Tobias Fink

Ion mobility spectrometry in breath research.

The number of publications in the field of breath analysis using different types of ion mobility spectrometers (IMS) has increased over the last few years. In this paper, the publications between 2010 and 2013 are reviewed with respect to different types of IMS such as differential mobility spectrometers, high-field asymmetric waveform ion mobility spectrometers and multi-capillary columns coupled to conventional IMS. The analytes detected by IMS and declared with significance to a specific medical question were considered further with respect to medical and analytical questions. In total, 42 different analytes were found to be detected using IMS on a high significance level and were compared to findings using other analytical methods with respect to the individual analyte.

Melatonin receptors mediate improvements of survival in a model of polymicrobial sepsis.

Objectives:Melatonin has been demonstrated to improve survival after experimental sepsis via antioxidant effects. Yet, recent evidence suggests that this protective capacity may also rely on melatonin receptor activation. Therefore, the present study was designed to investigate whether selective melatonin receptor-agonist ramelteon may influence survival and immune response in a model of polymicrobial sepsis in rats, wild-type and melatonin receptor MT1/MT2 double knockout mice. Design:Prospective, randomized, controlled study. Setting:University research laboratory. Subjects:Male Sprague-Dawley rats (200–250 g) and male C3H/HeN wild-type and MT1/MT2 receptor knockout mice (20–22 g). Interventions:Animals underwent cecal ligation and incision and remained anesthetized for evaluation of survival for 12 hours (rats: n = 15 per group) or 15 hours (mice: n = 10 per group). Analysis of immune response by means of enzyme-linked immunosorbent assay was performed before and 5 hours after cecal ligation and incision (rats only; n = 5 per group). After induction of sepsis, animals were treated IV with vehicle, different doses of melatonin (rats: 0.01/0.1/1.0/10 mg/kg; mice: 1.0 mg/kg), ramelteon, melatonin receptor-antagonist luzindole, ramelteon + luzindole, or melatonin + luzindole (each 1.0 mg/kg). Sham controls underwent laparotomy but not cecal ligation and incision. Measurements and Main Results:Compared with vehicle, administration of ramelteon or melatonin significantly improved median survival time in rats (sepsis/melatonin [0.1 mg/kg], 554 min, [1.0 mg/kg] 570 min, [10 mg/kg] 579 min; sepsis/ramelteon, 468 min; each p < 0.001 vs sepsis/vehicle, 303 min) and wild-type mice (sepsis/melatonin, 781 min; sepsis/ramelteon, 701 min; both p < 0.001 vs sepsis/vehicle, 435 min). This effect was completely antagonized by coadministration of luzindole in all groups. Melatonin, ramelteon, or luzindole had no significant effect on survival time in knockout mice. Significantly elevated concentrations of tumor necrosis factor-&agr;, interleukin-6, and interleukin-10 were observed 5 hours after cecal ligation and incision in rats (p < 0.05 vs baseline and corresponding sham); neither ramelteon nor melatonin treatment significantly affected immune response. Conclusions:Melatonin receptors mediate improvements of survival after polymicrobial sepsis in rats and mice; this effect appears to be independent from major alterations of cytokine release.

Dobutamine pretreatment improves survival, liver function, and hepatic microcirculation after polymicrobial sepsis in rat.

ABSTRACT Dobutamine is recommended for the treatment of sepsis-related circulatory failure in international guidelines. Furthermore, dobutamine has been demonstrated to improve liver function and hepatic perfusion after experimental hemorrhagic shock. Yet, it is unknown whether dobutamine may also induce hepatoprotective effects in sepsis. This study was designed to investigate the effect of dobutamine on survival, hepatic function, and microcirculation after polymicrobial sepsis in rat. Under general anesthesia, male Sprague-Dawley rats (n = 25/group) underwent pretreatment with dobutamine (10 &mgr;g/kg per minute) in the presence or absence of &bgr;1-receptor antagonist esmolol (500 &mgr;g/kg per minute), esmolol alone, or vehicle for 6 h, before induction of sepsis (cecal ligation and incision [CLI]). Sham-operated animals were treated likewise but underwent no CLI. Five hours after CLI, either liver function was assessed by plasma disappearance rate of indocyanine green (n = 5/group), or intravital microscopy was performed (n = 5/group) for evaluation of hepatic perfusion index and hepatic integrity (as propidium iodide–stained cells per field). Alternatively, survival time after induction of CLI was monitored under general anesthesia (n = 15/group). Compared with controls, dobutamine pretreatment significantly improved plasma disappearance rate of indocyanine green (13.8% ± 4.1% vs. 20.6% ± 4.6%; P = 0.029), hepatic perfusion index (275.0 ± 126.1 vs. 703.5 ± 177.4 pL/s per mm; P < 0.001), hepatocellular injury (22.2 ± 6.7 vs. 6.4 ± 3.1 cells per field; P < 0.001), and survival time (326 ± 20 vs. 603 ± 41 min; P < 0.001). Coadministration of esmolol abolished the protective effect of dobutamine completely. Our results indicate that pretreatment with dobutamine may improve survival, liver function, and hepatic microcirculation after polymicrobial sepsis in rat via &bgr;1-adrenoceptor activation. Dobutamine could therefore play a relevant role for hepatoprotection under septic conditions.

