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The metabolism can be explored via 13C labeling of biological active substances and subsequent quantification of 13C enrichment in the exhaled carbon dioxide in breath. The resulting tracer enrichment values can be determined by Fourier-transform Infrared Spectroscopy (FTIR), since different CO2 isotopologues result in distinct absorption lines in the spectrum.The corresponding determination poses two challenges: first, FTIR absorbance can contain a nonlinear relationship between analyte amount and spectral signal and second, the spectral peaks for the different isotopologues overlap. The overlap precludes a separate calibration to asses the isotopologue concentration values and with it a determination of enrichments from concentration values. We propose here, first, a data reduction step like Principal Component Analysis (PCA) to convert the spectral information into one score pertaining to the 13CO2 enrichment. In a second step, a calibration function between score and enrichment values was established. The enrichment score can be derived by normalizing a subset of the spectrum by some measure for the 12CO2 sample content. Alternatively, the overlapping spectra were decomposed into two isotopologue spectra and the intensity of the separated spectra was used to form an enrichment score. For spectral separation, either Multivariate Curve Resolution Alternating Least Squares (MCR-ALS) was used or a novel decomposition strategy developed for this paper called Rotation and Angle-Bending Bayesian induced Transformation - Multivariate Curve Resolution (RABBIT – MCR) that operates in a Principal Component Analysis (PCA) subspace and is derived from MCR. We compared 13C enrichment estimates from FTIR CO2 spectra using different normalization variants with the two spectral separation models. In conclusion, the two spectral separation variants performed nearly equal, but better than any normalization variant.

Resume L’administration liberale d’oxygene etait initialement consideree comme l’un des piliers de la prise en charge des patients admis en reanimation dans un contexte d’etat de choc, dans l’objectif de corriger l’inadequation entre besoins et apports en oxygene. Depuis, cette approche est largement controversee, de nombreuses etudes ayant demontre un impact negatif de l’hyperoxie. En effet, la toxicite de l’hyperoxie a ete prouvee dans plusieurs situations de reanimation, avec une surmortalite dans les contextes de reanimation post-arret cardiaque, d’accident vasculaire cerebral ischemique et de traumatisme crânien severe. Plus recemment, une augmentation de la mortalite a ete mise en evidence chez les patients en etat de choc exposes a l’hyperoxie. A ce jour, en dehors d’indications particulieres (intoxications au CO, embolies gazeuses, accident de decompression), l’administration d’oxygene avec une strategie restrictive et non plus liberale (objectif prescrit de ne pas exceder une saturation pulsee en oxygene superieure a 95 %) semble a privilegier. Cette revue a pour objectif, par une approche physiopathologique, de synthetiser les donnees disponibles de la litterature concernant l’effet de l’hyperoxie chez le patient de reanimation dans diverses situations, et de faire apprecier au clinicien l’impact potentiel d’une administration non raisonnee d’oxygene.

Objectives Glucocorticoids (GCs) are one of the most widely prescribed anti-inflammatory drugs. By acting through their cognate receptor, the glucocorticoid receptor (GR), GCs downregulate the expression of pro-inflammatory genes and upregulate the expression of anti-inflammatory genes. Metabolic pathways have recently been identified as key parts of both the inflammatory activation and anti-inflammatory polarization of macrophages, immune cells responsible for acute inflammation and tissue repair. It is currently unknown whether GCs control macrophage metabolism, and if so, to what extent metabolic regulation by GCs confers anti-inflammatory activity. Methods Using transcriptomic and metabolomic profiling of macrophages, we identified GC-controlled pathways involved in metabolism, especially in mitochondrial function. Results Metabolic analyses revealed that GCs repress glycolysis in inflammatory myeloid cells and promote tricarboxylic acid (TCA) cycle flux, promoting succinate metabolism and preventing intracellular accumulation of succinate. Inhibition of ATP synthase attenuated GC-induced transcriptional changes, likely through stalling of TCA cycle anaplerosis. We further identified a glycolytic regulatory transcription factor, HIF1α, as regulated by GCs, and as a key regulator of GC responsiveness during inflammatory challenge. Conclusions Our findings link metabolism to gene regulation by GCs in macrophages., Highlights • Glucocorticoids regulate a network of genes associated with cellular metabolism in macrophages. • Glucocorticoids affect the macrophage metabolome, converging on reactive oxygen species and tricarboxylic acid. • Glucocorticoids mediate Tricarboxylic acid cycle anaplerosis at the glutamine-α-ketoglutarate pathway and this is required for anti-inflammatory gene regulation by glucocorticoids. • Glucocorticoids prevent Lipopolysaccharide-induced succinate accumulation, and HIF1α stabilization.

