Enzyme kinetic modelling as a tool to analyse the behaviour of cytochrome P450 catalysed reactions: application to amitriptyline N-demethylation

1. To determine kinetic parameters (Vmax, K(m)) for cytochrome P450 (CYP) mediated metabolic pathways, nonlinear least squares regression is commonly used to fit a model equation (e.g., Michaelis Menten [MM]) to sets of data points (reaction velocity vs substrate concentration). This method can also be utilized to determine the parameters for more complex mechanisms involving allosteric or multi-enzyme systems. Akaike's Information Criterion (AIC), or an estimation of improvement of fit as successive parameters are introduced in the model (F-test), can be used to determine whether application of more complex models is helpful. To evaluate these approaches, we have examined the complex enzyme kinetics of amitriptyline (AMI) N-demethylation in vitro by human liver microsomes. 2. For a 15-point nortriptyline (NT) formation rate vs substrate (AMI) concentration curve, a two enzyme model, consisting of one enzyme with MM kinetics (Vmax = 1.2 nmol min-1 mg-1, K(m) = 24 microM) together with a sigmoidal component (described by an equation equivalent to the Hill equation for cooperative substrate binding; Vmax = 2.1 nmol min-1 mg-1, K' = 70 microM; Hill exponent n = 2.34), was favoured according to AIC and the F-test. 3. Data generated by incubating AMI under the same conditions but in the presence of 10 microM ketoconazole (KET), a CYP3A3/4 inhibitor, were consistent with a single enzyme model with substrate inhibition (Vmax = 0.74 nmol min-1 mg-1, K(m) = 186 microM, K1 = 0.0028 microM-1). 4. Sulphaphenazole (SPA), a CYP2C9 inhibitor, decreased the rate of NT formation in a concentration dependent manner, whereas a polyclonal rat liver CYP2C11 antibody, inhibitory for S-mephenytoin 4'-hydroxylation in humans, had no important effect on this reaction. 5. Incubation of AMI with 50 microM SPA resulted in a curve consistent with a two enzyme model, one with MM kinetics (Vmax = 0.72 nmol min-1 mg-1, K(m) = 54 microM) the other with 'Hill-kinetics' (Vmax = 2.1 nmol min-1 mg-1, K' = 195 microM; n = 2.38). 6. A fourth data-set was generated by incubating AMI with 10 microM KET and 50 microM SPA. The proposed model of best fit describes two activities, one obeying MM-kinetics (Vmax = 0.048 nmol min-1 mg-1, K(m) = 7 microM) and the other obeying MM kinetics but with substrate inhibition (Vmax = 0.8 nmol min-1 mg-1, K(m) = 443 microM, K1 = 0.0041 microM-1). 7. The combination of kinetic modelling tools and biological data has permitted the discrimination of at least three CYP enzymes involved in AMI N-demethylation. Two are identified as CYP3A3/4 and CYP2C9, although further work in several more livers is required to confirm the participation of the latter.     

Costs and charges associated with three alternative techniques of hysterectomy

BACKGROUND: Many hysterectomies are now performed by a laparoscopically assisted vaginal technique. This procedure is controversial, partly because of concern about cost. We studied hospital charges and costs for the procedure as compared with those for total abdominal hysterectomy and total vaginal hysterectomy in clinically similar groups of patients. METHODS: From hospital-discharge data and patients' charts, we identified hysterectomies performed in 1993 and 1994 by 96 surgeons at a community teaching hospital to treat benign conditions. The patients were grouped according to the surgical procedures performed in conjunction with the hysterectomy. Data on hospital charges and cost-to-charge ratios for 64 hospital cost centers were used to assess charges and costs for specific resources, as well as for the hospitalization overall. RESULTS: Of 1049 patients studied, 26 percent underwent laparoscopically assisted vaginal hysterectomy, 54 percent underwent abdominal hysterectomy, and 20 percent underwent vaginal hysterectomy. The average hospital stays were 2.6, 3.9, and 2.9 days, respectively, and the mean total charges (facility charges plus professional fees) for the hospitalizations were $6,116, $5,084, and $4,221 (P<0.001 for the comparison of the laparoscopic technique with both other techniques). The mean facility costs were $4,914, $3,954, and $3,116, respectively (P<0.001 for the same comparison), with similar findings in all subgroups. The higher charges and costs for laparoscopically assisted vaginal hysterectomy were due to higher supply costs, particularly when disposable supplies were used, and to longer operating-room time. CONCLUSIONS: Despite shorter hospital stays, in-hospital charges and costs for laparoscopically assisted vaginal hysterectomy are higher than for either alternative procedure, because of the disposable supplies that are typically used and the longer operating-room time.     