Minimal retarded Propofol signals in human breath using ion mobility spectrometry

Propofol is a short-acting, intravenously administered hypnotic agent. Since the introduction of propofol into clinical practice, it has become the intravenous induction agent of choice for anaesthesia providers (Fig. 1). The physicochemical properties of Propofol are given in Table 1. Currently, there are no simple, reliable and clinically useful measures available to quantify Propofol in humans at the bedside. For investigations in pharmacokinetics and dynamics Propofol is measured in serum samples, which are invasive, time-consuming and therefore results are not directly available. The level of Propofol in the exhaled air can potentially be used as an indicator of patients’ hypnotic depth and would provide the possibility to measure the Propofol concentration on-line. Generally, several methods are under investigation to monitor the concentration of Propofol in exhaled breath. For example, mass spectrometry with emphasis on proton transfer reaction mass spectrometry, ion-molecule reaction mass spectrometry or GC/MS have been described [1–7]. The detection of Propofol in exhaled air using ion mobility spectrometry coupled to multi-capillary (MCC) columns was shown most recently [8–11]. The MCC provides the possibility to work within humid environment. The reactant ion peak (RIP) is clearly separated from the peak of Propofol using MCC combined with an IMS. The identification uses the drift time of the ions formed from Propofol and the retention time within the MCC. The signal intensity can be related to the concentration range with relevance for clinical diagnostics[8] . Further details with respect to the advantages and disadvantages of MCC [12–17] and the IMS [18–25] are well known. With respect to the detection of Propofol, a retention time of about 10 min was realized keeping the MCC adjusted at 40 °C. Perl et al. [9] estimated, that at higher MCC temperatures, retention times in the range of 1 min could be achieved. Zhou et al. [26] used a membrane inlet system for on-line measurement of Propofol in an anesthetized mouse and showed direct Propofol concentration in a time series. In their mouse model, the time profile showed a gap of about 10 min before the Propofol signal was observable. A close relation to serum Propofol concentration in air was found using mass spectrometric methods [4, 7]. Recently, a strong correlation between the serum Propofol concentration and signal intensity of ion mobility spectrometry has also been shown [8, 9, 11]. The goal of the present pilot is to show that by modifying the MCC/IMS peak of Propofol values can be obtained online within 60 s. To demonstrate biologically meaningful variations the MCC/IMS was applied in a conventional operation room. The peak intensities delivered by BioScout could then directly be correlated to the BIS-values of brain activity, where Propofol is expected to have its major effects in vivo.

Volatile Organic Compounds during Inflammation and Sepsis in Rats: A Potential Breath Test Using Ion-mobility Spectrometry