Childhood maltreatment (CM) is associated with an increased risk for the development of psychiatric and somatic diseases in later life. Individual risk and resilience factors may, however, influence how deep psychological stress gets under the skin. We hypothesized that the stress-related hormone cortisol and the attachment-related hormone oxytocin constitute biological factors that might moderate the biological sequelae and long-term health outcomes associated with CM. As biological outcome, we thereby focused on immunocellular oxygen consumption, which we previously found to be increased with a higher severity of CM experiences. In a study cohort of N = 49 postpartum women, we investigated the interaction between CM experiences, serum cortisol and plasma oxytocin levels, and the cellular oxygen consumption of intact peripheral blood mononuclear cells (PBMC) by high-resolution respirometry. Regression analyses revealed a significant interaction between the severity of CM experiences and cortisol as well as oxytocin on cellular oxygen consumption of PBMC three months postpartum: higher cortisol levels were thereby associated with an increase in oxygen consumption related to basal mitochondrial respiration and ATP turnover, while oxygen consumption related to basal mitochondrial respiration and ATP turnover were reduced with higher oxytocin levels in individuals with higher CM severity. These associations were not seen among women with no or low CM experiences. Together, the results suggest that cortisol and oxytocin might be associated with opposite effects on CM-related alterations in the bioenergetic profile of peripheral immune cells.

Polytrauma (PT) is frequently associated with hemorrhagic shock (HS), which increases morbidity and mortality. Although various aspects of HS have been addressed in PT patients, the impact of an additional HS is largely unknown regarding the development of multiple organ dysfunction associated with disturbed glycocalyx and barrier function early after trauma. A prospective, longitudinal, mono-centered, observational study enrolling severely injured patients (Injury Severity Score, ISS=38.1±2.6) served for an in-depth analysis of blood (drawn on days 0, 1, 2, 3 and 5) and clinical data (up to 21days) of 30 patients who were then stratified into PT with and without HS. HS significantly enhanced signs of acute organ injury, assessed by increased serum concentrations of novel damage markers. Moreover, indicators of glycocalyx and tight-junction dysfunction were found in PT patients all of which were significantly enhanced in co-presence of HS. These markers revealed multiple significant correlations with specific barrier, fluid-balance, coagulation, inflammation, and clinical-outcome parameters. Strikingly, mucosa fragments, which affected clotting, could be detected in serum after PT/HS. The results point to HS as a main driver for glycocalyx and barrier breakdown and suggest novel tools for the monitoring of organ dysfunction in the early course after PT.

During routine Fourier-Transform Infrared Spectroscopy (FTIR) based quantification of carbon dioxide in breath, it is necessary to account for a non-linear signal response to the analyte concentration and disturbance factors arising from the gas background matrix. These factors as well as day-to-day fluctuation should be corrected via calibration. We present a novel strategy to combine the information of previous calibrations with a minimal number of actual calibration measurements to obtain a precise calibration. After decomposition of the FTIR spectra via principal component analysis (PCA) into scores (corresponding to intensity) and loadings (corresponding to spectral curves), an empirical response surface fit equation between scores, analyte concentration and disturbance factors is established. The fit equation can be characterized via the coefficients determined by calibration. Out of a pool of coefficients gained from several calibrations, a multivariate inter-day distribution is generated. By requiring the coefficient set of the actual calibration to be a sample of the multivariate inter-day distribution, the number of necessary routine calibration samples is reduced to two. The corresponding coefficients are determined using the Lagrange Multipliers approach and the inter-day variability of coefficients is estimated using Bayesian statistics and Hierarchical models. The best calibration parameters in terms of calibration equation, wavelength region, preprocessing options and choice of routine calibration samples were determined; optimized for minimal number of calibration samples.

