Vapor diffusion-controlled meso crystallization of membrane proteins.

The presented method to crystallize membrane proteins combines the advantages of the meso-phase crystallization method and the classical vapor diffusion crystallization. It allows fast screening of crystallization conditions employing automated liquid handlers suited for the 96-well crystallization format.

Direct oxymetric peripheral tissue perfusion monitoring during open heart surgery with the use of cardiopulmonary bypass: preliminary experience.

BACKGROUND: Regional hypoperfusion has been associated with the development of postoperative organ dysfunction in cardiac surgery involving cardiopulmonary bypass (CPB). Direct tissue oxymetry is a potentially new method for monitoring the quality of the peripheral tissue perfusion during CPB. The aim of this study was to assess the effects of CPB in skeletal muscle oxygenation when measured in the deltoid muscle by direct oxymetry during perioperative period. METHOD: Seven patients underwent on-pump coronary artery bypass grafting. Direct oxymetry was performed by an optical cathether introduced into the deltoid muscle. Continuous measurement was made during the surgical procedure and the postoperative period. Mean arterial blood pressure, blood flow during CPB, laboratory markers of tissue hypoperfusion, blood gases and body temperature were also recorded. RESULTS: Interstitial muscle tissue oxygen tension (pO(2)) decreased after the introduction to anaesthesia and, more significantly, during CPB. After the disconnection from CPB at the end of the operation, the pO(2) returned to pre-anaesthetic values. During the first hours after admission of the patients to the intensive care unit, the pO(2) progressively decreased, reached a minimum value after four hours, and increased slowly thereafter. There was a significant correlation of pO(2) with mean arterial blood pressure and blood flow during that time. CONCLUSION: The result of this first measurement seems to demonstrate that the standard technique of conducting cardiopulmonary bypass produces low muscle oxygen tension and, thus, little perfusion of skeletal muscle. The data also indicate that both high mean arterial blood pressure and high flow are necessary during CPB to ensure skeletal muscle perfusion. The investigation is continuing.

[The effect of preoperative administration of heparin on the heparin resistance development]. / Vliv predoperacního podání heparinu na vznik heparinové rezistence.

BACKGROUND: Heparin resistance is relatively frequent problem in cardio surgery. We were try to determine real occurrence heparin resistance before operation. AIM OF THE STUDY: Purpose of the project--to find the real frequency of heparin resistance in patients who will undergo a cardio surgical operation. To find out the dependence between the pre-operational application of heparin and the development of heparin resistance. METHODS: We recorded pre-operative administration heparin in patients. If the dose of heparin was 5 mg/kg and more then we insert patients to the group heparin resistant. RESULTS: In our collection was heparin resistance in 203 patients from 624, it was 32.5%. Test agreement relative frequency with 22% was throw out--p < 0.001--heparin resistance in our group statistically different from 22% heparin resistance was higher than hypothesis. Heparin before operation was administrate 181 patients, which make to 29%. For administration of heparin was hypothesis of independence thrown (p < 0.001). CONCLUSION: Results of our works confirmed statistically significant occurrence of heparin resistance in patients that was administration heparin pre-operative. Heparin resistance occurred against presumption 22% in 32.5% in our group. It is statistic significant difference.

Impact of cardiopulmonary bypass on peripheral tissue metabolism and microvascular blood flow.