Background:Multicapillary column ion-mobility spectrometry (MCC-IMS) may identify volatile components in exhaled gas. The authors therefore used MCC-IMS to evaluate exhaled gas in a rat model of sepsis, inflammation, and hemorrhagic shock. Methods:Male Sprague–Dawley rats were anesthetized and ventilated via tracheostomy for 10 h or until death. Sepsis was induced by cecal ligation and incision in 10 rats; a sham operation was performed in 10 others. In 10 other rats, endotoxemia was induced by intravenous administration of 10 mg/kg lipopolysaccharide. In a final 10 rats, hemorrhagic shock was induced to a mean arterial pressure of 35 ± 5 mmHg. Exhaled gas was analyzed with MCC-IMS, and volatile compounds were identified using the BS-MCC/IMS-analytes database (Version 1209; B&S Analytik, Dortmund, Germany). Results:All sham animals survived the observation period, whereas mean survival time was 7.9 h in the septic animals, 9.1 h in endotoxemic animals, and 2.5 h in hemorrhagic shock. Volatile compounds showed statistically significant differences in septic and endotoxemic rats compared with sham rats for 3-pentanone and acetone. Endotoxic rats differed significantly from sham for 1-propanol, butanal, acetophenone, 1,2-butandiol, and 2-hexanone. Statistically significant differences were observed between septic and endotoxemic rats for butanal, 3-pentanone, and 2-hexanone. 2-Hexanone differed from all other groups in the rats with shock. Conclusions:Breath analysis of expired organic compounds differed significantly in septic, inflammation, and sham rats. MCC-IMS of exhaled breath deserves additional study as a noninvasive approach for distinguishing sepsis from inflammation.

Multi-capillary column-ion mobility spectrometer (MCC-IMS) breath analysis in ventilated rats: a model with the feasibility of long-term measurements

Rats are commonly used in medical research as they enable a high grade of standardization. The exhalome of ventilated rats has not as yet been investigated using an ion mobility spectrometer coupled with a multi-capillary column (MCC-IMS). As a first step, a rat model has to be established to measure potential biomarkers in the exhale with long-term settings, allowing constant and continuous analysis of exhaled air in time series. Therefore, eight animals were anaesthetized, prepared and ventilated for 1 h. A total of 73 peaks were directly detected with the IMS chromatogram. Thirty five of them were assigned to the ventilator system and 38 to the animals. Peak intensity varied within three measurements. The intensity of analytes of individual rats varied by a factor of up to 18. This new model will also enable continuous measurements of volatile organic compounds (VOCs) from rats breath in long-term experiments. It is hoped that, in the future, variability and progression of VOCs can be monitored in different models of diseases using this set-up.

Two different approaches for pharmacokinetic modeling of exhaled drug concentrations

Online measurement of drug concentrations in patients breath is a promising approach for individualized dosage. A direct transfer from breath- to blood-concentrations is not possible. Measured exhaled concentrations are following the blood-concentration with a delay in non-steady-state situations. Therefore, it is necessary to integrate the breath-concentration into a pharmacological model. Two different approaches for pharmacokinetic modelling are presented. Usually a 3-compartment model is used for pharmacokinetic calculations of blood concentrations. This 3-compartment model is extended with a 2-compartment model based on the first compartment of the 3-compartment model and a new lung compartment. The second approach is to calculate a time delay of changes in the concentration of the first compartment to describe the lung-concentration. Exemplarily both approaches are used for modelling of exhaled propofol. Based on time series of exhaled propofol measurements using an ion-mobility-spectrometer every minute for 346 min a correlation of calculated plasma and the breath concentration was used for modelling to deliver R2 = 0.99 interdependencies. Including the time delay modelling approach the new compartment coefficient ke0lung was calculated to ke0lung = 0.27 min−1 with R2 = 0.96. The described models are not limited to propofol. They could be used for any kind of drugs, which are measurable in patients breath.

Inhibition of glycogen synthase kinase (GSK)-3-β improves liver microcirculation and hepatocellular function after hemorrhagic shock.

Ischemia and reperfusion may cause liver injury and are characterized by hepatic microperfusion failure and a decreased hepatocellular function. Inhibition of glycogen synthase kinase (GSK)-3β, a serine-threonine kinase that has recently emerged as a key regulator in the modulation of the inflammatory response after stress events, may be protective in conditions like sepsis, inflammation and shock. Therefore, aim of the study was to assess the role of GSK-3β in liver microcirculation and hepatocellular function after hemorrhagic shock and resuscitation (H/R). Anesthetized male Sprague-Dawley rats underwent pretreatment with Ringer´s solution, vehicle (DMSO) or TDZD-8 (1 mg/kg), a selective GSK-3β inhibitor, 30 min before induction of hemorrhagic shock (mean arterial pressure 35±5 mmHg for 90 min) and were resuscitated with shed blood and Ringer´s solution (2h). 5h after resuscitation hepatic microcirculation was assessed by intravital microscopy. Propidium iodide (PI) positive cells, liver enzymes and alpha-GST were measured as indicators of hepatic injury. Liver function was estimated by assessment of indocyanine green plasma disappearance rate. H/R led to a significant decrease in sinusoidal diameters and impairment of liver function compared to sham operation. Furthermore, the number of PI positive cells in the liver as well as serum activities of liver enzymes and alpha-GST increased significantly after H/R. Pretreatment with TDZD-8 prevented the changes in liver microcirculation, hepatocellular injury and liver function after H/R. A significant rise in the plasma level of IL-10 was observed. Thus, inhibition of GSK-3β before hemorrhagic shock modulates the inflammatory response and improves hepatic microcirculation and hepatocellular function.

