Jan Hirsch

Mass Spectrometric Analysis of Protein Mixtures at Low Levels Using Cleavable 13C-Isotope-coded Affinity Tag and Multidimensional Chromatography

In order to identify and compare the protein content of very low quantity samples of high complexity, a protocol has been established that combines the differential profiling strength of a new cleavable 13C isotope-coded affinity tag (cICAT) reagent with the high sequence coverage provided by multidimensional liquid chromatography and two modes of tandem mass spectrometry. Major objectives during protocol optimization were to minimize sample losses and establish a robust procedure that employs volatile buffer systems that are highly compatible with mass spectrometry. Cleavable ICAT-labeled tryptic peptides were separated from nonlabeled peptides by avidin affinity chromatography. Subsequently, peptide samples were analyzed by nanoflow liquid chromatography electrospray ionization tandem mass spectrometry and liquid chromatography matrix-assisted laser desorption/ionization tandem mass spectrometry. The use of two ionization/instrumental configurations led to complementary peptide identifications that increased the confidence of protein assignments. Examples that illustrate the power of this strategy are taken from two different projects: i) immunoaffinity purified complexes containing the prion protein from the murine brain, and ii) human tracheal epithelium gland secretions. In these studies, a large number of novel proteins were identified using stringent match criteria, in addition to many that had been identified in previous experiments. In the latter case, the ICAT method produced significant new information on changes that occur in protein expression levels in a patient suffering from cystic fibrosis.

Impact of intraoperative hypotension and blood pressure fluctuations on early postoperative delirium after non-cardiac surgery

INTRODUCTION Postoperative delirium is common in older patients. Despite its prognostic significance, the pathophysiology is incompletely understood. Although many risk factors have been identified, no reversible factors, particularly ones potentially modifiable by anaesthetic management, have been identified. The goal of this prospective cohort study was to investigate whether intraoperative hypotension was associated with postoperative delirium in older patients undergoing major non-cardiac surgery. METHODS Study subjects were patients >65 years of age, undergoing major non-cardiac surgery, who were enrolled in an ongoing prospective observational study of the pathophysiology of postoperative delirium. Intraoperative blood pressure was measured and predefined criteria were used to define hypotension. Delirium was measured by the Confusion Assessment Method on the first two postoperative days. Data were analysed using t-tests, two-sample proportion tests and ordered logistic regression multivariable models, including correction for multiple comparisons. RESULTS Data from 594 patients with a mean age of 73.6 years (sd 6.2) were studied. Of these 178 (30%) developed delirium on day 1 and 176 (30%) on day 2. Patients developing delirium were older, more often female, had lower preoperative cognitive scores, and underwent longer operations. Relative hypotension (decreases by 20, 30, or 40%) or absolute hypotension [mean arterial pressure (MAP)<50 mm Hg] were not significantly associated with postoperative delirium, nor was the duration of hypotension (MAP<50 mm Hg). Conversely, intraoperative blood pressure variance was significantly associated with postoperative delirium. DISCUSSION These results showed that increased blood pressure fluctuation, not absolute or relative hypotension, was predictive of postoperative delirium.

Warm Ischemia-induced Alterations in Oxidative and Inflammatory Proteins in Hepatic Kupffer Cells in Rats

The aim of the study was to investigate the impact of ischemia/reperfusion injury on the proteome of Kupffer cells. Lean Zucker rats (n = 6 each group) were randomized to 75 min of warm ischemia or sham operation. After reperfusion for 8 h, Kupffer cells were isolated by enzymatic perfusion and density gradient centrifugation. Proteins were tryptically digested into peptides and differentially labeled with iTRAQ (isobaric tags for relative and absolute quantitation) reagent. After fractionation by cation exchange chromatography, peptides were identified by mass spectrometry (ESI-LC-MS/MS). Spectra were interrogated against the Swiss-Prot database and quantified using ProteinProspector®. The results for heat shock protein 70 and myeloperoxidase were validated by ELISA. Quantitative information for more than 1559 proteins was obtained. In the ischemia group proteins involved in inflammation were significantly up-regulated. The ratio for calgranulin B in the ischemia/sham group was 1.81 ± 0.97 (p < 0.0001), for complement C3 the ratio was 1.81 ± 0.49 (p < 0.0001), and for myeloperoxidase the ratio was 1.30 ± 0.32. Myeloperoxidase was only recently documented in Kupffer cells. The antioxidative proteins Cu,Zn-superoxide dismutase (1.34 ± 0.19; p < 0.001) and catalase (1.23 ± 0.43; p < 0.001) were also elevated. In conclusion, ischemia/reperfusion injury induces alterations in the Kupffer cell proteome. Isotope ratio mass spectrometry is a powerful tool to investigate these reactions. The ability to simultaneously monitor several pathways involved in reperfusion stress may result in important mechanistic insight and possibly new treatment options.