Summary Background There is insufficient research into the use of mechanical ventilation with increased inspiratory oxygen concentration (FiO 2 ) and fluid resuscitation with hypertonic saline solution in patients with septic shock. We tested whether these interventions are associated with reduced mortality. Methods This two-by-two factorial, multicentre, randomised, clinical trial (HYPERS2S) recruited patients aged 18 years and older with septic shock who were on mechanical ventilation from 22 centres in France. Patients were randomly assigned 1:1:1:1 to four groups by a computer generated randomisation list stratified by site and presence or absence of acute respiratory distress syndrome by use of permuted blocks of random sizes. Patients received, in an open-labelled manner, mechanical ventilation either with FiO 2 at 1·0 (hyperoxia) or FiO 2 set to target an arterial haemoglobin oxygen saturation of 88–95% (normoxia) during the first 24 h; patients also received, in a double-blind manner, either 280 mL boluses of 3·0% (hypertonic) saline or 0·9% (isotonic) saline for fluid resuscitation during the first 72 h. The primary endpoint was mortality at day 28 after randomisation in the intention-to-treat population. This study was registered with ClinicalTrials.gov, number NCT01722422. Findings Between Nov 3, 2012, and June 13, 2014, 442 patients were recruited and assigned to a treatment group (normoxia [n=223] or hyperoxia [n=219]; isotonic [n=224] or hypertonic [n=218]). The trial was stopped prematurely for safety reasons. 28 day mortality was recorded for 434 patients; 93 (43%) of 217 patients had died in the hyperoxia group versus 77 (35%) of 217 patients in the normoxia group (hazard ratio [HR] 1·27, 95% CI 0·94–1·72; p=0·12). 89 (42%) of 214 patients had died in the hypertonic group versus 81 (37%) of 220 patients in the isotonic group (HR 1·19, 0·88–1·61; p=0·25). We found a significant difference in the overall incidence of serious adverse events between the hyperoxia (185 [85%]) and normoxia groups (165 [76%]; p=0·02), with a clinically relevant doubling in the hyperoxia group of the number of patients with intensive care unit-acquired weakness (24 [11%] vs 13 [6%]; p=0·06) and atelectasis (26 [12%] vs 13 [6%]; p=0·04) compared with the normoxia group. We found no statistical difference for serious adverse events between the two saline groups (p=0·23). Interpretation In patients with septic shock, setting FiO 2 to 1·0 to induce arterial hyperoxia might increase the risk of mortality. Hypertonic (3%) saline did not improve survival. Funding The French Ministry of Health.

The calibration of analytical systems is time-consuming and the effort for daily calibration routines should therefore be minimized, while maintaining the analytical accuracy and precision. The ‘calibration transfer’ approach proposes to combine calibration data already recorded with actual calibrations measurements. However, this strategy was developed for the multivariate, linear analysis of spectroscopic data, and thus, cannot be applied to sensors with a single response channel and/or a non-linear relationship between signal and desired analytical concentration. To fill this gap for a non-linear calibration equation, we assume that the coefficients for the equation, collected over several calibration runs, are normally distributed. Considering that coefficients of an actual calibration are a sample of this distribution, only a few standards are needed for a complete calibration data set. The resulting calibration transfer approach is demonstrated for a fluorescence oxygen sensor and implemented as a hierarchical Bayesian model, combined with a Lagrange Multipliers technique and Monte-Carlo Markov-Chain sampling. The latter provides realistic estimates for coefficients and prediction together with accurate error bounds by simulating known measurement errors and system fluctuations. Performance criteria for validation and optimal selection of a reduced set of calibration samples were developed and lead to a setup which maintains the analytical performance of a full calibration. Strategies for a rapid determination of problems occurring in a daily calibration routine, are proposed, thereby opening the possibility of correcting the problem just in time.

