Determination of serum cortisol by isotope-dilution liquid-chromatography electrospray ionization tandem mass spectrometry with on-line extraction

A liquid chromatographic-mass spectrometric method for the determination of cortisol in serum using atmospheric pressure electrospray ionization and tandem mass spectrometry is described. During sample preparation, 150 mul of serum were deproteinized with methanol/zinc sulfate followed by on-line solid phase extraction employing column switching. Tri-deuterated cortisol was used as the internal standard. The following transitions were monitored: cortisol, 363 > 309 m/z; d3-cortisol, 366 > 312 m/z. The total run-time was 5 minutes. The method proved linear (0-500 mug/l; r=0.999), precise (total coefficient of variation between 5.0% and 3.2% at a mean cortisol concentration of 15.1 mug/l and 269 mug/l, respectively; n=16) and specific with regard to relevant endogenous and exogenous steroids

Free serum cortisol: quantification applying equilibrium dialysis or ultrafiltration and an automated immunoassay system

Background: Quantification of bioactive, free serum cortisol concentrations can characterize adrenocortical function more appropriately compared to total serum cortisol measurement. Ultrafiltration or equilibrium dialysis of serum samples allow direct measurement of free serum cortisol concentrations but respective methods have poorly been validated so far. The aim of our study was to investigate the analytical performance of free serum cortisol measurement employing equilibrium dialysis and ultrafiltration. Methods: Two commercially available ultrafiltration devices and self-assembled dialysis cells, respectively, were studied. Cortisol was quantified in filtrate or dialysate using an automated immunoassay system. Using two serum pools, the inter-assay coefficient of variation was determined for the three methods and a method comparison was performed. Results: Inter-assay coefficients of variation (n=10) between 3.2% and 14.8% were observed in the imprecision study. Method comparison demonstrated close agreement between free serum cortisol results obtained by ultrafiltration and equilibrium dialysis, respectively (equilibrium dialysis = 1.2x ultrafiltration+3.9 nmol/L; r=0.99; n=35). Conclusions: Direct quantification of free serum cortisol after equilibrium dialysis or ultrafiltration of the samples offers acceptable reproducibility and results in close agreement can be obtained. Both methods can potentially be introduced into a routine laboratory setting

Down-regulation of MicroRNA-31 in CD4(+) T Cells Contributes to Immunosuppression in Human Sepsis by Promoting T(H)2 Skewing

Background: Immunosuppression has been recognized as a major cause of sepsis-related mortality. Currently, there is much interest in identifying central hubs controlling septic immunoparalysis. In this context, in this study, the authors investigate the role of microRNA-31 (miR-31) as a regulator of T cell functions. Methods: Primary human T cells were separated from healthy volunteers (n = 16) and from sepsis patients by magnetic beads (n = 23). Expression of mRNA/microRNA (miRNA) was determined by real-time polymerase chain reaction. Gene silencing was performed by small interfering RNA transfection, and miRNA-binding sites were validated by reporter gene assays. Effects of miR-31 or anti-miR-31 transfection were analyzed by real-time polymerase chain reaction, Western blotting, and flow cytometry. Results: Overexpression of miR-31 in stimulated CD4(+) T cells promoted a proinflammatory phenotype with increased levels of interferon- (1.63 0.43;P = 0.001;means +/- SD) and reduced expression of interleukin (IL)-2 (0.66 +/- 0.19;P = 0.005) and IL-4 (0.80 +/- 0.2;P = 0.0001). In contrast, transfection of anti-miR-31 directed cells toward a T(H)2 phenotype. Effects on IL-2 and IL-4 were mediated by targeting of nuclear factor-kappa B-inducing kinase and factor-inhibiting hypoxia-inducible factor-1. Interferon-, however, was influenced via control of signaling lymphocytic activation molecule (SLAM)-associated protein, an essential adaptor molecule of immunomodulatory SLAM receptor signaling, which was identified as a novel target gene of miR-31. In sepsis patients, an epigenetically driven down-regulation of miR-31 was found (0.44 +/- 0.25;P = 0.0001), associated with increased nuclear factor-kappa B-inducing kinase, factor-inhibiting hypoxia-inducible factor-1, SLAM-associated protein expression, and a cytokine shift toward T(H)2. Conclusions: In this study, the authors provide novel evidence of miR-31 as an emerging key posttranscriptional regulator of sepsis-associated immunosuppression. The study results contribute to a further understanding of septic immunoparalysis and provide new perspectives on miRNA-based diagnostic approaches

Survival after Cardiac Arrest and Changing Task Profile of the Cardiac Arrest Team in a Tertiary Care Center

Background. The characteristics of in-hospital emergency response systems, survival rates, and variables associated with survival after in-hospital cardiac arrest vary significantly among medical centers worldwide. Aiming to optimize in-hospital emergency response, we performed an analysis of survival after in-hospital cardiopulmonary resuscitation and the task profile of our cardiac arrest team. Methods. In-hospital emergencies handled by the cardiac arrest team in the years 2004 to 2006 were analyzed retrospectively, and patient and event characteristics were tested for their associations with survival after cardiopulmonary resuscitation. The results were compared to a similar prior analysis for the years 1995 to 1997. Results. After cardiopulmonary resuscitation, the survival rate to discharge was 30.2% for the years 2004 to 2006 compared to 25.1% for the years 1995 to 1997 (difference not statistically significant). Survival after one year was 18.5 %. An increasing percentage of emergency calls not corresponding to medical emergencies other than cardiac arrest was observed. Conclusions. The observed survival rates are considerably high to published data. We suggest that for further improvement of in-hospital emergency response systems regular training of all hospital staff members in immediate life support is essential. Furthermore, future training of cardiac arrest team members must include basic emergency response to a variety of medical conditions besides cardiac arrest

