B. Stinner

Carbonyl reduction of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by cytosolic enzymes in human liver and lung.

The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen, independent of the route and type of administration. There are competing metabolic activation and detoxification pathways. NNK is activated by alpha-hydroxylation at either the methyl or methylene carbonyl adjacent to the N-nitroso group to yield intermediates that methylate and pyridyloxobutylate DNA. Detoxification of NNK in humans usually occurs via carbonyl reduction to its hydroxy product NNAL, which undergoes glucuronosylation and final excretion. In vitro studies on NNK metabolism have usually been performed with tissue homogenates, microsomal fractions and/or purified microsomal enzymes, but cytosolic metabolism of NNK has been ignored until today. The results of this study demonstrate that cytosolic fractions of human liver and lung also participate in NNK metabolism. We provide evidence that a substantial degree of NNK carbonyl reduction occurs by cytosolic enzymes and that these enzymes may contribute to NNK detoxification in human liver and lung. The relative contribution of cytosolic vs. microsomal NNK carbonyl reduction is nearly identical in liver, whereas it is more than 3-fold higher in lung microsomes compared to lung cytosol. The inhibition profile suggested that mainly carbonyl reductase (EC 1.1.1.184) was active in cytosol of both organs. The expression of carbonyl reductase mRNA in liver and lung was proven by reverse transcription-(RT)-PCR. In conclusion, the results of this study provide the first data on cytosolic enzymes participating in NNK detoxification in human liver and lung.

Parathyroid Xenotransplantation without Immunosuppression in Experimental Hypoparathyroidism: Long-term In Vivo Function following Microencapsulation with a Clinically Suitable Alginate

Permanent hypoparthyroidism is one of the most difficult of all endocrine disorders to treat medically. Because this deficiency syndrome rarely is a life-threatening condition, systemic immunosuppression for recipients of transgenic transplants is not justified. An alternative would be protecting the tissue to be transplanted from the immunologic response (immunoisolation) by coating it with a semipermeable membrane- microencapsulation. Unfortunately, prior to the first clinical use, further analysis of the coating substance (alginate) demonstrated that it has potential cancerogenic properties. Using a purified amitogenic alginate suitable for clinical use, we accomplished allotransplantation in a long-term animal model and reported the first clinical cases without postoperative immunosuppression recently. In view of the potential clinical use, we investigated the ability of the microencapsulation technology with the novel amitogenic alginate in experimental hypoparathyroidism (80 parathyroidectomized rats) to enable transgenic transplantation across the highest immunologic barrier (xenotransplantation: human to rat) to ensure intact transplant function without immunosuppression. In a controlled, long-term animal study, the effect of microencapsulation on xenotransplanted human parathyroid tissue was evaluated over a period of 30 weeks (microencapsulated and naked hyperplastic parathyroid tissue, respectively). Functionally, human parathyroid tissue was able to replace that of rats. More than 6 months after xenotransplantation 32 of 40 animals that had received microencapsulated transplants were normocalcemic. In contrast, serum calcium concentrations dropped to postparathyroidectomy levels within 3 weeks in the animals that had received naked tissue only. Correspondingly, normocalcemic animals showed vital parathyroid tissue inside the microcapsules, which were surrounded by a small rim of fibroblasts. When combining microencapsulation with an improved tissue culture method, xenotransplantation of human parathyroid tissue and maintenance of its physiologic function is reproducibly achieved over the highest transplantation barrier. Using the amitogenic alginate may be a crucial step toward the first clinical use of this technique for parathyroid xenotransplantation without immunosuppression.

