Postconditioning with a volatile anaesthetic in alveolar epithelial cells in vitro.

Acute lung injury is a common complication in critically ill patients. The present study examined possible immunomodulating effects of the volatile anaesthetic sevoflurane on lipopolysaccharide (LPS)-stimulated alveolar epithelial cells (AEC) in vitro. Sevoflurane was applied after the onset of injury, simulating a "postconditioning" scenario. Rat AEC were stimulated with LPS for 2 h, followed by a 4-h co-exposure to a CO(2)/air mixture with sevoflurane 2.2 volume %; control cells were exposed to the CO(2)/air mixture only. Cytokine-induced neutrophil chemoattractant-1, monocyte chemoattractant protein-1, intercellular adhesion molecule-1, as well as the potential protective mediators inducible nitric oxide synthase (iNOS)2 and heat shock protein (HSP)-32, were analysed. Additionally, functional assays (chemotaxis, adherence and cytotoxicity assay) were performed. A significant reduction of inflammatory mediators in LPS-stimulated, sevoflurane-exposed AEC was found, leading to reduced chemotaxis, neutrophil adherence and neutrophil-induced AEC killing. While iNOS2 was increased in the sevoflurane group, blocking experiments with iNOS2 inhibitor did not affect sevoflurane-induced decrease of inflammatory mediators and AEC killing. Interestingly, sevoflurane treatment also resulted in an enhanced expression of HSP-32. The data presented in the current study provide strong evidence that anaesthetic postconditioning with sevoflurane mediates cytoprotection in the respiratory compartment in an in vitro model of acute lung injury.

In vitro evaluation of the CeVOX continuous central venous oxygenation monitoring system.

We investigated the in vitro performance of the CeVOX system for continuous monitoring of central venous oxygen saturation by spectrophotometry (Pulsion Medical Systems, Munich, Germany). Oxygen inflow into the system was varied, and oxygen saturation values measured by CeVOX were documented. Blood samples were simultaneously taken to assess oxygen saturation by co-oximetry, and values were compared by Bland-Altman analysis. Sixty-six data pairs were obtained at CeVOX and co-oximetry values of 16-99% and 5.5-100%, respectively. Overall, CeVOX values only slightly overestimated co-oximetry values (mean bias +2.4%), but limits of agreement (2 SD of bias) were wide (-11.8 to +16.6%). Saturation measured by CeVOX underestimated that measured by co-oximetry at higher oxygen concentrations and overestimated it at lower oxygen concentrations. There was a nearly linear correlation of the mean bias, suggesting a systematic error. We conclude that the current version of the CeVOX system does not reliably reflect oxygen saturation.

The amino acid sequence of AQN-3, a carbohydrate-binding protein isolated from boar sperm. Location of disulphide bridges.

Gamete recognition and adhesion are essential steps in fertilization. Among others, carbohydrate-binding proteins on the sperm surface have been recognized to play a central role in the initial interaction of the male gamete with components of the zona pellucida of the homologous investing oocyte. We have isolated several members of a carbohydrate- and zona pellucida-binding protein family from ejaculated sperm. Here we report the biological origin and structural characterization of AQN-3, a component of this carbohydrate-binding family. The molecular weight of purified AQN-3 was determined by plasma desorption mass spectrometry. The protein was chemically and enzymatically degraded, the proteolytic fragments isolated and characterized by N-terminal sequencing and fast atom bombardment mass spectrometry. In this manner we established the complete amino acid sequence of AQN-3 and the location of its two disulphide bonds. No analogous protein sequence could be found in the MIPS protein sequence data bank, indicating that AQN-3 may belong to a novel mammalian carbohydrate-binding protein family.

Molecular weight-determination of biosynthetically modified monomeric and oligomeric muropeptides from Escherichia coli by plasma desorption-mass spectrometry.

The presence of certain D-amino acids in the growth media of Escherichia coli results in the accumulation of 2 major and 3-5 minor new muropeptides in the murein sacculus. Preliminary data suggested that the major muropeptides correspond to a monomer and a cross-linked dimer with one residue of D-amino acid per molecule. We have analyzed several D-amino acid-modified muropeptides by plasma desorption-mass spectrometry. Our results confirmed that the general structures of the major modified muropeptides are: GlucNAc-MurNAc-L-Ala-D-Glu-m-A2pm-D-X, and GlucNAc-MurNAc-L-Ala-D-Glu-m-A2pm-D-Ala; GlucNAc-MurNAc-L-Ala-D-Glu-m-A2pm-D-X, being X a residue of the D-amino acid. These results corroborate the usefulness of this technique for the structural analysis of muropeptides.

