Dried Blood Spot Multiplexed Steroid Profiling Using Liquid Chromatography Tandem Mass Spectrometry in Korean Neonates.

BACKGROUND: Screening for congenital adrenal hyperplasia (CAH) using immunoassays for 17α-hydroxyprogesterone generates many false-positive results. We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for simultaneous quantification of nine steroid hormones in dried blood spot (DBS) samples, and established reference intervals for these hormones. METHODS: We examined our method for linearity, precision, accuracy, extraction recovery, and matrix effects and determined the reference intervals of cortisol, 17α-hydroxyproges-terone, 11-deoxycortisol, 21-deoxycortisol, androstenedione, corticosterone, 11-deoxycorticosterone, testosterone, and progesterone in 1,146 DBS samples (from 272 preterm and 874 full-term neonates). Immunoassay and LC-MS/MS methods were compared for 17α-hydroxyprogesterone. Fourteen additional samples were tested to validate the clinical applicability of the LC-MS/MS method. RESULTS: The linearity range was 2.8-828.0 nmol/L for cortisol and 0.9-40.0 nmol/L for the other steroids (R²>0.99). Intra-day and inter-day precision CVs were 2.52-12.26% and 3.53-17.12%, respectively. Accuracy was 80.81-99.94%, and extraction recovery and matrix effects were 88.0-125.4% and 61.7-74.2%, respectively. There was a negative bias, with higher values measured by immunoassay compared with LC-MS/MS (r=0.8104, P<0.0001). The LC-MS/MS method was successfully applied to the analysis of nine steroids in DBS for screening and diagnosis of CAH using the 14 additional samples. CONCLUSIONS: Our method enables highly sensitive and specific assessment of nine steroids from DBS and is a promising tool for clinical analysis of CAH.

Incorporation of antibiotics and systemic steroids by sinus surgeons: is there widespread consensus?

BACKGROUND: The objective of this work was to evaluate factors associated with antibiotic and oral corticosteroid (OCS) prescription among otolaryngologists regularly performing sinus surgery. METHODS: Fellowship-trained rhinologists, including fellowship directors, were identified via the American Rhinologic Society (ARS) website. Non-fellowship-trained otolaryngologists performing ≥25 balloons (frontal/maxillary) or ≥25 functional endoscopic sinus surgeries (FESSs) (frontal/maxillary/ethmoids) were also included in "balloon surgeons" and "sinus surgeon" cohorts, respectively. Prescribing data for Medicare Part D beneficiaries was obtained for 2015. RESULTS: Otolaryngologists included in this analysis wrote a median of 54 scripts for antibiotics, with a 15.1% antibiotic prescription rate. The overall script length per antibiotic was 11.1 days. Of fellowship-trained rhinologists, 90.2% wrote fewer than 100 scripts, compared to 25.6% and 32.5% of sinus surgeons and balloon surgeons, respectively. Fellowship-trained rhinologists wrote lengthier antibiotic scripts (14.1 vs 10.3 days, p < 0.05). Clinicians who have been in practice longer prescribed antibiotics significantly more frequently. Fellowship-trained rhinologists had a greater OCS rate (8.9%) than balloon and sinus surgeons (7.1%), also writing lengthier courses (15.0 vs 8.1 days). Early-career otolaryngologists wrote lengthier steroid prescriptions than those with 11 to 20 years and >20 years in practice. CONCLUSION: Antibiotic and OCS utilization varies by type of training, as non-fellowship-trained sinus surgeons and balloon surgeons tend to utilize antibiotics more aggressively, and fellowship-trained rhinologists utilize OCS more frequently. Otolaryngologists with more years in practice are more likely to incorporate antibiotics in the management of sinus disorders, although these conclusions must be considered in the context of this resource's limitations. Further clarification of guidelines may be helpful for minimizing divergent practices and maintaining a consensus.

High-volume steroid isotopic standards developed as working standards for gas chromatography-combustion-isotope ratio mass spectrometry.