Melatonin modifies cellular stress in the liver of septic mice by reducing reactive oxygen species and increasing the unfolded protein response

BACKGROUND & AIMS Melatonins hepatoprotective actions have numerously been demonstrated in the past but the underlying molecular mechanisms are widely unknown. For a better understanding of melatonins effects on hepatic stress response this study aimed to elucidate alterations in oxidative stress, unfolded protein response and acute phase response in septic mice. METHODS Male C3H/HeN mice underwent sham operation or cecal ligation and incision and remained anesthetized for 5h. Production of reactive oxygen species was determined by electron spin resonance spectroscopy. Protein and mRNA expression levels were determined by western blot analysis and quantitative real-time PCR, respectively. RESULTS Production of reactive oxygen species was strongly increased in the aorta and liver after 5h of polymicrobial sepsis which was entirely inhibited by treatment with melatonin. SOD-1 levels did not differ between the groups. Sepsis also induced the upregulation of VCAM-1 and ICAM-1 independent of melatonin treatment but probably regulated via ERK1/2 signaling. Melatonin triggered the transcriptional upregulation of PERK in septic animals which seems to be independent on ERK1/2 signaling and NR4A1 activation. Melatonin therapy also engendered an increased expression of CHOP, but apoptosis was not initiated. Furthermore, sepsis reduced the expression of the transcription factor CREBH which was entirely suppressed by melatonin. CONCLUSIONS This study gives new insight into the mechanisms by which melatonin might confer its hepatoprotective actions during polymicrobial sepsis. The results clearly show the melatonin-mediated amelioration of oxidative stress as well as alterations in the cellular stress mechanisms via the unfolded protein response and the acute phase response.

Exhalation of volatile organic compounds during hemorrhagic shock and reperfusion in rats: An exploratory trial

Ischemia and reperfusion alter metabolism. Multi-capillary column ion-mobility spectrometry (MCC-IMS) can identify volatile organic compounds (VOCs) in exhaled gas. We therefore used MCC-IMS to evaluate exhaled gas in a rat model of hemorrhagic shock with reperfusion. Adult male Sprague-Dawley rats (n  =  10 in control group, n  =  15 in intervention group) were anaesthetized and ventilated via tracheostomy for 14 h or until death. Hemorrhagic shock was maintained for 90 min by removing blood from the femoral artery to a target of MAP 35  ±  5 mmHg, and then retransfusing the blood over 60 min in 15 rats; 10 control rats were evaluated without shock and reperfusion. Exhaled gas was analyzed with MCC-IMS, VOCs were identified using the BS-MCC/IMS analytes database (Version 1209). VOC intensities were analyzed at the end of shock, end of reperfusion, and after 9 h. All normotensive animals survived the observation period, whereas mean survival time was 11.2 h in shock and reperfusion animals. 16 VOCs differed significantly for at least one of the three analysis periods. Peak intensities of butanone, 2-ethyl-1-hexanol, nonanal, and an unknown compound were higher in shocked than normotensive rats, and another unknown compound increased over the time. 1-butanol increased only during reperfusion. Acetone, butanal, 1.2-butandiol, isoprene, 3-methylbutanal, 3-pentanone, 2-propanol, and two unknown compounds were lower and decreased during shock and reperfusion. 1-pentanol and 1-propanol were significant greater in the hypotensive animals during shock, were comparable during reperfusion, and then decreased after resuscitation. VOCs differ during hemorrhagic shock, reperfusion, and after reperfusion. MCC-IMS of exhaled breath deserves additional study as a non-invasive approach for monitoring changes in metabolism during ischemia and reperfusion.