The complete primary structure of three isoforms of a boar sperm-associated acrosin inhibitor.

Acrosin inhibitors of seminal vesicle origin, after binding to their acceptor molecules on the anterior part of ejaculated sperm, are thought to be important capacitation factors, protecting zona binding sites during sperm uterine passage, and then dissociating to allow sperm binding to the zona pellucida of the oocyte. Each species so far tested possess an heterogeneous population of isoinhibitors which may display overlapping but not identical biological functions. Here we report the complete primary structure of three isoforms of a boar sperm-associated acrosin inhibitor, whose sequences are 90% identical to the seminal plasma counterpart. Despite this high analogy, the differences between the sperm-associated and the seminal plasma inhibitors may confer to them different physico-chemical properties which are postulated to be of functional importance.

The complete primary structure of the spermadhesin AWN, a zona pellucida-binding protein isolated from boar spermatozoa.

AWN is a boar protein which originates in secretions of the male accessory glands and which becomes sperm surface-associated upon ejaculation. It is one of the components thought to mediate sperm adhesion to the egg's zona pellucida through a carbohydrate-recognition mechanism. AWN may, thus, participate in the initial events of fertilization in the pig. In this report we describe its complete primary structure by combination of protein-chemical and mass spectrometric methods. AWN exists as two isoforms, AWN-1 and AWN-2, which differ in that AWN-2 is N-terminally acetylated. The amino acid sequence of AWN contains 133 amino acid residues and two disulphide bridges between nearest-neighbour cysteine residues. Analysis of the amino acid sequence of the AWN proteins showed significant similarity only to AQN-1 and AQN-3, two other boar spermadhesins.

Characterization of braun's lipoprotein and determination of its attachment sites to peptidoglycan by (252)Cf-PD and MALDI time-of-flight mass spectrometry.

A strategy for the characterization of bacterial lipoprotein-in this case Braun's lipoprotein (an outer membrane 7-ku lipoprotein) isolated from Escherichia coli-is described by time-of-flight mass spectrometric (TOF/MS) techniques [(252)Cf plasma desorption (PD) TOF/MS and matrix-assisted laser desorption-ionization (MALDI) TOF/MS]. Covalent linkage of lipid at the N-terminal cysteine (posttranslationally modified to a S-[2,3-bis(acyloxy)-propyl]-N-acylcysteine) and, therefore, strict insolubility in aqueous solution constitute common features for this class of proteins. Relative molecular mass determination of the major molecular species of Braun's lipoprotein was obtained by selection of an appropriate mixture of organic solvents compatible with matrix/support materials useful for the mass spectrometric techniques applied. Minor components of this lipoprotein that differ only in the fatty acid composition of the lipid anchor were detected by PD TOF/MS after enzymatic release of the extremely hydrophobic N-terminal amino acid followed by selective extraction with chloroform. Part of the primary sequence of this lipoprotein was confirmed based on peptide fragment ions observed in the positive ion PD mass spectra of cyanogen bromide-generated peptide fragments that had been isolated previously by reverse phase high-performance liquid chromatography (HPLC). Peptidoglycan fragments that represent the attachment sites of lipoprotein to peptidoglycan were enzymatically released, separated by reverse phase HPLC, and finally characterized by time-of-flight mass spectrometric techniques ((252)Cf-PD TOF/MS, MALDI TOF/MS). The results obtained with both techniques differed only in the better sensitivity obtained with MALDI TOF/MS, which consumed a factor of 100 to 1000 less material than with PD TOF/MS.

Acute lung injury during cardiopulmonary bypass. Are the neutrophils responsible?

To test the hypothesis that acute lung injury during cardiopulmonary bypass (CPB) is related to the activation of neutrophils and the body temperature during bypass, we determined the differential WBC count, plasma elastase concentrations, and lung function before, during, and after CPB in 38 patients undergoing elective coronary artery bypass surgery. The patients were randomly assigned to receive either normothermic (n = 19, rectal temperature: 35.9 +/- 0.1 degrees C, mean +/- SE) or hypothermic (n = 19, 29.2 +/- 0.5 degrees C) CPB. The cellular response to the extracorporeal circulation was significantly delayed in the hypothermic group with a later onset of neutrophilia and a later increase in plasma elastase levels during bypass. Lung function deteriorated significantly after CPB as assessed by respiratory index, alveolar-arterial oxygen gradient, and intrapulmonary shunt, independent of bypass temperature. There was a positive correlation between peak elastase concentrations and postoperative respiratory index as well as intrapulmonary shunt (R2 = 0.5, p = 0.002 and R2 = 0.45, p = 0.003, respectively). Besides peak plasma elastase levels, multiple regression revealed no significant influence of other independent factors on postoperative lung dysfunction in our patients.