Expression and NNK reducing activities of carbonyl reductase and 11β-hydroxysteroid dehydrogenase type 1 in human lung

The tobacco specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK), which is found in high amounts in tobacco products, is believed to play an important role in lung cancer induction in smokers. NNK requires metabolic activation by cytochrome P450 mediated alpha-hydroxylation to exhibit its carcinogenic properties. On the other hand, NNK is inactivated by carbonyl reduction to its alcohol-equivalent 4-methylnitrosamino-1-(3-pyridyl)-1-butanol (NNAL) followed by glucuronidation and final excretion into urine or bile. Carbonyl reduction and alpha-hydroxylation are the predominant pathways in man, and it has been postulated that the extent of these competing pathways determines the individual susceptibility to lung cancer. Moreover, only a minor part of all habitual smokers develop lung cancer, suggesting the existence of susceptibility genes. Microsomal 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD 1) (EC 1.1.1.146) and cytosolic carbonyl reductase (CR) (EC 1.1.1.184) have been shown to be mainly responsible for NNAL formation in liver and lung. In the present study, we performed comparative investigations of human lung tissue samples from several patients with respect to the expression and activity of 11beta-HSD 1 and carbonyl reductase. We observed varying levels in 11beta-HSD 1 and carbonyl reductase expression in these patients, as revealed by RT-PCR and ELISA. Also, the tissue samples showed a different activity and inhibitor profile for both enzymes. According to our results, variations in the expression and activity of NNK carbonyl reducing enzymes may constitute a major determinant in the overall NNK detoxification capacity and thus may be linked to the great differences observed in the individual susceptibility of tobacco-smoke related lung cancer.

Granulocyte-colony stimulating factor in the prevention of postoperative infectious complications and sub-optimal recovery from operation in patients with colorectal cancer and increased preoperative risk (ASA 3 and 4)¶ Protocol of a controlled clinical trial developed by consensus of an international study group¶ Part one: rationale and hypothesis

Abstract:General design: Presentation of a new type of a study protocol for evaluation of the effectiveness of an immune modifier (rhG-CSF, filgrastim): prevention of postoperative infectious complications and of sub-optimal recovery from operation in patients with colorectal cancer and increased preoperative risk (ASA 3 and 4). This part describes the design of the randomised, placebo controlled, double-blinded, single-centre study performed at an university hospital (n = 40 patients for each group).¶Objective: The trial design includes the following elements for a prototype protocol:¶ - The study population is restricted to patients with colorectal cancer, including a left sided resection and an increased perioperative risk (ASA 3 and 4).¶ - Patients are allocated by random to the control or treatment group.¶ - The double blinding strategy of the trial is assessed by psychometric indices.¶ - An endpoint construct with quality of life (EORTC QLQ-C30) and a recovery index (modified Mc Peek index) are used as primary endpoints. Qualitative analysis of clinical relevance of the endpoints is performed by both patients and doctors.¶ - Statistical analysis uses an area under the curve (AUC) model for improvement of quality of life on leaving hospital and two and six months after operation. A confirmatory statistical model with quality of life as the first primary endpoint in the hierarchic test procedure is used. Expectations of patients and surgeons and the negative affect are analysed by social psychological scales.¶Conclusion: This study design differs from other trials on preoperative prophylaxis and postoperative recovery, and has been developed to try a new concept and avoid previous failures.¶

Early and late histamine release induced by albumin, hetastarch and polygeline: some unexpected findings

AbstractObjective: The perioperative use of colloidal plasma substitutes is still under discussion. We therefore conducted a prospective randomised study with three commonly used plasma substitutes to examine their histamine releasing effects in 21 volunteers. Material or subjects: 21 male volunteers were enrolled in this prospective, randomised, controlled clinical study. Endpoints were the incidence of early and late histamine release and the time course of the release kinetics. Normovolemic hemodilution technique was used with hydroxyethyl starch (n = 6), human albumin (n = 6) and polygeline (n = 9). Measurement and observation period was 240 min after the start of the plasma substitute infusion. Heart rate, blood pressure, SaO2, clinical symptoms/signs and plasma histamine were measured during the observation period. Results: The incidence of histamine release over the whole observation period in all three groups was 100%. Histamine release occurred frequently in all three groups until 30 min (50%–78%) and up to 240 min (late release reaction: 67%–83%) after the start of infusion. Surprisingly even hydroxyethyl starch, which is regarded as a generally safe and effective plasma substitute, caused high incidences of late histamine release (67%). Histamine release is a well known side effect of polygeline and – to a lesser extent – also of albumin, but was a novel finding for hydroxyethyl starch. Conclusions: We demonstrated for the first time histamine releasing effects of hydroxyethyl starch over a long period of time after administration. This perioperatively and for intensive care possibly relevant finding should make clinicians aware of late side effects not yet connected with the clinical use of these colloidal plasma substitutes.