The aim of this study was to monitor and compare the changes in metabolism and blood flow in the skeletal muscles during cardiac operations performed with cardiopulmonary bypass (CPB) and operations without CPB (off-pump) by means of interstitial microdialysis (Figure 1). Surgical revascularization, coronary artery bypass grafting (CABG), was performed in 40 patients randomized to two groups. Twenty patients (On-Pump Group) were operated on using CPB, 20 patients (Off-Pump Group) were operated on without CPB. Interstitial microdialysis was performed by 2 probes of a CMA 60 (CMA Microdialysis AB, Solna, Sweden) inserted into the patient's deltoid muscle. Microdialysis measurements were performed at 30-minute intervals. Glucose, lactate, pyruvate and glycerol as markers of basic metabolism and tissue perfusion were measured in samples from the first probe, using a CMA 600 Analyzer (CMA Microdialysis AB). Blood flow through the interstitium was monitored by means of dynamic microdialysis of ethanol as a flow-marker in the dialysates taken from the second probe (ethanol dilution technique). Results in both the groups were statistically processed and compared. Both the groups were similar in respect of preoperative characteristics. Dynamic changes of interstitial concentrations of the measured analytes were found in both the patient groups (on-pump vs. off-pump) during the operation. There was no significant difference in dialysate concentrations of glucose and lactate between the groups. Significant differences were detected in pyruvate and glycerol interstitial concentrations, lactate/pyruvate ratio and lactate/glucose ratio between the on-pump vs. off-pump patients. In the Off-Pump Group, pyruvate concentrations were higher and the values of concentrations of glycerol lower. The lactate/pyruvate ratio and the lactate/glucose ratio, indicating the aerobic and anaerobic tissue metabolism status, were lower in the Off-Pump Group. There was no significant difference in dialysate concentrations of ethanol as a flow-marker during the surgery in either of the groups. There was no statistically significant difference between the groups (On-Pump Group vs. Off-Pump Group) comparing the postoperative clinical outcome (ICU stay, ventilation duration, length of hospital stay). The dynamic changes in the interstitial concentrations of the glucose, glycerol, pyruvate and lactate were found in both the groups of patients (On-Pump Group and Off-Pump Group), but there was no difference in local blood flow when the ethanol dilution technique was used. These results showed significantly higher aerobic metabolic activity of the peripheral tissue of patients in the Off-Pump Group vs. the On-Pump Group during the course of cardiac revascularization surgery. Results suggest that extracorporeal circulation, cardiopulmonary bypass, compromises peripheral tissue (skeletal muscles) energy metabolism. These changes have no impact on the postoperative clinical outcome; no significant difference between the groups was found.

Changes in metabolism and blood flow in peripheral tissue (skeletal muscle) during cardiac surgery with cardiopulmonary bypass: the biochemical microdialysis study.

The aim of this study was to monitor the metabolism and blood flow in the interstitium of the skeletal muscle during cardiac surgery with cardiopulmonary bypass (CPB) and in the early postoperative period by means of microdialysis and to compare metabolic changes during CPB at normothermia (NT) and hypothermia (HT). Surgical revascularization using CPB was performed in 50 patients, 25 patients (group HT) were operated using hypothermic CPB, 25 (group NT) using normothermic CPB. Interstitial microdialysis was performed by two CMA 60 probes (CMA Microdialysis AB, Solna, Sweden) inserted into the patient's deltoid muscle. Constituents analysed in the obtained dialysates, collected at intervals, were glucose, urea, glycerol and lactate. Tissue blood flow was monitored by dynamic microdialysis with gentamicin as a marker. In both groups, NT versus HT, similar dynamics of concentrations were found. Low initial concentrations were followed by gradual increases during CPB and in the following phase of the operation. Concentrations were higher in the NT group. Immediately after the operation, the decrease in values continued, with a gradual increase in the succeeding postoperative period in both groups. Similar dynamic changes in the lactate concentration were found in both groups. The gentamicin concentrations were lower in the NT group (versus the HT group). The results showed dynamic changes in the interstitial concentrations of glucose, urea, glycerol and lactate, which depend on the phase of the surgery in the CPB and early postoperative phase in the both groups of patients. Higher tissue perfusion of the skeletal muscle was noted in those patients operated on in normothermia. The dynamics of the concentration changes of these substances in the interstitium of the skeletal muscle has been proven to be caused by both the metabolic activity of the tissue and by the blood flow through the interstitium of the muscle.

[Interstitial microdialysis study of changes in metabolism and blood flow in skeletal muscles during cardiac surgery with normothermic and hypothermic extracorporeal circulation]. / Sledování zmen metabolismu a krevního prutoku v kosterním svalu behem srdecní operace v mimotelním obehu v normotermii a hypotermii pomocí intersticiální mikrodialýzy.