Fatal fungal pericarditis after cardiac surgery and immunosuppression.

The cases of two patients with fulminant pericarditis after cardiac surgery are reported. Both fungal infections developed after rethoracotomy for open-chest cardiac resuscitation and high-dose glucocorticoid treatment. Although the time course of both infections from the inoculation of fungi during rethoracotomy and immunosuppression with glucocorticoids to the lethal outcome was strikingly similar, histopathologic studies disclosed the disparate character of the two fungal pathogens responsible: the yeast Candida albicans and the angiotropic mold Aspergillus fumigatus.

Continuous thermodilution cardiac output: clinical validation against a reference technique of known accuracy.

OBJECTIVE: To evaluate the accuracy and precision of continuous thermodilution (CCO) by using a validated bolus thermodilution (BCO) reference technique as criterion standard. DESIGN: Under circulatory steady state conditions, a CCO system (Vigilance, software versions 4.35 and 4.39) was validated with regard to CCO as well as iced and room temperature BCO. SETTING: Intensive care unit at a university hospital. PATIENTS: Method comparison was conducted in 56 cardiac surgical patients, 28 patients being allocated to one of the two software versions, and 14 within each group to either iced or room temperature BCO. MEASUREMENTS AND RESULTS: CCO readings were registered in duplicate before and after three to five bolus injections conducted with both the Vigilance and reference systems. Iced BCO showed excellent agreement between the Vigilance and reference systems, yielding SDs of bias of 0.41 and 0.37 l/min and linear correlation coefficients (r) of 0.97 and 0.96. With room temperature BCO, agreement was significantly less. CCO, irrespective of software version, showed higher SDs of bias (0.90 and 0.84 l/min) and lower r values (0.84 and 0.81) than iced BCO (p < 0.0001). CCO measurements with software version 4.39 yielded a similar SD of bias to that with room temperature BCO. CONCLUSION: Decreased precision of CCO as compared to iced BCO may, in clinical settings, be outweighed by the advantages of automated and continuous monitoring. Under research conditions, however, iced BCO remains the method of choice.

Treatment of left atrial dissection after mitral repair: internal drainage.

Two patients with intraoperative dissection of the entire left atrium after mitral valve repair are presented. Intraoperative transesophageal echocardiography detected left atrial dissection with formation of a large cavity compressing the left atrium. The false lumen was opened and widely connected to the right atrium to perform the decompression. This technique permits the runoff into the low pressure system in case of persisting hemorrhage from the unknown entry, and eliminates the risk of systemic embolization from the cavity.

Normothermia versus hypothermia during cardiopulmonary bypass: a randomized, controlled trial.

To evaluate the influence of perfusion temperature on systemic effects of cardiopulmonary bypass (CPB), 30 patients undergoing elective coronary artery bypass grafting were randomly assigned to either normothermic (warm, n = 14, 36 degrees C) or hypothermic (cold, n = 16, 28 degrees C) CPB. Serial hemodynamic measurements and blood samples were obtained before, during and after the CPB procedure. During CPB, there were no differences between both groups in the need for vasopressors (norepinephrine, phenylephrine), urinary output, or fluid balance. In the early postoperative period, normothermic CPB patients had significantly lower systemic vascular resistance and higher cardiac index measurements (mean +/- standard error: systemic vascular resistance, 880 +/- 27 versus 1,060 +/- 57 dyne.s.cm-5, p = 0.025; cardiac index, 3.6 +/- 0.1 versus 2.9 +/- 0.1 L.min-1.m-2, p = 0.01) without differences in the administration of vasoactive drugs. Blood loss was significantly higher in patients after hypothermic CPB (median [range] body surface area: 370 [180-560] versus 490 [280-2,120] mL/m2, p = 0.0006), with a greater need for transfusion of erythrocytes and fresh frozen plasma. Plasma levels of tumor necrosis factor and soluble tumor necrosis factor receptors increased during and after CPB, independent of perfusion temperature. This study suggests a significant influence of CPB temperature and respective perfusion management on postoperative hemodynamics and blood loss. Normothermic CPB is not associated with additional systemic adverse effects.

Outcome and risk factors in surgery of descending thoracic aneurysms.