Reactive oxygen species (ROS) play a crucial role in both physiological and pathological process such as intracellular signaling and host defense. However, elevated ROS levels lead to protein oxidation, lipid peroxidation, and DNA damage. Inflammation and tissue injury are associated with increased ROS production. The role of hydrogen peroxide (H2O2), which is an important representative of ROS within these physiological processes, is still not fully understood. Here, we present microelectrodes modified with electrocatalytically active platinum-black (Pt-black) or Prussian blue (PB) films for the detection of extracellular H2O2. Detection limits in the nanomolar range with limits of detection (LOD) of 201 ± 10 nM for PB and of 214 ± 2 nM for Pt-black, respectively, could be achieved, thereby enabling the detection/quantification of extracellular H2O2 levels at porcine peripheral blood mononuclear cells (PBMCs) and at granulocytes from swine undergoing hemorrhagic shock and resuscitation or undergoing hemorrhagic shock subsequent to an acute subdural hematoma and resuscitation. Increased levels of H2O2 could be observed due to increased oxidative stress, especially during the acute trauma phase. The simultaneous detection of catecholamines such as norepinephrine administered during the recovery phase using a dual microelectrode assembly and carbon fiber electrodes was also investigated.

Ambroxol (Ax) is a frequently prescribed drug used to facilitate mucociliary clearance, but its mode of action is yet poorly understood. Here we show by X-ray spectroscopy that Ax accumulates in lamellar bodies (LBs), the surfactant storing, secretory lysosomes of type II pneumocytes. Using lyso- and acidotropic substances in combination with fluorescence imaging we confirm that these vesicles belong to the class of acidic Ca2+ stores. Ax lead to a significant neutralization of LB pH, followed by intracellular Ca2+ release, and to a dose-dependent surfactant exocytosis. Ax-induced Ca2+ release was significantly reduced and slowed down by pretreatment of the cells with bafilomycin A1 (Baf A1), an inhibitor of the vesicular H+ ATPase. These results could be nearly reproduced with NH3/NH4+. The findings suggest that Ax accumulates within LBs and severely affects their H+ and Ca2+ homeostasis. This is further supported by an Ax-induced change of nanostructural assembly of surfactant layers. We conclude that Ax profoundly affects LBs presumably by disordering lipid bilayers and by acting as a weak base. The pH change triggers – at least in part – Ca2+ release from stores and secretion of surfactant from type II cells. This novel mechanism of Ax as a lysosomal secretagogue may also play a role for its recently discussed use for lysosomal storage and other degenerative diseases.

Controversial data are available on hydrogen sulfide (H2S) during hemorrhage and resuscitation, depending on timing, dosing, mode of application, and the H2S donor used. Sodium thiosulfate (Na2S2O3) is a recognized drug devoid of major side effects, which attenuated murine acute lung injury and cerebral ischemia/reperfusion injury. Therefore, we tested the hypothesis whether Na2S2O3 would mitigate organ dysfunction in porcine hemorrhage-and-resuscitation. We studied animals with pre-existing coronary artery disease because of the reduced coronary arterial expression of the H2S producing enzyme cystathionine-γ-lyase (CSE) in this prospective, randomized, controlled, blinded experimental study. 20 anesthetized and instrumented pigs underwent 3 h of hemorrhage (removal of 30 % of the blood volume and subsequent titration of mean arterial pressure to 40 mmHg). Resuscitation (72 h) comprised re-transfusion of shed blood, crystalloids, and continuous i.v. norepinephrine. Animals randomly received vehicle or Na2S2O3 (0.1 g·kg-1 h-1) for 24 h. Before, at the end of and every 24 h after shock, hemodynamics, metabolism, blood gases, lung, heart, kidney, and liver function and injury were evaluated together with cytokines and parameters of oxidative and nitrosative stress. Immediate post mortem lung, kidney, heart, and liver specimen were analyzed for marker proteins of inflammation and oxidative and nitrosative stress and mitochondrial respiratory activity in the heart, kidney, and liver. Immuno-histochemical analysis comprised lung extra-vascular albumin accumulation, nitrotyrosine formation, and CSE and glucocorticoid receptor (GCR) expression. Na2S2O3 significantly attenuated shock-induced impairment of lung mechanics and gas exchange (plateau and positive end-expiratory pressure at 72 h p = 0.0006/p = 0.0264; Horovitz index at 48 h p = 0.0261), which coincided with a higher tissue GCR expression (p = 0.0415). During resuscitation from hemorrhagic shock Na2S2O3 attenuated shock-induced acute lung injury in co-morbid swine, most likely due to a GCR expression related mechanism.