High-precision carbon isotope ratio analysis of urinary steroids by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is the official test to detect illicit doping of synthetic versions of endogenous steroids, such as testosterone. Our group created the first steroid isotopic standards (SIS) specifically for World Anti-Doping Agency (WADA) accredited laboratories. The standards contain mixtures of steroids as acetates or free steroids at ~400 µg each per ampoule and have been widely distributed to anti-doping laboratories to facilitate comparability of inter-laboratory results. Here we report on the creation and characterization of 3 new high-volume single component SIS suitable for use as working standards. They contain ~50 times more steroid mass per ampoule than previous SIS. The new SIS, coded CU/PCC 40-1, CU/PCC 41-1, & CU/PCC 42-1, contain ~20 mg of androsterone, androsterone-AC, and 5α-cholestane, with determined isotopic values of -27.09 ± 0.07 mUr, -32.82 ± 0.01 mUr, -25.03 ± 0.01 mUr, respectively. We used our previously developed protocol to calibrate the isotopically uniform steroids against the isotopic standard gases methane and ethane in NIST RM 8559 that are traceable to the international standard Vienna PeeDee Belemnite (VPDB). Two sets of data, acquired 7 months apart, of absolute δ13 CVPDB and ∆Δδ13 CVPDB values from 8 randomly selected ampoules of all 3 SIS indicate uniformity of steroid isotopic composition within measurement reproducibility, SD(δ13 C) < 0.2 mUr Our results show that protocols for SIS extend to creation of high volume working standards that can also be used as internal standards under appropriate GC conditions.

Fever as an initial manifestation of spondyloarthritis: A retrospective study.

OBJECTIVES: We aimed to evaluate a wide spectrum of clinical features of adult patients with spondyloarthritis (SpA) whose initial manifestation was fever, using the Assessment of SpondyloArthritis international Society (ASAS) classification criteria. METHODS: We retrospectively collected the electronic medical records of hospitalized SpA patients who initially presented to the Severance Hospital (Seoul, Korea) with fever from January 2010 to May 2016. As a control group, we also recruited one-hundred consecutive patients who were diagnosed with SpA in our outpatient clinic. Clinical features and laboratory findings were compared in two patient groups. RESULTS: There were 26 patients who had fever as initial presentation of SpA (reactive arthritis 50%, undifferentiated SpA 26.9%, ankylosing spondylitis 15.4%, enteropathic arthritis 3.8%, psoriatic arthritis 3.8%). Peripheral SpA was more common in febrile SpA patients than in control SpA patients (65.4% vs 24.0%, p<0.001). Febrile SpA patients were less frequently HLA-B27 positive than control SpA patients (52.2% vs 77.0%, p<0.05). At baseline, systemic inflammatory markers were significantly higher in the febrile SpA patients (white blood cell count, 11.57 vs 7.81 cells/µL, p<0.001; erythrocyte sedimentation rate, 69.2 vs 41.0 mm/h, p<0.001; C-reactive protein, 109.6 vs 15.3 mg/L, p<0.001). The proportion of patients treated with systemic steroids was significantly higher in febrile SpA patients (57.7% vs. 11.0%, p<0.001). The proportion of patients who visited rheumatology specialty was significantly lower in febrile SpA patients than in control SpA patients (7.7% vs 59.0%, p<0.001). CONCLUSION: Various subgroups of SpA can be presented with fever as an initial manifestation. Febrile SpA patients demonstrated higher systemic inflammation and a lower chance to visit rheumatology in early stage. When evaluating febrile patients with any clinical features of SpA, clinicians are advised to consider performing SpA-focused evaluation including HLA-B27 or a simple sacroiliac joint radiograph.

Gas chromatography tandem mass spectrometry offers advantages for urinary steroids analysis.