Do higher alarm thresholds for arterial blood pressure lead to less perioperative hypotension? A retrospective, observational cohort study

Arterial blood pressure is one of the vital signs monitored mandatory in anaesthetised patients. Even short episodes of intraoperative hypotension are associated with increased risk for postoperative organ dysfunction such as acute kidney injury and myocardial injury. Since there is little evidence whether higher alarm thresholds in patient monitors can help prevent intraoperative hypotension, we analysed the blood pressure data before (group 1) and after (group 2) the implementation of altered hypotension alarm settings. The study was conducted as a retrospective observational cohort study in a large surgical centre with 32 operating theatres. Alarm thresholds for hypotension alarm for mean arterial pressure (MAP) were altered from 60 (before) to 65 mmHg for invasive measurement and 70 mmHg for noninvasive measurement. Blood pressure data from electronic anaesthesia records of 4222 patients (1982 and 2240 in group 1 and 2, respectively) with 406,623 blood pressure values undergoing noncardiac surgery were included. We analysed (A) the proportion of blood pressure measurements below the threshold among all measurements by quasi-binomial regression and (B) whether at least one blood pressure measurement below the threshold occurred by logistic regression. Hypotension was defined as MAP < 65 mmHg. There was no significant difference in overall proportions of hypotensive episodes for mean arterial pressure before and after the adjustment of alarm settings (mean proportion of values below 65 mmHg were 6.05% in group 1 and 5.99% in group 2). The risk of ever experiencing a hypotensive episode during anaesthesia was significantly lower in group 2 with an odds ratio of 0.84 (p = 0.029). In conclusion, higher alarm thresholds do not generally lead to less hypotensive episodes perioperatively. There was a slight but significant reduction of the occurrence of intraoperative hypotension in the presence of higher thresholds for blood pressure alarms. However, this reduction only seems to be present in patients with very few hypotensive episodes

Decreased Linezolid Serum Concentrations in Three Critically Ill Patients: Clinical Case Studies of a Potential Drug Interaction between Linezolid and Rifampicin

Linezolid is a valuable treatment option for treating infections caused by multi-resistant gram-positive pathogens. Lack of effective linezolid levels due to the co-administration of rifampicin has been described in healthy subjects. However, the clinical significance of this potential drug interaction (DI) for critically ill patients is still unclear. This was a retrospective analysis of 3 critically ill patients with the combination therapy of linezolid and rifampicin or rifampicin pre-treatment. Despite increasing the dose of linezolid, the majority of observed linezolid trough concentrations in all 3 patients were below 2 mg/l. Furthermore, linezolid trough concentrations remained below 2 mg/l after discontinuation of rifampicin. This potential DI between linezolid and rifampicin could lead to treatment failure. Therefore, we strongly recommend that linezolid serum concentrations be monitored in patients with rifampicin co-administration or rifampicin pretreatment. (C) 2016 S. Karger AG, Base

Cerebrospinal fluid penetration of meropenem in neurocritical care patients with proven or suspected ventriculitis: a prospective observational study

Background: Ventriculitis is a complication of temporary intraventricular drains. The limited penetration of meropenem into the cerebrospinal fluid (CSF) is well known. However, ventricular CSF pharmacokinetic data in patients with ventriculitis are lacking. The aim of this study was to evaluate meropenem pharmacokinetics in the serum and CSF of neurocritical care patients with proven or suspected ventriculitis. Methods: We conducted an observational pharmacokinetic study of neurocritical care patients with proven or suspected ventriculitis receiving meropenem. Multiple blood and CSF samples were taken and were described using nonparametric pharmacokinetic modelling with Pmetrics. Results: In total, 21 patients (median age 52 years, median weight 76 kg) were included. The median (range) of peak and trough concentrations in serum were 20.16 (4.40-69.00) mg/L and 2.54 (0.00-31.40) mg/L, respectively. The corresponding peak and trough concentrations in CSF were 1.20 (0.00-6.20) mg/L and 1.28 (0.00-4.10) mg/L, respectively, with a median CSF/serum ratio (range) of 0.09 (0.03-0.16). Median creatinine clearance ranged from 60. 7 to 217.6 ml/minute (median 122.5 ml/minute). A three-compartment linear population pharmacokinetic model was most appropriate. No covariate relationships could be supported for any of the model parameters. Meropenem demonstrated poor penetration into CSF, with a median CSF/serum ratio of 9 % and high interindividual pharmacokinetic variability. Conclusions: Administration of higher-than-standard doses of meropenem and therapeutic drug monitoring in both serum and CSF should be considered to individualise meropenem dosing in neurocritical care patients with ventriculitis