AIM: Hypoperfusion of peripheral tissues and splanchnic organs during cardiac surgery in extracorporeal circulation may lead to the origin of serious complications. The aim of the study was to monitor metabolism and blood pressure in interstital peripheral tissue, skeletal muscle, during the operation on the patient with extracorporeal circulation (ECC) in an early post-operation period by means of microdialysis. METHODS: The interstitial microdialysis is a minimally invasive method for the biochemical monitoring of metabolic changes and blood pressure in extracellular space of tissue. The substances in interstitium pass across a semipermeable membrane of the inserted microdialysis probe and may be analyzed. Microdialysis in this study was performed by means of two microdialysis probes CMA (CMA Microdialysis AB, Sweden) inserted into the deltoid muscle of the surgically treated patient. The probes were perfused by the Ringer solution at the rate of 0.3 ml/hour. The dialysates were sampled in the following intervals: beginning of the operation, beginning of ECC, end of ECC, end of the operation, two hours during the post-operation period. Standard biochemical methods were to evaluate, in the dialysates, glucose, urea, glycerol and lactate. The blood flow in the interstitium was monitored by means of dynamic microdialysis of gentamycine as a marker. Microdialysis was performed in 40 patients with ischemic heart disease, operated on in the extracorporeal circulation. In 20 patients the ECC was performed in normothermia (NT), while in the other 20 patients it was made in hypothermia (HT). RESULTS: In both groups, NT versus HT, a similar dynamism of interstitial concentration of the observed substances in relation to the operation phase and in early post-operation period. Low initial concentrations were gradually increasing during the extracorporeal circulation and increased further after the end of extracorporeal circulation and also in the subsequent phase of the operation. The concentration values of the analytes under observation were higher in the groups operated on under normothermia, apparently due to normal cellular activity during normothermia (versus values in hypothermia). Immediately after the operation the observed values decreased in the both groups and subsequently gradually increased in the post-operation period in the both groups. The trend of dynamic changes of the observed analytes, selected as compounds indicating metabolic activity of skeletal muscles during hypothermia documents a lower metabolic activity of the cells during hypothermia and its marked increase (against NT) in the phase of subsequent normalization of the tissue temperature. Analysis of the concentrations of lactate, as a compounds mapping anaerobic metabolism of skeletal muscle, revealed similar dynamic changes in the both groups (NT vs. HT). There were no significant differences, related to the phase of the operation or the phase of immediate post-operation course when the both groups were compared. The analysis of gentamycine concentrations as a flow marker revealed lower gentamycine concentrations in dialysate during the operation, ECC and the early post-operation course in the group operated on in normotheramia (vs. HT), indicating a higher tissue flow in skeletal muscle against the group of patients operated on under hypothermia. CONCLUSION: The results of the microdialysis study demonstrated dynamic changes in interstitial concentrations of the observed compounds (glucose, urea, glycerol and lactate) related to the phase of operation on the heart in extracorporeal circulation and in early post-operation period. A higher perfusion of skeletal muscle was documented in patients operated on under normothermia. It became obvious that the dynamism in the changes of the compounds observed in the interstitium of skeletal muscle was determined by metabolic activity of the tissue as well as by blood flow in the muscle interstitium.

[Surgical myocardial revascularization in a female patient with bilateral occlusion of the common carotid arteries]. / Chirurgická revaskularizace myokardu u nemocné s oboustranným uzáverem spolecných krkavic (kazuistika).

Concurrent stenosis of the coronary and carotid circulation is one of the most serious disease. The authors describe the case of a 74-year old female patient with ischaemic heart disease with concurrent bilateral occlusion of the common carotid arteries. Surgical revascularization of the myocardium was made using extracorporeal circulation under general normothermia with protection of the heart muscle from ischaemia by the method of warm blood cardioplegia. In the discussion the authors analyze contemporary possibilities of surgical treatment of ischaemic disease (MIDCAB, OPCAB, operations with extracorporeal circulation) and emphasize the advantages and risks of different procedures.

Changes of the serum antibiotic levels during open heart surgery (ceftazidim, ciprofloxacin, clindamycin).