Our aim was to study the ability of an immortalized cell line (AMJ2-C11) to sustain aerobic cell respiration at decreasing oxygen concentrations under continuous sulfide exposure. We assumed that the rate of elimination of sulfide through the pathway linked to the mitochondrial respiratory chain and therefore operating under aerobic conditions, should decrease with limiting oxygen concentrations. Thus, sulfide’s inhibition of cellular respiration would occur faster under continuous sulfide exposure when the oxygen concentration is in the very low range. The experiments were performed with an O2K-oxygraph (Oroboros Instruments) by suspending 0.5–1 × 106 cells in 2 ml of continuously stirred respiration medium at 37 °C and calculating the oxygen flux (JO2) as the negative derivative of the oxygen concentration in the medium. The cells were studied in two different metabolic states, namely under normal physiologic respiration (1) and after uncoupling of mitochondrial respiration (2). Oxygen concentration was controlled by means of a titration-injection pump, resulting in average concentration values of 0.73 ± 0.05 μM, 3.1 ± 0.2 μM, and 6.2 ± 0.2 μM. Simultaneously we injected a 2 mM Na2S solution at a continuous rate of 10 μl/s in order to quantify the titration-time required to reduce the JO2 to 50% of the initial respiratory activity. Under the lowest oxygen concentration this effect was achieved after 3.5 [0.3;3.5] and 11.7 [6.2;21.2] min in the uncoupled and coupled state, respectively. This time was statistically significantly shorter when compared to the intermediate and the highest O2 concentrations tested, which yielded values of 24.6 [15.5;28.1] min (coupled) and 35.9 [27.4;59.2] min (uncoupled), as well as 42.4 [27.5;42.4] min (coupled) and 51.5 [46.4;51.7] min (uncoupled). All data are medians [25%, and 75% percentiles]. Our results confirm that the onset of inhibition of cell respiration by sulfide occurs earlier under a continuous exposure when approaching the anoxic condition. This property may contribute to the physiological role of sulfide as an oxygen sensor.

Numerous papers have been published on the role of H2S during circulatory shock. Consequently, knowledge about vascular sulfide concentrations may assume major importance, in particular in the context of "acute on chronic disease", i.e., during circulatory shock in animals with pre-existing chronic disease. This review addresses the questions (i) of the "real" sulfide levels during circulatory shock, and (ii) to which extent injury and pre-existing co-morbidity may affect the expression of H2S producing enzymes under these conditions. In the literature there is a huge range on sulfide blood levels during circulatory shock, in part as a result of the different analytical methods used, but also due to the variable of the models and species studied. Clearly, some of the very high levels reported should be questioned in the context of the well-known H2S toxicity. As long as "real" sulfide levels during circulatory shock are unknown and/or undetectable "on line" due to the lack of appropriate techniques, it appears to be premature to correlate the measured blood levels of hydrogen sulfide with the severity of shock or the H2S therapy-related biological outcomes. The available data on the tissue expression of the H2S-releasing enzymes during circulatory shock suggest that a "constitutive" CSE expression may play a crucial role of for the maintenance of organ function, at least in the kidney. The data also indicate that increased CBS and CSE expression, in particular in the lung and the liver, represents an adaptive response to stress states.

Hypothermia in near-drowning victims is a serious problem that impacts clinical decision-making. The purpose of this trial was to determine the reliability of tympanic temperature measurements compared to oral temperature measurements after immersion in water. After ethical approval was obtained, we studied oral and tympanic temperature in 25 volunteer swimmers (aged 18-49 years). Sublingual (Fixotherm; Tradesell Europe, Eglharting, Germany) and tympanic (First Temp Genius; Sherwood Medical, Sulzbach, Germany) temperature measurements were performed before entering the water, after 45 min of immersion in water, and 15 min after leaving the water. During the immersion phase, the ears were temporarily immersed. A control group (the same 25 volunteers) had to swim for the same amount of time without ever immersing their heads in the water. The trial was performed in an indoor swimming pool at 28 degrees C water and 30 degrees C air temperature. The oral temperature did not change over time in either group. The tympanic temperature was significantly lower after immersion compared to baseline in the "immersed" group (33.7 degrees C vs. 37.5 degrees C, p0.001), increased significantly in the recovery period, but remained significantly lower than baseline (36.0 degrees C vs. 37.5 degrees C, p0.001). At baseline, the oral temperature was lower compared to the tympanic temperature. This relationship reversed after immersion and remained reversed until the end of the trial in the immersion group. The control group maintained oral temperatures lower than tympanic throughout the study; furthermore, the control group had no clinically relevant change in oral or tympanic temperature over the time (tympanic temperature: 37.4 degrees C vs. 37.2 degrees C, p = 0.06). Our data suggest that in water-related accidents such as near drowning, the values of body (core) temperature obtained via use of infrared ear thermometry are unreliable, and should not be used for clinical decision-making.