Dependence of Positive Effects of Granulocyte Colony-stimulating Factor on the Antibiotic Regimen: Evaluation in Rats with Polymicrobial Peritonitis

We tested the hypothesis that the ability of granulocyte colony-stimulating factor (G-CSF) to prevent death from fecal peritonitis is influenced by the composition of the antibiotic regimen with which it is administered. We used a rodent model of polymicrobial peritoneal contamination and infection and the concept of clinical modeling randomized trials (CMRTs), which includes the conditions of randomized, clinical trials and complex clinical interventions (e.g., anesthesia, volume substitution, antibiotics, surgery, postoperative analgesia). With the peritonitis model we obtained a mortality dose-response curve that was sensitive to antibiotic prophylaxis. G-CSF was most efficacious when it was administered both prophylactically and after the onset of peritonitis. Cefuroxime/metronidazole, ofloxacin/metronidazole, and amoxicillin/clavulanate improved survival in combination with G-CSF best, whereas cefotaxime or ceftriaxone with and without metronidazole did not. G-CSF administration was associated with improved polymorphonuclear neutrophil phagocytosis and enhanced bacterial clearance. Pro-inflammatory cytokine release (tumor necrosis factor-a, interleukin-6, macrophage inflammatory protein-2) was decreased in plasma and in the peritoneal fluid. Their expression was lowered in various organs on the protein and mRNA level. The results were used to design a clinical trial to test the ability of G-CSF to prevent serious infections in patients with colorectal cancer surgery. In this trial G-CSF application and antibiotic prophylaxis were performed with the most effective scheduling and combinations (cefuroxime/metronidazole and ofloxacin/metronidazole) as defined here.

Histamine release during induction of anaesthesia and preparation for operation in patients undergoing general surgery: Incidence and clinically severe cases

Histamine release events were shown in a prospective randomized controlled trial in patients undergoing elective general surgery with an extraordinarily high incidence: 73 per cent. This high incidence was explained by several factors: — the sample size which was much greater than in previous studies — the improved plasma histamine assay — the precise definition of histamine release in clinical conditions and its measurement at the top of Bateman functions — the standardized induction of anaesthesia and preparation of the surgical patient — and finally the considerable number of cancer patients since more than 60% of the reactions >5 ng/ml occurred in this group which comprised only 20% of the study population.Two cases of life-threatening anaphylactoid reactions occurred in this trial corresponding to an incidence of 1 per cent. This was — again — very high compared to previous epidemiological studies. Both cases were again cancer patients and occurred in the placebo group — information given by the external study advisory group for further treatment of the individual patient.The data on the high incidence of histamine release including the high incidence of life-threatening reactions favourrationally a preoperative H1−+H2-prophylaxis with the drugs used in this study: dimetindene and cimetidine. The question of the incidence was one of the unsettled problems which led to this trial. Analysis of the first 180 patients already answered this question more than we had ever expected.

Histamine release in sepsis : A prospective, controlled, clinical study

OBJECTIVE To determine if histamine release occurs in clinical sepsis. DESIGN Prospective, controlled, clinical study. SETTING Interdisciplinary intensive care unit and trauma ward. PATIENTS Sepsis was confirmed in 20 patients (test group) by the criteria of the Veterans Administration Systemic Sepsis Cooperative Study Group (1987) and was verified by positive blood culture. In addition, patients were scored by the Elebute and Stoner Sepsis Score (1983), as modified by Dionigi et al (1985). A concomitant control group consisted of 20 postoperative patients with non-life-threatening trauma to the extremities and without signs of local or systemic infection. INTERVENTIONS Observational study. Blood samples were collected for determination of plasma histamine concentrations in both groups at the time of study entry and on five succeeding days. MEASUREMENTS AND MAIN RESULTS The patients were well matched, and the groups were not significantly different for all criteria known to influence histamine release. Comparison of the median values of each group on days 1 through 5 demonstrated significantly higher plasma histamine values in the test group on days 1 through 4, but these values were no longer significantly higher on day 5. While none of the nonseptic control patients achieved a plasma histamine concentration of > 1 ng/mL (the concentration of which was considered to be the pathologic cutoff point representing histamine release), these values (i.e., > 1 ng/ mL) were found in nine of 20 test group patients. In the test group, nonsurvivors (n = 9) had significantly higher plasma histamine concentrations than survivors (n = 11) throughout the whole study and eight of nine nonsurvivors showed a plasma histamine concentration of > 1 ng/mL. Correlation of plasma histamine concentrations on day 1 to sepsis severity (initial Sepsis Score) showed that all but one patient with a combined low Sepsis Score (< 20 points) and histamine concentration of < 1 ng/mL survived, while all patients with a Sepsis Score of > 20 points and histamine release (plasma histamine concentration of > 1 ng/mL) died. CONCLUSION Increased histamine concentrations were shown to be causally associated (contributory determinant) with sepsis.