BACKGROUND: Wound, mediastinal and intracardiac infections are still very serious complications of open-heart surgery. The incidence of it is still in the range of 0.4%-5%. The aims of our study were to assess the adequacy of regimen using ceftazidim (CTZ), ciprofloxacin (CPF) and clindamycin (CLIN) as prophylactic antibiotics and to verify whether cardiopulmonary bypass (CPB) can modify the time of antibiotic serum concentrations. That is why the serum levels of them were measured during open heart procedures. METHODS: The prospective study comprised 75 consequent coronary patients randomized in to three groups receiving 1 g of CTZ or 400 mg of CPF or 900 mg of CLIN i.v. with anesthesia induction. Routine coronary surgery with left internal mammary artery harvesting, moderate body hypothermic (30 degrees C) CPB with crystaloid cardioplegia was performed. Serum antibiotic levels were determined before application, with skin incision, prior CPB induction, after cardioplegia infusion, every 20 minutes of CPB, prior end of CPB, in time of chest closure. Conventional cylinder-plate microbiological assay was used for antibiotic level measurement. RESULTS: All serum antibiotic concentrations showed a sharp decrease immediately after starting CPB and lasted until CPB ended. After initiating of CPB after cardioplegia administration serum concentrations of CTZ (105 min after initial dose) decreased by, on average 55%, CPF (97 min) by 42% and CLIN (116 min) by 78%. CONCLUSION: CPB can modify the time course of antibiotic serum concentrations. The serum levels of CTZ at the end of the longest procedures were found to be below the MICs for some of the suspected pathogens. We recommend to use higher antibiotic doses for prophylaxis and to administer the second dose with protamin sulphate to obtain maximum concentration in newly formed blood clots.

Pyridine does not induce unscheduled DNA synthesis (UDS) in hepatocytes of male B6C3F1 mice treated in vivo.

Pyridine was evaluated in an in vivo/in vitro mouse DNA repair assay. Unscheduled DNA synthesis (UDS) was used as an indicator of DNA damage to hepatocytes from male B6C3F1 mice. Test animals were exposed by oral gavage to pyridine or to the vehicle or positive control articles, and hepatocytes were collected and labeled by incubation in media supplemented with [3H]thymidine. Following labeling, the cultures were processed for autoradiographic analysis. Doses were selected based on a pilot study in which 0, 250, 500, 750, 1000 or 2000 mg kg(-1) pyridine in water was administered by gavage. Mice in the 1000 and 2000 mg kg(-1) dose groups were comatose following dosing and died within 24 h of dose administration. Pyridine dose levels for the UDS determination were set at 175, 350 and 700 mg kg(-1). Pyridine solutions in water were administered to mice 2 or 16 h prior to the scheduled sacrifice. The vehicle control group received water 16 h before sacrifice and the positive control group received 10 mg kg(-1) dimethylnitrosamine (DMN) 2 h before sacrifice. Pyridine did not significantly increase the UDS response in hepatocytes isolated from the treated animals, as measured by the incorporation of [3H]thymidine, using standard criteria for a negative response: less than zero mean net grains in repair (NG) and <20% of cells in repair (% IR; cells in repair have at least 5 NG). The vehicle control group and the low, mid- and high pyridine dose groups yielded less than -8.3 NG and < or =1% IR. The positive control group yielded a positive UDS response, with 10.8 NG and 62% IR. These results indicate that pyridine is non-genotoxic in B6C3F1 mouse liver using the UDS endpoint.

[Kinetics of ceftazidime in prophylactic administration during cardiopulmonary bypass]. / Kinetika ceftazidimu pri profylaktické aplikaci u kardiopulmonálního bypassu.

Serum ceftazidime levels were followed in 21 patients in which routine coronary bypass surgery with cardiopulmonary bypass was performed. Each patient received one gram of ceftazidime intravenously with anesthesia induction. Antibiotic concentrations were estimated using the microbiologic assay diffusion plate method. The average operation time was 220 +/- 41 minutes (range 130-310). The start of cardiopulmonary bypass was 86 +/- 21 minutes and the full flow time was 104 +/- 21 minutes after starting of ceftazidime application. It can be stated that the decline of ceftazidime serum levels after starting of cardiopulmonary bypass was faster in comparison with standard serum curves of this antibiotic. The concentrations of ceftazidime at the end of some operations were under the supposed minimal inhibitory concentrations for some microorganisms possibly implicated. No infection was recorded.

[The post-perfusion syndrome after operations performed with extracorporeal circulation]. / Postperfuzní syndrom po operaci v mimotelním obehu.

Postperfusion syndrome (PPS) is a dreaded complication of cardiac surgery operation in extracorporeal circulation (ECC). Four factors play a key role in its pathophysiology: 1. contact of blood with the material of ECC, 2. release of activated leucocytes from pulmonary bed after the release of aortic cross-clamp, 3. translocation of endotoxin due to gut ischemia and its consequent reperfusion, 4. activation of coagulation, fibrinolytic, kallikrein-kinin and complement systems. The occurrence of PPS can be limited by reducing the ECC time and/or cross-clamp time, by using membrane oxygenator in the system of ECC, by using polyester or polypropylene in the set of ECC, by using heparin-coated set or leucocyte filter in the system of ECC, by application of pharmacological dosis of corticosteroids prior to the ECC, by early enteral nutrition. In eligible patients it is possible to eliminate the risk of PPS completely by using the operation without ECC. The experience of the authors with above mentioned problems is given and confronted with literature.