This article summarizes the effects of catecholamines and vasopressin on the cardiovascular system, focusing on their metabolic and immunologic properties. Particular attention is dedicated to the septic shock condition.

In critical care medicine, catecholamines are most widely used to reverse circulatory dysfunction and thus to restore tissue perfusion. However, catecholamines not only influence systemic and regional hemodynamics, but also exert a variety of significant metabolic, endocrine, and immunologic effects. Arginine vasopressin is a vasomodulatory hormone with potency to restore vascular tone in vasodilatory hypotension. Although the evidence supporting the use of low doses of vasopressin or its analogs in vasodilatory shock is increasing, lack of data regarding mortality and morbidity prevent their implementation in critical care protocols.

Hyperbaric oxygen (HBO) treatment is applied as a therapy for a wide variety of diseases with symptoms caused by lack of oxygen in the target tissues. However, it is known that exposure to high concentrations of oxygen may lead to oxidative stress and cause cell and tissue damage. Oxygen toxicity and possible cancer-promoting effects of HBO therapy have been a matter of serious concern. Although a cancer-inducing effect of HBO was not found to date, recent studies clearly indicated an induction of oxidative DNA damage in blood cells of healthy subjects after HBO under therapeutic conditions. The biological significance of this finding has been investigated in a series of in vitro and in vivo tests. This review summarizes these studies and critically discusses potential adverse genetic effects of HBO therapy. Furthermore, since an induction of anti-oxidative defense mechanisms has been determined after HBO exposure, a modified treatment regimen of HBO therapy is proposed which avoids genotoxic effects.

We compared the effects of weaning using synchronized intermittent mandatory ventilation (SIMV) with the use of biphasic positive airway pressure (BIPAP) on the stress response, oxygen uptake (V˙O2) and work of breathing (WOB) in 10 patients after aortocoronary bypass surgery. All three ventilatory settings were investigated in each patient, for example, volume-controlled mechanical ventilation immediately before weaning was followed, in randomized order, by both SIMV and BIPAP. In addition to routine monitoring of continuous and respiratory state, we measured V˙O2, WOB, and pressure–time product (PTP) as well as the plasma concentrations of epinephrine, norepinephrine, ACTH, cortisol, vasopressin, and prolactin. Although respiratory rate (f), WOB and PTP were greater with both SIMV and BIPAP when compared with control, other variables did not change with the ventilatory mode. In conclusion, weaning from mechanical ventilation using partial support modes does not affect the postoperative stress response in patients who have had uncomplicated cardiac surgery.

Disturbed endogenous H2S production as a result down-regulation of the H2S-catalyzing enzymes cysthathionine-γ-lyase (CSE) and cysthathione-β-synthase (CBS) is associated with chronic cardiovascular pathology, e.g. hypertension, atherosclerosis, and chronic kidney disease [1] , [2] , [3] . CSE and CBS up-regulation was found during acute hyperinflammatory states resulting from circulatory shock [4] , [5] , [6] , [7] , [8] , [9] , [10] , [11] , but little is known on the effect of acute stress states on CSE and CBS expression during chronic disease. Therefore we measured CBS and CSE expression before and after porcine kidney ischemia/reperfusion (I/R) injury comparing otherwise healthy animals and swine with ubiquitous atherosclerosis resulting from a mutation of the LDL receptor together with a cholesterol-enriched diet [12] , [13] . CBS expression was not present in native biopsies, and minimal only post I/R, i.e. in necrotic tubules and cells only. Atherosclerotic swine had lower CSE expression in native biopsies. I/R injury caused a down-regulation of the CSE enzyme in both groups, which was more pronounced in the atherosclerotic animals. In the atherosclerotic swine fecal peritonitis-induced septic shock also caused marked down-regulation of kidney CSE expression when compared to sham-operated animals. Therefore, the effect of H2S-supplementation using the slow-releasing H2S donor GYY4137 [14] in atherosclerotic swine with septic shock will be shown. GYY4137 is used to avoid the short and high and therefore potentially deleterious peak sulfide concentrations associated with NaSH or Na2S administration. Equivocal data are available on H2S-catalyzing enzymes during cigarette smoke-induced lung disease: both reduced [15] , [16] and increased [16] , [17] CSE expression were reported. So far, no data are available whether chronic lung disease affects the otherwise pronounced increase in CSE and CBS expression after trauma [18] or sepsis [4] , [5] , [6] , [7] , [10] , [11] . Therefore, the effects of genetic CSE deletion and H2S-supplementation using the slow-releasing H2S donor GYY4137 in mice with cigarette smoke-induced COPD undergoing pressure wave-generated blunt chest trauma [18] will be shown.

