LC-HRMS and GC-MS Profiling of Urine Free Cortisol, Cortisone, 6Β-, and 18-Hydroxycortisol for the Evaluation of Glucocorticoid and Mineralocorticoid Disorders.

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, 6ß-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 6ß-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.

Determination of three C18-oxygenated steroids in adrenal lesion segments in primary aldosteronism by super-selective adrenal venous sampling and LC/ESI-MS/MS.

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.

Efficacy and safety of a hydrocortisone aceponate-containing ear spray solution in dogs with erythemato-ceruminous otitis externa: A randomised, multicentric, single-blinded, controlled trial.

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.

Plasma 6ß-hydroxycortisol to cortisol ratio as a less invasive cytochrome P450 3A phenotyping method.

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 6ß-hydroxycortisol to cortisol concentration (6ß-OHF/F) ratio as a minimally invasive CYP3A phenotyping method. METHODS: Plasma 6ß-OHF and cortisol concentrations were measured via liquid chromatography/tandem mass spectrometry. The plasma 6ß-OHF/F ratio was compared with 6ß-hydroxylation clearance of endogenous cortisol (CLm(6ß); which we previously developed as an index of CYP3A activity) before, during and after oral contraceptive administration in 3 healthy women. The plasma 6ß-OHF/F ratio was observed during oral clarithromycin administration. The plasma 6ß-OHF/F ratio was also measured in 39 healthy participants. RESULTS: The plasma 6ß-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 CLm(6ß) values (43, 54 and 59%, respectively). Plasma 6ß-OHF/F ratio and CLm(6ß) exhibited a good correlation (r = .9053). The 6ß-OHF/F ratio decreased from 0.00921 to 0.00577 only 3 h following clarithromycin administration. The plasma 6ß-OHF/F ratio ranged 0.00565-0.01556 in 39 healthy participants. CONCLUSION: Based on its close relationship with CLm(6ß) and its decrease upon inhibition by clarithromycin, the plasma 6ß-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.

A dilute-and-shoot liquid chromatography-tandem mass spectrometry method for urinary 18-hydroxycortisol quantification and its application in establishing reference intervals.

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 × 103 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.

Origin of circulating 18-oxocortisol in the normal human adrenal.

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 3ß-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.

Development and validation of HPLC-DAD and LC-(ESI)/MS methods for the determination of sulfasalazine, mesalazine and hydrocortisone 21-acetate in tablets and rectal suppositories: In vitro and ex vivo permeability studies.

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 (LODSLF = 12 ng/mL, LODHA = 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 C18 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%, LODSLF = 1.28 ng/mL, LODHA = 1.07 ng/mL, LODMSL = 3.16 ng/mL). Based on the results, important conclusions were drawn concerning the role of excipients and SLF metabolism.

Successful management of feline pemphigus foliaceus with pentoxifylline and topical hydrocortisone aceponate.

The treatment regimen for feline pemphigus foliaceus (PF), an autoimmune disease caused by auto-antibodies against proteins of the desmosome junction, usually includes high doses of oral or parenteral immunosuppressive drugs, typically glucocorticoids. This case adds to a growing body of evidence that topical hydrocortisone aceponate is effective for the treatment of feline PF, and demonstrates the practical use of a non-invasive diagnostic method for histopathology when owners refuse a biopsy to support a clinical diagnosis of PF. Finally, this case highlights an international trend of owner-initiated treatment of feline infectious peritonitis (FIP) using unlicensed, unregistered drugs.

Limited Role of Hair Cortisol and Cortisone Measurement for Detecting Cortisol Autonomy in Patients With Adrenal Incidentalomas.

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 20ß-dihydrocortisol concentrations between healthy controls and AI patients, while 6ß-hydroxycortisol and 11-deoxycortisol were undetectable. Moreover, no significant difference was found in hair cortisol, cortisone, and 20ß-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).

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) based assay for the simultaneous quantification of 18-hydroxycorticosterone, 18-hydroxycortisol and 18-oxocortisol in human plasma.

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 (r2 = 0.999) and 20 to 3000 pg/ml for 18-OHB (r2 = 0.997) and 18-OHF (r2 = 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.

Dried blood spots analysis of 6ß-hydroxycortisol and cortisol using liquid chromatography/tandem mass spectrometry for calculating 6ß-hydroxycortisol to cortisol ratio.

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 6ß-hydroxycortisol (6ß-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 6ß-OHF/cortisol. The lower limits of quantification obtained using four DBS were 1.08 pg/50 µl for 6ß-OHF and 1.01 pg/50 µl for cortisol. The 6ß-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 6ß-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 6ß-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 6ß-OHF/cortisol ratio in DBS decreased by approximately 50% on administering clarithromycin compared with that before the administration of clarithromycin. The 6ß-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 6ß-OHF and cortisol in DBS. It also allows the use of DBS instead of plasma for calculating the 6ß-OHF/cortisol ratio. The 6ß-OHF/cortisol ratio could be an index of CYP3A activity in clinical setting.

Effective treatment of atopic dermatitis in small children significantly improves the quality of life of patients and their families.

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.

Kinetics of hydrocortisone sodium phosphate penetration into the human aqueous humor after topical application.

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.

Studies on CYP3A activity during the menstrual cycle as measured by urinary 6ß-hydroxycortisol/cortisol.

The 6ß-OH-cortisol/cortisol ratio (6ß-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 6ß-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. 6ß-OHC/C were analyzed in urine and sex hormones, TMAO and miRNA-27b were analyzed in serum at the same time points. 6ß-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 6ß-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. 6ß-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 6ß-OHC/C in urine and levels of TMAO in serum suggest that gut microbiota may affect CYP3A activity.

Antioxidant effects of piperine on steroid-induced hepatotoxicity.

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.

Metabolite Biomarkers of CKD Progression in Children.

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 m2; 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 m2, 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 m2, 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.

ACTH Stimulation Maximizes the Accuracy of Peripheral Steroid Profiling in Primary Aldosteronism Subtyping.

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.

Lichen Planus Pigmentosus Associated with Topical Olive Oil Application.

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).

Efficacy and safety of Soshiho-tang in atopic dermatitis patients with gastrointestinal disorders: A double-blinded, randomized, and placebo-controlled clinical trial.

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.