3 μg/mL for HQ, HCA and TRN, respectively, and a correlation coefficient higher than 0.9998. All validation requirements were satisfied by proving its linearity, precision, accuracy, robustness and specificity. The method was extended for application in triple combination cream, HQ/TRN co-formulated cream and HQ and TRN single ingredient cream with a recovery >97%., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Purpose We retrospectively studied the efficacy of intratympanic steroid administration in comparison with hyperbaric oxygen (HBO) therapy for idiopathic sudden sensorineural hearing loss (ISSNHL) with negative prognostic factors. Method We enrolled 301 patients (302 ears) with ISSNHL (average hearing level at 250-4000 Hz ≥ 40 dB; time from onset to treatment ≤ 30 days). From August 2002 to March 2009, 174 patients (174 ears) received systemic steroid plus HBO therapy (HBO group), and from June 2015 to January 2018, 127 patients (128 ears) received systemic plus intratympanic steroid (IT group). Hearing outcomes were evaluated by 6 indices: cure rate, marked-recovery rate (percent of patients with hearing gain ≥ 30 dB), recovery rate (percent of patients with hearing gain ≥ 10 dB), hearing gain, hearing level after treatment, and percent hearing improvement compared to the unaffected contralateral ear. Results The recovery rate was significantly higher in the IT group than in the HBO group (80.5% vs. 68.4%, p = .019). The IT group showed a higher recovery rate than the HBO group in patients aged ≥ 60 years ( p = .010), patients with early (≤ 7 days from onset) treatment ( p = .005), patients with initial hearing levels ≥ 90 dB ( p = .037), and patients with vertigo/dizziness ( p = .040). The IT group also showed higher hearing gain and percent hearing improvement than the HBO group in patients with vertigo/dizziness ( p = .046 and p = .026, respectively). Conclusions Systemic plus intratympanic steroid is more effective for ISSNHL than systemic steroid plus HBO, particularly in patients with negative prognostic factors, such as old age, profound hearing loss, and/or presence of vertigo/dizziness.

Purpose: The etiopathogenesis of steroid-induced cataracts is unknown. One hypothesis is that the higher reactive oxygen species (ROS) levels play an important role in the pathogenesis of several disorders, including the evolution of cataracts. This study investigated the antioxidant effects of piperine in our steroid-induced chick embryo lens model., Methods: The study included 36 specific pathogen-free (SPF) fertilized eggs divided into six groups: phosphate buffer saline (PBS, pH 7.4 Saline Solution (0.9%) isotonic) group (G1), hydrocortisone succinate sodium (HC)-treated group (G2), 100 mg/kg piperine and HC treated group (G3), 50 mg/kg piperine and HC treated group (G4), 25 mg/kg piperine and HC treated group (G5), and 10 mg/kg piperine and HC treated group (G6). On the 15th day of incubation, the SPF eggs in the six groups were removed from the incubator; all were injected using insulin injectors into the chorioallantoic membrane. On day 17, all of the chick embryos were removed from the eggs and all lenses were dissected from the embryos. Cataract formation was evaluated in all lenses, and total antioxidant status (TAS), total oxidant status (TOS), reduced glutathione (GSH), and lipid peroxidation (MDA, malondialdehyde) levels were measured in all lens., Results: The lenses in the G1 group had higher levels of GSH and TAS (p < 0.01), and lower levels of MDA and TOS than the G2 group (p < 0.05 and p < 0.01, respectively). Group 3 had higher levels of GSH and TAS (p < 0.001 and p < 0.001 respectively), and lower levels of MDA and TOS than the G2 group (p < 0.01 and p < 0.001, respectively)., Conclusion: Steroid therapy causes a decrease in GSH and TAS levels and an increase in TOS and MDA levels in lens tissues, indicating increased oxidative stress. Piperine exerts its effects as an antioxidant substance, in increasing doses.

Peripheral 18-oxocortisol (18oxoF) level could contribute to the detection of aldosterone-producing adenoma (APA) in patients with primary aldosteronism. However, peripheral 18oxoF varies among such patients, which is a big drawback concerning its clinical application. We studied 48 cases of APA, 35 harboring KCNJ5 mutation, to clarify the significance of clinical and pathological parameters about peripheral 18oxoF. Peripheral 18oxoF concentration ranged widely from 0.50 to 183.13 ng/dL and correlated positively with intratumoral areas stained positively for steroidogenic enzymes ( P<0.0001). The peripheral 18oxoF level also correlated significantly with that of circulating aldosterone ( P<0.0001) but not with that of cortisol, a precursor of 18oxoF. However, a significant correlation was detected between peripheral 18oxoF and intratumoral glucocorticoids ( P<0.05). In addition, peripheral 18oxoF correlated positively with the number of hybrid cells double positive for 11β-hydroxylase and aldosterone synthase ( P<0.0001). Comparing between the cases with and those without KCNJ5 mutation, the KCNJ5-mutated group demonstrated a significantly higher concentration of peripheral 18oxoF (28.4±5.6 versus 3.0±0.9 ng/dL, P<0.0001) and a larger intratumoral environment including the hybrid cells ( P<0.001), possibly representing a deviation from normal aldosterone biosynthesis. After multivariate analysis, KCNJ5 mutation status turned out to be the most associated factor involved in 18oxoF synthesis in APA ( P<0.0001). Results of our present study first revealed that enhanced 18oxoF synthesis in APA could come from a functional deviation of aldosterone biosynthesis from the normal zona glomerulosa and the utility of peripheral 18oxoF measurement could be influenced by the prevalence of KCNJ5 mutation in an APA.

We used rapid one-step derivatization of 6β-hydroxylated hydrocortisone by sulfuric acid for fluorimetric determination of CYP3A4-dependent hydroxylase reaction in the electrochemical system. We have shown that CYP3A4 substrate - hydrocortisone - and its 6β-hydroxylated product have different emission wavelengths at an excitation λ ex = 365 nm after treatment with sulfuric acid:ethanol (3:1) mixture (λ em = 525 ± 2 nm and λ em = 427 ± 2 nm, respectively). The detection limit for 6β-hydroxycortisol was estimated to be 0.32 μM (corresponding to 0.095 nmol in 300 μL sample) (S/N = 3). Using the fluorimetric method of 6β-hydroxycortisol detection following the electrolysis of hydrocortisone with CYP3A4 immobilized on a screen-printed graphite electrode modified by didodecyldimethylammonium bromide we have calculated the steady-state kinetic parameters of CYP3A4 for hydrocortisone: the maximal rate of the reaction (V max ) as 89 ± 5 pmol of product per min per pmol of electroactive enzyme and the Michaelis constant (K M ) as 10 ± 2 μM. In our system, ketoconazole inhibited hydroxylase activity of CYP3A4 towards hydrocortisone with the IC 50 value of 70 ± 5 nM. The approach proposed for determination of the CYP3A4 electrocatalytic activity can be used for throughput screening of different modulators of this cytochrome P450 isozyme during drug development., (Copyright © 2018 Elsevier B.V. All rights reserved.)