In a series of 100 consecutive patients operated upon for aneurysmatic lesions of the descending thoracic aorta, the mean age was 52 +/- 16 years (male = 81, female = 19). There were 31 dissections (acute 10, chronic 21); 28 post-traumatic aneurysms (ruptured 5, acute 7, chronic 16); 22 arteriosclerotic aneurysms (ruptured 1, chronic 21); 11 thoracoabdominal aneurysms, 5 anastomotic aneurysms and 3 mycotic aneurysms. No symptomatic patient was refused. Preoperative risk factors were graded on a scale of 6 by counting 1 point for each of the following elements: central nervous system disease; heart, pulmonary, and renal disease; arterial hypertension, age greater than 60. In-hospital mortality and paraplegia for the whole series were 25% and 7% respectively. In acute dissection, the mortality was 6/10 patients versus 2/21 in chronic events. In post-traumatic aneurysms, mortality was 2/5 in ruptured, 2/7 in acute and 0/16 in chronic events. In arteriosclerotic aneurysms, mortality was 1/2 in ruptured and 6/20 in chronic events. In thoracoabdominal aneurysms, mortality was 5/11, in anastomotic 1/5 and in mycotic 0/3 patients. The mean number of risk factors in non-survivors versus survivors was significantly higher in acute dissection, chronic dissection, chronic arteriosclerotic aneurysms and thoracoabdominal aneurysms. Rupture and acute events are related to a high surgical mortality. Non-survivors appear to have significantly more preoperative risk factors.

Left ventricular assist device as bridge to heart transplantation--lessons learned with the MicroMed DeBakey axial blood flow pump.

OBJECTIVE: The MicroMed DeBakey left ventricular assist device (LVAD) axial blood flow pump was used as bridge to heart transplantation (HTx) in patients with terminal heart failure. The aim was to evaluate this novel mechanical circulatory support system in regard to overall outcome. METHODS: Prospective study in 15 HTx candidates (mean age 40+/-7 years) with terminal heart failure and maximal medical treatment due to ischemic cardiomyopathy (CMP, n=5), dilated CMP (n=3), restrictive CMP (n=2), unclassified CMP (n=1), metabolic CMP (n=1), valvular CMP (n=1) and congenital CMP (n=2). All patients were implanted with a MicroMed DeBakey LVAD. A rescue procedure was necessary in eight critical patients, while seven underwent elective LVAD implantation. Procedures were performed via median sternotomy, in normotherm femoro-femoral CPB (mean duration 59+/-1 min). Oral Marcoumar (INR 2.0-3.0) and Aspirin (100 mg daily) were started as soon as possible. Patients were discharged into a specialized rehabilitation clinic from which it was possible to release them home after a few weeks. RESULTS: Successful implantation and discharge from ICU (mean stay 10+/-7 days) was possible in 11 patients. Seven were transplanted (mean support 50.7 days) and one is awaiting HTx (support >310 days) in the comfort of his home (NYHA I). Survival was 100% among the transplanted patients. Of the seven elective implants, five, and of the eight rescue procedures three patients underwent successful HTx. Four patients died early, while three patients died late on pump support due to intracranial hemorrhage (n=2, 73 and 76 days) and chest infection (n=1, 124 days). All survivors were discharged from hospital, with significant decrease in NYHA class (mean 3.8-2.4 (n=11)). Treadmill testing showed increased exercise tolerance, from 35 to 71W (n=4). Plasma BNP values (mean 950-162 ng/l (n=4)) and pulmonary resistance (mean 316-194.5 dyne s/cm(5) (n=3)) decreased significantly during LVAD support. CONCLUSIONS: The MicroMed DeBakey LVAD is simple to implant; outpatient treatment is safe and efficient. Patients' condition and pulmonary resistances normalize within 6 weeks, making previously considered inoperable patients amenable for HTx. HTx can be performed in low-risk situation, allowing better donor-recipient matching and improving overall outcome.

Expression of adhesion molecules and cytokines after coronary artery bypass grafting during normothermic and hypothermic cardiac arrest.

