An automatic system for the assessment of complex medium additives under cultivation conditions.

Complex medium additives such as yeast extract or peptone are often used in industrial cell culture processes to prolong cell growth and/or to improve product formation. The quality of those supplements is dependent on the preparation method and can differ from lot to lot. To guarantee consistent production these different lots have to be tested prior to use in fermentation processes. Because a detailed qualitative and quantitative analysis of all components of such a complex mixture is a very difficult task, another assessment method has to be chosen. The best way to evaluate the effect of such supplements is to monitor cell activity during real cultivation conditions with and without the added supplement lot. A bioreactor-based test system has been developed to determine the oxygen requirement of the cells as a response to the addition of a supplement to be tested under standardized conditions. Investigations were performed with a mouse-mouse hybridoma cell line and yeast extracts as an example for complex medium additives. The results showed differences in the impact between different extract lots and between different concentrations of an extract.

[Qualitative comparison of modified neurolept-, balanced and intravenous anesthesia. 2. Results of a clinical study, 1992]. / Qualitätsvergleich von modifizierter Neurolept-, balancierter und intravenöser Anästhesie. Teil 2. Ergebnisse der Krefelder Studie 1992.

The safety and tolerance of neuroleptanaesthesia (NLA), balanced anaesthesia (BAL), and intravenous anaesthesia with propofol (IVA) were analysed for the first time in a prospective, randomised clinical trial. METHODS. In all, 1318 surgical patients received either NLA, BAL, or IVA. Patients who had regional anaesthesia, were aged under 18 years, or were non-cooperative or vitally threatened (ASA class i.v. to V) did not participate in the study. Premedication and anaesthetic course were set up at a standard of 30% oxygen and 70% nitrous oxide. Incidents, events, and complications due to anaesthesia were obtained (IEC key of the German Society of Anaesthesia and Critical Care Medicine, DGAI). Furthermore, postanaesthetic alertness based on specific recovery tests and the quality of anaesthesia from the patient's viewpoint, rated by patient questionnaires from the DGAI were evaluated. All parameters were calculated and checked for statistical significance using the chi-square test. RESULTS AND DISCUSSION. The groups were broadly comparable with respect to age (P = 0.91), ASA class (P = 0.42), preoperative blood pressure (P = 0.36), and length of anaesthesia (P = 0.82). The anaesthesia, which averaged 103 min, comprised the following regimens: (1) NLA: 7.1 mg droperidol and 0.008 mg/kg body weight fentanyl, (2) IVA: 493.4 mg propofol and 0.004 mg/kg body weight fentanyl, and (3) BAL: 2.6 mg droperidol and 0.004 mg/kg body weight fentanyl with 0.4 vol.% isoflurane. With respect to anaesthetic risk, the following reactions were observed: the use of NLA led to a high incidence of tachycardia (P = 0.001), arrhythmias (P = 0.05), and hypertensive reactions (P = 0.001), whereas in the IVA group only hypotension (P = 0.0001) occurred. However, after the use of BAL none of the aforementioned complications were detectable to any considerable degree. Similarly, patients who had cardiac disease showed greater IEC changes after the use of NLA than after BAL or IVA (P = 0.02) (Tables 1 and 2). The heart rates and blood pressures during BAL and IVA were extremely stable, and therefore, vasoactive therapy was required considerably less in comparison to NLA (P = 0.001) (Table 4). Recovery after the use of IVA was strikingly rapid: the patient's responsiveness, orientation, and ability to concentrate was significantly better than after the other anaesthetic regimen (P = 0.01) (Table 5). With regard to the typical discomforts after anaesthesia, IVA was highly superior to BAL and NLA: nausea (P = 0.0003) and retching (P = 0.03) hardly ever occurred (Table 6). Due to the tolerable manner of waking up and rapid return of orientation and the ability to concentrate, IVA was highly favoured by the patients (P = < 0.01) (Table 7). CONCLUSION. The present results show clear clinical advantages of BAL and IVA in contrast to neuroleptanaesthesia. Due to the very low incidence of side effects such as nausea and vomiting IVA was highly recommended by the patients, at least in part because of the rapid recovery time.

[Quality comparison of modified neurolept-, balanced and intravenous anesthesia. 1. Study design and patient analysis of the Krefelder study 1992]. / Qualitätsvergleich von modifizierter Neurolept-, balancierter und intravenöser Anästhesie. Teil 1. Studiendesign und Patientenanalyse der Krefelder Studie 1992.