Gas chromatography mass spectrometry has been the lynchpin of clinical assessment of steroid profiles for ∼3 decades. The improvements in assay performance offered by tandem mass spectrometry were assessed. Across the spectrum of glucocorticoid and androgen analytes tested, limits of detection and quantitation were ∼20 fold lower with triple than single quadrupole systems, but the more noticeable improvement was that signal to noise was substantially improved and the linear range wider. These benefits allowed more reliable and concomitant measurement of steroids with substantially different abundances and in smaller volumes of urine.

[Diagnosis and treatment of sarcoidosis. Current standards]. / Diagnostik und Therapie der Sarkoidose. Was ist gesichert?

Sarcoidosis is a granulomatous disease that mainly affects the lungs and intrathoracic lymph nodes; however, virtually any organ can be affected. As an orphan disease, recommendations are mainly based on observational or small randomized studies as well as experts' opinion. Diagnosing sarcoidosis requires proof of non-necrotizing granulomas in patients with a compatible symptomatic pattern and the exclusion of other granulomatous diseases. Granulomas can be detected best in the lungs or intrathoracic lymph nodes. Therefore, bronchoscopy and endobronchial ultrasound with biopsies of lymph nodes are the major tools to diagnose sarcoidosis. Frequently, close follow-up and symptomatic therapy are sufficient to allow for spontaneous resolution. In case of functional organ impairment, cardial or CNS involvement, or other complications, steroid therapy is necessary with a starting dose of 0.5 mg/kg body weight that should be tapered-off over 6-12 months. Steroid-refractory disease can be treated by adding methotrexate or azathioprine, two drugs long known in sarcoidosis treatment. Monoclonal antibodies against TNF and lung transplantation are further therapeutic options.

Dried blood spot testing for seven steroids using liquid chromatography-tandem mass spectrometry with reference interval determination in the Korean population.

BACKGROUND: Conventional screening for congenital adrenal hyperplasia (CAH) using immunoassays generates a large number of false-positive results. A more specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been introduced to minimize unnecessary follow-ups. However, because of limited data on its use in the Korean population, LC-MS/MS has not yet been incorporated into newborn screening programs in this region. The present study aims to develop and validate an LC-MS/MS method for the simultaneous determination of seven steroids in dried blood spots (DBS) for CAH screening, and to define age-specific reference intervals in the Korean population. METHODS: We developed and validated an LC-MS/MS method to determine the reference intervals of cortisol, 17-hydroxyprogesterone, 11-deoxycortisol, 21-deoxycortisol, androstenedione, corticosterone, and 11-deoxycorticosterone simultaneously in 453 DBS samples. The samples were from Korean subjects stratified by age group (78 full-term neonates, 76 premature neonates, 89 children, and 100 adults). RESULTS: The accuracy, precision, matrix effects, and extraction recovery were satisfactory for all the steroids at three concentrations; values of intra- and inter-day precision coefficients of variance, bias, and recovery were 0.7-7.7%, -1.5-9.8%, and 49.3-97.5%, respectively. The linearity range was 1-100 ng/mL for cortisol and 0.5-50 ng/mL for other steroids (R²>0.99). The reference intervals were in agreement with the previous reports. CONCLUSIONS: This LC-MS/MS method and the reference intervals validated in the Korean population can be successfully applied to analyze seven steroids in DBS for the diagnosis of CAH.

Setting a standard for the initiation of steroid therapy in refractory or severe Mycoplasma pneumoniae pneumonia in adolescents and adults.