How long can the previously assembled cardiopulmonary bypass circuit stay sterile?

The sterility of previously assembled cardiopulmonary bypass circuits was investigated for 100 extracorporeal circuits. The closed circuits were assembled using aseptic technique and remained in the pump room until time of use. The mean time from point of setup to point of priming for the 100 consecutive circuits was 32 hours, with a range of 19 to 89 hours. Circuits were primed with the calculated volume of priming solution, circulated for 5 minutes and tested for microbial contamination by withdrawing 20 ml of the priming solution and 10 days incubated in Thioglycolate and Sabouraud culture mediums. All were found to be free of microbial contamination. The results of this investigation demonstrate that the sterility of the extracorporeal circuit, pre-assembled in advance of actual priming, can be maintained over an extended interval when standard aseptic technique is used. This allows the utilization of a pre-assembled circuit for emergency cardiopulmonary support.

[Personal experience with management of extracorporeal circulation in patients with asymptomatic carotid arteriosclerosis]. / Nase zkusenosti s vedením mimotelního obehu u nemocných s asymptomatickým arteriosklerotickým postizením karotického reciste.

The authors describe in following paper their own group of patients with asymptomatic but hemodynamically significant carotid disease operated under cardiopulmonary bypass on. Current studies recommend the "alpha-stat" regime as the optimal strategy of CPB conduction to ensure intraoperative cerebral protection. This study was done to verify this idea. 284 consecutive patients admitted for elective coronary surgery underwent a non-invasive Doppler scanning of extracranial carotid arteries. Twenty seven patients (10%) had significant carotid disease. All patients underwent coronary surgery as the first procedure. There was no cerebral complication in this group of patients and neurological status of these patients postoperatively was the same as it was preoperatively. This study shows that CPB in patients with hemodynamically significant carotid disease can be achieved without impairment of central nervous system with "alpha-stat" regime of perfusion.

A modified technique for the production of an arterio-venous shunt in sheep, allowing a comparison of biocompatibility of synthetic materials.

Polytetrafluorethylene (PTFE) prostheses were implanted in 12 sheep as a shunt between the carotid artery and the jugular vein using an end-to-side anastomosis technique. This technique allows repeated tests of the pharmacological and toxicological safety of artificial kidney units after both single and multiple administration. Furthermore, it enables the investigation of detoxification of compounds via dialysis, thus contributing to drug safety. Implantation of the prosthesis was uncomplicated. Connection to the extracorporeal circulation was achieved via catheters and maintained using a pump with an output of up to 300 ml/min. This enabled maintenance of extracorporeal circulation for several hours without clinical impairment to the animals. The AV-shunts remained functional for between 8 and 253 days (mean 112.3 days).

[Evaluation of statistical tables in surgery using a computer]. / Vyhodnocovanie statistickych tabuliek v chirurgii pocítacom.

The authors describe the possibility to use a computer in surgery for evaluation of classification tables of child injuries and the six-month report on the activity of policlinical and in-patient surgical departments. The solution of the problem was elaborated using system DOS-RV on a SM 4/20 computer.

Pyrophosphate scintigraphy and other non-invasive methods in the detection of cardiac involvement in some systemic connective tissue diseases.

Thirteen patients with systemic lupus erythematosus, 8 patients with polymyositis, and 6 patients with spondylitis ankylopoetica (Bechterew's disease) underwent clinical cardiologic examination and scintigraphy of the myocardium (99mTc-pyrophosphate), ECG, echocardiography, polygraphy, and their blood pressure was taken. The aim of the study was to ascertain how such a combination of non-invasive examinations can help in recognizing a cardiac involvement. In systemic lupus erythematosus cases one or more positive findings were revealed in 9 patients (69%), in 4 patients all examinations were negative (31%). Four patients (50%) with polymyositis had positive findings. In patients with spondylitis ankylopoetica positive findings occurred in 2 cases (33%). The study has shown that a combination of non-invasive cardiologic methods increases the probability of detecting cardiac involvement in systemic connective tissue diseases.