Hearing loss after spinal and general anesthesia: A comparative study.

Hearing loss has been described after spinal anesthesia. We examined the hearing in patients before and after spinal and general anesthesia by pure tone audiometry (LdB: 125-1500 Hz; HdB: 2000–8000 Hz). Tympanic membrane displacement analysis was used to noninvasively monitor the intralabyrinthine and intracranial pressure. Eighteen patients received spinal anesthesia (GSA); 19 patients general anesthesia (GGA). Pure tone audiometry and TMD data were obtained preoperatively (0) and postoperatively on day 1 (1) and 2 (2). The mean threshold differences (&Dgr;) in LdB10 and LdB20 were significantly different in GSA compared with GGA (&Dgr;LdB10 + 0.15 ± 3.07 dB vs −1.34 ± 3.77 dB, P = 0.05; &Dgr;LdB20 −0.54 ± 2.24 dB vs −2.45 ± 3.39 dB, P < 0.01). However, there were no differences in &Dgr;HdB10 between GSA and GGA, but in &Dgr;HdB20 (−1.40 ± 3.95 dB vs −5.12 ± 6.35 dB, P = < 0.01). We found a significant correlation between the magnitude of intraoperative intravascular volume replacement and low-frequency hearing loss. Tympanic membrane displacement values were not different pre- and postoperatively. Hearing was impaired after spinal and general anesthesia. Low-frequency hearing loss was correlated with intraoperative volume replacement. Tympanic membrane recordings did not reveal significant changes. Implications The results of this study imply that hearing loss occurs after spinal as well as after general anesthesia. The positive correlation of low-frequency hearing loss and intraoperative fluid administration replacement suggests that cerebrospinal fluid leakage via the spinal puncture hole is not the only factor involved.

The efficacy of skin temperature for block assessment after infraclavicular brachial plexus block.

BACKGROUND: Although it has been reported that an increase in skin temperature indicates block success with higher specificity and sensibility than skin sensitivity to pinprick and cold, the methodology previously used computer-assisted infrared thermography, a technique that is expensive and requires substantial personnel training. In this prospective observational study, we evaluated whether a simple infrared thermometer can reliably predict block effectiveness after infraclavicular brachial plexus blockade. METHODS: Thirty consecutive patients undergoing upper limb surgery under infraclavicular block were enrolled. From the end of the local anesthetic injection, skin temperature was measured in all four major nerve distribution areas, and the sensory block onset (using cold and pinprick with 0 = no sensation to 2 = normal) were evaluated every 5 min for 30 min. A successful block was defined as the absence of sensation to cold (swab soaked with alcohol) and pinprick (needle) with a score of “0” within 30 min after the injection in the 4 major nerve distribution areas (radial, ulnar, median and musculocutaneous). Skin temperature measurements were performed using a noncontact temperature probe. RESULTS: One-hundred-twenty nerves (30 patients, 4 nerves per patient) were anesthetized. Twenty-five patients had a successful block. Four patients required supplementation for block failure. General anesthesia was performed in one patient. Skin temperature variation was not different among different nerves. There was a statistically significant increase in cutaneous temperature after nerve block compared to the same skin area before the procedure (P < 0.0001 from T5 to T30). Average temperature variations in blocked versus unblocked nerves at the same time were significantly different (P < 0.05 at T5 then P < 0.0001 from T10 to T30). When temperature in a specific sensory territory increased 1°C or more, at 5 and 10 min, the specific nerve was blocked (the score was “0”). Thus, when temperature changes in all 4 nerves were noted at 5 and 10 min, the block was successful at 30 min. No change in temperature in the contralateral arm or in the core temperature was observed. CONCLUSION: Skin temperature assessment with an infrared thermometer is a reliable, simple and early indicator of a successful nerve block.