H2S production in the kidney, both before and after ischemic injury, and its regulation via endogenous synthesizing enzymes: cystathionine β synthase (CBS), γ lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MST) remains controversial. The conflicting results were all reported in non-resuscitated young and healthy rodent models that have come into question with regard to their applicability to humans, let alone the co-morbidity frequently present in ICU patients. To understand the endogenous H2S synthesis in disease states, we compared kidneys from swine strains with and without pre-existing cardiovascular co-morbidity. CSE, CBS and MST expression was quantified by immunohistochemistry (densitometric image analysis, presented as mean sum of densitometric units x105) of formalin-fixed paraffin sections from both pre-injury and post-ischemia/reperfusion (I/R) kidney biopsies taken in young and healthy pigs (German Landrace, n = 8 ) as well as swine strain with familial hypercholesterolemia (FBM, n = 8) (11.1 (7.4;12.3) vs. 1.4 (1.3;1.5) mmol/L, p

Background Even in higher species sulfide is quickly metabolised and thus degraded by an ancient metabolic pathway linked to the mitochondrial respiratory chain [1] . Since this metabolic process is strictly aerobic, sulfide concentration is expected to increase at low oxygen, provided that the production rate remains constant. This mechanism has been suggested as an explanation of how sulfide acts as an oxygen sensor within cells [2] . However, one may argue that the efficiency of sulfide as an O 2 -sensor requires that the capacity of the cells to metabolise this molecule already decreases at oxygen concentrations still not affecting the aerobic cell metabolism. In fact, only in that way sulfide concentration would start rising, thus signaling the imminent hypoxia, before low oxygen per se becomes a seriously limiting factor for the cell. Therefore, in a series of preliminary experiments we quantified the sulfide metabolism in a stable cell line derived from alveolar macrophages (AMJ2-C11), which previously proved to efficiently metabolise sulfide under aerobic conditions [3] , at O 2 partial pressures approaching hypoxia from 0.8 kPa down to 0.4 and 0.1 kPa. Methods All Measurements were conducted using an O2K ® -Oxygraph together with a TIP-2K ® titration pump (Oroboros Instruments, Austria). For each O 2 -partial pressure we performed 6–8 separate experiments. Before performing the experiments we recorded several trasitions to anoxia in order to quantify the un-inhibited respiratory capacity close to anoxia under the O 2 -partial pressures of 0.1, 0.4, and 0.8 kPa. The inhibition of mitochondrial respiration was quantified in terms of the total amount of sulfide required to reduce the routine oxygen flux (JO2) to 50% by means of a continuous sulfide injection at a rate of 10 nM/s. A second titration pump was used to simultaneously maintain the oxygen concentration at the predefined level adopting a previously described technique [4] . Results Fig. 1 shows representative records of 3 anoxic transition experiments in AMJ2-C11 cells; the data suggest that the JO2 only moderately declines up to a O 2 -partial pressure of 0.1 kPa. In contrast, the total amount of sulfide required for achieving a 50%-inhibition of mitochondrial respiration was about 10% at 0.1 kPa when compared to that needed at 0.8 kPa (0.2 ± 0.03 μmol at 0.1 kPa vs. 1.3 ± 0.4 μmol at 0.4 kPa, and 2.2 ± 0.1 μmol at 0.8 kPa). This result suggests a much less efficient degradation of sulfide at the lower oxygen level. Fig.2 plots the injected amount of sulfide against the JO 2 at all 3 partial pressures. Conclusions Our preliminary data indicate that the capacity to metabolise sulfide is already affected at oxygen concentrations still not severily limiting cell respiration. This result further emphasize the potential role of sulfide as an oxygen sensor in the cell.