Serum interleukin (IL)-18 level was thought to be a useful as a predictor of refractory or severe Mycoplasma pneumoniae pneumonia, and steroid administration is reported to be effective in this situation. The serum levels of IL-18 correlated significantly with those of lactate dehydrogenase (LDH). The purpose of this study was to set a standard for the initiation of steroid therapy in M. pneumoniae pneumonia using a simple serum marker. We analyzed 41 adolescent and adult patients with refractory or severe M. pneumoniae pneumonia who received steroid therapy, and compared them with 108 patients with M. pneumoniae pneumonia who responded to treatment promptly (control group). Serum LDH levels were significantly higher in the refractory and severe group than in the control group at the initiation of steroid therapy (723 vs 210 IU/L, respectively; p < 0.0001). From receiver operating characteristic curve analysis, we calculated serum LDH cut-off levels of 364 IU/L at initiation of steroid therapy and 302 IU/L at 1-3 days before the initiation of steroid therapy. The administration of steroids to patients in the refractory and severe group resulted in the rapid improvement of symptoms and a decrease in serum LDH levels in all patients. Serum LDH level can be used as a useful parameter to determine the initiation of steroid therapy in refractory or severe M. pneumoniae pneumonia. A serum LDH level of 302-364 IU/L seems to be an appropriate criterion for the initiation of steroid therapy.

High performance enzyme-linked immunosorbent assay for determination of miroestrol, a potent phytoestrogen from Pueraria candollei.

Pueraria candollei associated preparation is widely applied in folk Thai medicine for rejuvenating purpose in aged people, which correlated with its pharmacological activities reported by pre-clinical and clinical trials. Therefore, standardized products of this plant are needed by consumers and health care personnel. Miroestrol, a potent and stable phytoestrogen in P. candollei, exhibited potential to be biomarker for quality control of P. candollei samples in research or industrial levels. Indirect competitive enzyme-linked immunosorbant assay (ELISA) for miroestrol determination was developed and validated by using polyclonal antibody from rabbit immunization. The polyclonal antibody recognized specifically to miroestrol, which exhibited cross-reactivity to deoxymiroestrol and isomiroestrol with 6.68% and 1.05%, respectively. The linearity range of measurement was 0.73-3000 ng mL(-1), which coefficient of variation (CV) of both intra- and inter-plate determination was less than 5%. With spiked samples of known amount miroestrol, the percentages of recovery were 98.80-104.37% and 98.31-106.69% in P. candollei and its involved product samples, respectively. Validated ELISA was comparable with published HPLC method (R(2)=0.9996) (Yusakul et al.) in samples with various miroestrol contents. For application, the P. candollei involved preparations contained miroestrol 0.695±0.037-12.108±0.285 µg g(-1) dry wt. The developed ELISA was high performance for miroestrol determination, which could be applied for P. candollei quality control in research fields and industrial productions.

Decreasing unnecessary utilization in acute bronchiolitis care: results from the value in inpatient pediatrics network.

BACKGROUND: Acute viral bronchiolitis is the most common diagnosis resulting in hospital admission in pediatrics. Utilization of non-evidence-based therapies and testing remains common despite a large volume of evidence to guide quality improvement efforts. OBJECTIVE: Our objective was to reduce utilization of unnecessary therapies in the inpatient care of bronchiolitis across a diverse network of clinical sites. METHODS: We formed a voluntary quality improvement collaborative of pediatric hospitalists for the purpose of benchmarking the use of bronchodilators, steroids, chest radiography, chest physiotherapy, and viral testing in bronchiolitis using hospital administrative data. We shared resources within the network, including protocols, scores, order sets, and key bibliographies, and established group norms for decreasing utilization. RESULTS: Aggregate data on 11,568 hospitalizations for bronchiolitis from 17 centers was analyzed for this report. The network was organized in 2008. By 2010, we saw a 46% reduction in overall volume of bronchodilators used, a 3.4 dose per patient absolute decrease in utilization (95% confidence interval [CI] 1.4-5.8). Overall exposure to any dose of bronchodilator decreased by 12 percentage points as well (95% CI 5%-25%). There was also a statistically significant decline in chest physiotherapy usage, but not for steroids, chest radiography, or viral testing. CONCLUSIONS: Benchmarking within a voluntary pediatric hospitalist collaborative facilitated decreased utilization of bronchodilators and chest physiotherapy in bronchiolitis.

Development of criteria for the detection of adrenosterone administration by gas chromatography-mass spectrometry and gas chromatography-combustion-isotope ratio mass spectrometry for doping control.