Bone Fracture Exacerbates Murine Ischemic Cerebral Injury

Background:Bone fracture increases alarmins and proinflammatory cytokines in the blood, and provokes macrophage infiltration and proinflammatory cytokine expression in the hippocampus. We recently reported that stroke is an independent risk factor after bone surgery for adverse outcome; however, the impact of bone fracture on stroke outcome remains unknown. We tested the hypothesis that bone fracture, shortly after ischemic stroke, enhances stroke-related injuries by augmenting the neuroinflammatory response. Methods:Tibia fracture (bone fracture) was induced in mice one day after permanent occlusion of the distal middle cerebral artery (stroke). High-mobility-group box chromosomal protein-1 (HMGB1) was tested to mimic the bone fracture effects. HMGB1 neutralizing antibody and clodrolip (macrophage depletion) were tested to attenuate the bone fracture effects. Neurobehavioral function (n = 10), infarct volume, neuronal death, and macrophages/microglia infiltration (n = 6–7) were analyzed after 3 days. Results:We found that mice with both stroke and bone fracture had larger infarct volumes (mean percentage of ipsilateral hemisphere ± SD: 30±7% vs.12±3%, n = 6, P < 0.001), more severe neurobehavioral dysfunction, and more macrophages/microglia in the periinfarct region than mice with stroke only. Intraperitoneal injection of HMGB1 mimicked, whereas neutralizing HMGB1 attenuated, the bone fracture effects and the macrophage/microglia infiltration. Depleting macrophages with clodrolip also attenuated the aggravating effects of bone fracture on stroke lesion and behavioral dysfunction. Conclusions:These novel findings suggest that bone fracture shortly after stroke enhances stroke injury via augmented inflammation through HMGB1 and macrophage/microglia infiltration. Interventions to modulate early macrophage/microglia activation could be therapeutic goals to limit the adverse consequences of bone fracture after stroke.

Indicators of Erythrocyte Damage after Microwave Warming of Packed Red Blood Cells

BACKGROUND Localized overheating of packed red blood cells (PRBCs) after microwave warming with consequent damage to erythrocytes has been reported. We therefore compared possible cellular markers of erythrocyte damage, as measured by flow cytometry, with laboratory indicators of hemolysis to evaluate the effects of microwave warming on PRBCs. METHODS PRBC samples were warmed to room temperature or to 37, 42, 47, 52, or 57 degrees C in a water bath. Flow cytometry was performed after fluorescein labeling using antibodies to spectrin, Ca(2+)-ATPase, and Na(+)-K(+)-ATPase. The forward-to-sideward scatter (FSC/SSC) ratio and antibody binding were evaluated. Plasma free hemoglobin (FHb) and alpha-hydroxybutyrate dehydrogenase (HBDH) were measured immediately after heating and after 48 h. In addition, all measurements were made before and after the heating of PRBCs to 35 degrees C by a microwave blood warmer. RESULTS Analysis of 15000 erythrocytes showed a decrease in the FSC/SSC ratio and antibody binding above 47 degrees C [at 37 degrees C, median (SD) of 94.2 (7.4) with 0.07 (0.05)% fluorescein-positive; at 52 degrees C, median (SD) of 177.0 (19.0) with 18.5 (6.4)% positively gated; P <0.001]. FHb [room temperature, 0.3 (0.2) g/L] was increased 2-fold at 37 and 42 degrees C, 4-fold at 47 degrees C, and 25-fold at 52 degrees C. HBDH increased in parallel. Hemolysis markers showed an additional twofold increase 48 h after heating to 42 and 47 degrees C. Microwave heating to 35 degrees C did not produce significant changes of any marker. CONCLUSIONS All markers of cellular damage were altered after heating to >47 degrees C, and a substantial part of hemolysis was delayed. The methodology can be used for future testing of other blood warming devices.

Temperature course and distribution during plasma heating with a microwave device.