The choice of appropriate anaesthesia in a more or less seriously ill patient requires detailed information on the risk and tolerance of each specific anaesthetic regimen. The objective of this prospective, randomised clinical trial was to test the hypothesis that three regimens of general anaesthesia--neurolept-(NLA), balanced (BAL), and intravenous propofol anaesthesia (IVA)--differ with regard to safety and comfort. The criteria for the intraoperative safety and postoperative comfort of the patients were the incidents, events and complications (IEC) that required medical treatment as well as the evaluation of postoperative complaints by the patients according to the IEC list and patient questionnaires of the German Society of Anaesthesia and Critical Care Medicine (DGAI). METHODS. The study duration was about 4 months, from January to April 1992. During this period the patients of all nine operative departments of the hospital received strictly randomised NLA, BAL, or IVA. Patients who had regional anaesthesia or were not capable of understanding the German language, were nonco-operative, or were seriously ill (ASA class IV to V) as well as children under 18 years of age did not participate in the study. All eligible patients provided their informed consent. ANAESTHESIA. For premedication 10 mg chlorazepate was administered the night before and on the day of surgery. Anaesthesia was conducted under normoventilation using a mixture of 70% nitrous oxide and 30% oxygen. NLA patients were induced intravenously with 0.2 mg/kg body weight etomidate and received 0.005 mg/kg fentanyl and 0.07 mg/kg droperidol before the start of surgery. The repetition dose was 0.2 mg fentanyl and 2.5 mg droperidol. In the BAL patients the dose of fentanyl and droperidol was reduced to 50% due to the addition of isoflurane up to 1 vol. %. IVA patients received 2 mg/kg propofol over 3 min followed by an infusion of 3-5 mg/kg per hour together with 0.2 mg fentanyl/h. Neuromuscular blockade was accomplished with vecuronium 0.1 mg/kg. If the blood pressure and heart rate increased by more than 20% of preoperative values, analgesia was reinforced by an additional fentanyl dose. Anaesthesia was subsequently enhanced by increasing the neurolept/propofol/isoflurane dose by up to 50%. DATA COLLECTION. The following parameters were registered: patients' personal data and physical condition according to ASA classification; the grade of risk according to the Munich risk checklist; the frequency of IEC during surgery; the patients' permanent medications; postanaesthetic vigilance and recovery; the acceptance of the assigned anaesthetic by the physician; the cost of the anaesthetic used; and pre- and post-operative complaints as well as the assessment of anaesthesia by the patient. The statistical evaluation was performed using the chi-square test. RESULTS. A total of 1,346 patients were enrolled in the study; 28 (2%) were excluded because the treatment protocol was changed by the anaesthesiologist. Seventy per cent were recruited from general, gynaecologic, or otorhinolaryngologic surgery. The three anaesthetic regimens (NLA, BAL, and IVA) were used in other departments with the same frequency with the exception of ophthalmology and urology (P > 0.1) (Fig. 1). Of the 1,318 eligible patients, 443 received NLA, 443 BAL, and 432 IVA (P = 0.8). The distribution of the various parameters was surprisingly similar among the three groups: the average age was 50 years (P = 0.91), body weight 71 kg (P = 0.33), reference or initial blood pressure 130/80 mm Hg (P = 0.36), average time of anaesthesia 103 min (P = 0.82), and all had the same risk score (P = 0.42). Sixty per cent were female. An average of 85% of the 18- to 89-year-old patients were considered to be healthy according to the ASA risk classification (P = 0.42). However, on applying the Munich risk checklist the average number of healthy individuals was 5% to 10% lower than that of the ASA risk classification.

Optimization of the growth conditions of Sf21 insect cells for high-density perfusion culture in stirred-tank bioreactors.

Spodoptera frugiperda insect cells (IPLB-Sf21-AE) (Sf21), infected with baculovirus expression vectors during their exponential growth phase, are commonly used to produce a variety of heterologous recombinant proteins. In the present study the culture conditions of these insect cells were studied to establish high-density suspension cultures with prolonged exponential growth phases. The Sf21 cells were grown in 125-ml spinner flasks using five different culture media supplemented with 5% fetal calf serum and four protein-free or low-protein culture media. The best results were achieved in EX-CELL 401 (protein-free media) and in IPL-41 modified with 2.5 g l-1 tryptose phosphate broth (serum-supplemented media), respectively. The latter was used for further batch and continuous cultivation of Sf21 cells in a perfused 1.4-l stirred-tank bioreactor with special attention to the oxygen requirement of these cells. Optimal growth was found at an oxygen concentration of 70% air saturation, resulting in a prolonged exponential growth phase that could be maintained for more than 16 days. A maximum cell density of 5.5 x 10(7) viable cells ml-1 was achieved.

[Double-guided vacuum trephine system "Asmotom"]. / Doppelt geführtes Vakuumtrepansystem "Asmotom".

A new, doubly guided cornea suction trephine system is presented. By means of a suction rim placed on the limbus corneal and a suction disc in the centre of the cornea the vault of the cornea is preserved throughout the trephination procedure. Cutting is performed by a motor trephine, which rotates between the inner and the outer suction. The system allows creation of an absolutely safe perforation in a 360 degrees cut. The cut edges are precise and sharp. Both donor and recipient are trephined from the epithelial side. The endothelial cell loss is very low, amounting to 0.170 mm. The suction stabilization prevents any tilting of the trephine and avoids elliptical forms. Preliminary clinical experiences are presented.

A comparative investigation of the metabolism of methyl bromide and methyl iodide in human erythrocytes.