Adrenosterone (androst-4-ene-3,11,17-trione, 11-oxoandrostenedione) is an endogenous steroid hormone that has been promoted as a dietary supplement capable of reducing body fat and increasing muscle mass. It is proposed that adrenosterone may function as an inhibitor of the 11beta-hydroxysteroid dehydrogenase type 1 enzyme (11beta-HSD1), which is primarily responsible for reactivation of cortisol from cortisone. The urinary metabolism of adrenosterone was investigated, after a single oral administration in two male subjects, by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Substantially increased excretion of 11beta-hydroxyandrosterone, 11beta-hydroxyetiocholanolone, 11-oxoandrosterone and 11-oxoetiocholanolone was observed. Minor metabolites such as 3alpha,17beta-dihydroxy-5beta-androstan-11-one, 3alpha-hydroxyandrost-4-ene-11,17-dione and 3alpha,11beta-dihydroxyandrost-4-en-17-one were also identified. The exogenous origin of the most abundant adrenosterone metabolites was confirmed by GC-C-IRMS according to World Anti-Doping Agency criteria. Through analysis of a reference population data set obtained from urine samples provided by elite athlete volunteers (n = 85), GC-MS doping control screening criteria are proposed: 11beta-hydroxyandrosterone concentration greater than 10 000 ng/mL (specific gravity adjusted to 1.020) or 11beta-hydroxyandrosterone/11beta-hydroxyetiocholanolone ratio greater than 20.Urine samples fulfilling these screening criteria may be subjected to GC-C-IRMS analysis for confirmation of adrenosterone administration.

Reference intervals of urinary steroid metabolites using gas chromatography-mass spectrometry in Chinese adults.

BACKGROUND: Urinary steroid profiling by GC or GC-MS are established clinical tools to complement other biochemical tests in the diagnosis and investigation of a wide range of adrenocortical disorders, but normative data on adults using the more specific GC-MS are lacking. Our objective was to set up the reference intervals of commonly detected urinary steroid metabolites as well as marker metabolites seen in disease states. METHOD: Apparently healthy adult Chinese males and females were recruited by completing health questionnaires. A 24-h urine specimen was collected from all the participants for urinary steroid profiling by GC-MS in cyclic scan mode. The analyzer was calibrated by using authentic steroid standards. Statistical methods recommended by the National Committee for Clinical Laboratory Standards were followed for setting up the reference intervals of various steroid metabolites. After outliers were excluded, the data were tested for the necessity to partition into sex-, menopausal status- and age-specific reference intervals. RESULTS: 83 males and 89 females were recruited for the study. Necessity to partition into sex-specific reference intervals was demonstrated for almost all steroid metabolites. Menopausal status and age also had a significant impact on steroid metabolite excretion, making separate reference intervals necessary. CONCLUSIONS: We have set up the normative data on the levels of urinary steroid metabolite excretion in Chinese adults for future reference in patient management and research in steroid metabolism.

Complete 1H and 13C NMR assignments of four new steroidal glycosides from a gorgonian coral Junceella juncea.

Four new cholest-type steroidal glycosides, junceellosides A-D, isolated from the EtOH/CH(2)Cl(2) extracts of the South China Sea gorgonian coral Junceella juncea, were identified. Complete assignments of the (1)H and (13)C NMR chemical shifts for these compounds were achieved by means of one- and two-dimensional NMR techniques, including (1)H-(1)H COSY, HSQC, HMBC and NOESY spectra.

Analysis of over-the-counter dietary supplements.