OBJECTIVE: Cardiac surgery with cardiopulmonary bypass (CPB) results in vascular injury and tissue damage which involves leukocyte-endothelial interactions mediated by cytokines and adhesion molecules. This study was designed to demonstrate the effect of normothermic and hypothermic CPB to cytokine and soluble adhesion molecule levels in adults and to determine whether these levels correlate to the patients postoperative course. DESIGN AND PATIENTS: In 25 patients after normothermic and in 25 patients after hypothermic coronary artery bypass grafting with cardiopulmonary bypass (CPB), blood samples for cytokine and soluble adhesion molecule analysis were taken preoperatively, 24, 36, 48 h, and 6 days postoperatively. Soluble adhesion molecules (sE-selectin, sICAM-1) were measured by ELISA and cytokines (TNF-alpha, IL-6, IL-8) by chemilumenscent-immunoassay. Clinical data were collected prospectively. RESULTS: Postoperatively, adhesion molecule and cytokine levels were significantly elevated after CPB. Mean plasma levels of sICAM-1 was 2.4-fold higher after 6 days. Mean plasma concentration of sE-selectin peaked after 48 h with a 2-fold increase compared to normothermic conditions. In the hypothermia group sICAM-1, sE-selectin, IL-6, and IL-8 showed significantly higher levels (P<0.0057, P<0.0012, P<0.0419, P<0.0145) after 24 h compared to the normothermia group. No clinical differences were seen. CONCLUSION: Adhesion molecules and cytokines are elevated after CPB. Patients after hypothermic CPB show significant higher sICAM-1, sE-selectin, IL-6, and IL-8 levels after 24 h compared to normothermic conditions. These results are mainly due to longer CPB and crossclamp times but do not alter the patient's postoperative course.

Serum and CSF levels of neuron-specific enolase (NSE) in cardiac surgery with cardiopulmonary bypass: a marker of brain injury?

We investigated whether neuron-specific enolase (NSE) in serum or cerebrospinal fluid (CSF) reflects subtle or manifest brain injury in children undergoing cardiac surgery using cardiopulmonary bypass (CPB). NSE was measured in serum (s-NSE) before, and up to, 102 h after surgery in 27 children undergoing cardiac surgery with CPB. In 11 children, CSF-NSE was also measured 48 or 66 h post-surgery. As erythrocytes contain NSE, hemoglobin concentration in the samples was determined spectrophotometrically at 550 nm (cut-off limit: absorbance 0.4 = 560 mg/l) in 14 children and in a further 13 children by spectroscopic multicomponent analysis (cut-off limit 5 micromol/l = 80 mg/l). One hundred and one of 214 post-operative serum samples (47%) had to be discarded because of hemolysis (18% spectrophotometrically at 550 nm and 88% with spectroscopic multicomponent analysis). On the first and second post-operative day, the median s-NSE values were significantly higher when compared with samples taken after 54 h or longer (P = 0.008 and P = 0.002). All CSF-NSE levels were within the normal range and below the s-NSE measured in the same patient. Although in our study elevated s-NSE seems to indicate brain injury in CPB-surgery, the low concentration of NSE in the post-operative CSF of 11 children puts the neuronal origin of s-NSE in question. NSE from other non-neuronal tissues probably contributes to the elevated s-NSE. Additionally, normal post-operative CSF-NSE values in two children with post-operative neurological sequelae might question the predictive value of CSF-NSE with regard to brain injury.

Determination of estrogenic activity in beer by biological and chemical means.

It has been suspected that beer drinking may change the hormonal status of men caused by phytoestrogens. Five different Austrian lager beers have been investigated for estrogenic activity by a yeast two-plasmid system harboring the human estrogen receptor alpha, after concentration by solid phase extraction. The beer concentrate was further fractionated by reversed phase HPLC, and then the fractions were characterized by the biological assay and GC-MS. The most potent fraction did not contain a known phytoestrogen. The total activity corresponded to an average of 43 ng of 17beta-estradiol/L of beer. It was concluded that the human health hazard of beer drinking originating from compounds activity on the estrogen receptor alpha is negligible.

MS-UTIL: a program for interpretation and manipulation of mass spectra and chemical structures.

A computer program for personal computers has been developed, which enables the user of interpret fragmentation processes in electron impact mass spectrometry more easily. Starting with a low resolution mass spectrum and a selected chemical structure, fragments of the molecule which are related to the spectral data can be created interactively. Some frequently occurring fragmentation reactions can be performed automatically. An included editor allows the user to draw chemical structures and to create or modify mass spectra. Fragments of a molecule can be displayed as a tree. One aim of the program is to facilitate learning of fragmentation reactions. Furthermore, the program serves as a tool for verifying fragmentation mechanisms proposed and for checking presumptive chemical structure formulas of unidentified compounds.

Epidural anesthesia for a cesarean section in a patient with pulmonary atresia and ventricular septal defect.

The perioperative management and the pathophysiology of a parturient with pulmonary atresia, ventricular septal defect, patent ductus arteriosus Botalli (PDA), and pulmonary hypertension are described. The patient previously had a cesarean section under general anesthesia and was currently managed with an epidural block. The outcome was successful for the mother. The postoperative period of the premature infant was characterized by hyaline membrane disease, with its typical sequelae.