Clinic modelling randomised trials (CMRT's) in animals as a new intermediate between biological experiments and randomised clinical trials: application to antihistamine prophylaxis in anaesthesia and surgery.

I. Celik, W. Lorenz, B. Stinner, D. Duda, H. Sitter, S. Sauer, A. Junge and M. Hoppe Institute of Theoretical Surgery, Philipps University, Baldingerstr., D-35033 Marburg, Germany, Fax +49 6421 288926 Department of General Surgery, Philipps University, Baldingerstr., D-35033 Marburg, Germany Clinic of Anaesthesiology, Johannes Gutenberg University, Langenbeckstr. 1, D-55131 Mainz, Germany Department of Trauma Surgery, Philipps University, Baldingerstr., D-35033 Marburg, Germany Department of Radiology, Philipps University, Baldingerstr., D-35033 Marburg, Germany

Perioperative nonspecific histamine release: a new classification by aetiological mechanisms and evaluation of their clinical relevance.

As a consequence of the performance of a randomized controlled clinical trial on perioperative histamine release and cardiovascular and respiratory disturbances, several types of increases in plasma histamine had to be distinguished instead of only two which existed at the beginning of the study: drug-induced allergic and pseudoallergic reactions. First of all, the new classification by aetiology (clinical epidemiology) was derived from a meta-analysis (secondary analysis) of the most recent literature. According to that histamine release in the perioperative period has several, different causes and is involved in several, different disease manifestations. A clear distinction (classification), however, is necessary if histamine release as an unwanted (adverse) effect has to be recognized, value judged according to its clinical relevance and therefore also prevented by histamine antagonists. Histamine release by neuro-endocrine and neuro-inflammatory mechanisms, cytotoxic histamine release and local, cytokine induced histamine release have been distinguished from pseudoallergic histamine release, but its functions are not yet clear. It has been analysed in prospective trials which used special clinical situations as models: patients on a normal ward or before and during upper GI endoscopy without premedication, but also in specific phases of laparoscopic cholecystectomy (trocar phase and dissection phase). Their existence in the clinical reality is now very likely, but new trials must investigate the pathophysiological effects such as in metabolism, coagulation, pulmonary haemodynamics (shunt volume) and gastric acid secretion. Histamine release by pseudoallergic mechanisms, however, was identified in the very vulnerable post-induction phase of anaesthesia up to skin incision. Its incidence was much higher than ever expected and its clinical relevance was demonstrated by the severity of reactions and the intervention strategies of the anaesthetists who were blinded concerning the type of the plasma substitute given and the prophylaxis with antihistamines. Pseudoallergic histamine release was clearly unwanted (adverse). Its occurrence in the other phases of anaesthesia has to be further evaluated in the tedious procedure of data analysis of the Mainz-Marburg-trial. The overall incidence of histamine release in the trial was so incredible high (72% of all patients, some of them with up to 4 episodes of histamine release) that a distinction between pseudoallergic (unwanted) and other types of histamine release (possibly less unwanted or even beneficial) is urgently needed. In the phase of steady state (maintenance) of anaesthesia the H1-(+)H2-prophylaxis was highly effective. Further analysis must show whether this is also the case during the phases of induction of anesthesia.(ABSTRACT TRUNCATED AT 400 WORDS)