OBJECTIVE: To determine if steroids containing over-the-counter (OTC) dietary supplements conform to the labeling requirements of the 1994 Dietary Supplement Health and Education Act (DSHEA). DESIGN: 12 brands of OTC supplements containing 8 different steroids were randomly selected for purchase in stores that cater to athletes. There are two androstenediones (4- and 5-androstene-3,17-dione), two androstenediols (4- and 5-androstene-3beta, 17beta-diol), and 4 more are 19-nor cogeners (19-nor-4- and 5-androstene-3,17-dione and 19-nor-4- and 5-androstene-3beta, 17beta-diol). MAIN OUTCOME MEASURES: 12 brands of OTC anabolic-androgenic supplements were analyzed by high-pressure liquid chromatography. RESULTS: We found that 11 of 12 brands tested did not meet the labeling requirements set out in the 1994 Dietary Supplement Health and Education Act. One brand contained 10 mg of testosterone, a controlled steroid, another contained 77% more than the label stated, and 11 of 12 contained less than the amount stated on the label. CONCLUSIONS: These mislabeling problems show that the labels of the dietary steroid supplements studied herein cannot be trusted for content and purity information. In addition, many sport organizations prohibit OTC steroids; thus, athletes who use them are at risk for positive urine test results. In this article we provide the details of the analyses, a summary of the steroids by name and structure, and information on the nature of the positive test results. Athletes and their physicians need this information because of the potential medical consequences and positive urine test results.

Quantitation of combinatorial libraries of small organic molecules by normal-phase HPLC with evaporative light-scattering detection.

The advantages of evaporative light-scattering detection over UV detection for the quantitation of combinatorial libraries composed of small organic compounds by HPLC are described. The detector's response is independent of the sample chromophore, which makes it well-suited to chromatographic analyses of mixtures of dissimilar solutes. Thus, HPLC with evaporative light-scattering detection offers to potential for reducing false positive or false negative results in screening assays, because of its ability to detect the presence of impurities that absorb poorly in the UV (e.g., those impurities originating from the polymeric support). Furthermore, the evaporative light-scattering detector exhibits a nearly equivalent response to compounds of similar structural class. Hence, rapid quantitation of compound libraries may be carried out with the use of a single external standard. For example, the quantitation errors, based on a single external standard, for a series of steroids, hydantoins, and BOC- and Fmoc-protected amino acids by normal-phase HPLC with evaporative light-scattering detection average approximately +/-10%. The application of the evaporative light-scattering detector to the quantitation of low-level sample impurities and the detector's compatibility with gradient elution are also described.

Therapy for feline dermatoses.

This article compiles information on various therapies used in feline dermatology. Information on the following therapeutic agents and devices is discussed: antibiotics, antifungals, antileprosy drugs, antiparasiticides, antivirals, antihistamines, behavior modification drugs, fatty acids, progestogens, steroids, immunomodulating drugs, chemotherapeutic/immunosuppressive agents, retinoids, mechanical devices, hyposensitization, immunotherapy, food elimination trials, hypoallergenic diets, and miscellaneous topical agents such as polyhydroxydine solution, tar, and benzocaine-containing creams.

Aldosterone metabolism in rat renal tissue in vitro. Formation of lipid soluble metabolites.

In the present study the formation of lipid soluble metabolites from 3H-aldosterone was investigated in vitro in isolated kidneys and kidney and liver slices of Sprague Dawley rats. The steroids were separated by HPLC (forward and reversed phase systems) and detected on-line as UV- or 3H-chromatograms. Apart from an unenzymatically formed substance, isoaldosterone, three less polar metabolites were traced (A1, A2, A3). The structure of the quantitatively most important metabolite (A1), was identified as 5 alpha-dihydroaldosterone using a combination of techniques such as chromatographic comparison with reference steroids, antibody binding and mass spectrometry. Evidence for further conversion of DHaldo to 3 alpha, 5 alpha-tetrahydroaldosterone was obtained in chromatographic and antibody binding studies. The formation of metabolites was not dependent on glomerular filtration. Furthermore it displayed regional heterogeneity with highest activity in the outer medulla. Finally it was observed that the in vitro metabolism of aldosterone was not saturable over a range of initial aldo concentration of 10(-9) to 10(-5) M.