The reported sulfide absorption values and baseline blood concentrations are still controversial [1] . We modified an existing gas chromatographic/mass spectrometric method of routine sulfide quantification using a bis-pentafluorobenzyl derivative [2] . This approach allows sensitive determination of sulfide in small sample volumes under mild chemical conditions. In addition, in in vitro experiments, it allows to distinguish between endogenous and exogenous sulfide by administration of the stable isotope 34 S 2− . A standard protocol utilises 100 μl of blood, but can be scaled down to 25 μl for rodent samples. In brief, a mixture, consisting of 400 μl internal standard (5 μg/ml tribromobenzene in isooctane), 200 μl of alkylation reagent (10 μ l/ml pentafluorobenzylbromide in isooctane) and 400 μl of phase transfer catalyst (2 mg/ml tetradecyldimethylbenzylammonium chloride in sodium tetraborate saturated water) was prepared. After addition of 100 μl sample, the mixture was vigorously shaken for 1 min, 400 μl of saturated potassium dihydrogenphosphate solution were added and the cup was shaken for another 10 s. 2 μl of the isooctane phase were injected into an Agilent 5890/5970 gas chromatography/mass spectrometry instrument, housing a 12 m Macherey–Nagel Optima-5MS capillary column. In sim mode ions m/z 313.7 for the internal standard and 394.0 for the sulfide derivative (or 396.0 for 34 S 2− ) were recorded. The method was used to determine basal sulfide blood concentrations in pigs and to measure the sulfide release from the sulfide releasing drug GYY4137 over 24 h in cell culture medium. In addition this method reproducibly was used to determine the absorption of a spike of 100 μM 34 S 2− at pH 7.4 in buffer, blood, plasma, cysteine, glutathione and albumine. Baseline blood sulfide concentrations in pigs were between 0.2 and 2.1 μM. Sulfide concentrations of a 300 μ M solution of GYY4137 in cell culture medium (in cell culture flask, gas exchange enabled) were 1.02 μM (10 min after GYY addition), 1.32 μM (2 h), 1.37 μM (4 h), 1.60 μM (8 h), 1.58 μM (12 h) and 1.81 μM (24 h). The sulfide concentration of the corresponding medium without GYY4137 ranged between 0.25 and 0.50 μM. An initial 100 μM spike of 34 S 2− in blood decreased to 23 μM after 1 min, 12 μM after 10 and 1 μM after 60 min. The corresponding values for plasma were 60, 41 and 3 μM, for phosphate buffer (pH 7.4) 103, 96 and 85 μM. In a 50 mg/ml solution of albumine the 34 S 2− concentration decreased to 22, 15, 3 μM, in 5 mg/ml oxidized glutathione to 32, 18, 3 μM, in 5 mg/ml reduced glutathione to 79, 76, 75 μM, in 120 μg/ml cystine to 91, 64, 12 μM and in 1 mg/ml cysteine to 110, 102, 99 μM. Our findings show, that sulfide baseline concentrations in blood are in the low micromolar range. Added sulfide is rapidly absorbed by blood, plasma and disulfide bridge containing peptides and proteins, but much lower by the reduced species. Adding the sulfide releasing drug GYY4137, increased the sulfide concentration of cell culture media for 24 h.

There are no data available on the kinetics of blood concentrations of xenon during the wash-in phase of an inhalation anaesthesia aiming at 1 MAC end-expiratory concentration. Therefore, we anaesthetized eight pigs with continuous propofol and fentanyl and measured arterial, mixed venous and end-expiratory xenon concentrations by gas chromatography-mass spectrometry 1, 2, 3, 4, 5, 7, 10, 15, 20, 30, 60 and 120 min after starting the anaesthetic gas mixture [67% xenon/33% oxygen; 3 litre x min(-1) during the first 10 min, thereafter minimal flow with 0.48 (SD 0.03) litre x min(-1)]. End-expiratory xenon concentrations plateaued (defined as