Summary In spite of the much shorter thawing times, the use of microwave devices for heating units of fresh frozen plasma is still being discussed. Concerns about general and localised overheating are the main arguments against the use of microwave devices. We evaluated the warming of fresh frozen plasma using the recently introduced Transfusio‐therm 2000® microwave blood warmer. Units of fresh frozen plasma were weighed and the heating times were recorded. The surface temperature of the fresh frozen plasma bags during heating was recorded every 10 s. Temperature variation on the surface was examined by measuring the difference between peripheral and centrally placed temperature sensors. After heating, plasma temperature was determined using a calibrated thermometer. There were no signs of overheating during the heating process. The surface temperature of three units of fresh frozen plasma heated simultaneously (n = 45) was 34.0°C (SD, 1.5°C) after a mean heating time of 23.2 min (SD, 1.1 min). The mean (SD) temperature difference was −0.6 (0.5)°C and the mean (SD) plasma temperature was 33.6 (0.8)°C. Heating one fresh frozen plasma unit at a time (n = 20), the mean (SD) heating time was 6.3 (0.4) min. The surface temperature after heating was 34.3 (0.2)°C, the mean (SD) temperature difference was −0.6 (0.4)°C and the mean (SD) plasma temperature after heating 33.1 (0.6)°C. We conclude that no general or localised overheating of fresh frozen plasma occurs during or after heating with the microwave blood warmer.

Point-of-care testing apparatus. Measurement of coagulation.

Point‐of‐care testing of coagulation parameters provides a more rapid assessment of test results compared with laboratory testing. A new coagulation monitor (GEM® PCL, Instrumentation Laboratory, Kirchheim, Germany) was evaluated. Point‐of‐care data for activated partial thromboplastin time and prothrombin time (expressed as the international normalised ratio) and turn‐around‐time were compared. Coagulation parameters were compared in the blood of 57 patients with and without heparin therapy. The point‐of‐care and laboratory test results showed a bias (SD) of − 0.26 (4.55) s for activated partial thromboplastin time and − 0.011 (0.150) s for prothrombin time. The average turn‐around‐time was 3 min for point‐of‐care testing vs. 52 min for laboratory testing. We conclude that the reliability of point‐of‐care testing is sufficient for clinical use.

Alterations in the proteome of pulmonary alveolar type II cells in the rat after hepatic ischemia-reperfusion.

Objective:Hepatic ischemia-reperfusion can be associated with acute lung injury. Alveolar epithelial type II cells (ATII) play an important role in maintaining lung homeostasis in acute lung injury. Design:To study potentially new mechanisms of hepatic ischemia-reperfusion-induced lung injury, we examined how liver ischemia-reperfusion altered the proteome of ATII. Setting:Laboratory investigation. Subjects:Spontaneously breathing male Zucker rats. Interventions:Rats were anesthetized with isoflurane. The vascular supply to the left and medial lobe of the liver was clamped for 75 mins and then reperfused. Sham-operated rats were used as controls. After 8 hrs, rats were killed. Measurements and Main Results:Bronchoalveolar lavage and differential cell counts were performed, and tumor necrosis factor-&agr; and cytokine-induced neutrophil chemotactic factor-1 in plasma were determined by enzyme-linked immunosorbent assay. ATII were isolated, lysed, tryptically digested, and labeled using isobaric tags (iTRAQ). The samples were fractionated by cation exchange chromatography, separated by high-performance liquid-chromatography, and identified using electrospray tandem mass spectrometry. Spectra were interrogated and quantified using ProteinProspector. Quantitative proteomics provided quantitative data for 94 and 97 proteins in the two groups. Significant changes in ATII protein content included 30% to 40% increases in adenosine triphosphate synthases, adenosine triphosphate/adenosine diphosphate translocase, and catalase (all p < .001). Following liver ischemia-reperfusion, there was also a significant increase in the percentage of neutrophils in bronchoalveolar lavage (48% ± 26%) compared with sham-operated controls (5% ± 3%) (p < .01), and plasma tumor necrosis factor-&agr; levels were also significantly increased. Conclusions:The proteins identified by quantitative proteomics indicated significant changes in moderators of cell metabolism and host defense in ATII. These findings provide new insights into possible mechanisms responsible for hepatic ischemia-reperfusion-related acute lung injury and suggest that ATII cells in the lung sense and respond to hepatic injury.