Human erythrocyte cytoplasm was incubated in head space vials with either methyl bromide or methyl iodide. The decline in concentration of the two methyl halides was monitored by gas chromatography. Simultaneously, the production of S-methylglutathione was determined by thin layer chromatography. In parallel experiments, boiled erythrocyte cytoplasm was used in order to determine non-enzymatic conjugation. Furthermore, inhibition experiments with sulfobromophthalein were performed. The results were compared with previous findings on the metabolism of methyl chloride. In contrast to methyl chloride, both methyl bromide and methyl iodide showed a significant non-enzymatic conjugation with glutathione. In addition, an enzymatic conjugation could be observed in the erythrocyte cytoplasm of the majority of the population, whereas a minority lacks this enzymatic activity. This is consistent with findings on methyl chloride. Inhibition experiments show that a minor form of the erythrocyte glutathione transferase may be responsible for the enzymatic conjugation. Of the three monohalogenated methanes, methyl bromide is the substrate with the highest affinity for the conjugating enzyme(s). In the case of methyl iodide, non-enzymatic reaction overweighs the enzymatic process. There are possible implications of the results for occupational health and the toxicity of the substances.

Glutathione conjugation and cytochrome P-450 metabolism of methyl chloride in vitro.

Possible carcinogenic properties of methyl chloride (CH(3)Cl) have been under discussion since an increase of renal tumours was observed in male B6C3F(1) mice after a 2-yr inhalation exposure to the substance. This was, however, only observed following exposure of male mice to the highest concentration level and not after exposure of females or F344 rats of both sexes. Accumulation of formaldehyde in the kidneys was thought to be responsible for tumour production. In the experiments presented here, cytosolic enzymes from the liver and kidneys of different mouse strains and F344 rats were incubated in head-space vials with methyl chloride or methyl bromide. Following equilibration, the decrease in the concentration of the gases was monitored by gas chromatography as a parameter for metabolic elimination. The metabolic turnover of the methyl halides was found to be significantly higher in female animals than in the males. In parallel experiments, the glutathione content of the liver and kidneys of mice exposed by inhalation to 1000 ppm methyl chloride was determined. In both organs, the glutathione content diminished rapidly after exposure to the methyl halide. The glutathione depletion was slightly greater in females than in males. Finally, the content of cytochromes P-450, P-420 and b5 was determined in liver and kidneys of different mouse strains by difference spectroscopy. Female mice were found to have a lower content of P-450 and b5 than males in the kidneys; there was no such sex difference in liver tissue. The results show that a sex difference in metabolism is unlikely to be responsible for the unique kidney tumour production in male B6C3F(1) mice. Other possible explanations are discussed.

Experiences with a new corneal suction trephine.

The importance of exact trephining for successful keratoplasty is well known. We employ a new suction trephine, thus obtaining exact and sharp cutting margines which are examined by scanning electron microscopy (SEM). The first observed clinical results in 25 cases are good.

Concentration-dependent depletion of non-protein sulfhydryl (NPSH) content in lung, heart and liver tissue of rats and mice after acute inhalation exposure to butadiene.

The effects of different exposure concentrations of butadiene on the cellular non-protein sulfhydryl (NPSH) content of liver, lung and heart tissue were investigated in B6C3F1 mice and Sprague-Dawley rats. Groups of male animals of both species were exposed for 7 h to 10, 50, 100, 250, 500, 1000 and 2000 ppm butadiene. Immediately after exposure, NPSH content of liver, lung and heart tissue was determined according to a modified Ellman procedure. A comparison of both species shows that a dose-dependent NPSH depletion can be observed in mice for all tissues examined. In rats, liver NPSH content shows a major reduction at high exposure concentrations only. In mice, depletion of NPSH content of liver, lung and heart tissue starts at exposure concentrations of about 250 ppm butadiene. A reduction in NPSH content of about 80% is observed for lung tissue at 1000 ppm and for liver and heart tissue at exposure concentrations of 2000 ppm butadiene. The data on tissue concentrations of NPSH obtained after exposure of rats and mice to butadiene reflect the quantitative differences in butadiene metabolism and in biological effectivity of reactive butadiene intermediates between both species.

Metabolism of methyl chloride by human erythrocytes.

Erythrocyte cytoplasm of rats, mice and humans was incubated in head space vials with methyl chloride and the decline in concentration of the substance monitored as a parameter of metabolism. The production of S-methylglutathione was controlled by tlc. Rats, mice, bovines, pigs, sheep and rhesus monkeys showed no conversion of methyl chloride in erythrocyte cytoplasm. About 60% of the human blood samples showed a significant metabolic elimination of the substance (conjugators), whereas about 40% did not (non-conjugators). The production of S-methylglutathione indicated enzymatic metabolism of the substance by glutathione S-transferases. In literature, a "major" and "minor" form of human erythrocyte glutathione S-transferase has been described. The results indicate that the "minor" form is probably responsible for the unique metabolism of methyl chloride in human erythrocytes.