Human T-cell receptor V beta gene polymorphism and multiple sclerosis

Population-based genetic associations have been reported between RFLPs detected with probes corresponding to the genes encoding the beta chain of the T-cell receptor for antigen (TCRB) and a variety of autoimmune disorders. In the case of multiple sclerosis (MS), these studies have localized a putative disease-associated gene to a region of approximately 110 kb in length, located within the TCRB locus. In the current study, all 14 known TCRBV (variable region) genes within the region of localization were mapped and identified. The nucleotide sequences of these genes were determined in a panel of six MS patients and six healthy controls, who were human-leukocyte antigen and TCRB-RFLP haplotype matched. Nine of the 14 TCRBV genes studied showed evidence of polymorphism. PCR-based assays for each of these polymorphic genes were developed, and allele and genotype frequencies were determined in a panel of DNA samples from 48 MS patients and 60 control individuals. No significant differences in allele, genotype, or phenotype frequencies were observed between the MS patients and controls for any of the 14 TCRBV-gene polymorphisms studied. In light of the extensive linkage disequilibrium across the region studied, the saturating numbers of polymorphisms examined, and the direct sequence analysis of all BV genes in the region, these results suggest that it is unlikely that germ-line polymorphism in the TCRBV locus makes a major contribution to MS susceptibility.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experience in the undergraduate training of ophthalmologists

The authors present their experience gained in the training of ophthalmologists at the Chair of Ocular Diseases of the Krasnoyarsk Medical Institute. In 1991 the interns, instead of traditional state examinations, maintained their diploma research, which was assessed as their skills and knowledge in the field of ocular diseases, social hygiene, and public health organization. The authors prove that such diploma research is an integral part of undergraduate training and one of the types of individual work at higher educational institutions, and enumerate the topics of diploma research.     

Catastrophic complications of the cervical esophagogastric anastomosis

Recent enthusiasm for the cervical esophagogastric anastomosis has arisen because of its perceived low morbidity. Although catastrophic complications of a cervical esophagogastric anastomosis are unusual, they can and do occur, and prevention is possible if the potential for them is recognized. Among 856 patients undergoing a cervical esophagogastric anastomosis after transhiatal esophagectomy, catastrophic cervical infectious complications occurred in 11 patients (1.3%): vertebral body osteomyelitis (1), epidural abscess with neurologic impairment (2), pulmonary microabscesses from internal jugular vein abscess (1), tracheoesophagogastric anastomotic fistula (1), and major dehiscence necessitating anastomotic takedown (6). These complications became manifest from 5 to 85 days after the esophageal resection and reconstruction (mean 19 days). Leakage from a gastric suspension stitch placed in the anterior spinal ligament over the vertebral bodies resulted in a posterior gastric leak and either osteomyelitis or an epidural abscess in three patients, none of whom had evidence of extravasation on the routine barium swallow 10 days after operation. Cervical exploration for a presumed anastomotic leak led to the unexpected discovery of an abscess formed by the stomach and the adjacent wall of the internal jugular vein, which was ligated and resected. One patient without symptoms who was discharged from the hospital with a contained anastomotic leak on the postoperative barium swallow was readmitted 7 days later with a cervical tracheoesophagogastric anastomotic fistula of which he ultimately died. In 6 patients (7% of those who had anastomotic leaks) there was sufficient gastric ischemia or necrosis, or both, to necessitate takedown of the anastomosis and intrathoracic stomach, cervical esophagostomy, and insertion of a feeding tube. As a result of this experience, it is recommended that cervical gastric suspension sutures either be omitted entirely or placed in the fascia over the longus colli muscles anterior to the spine, but not directly into the prevertebral fascia overlying the vertebral bodies or cervical disks. All but minute cervical anastomotic leaks, even if apparently contained, are best drained rather than treated expectantly. Patients who remain febrile and ill after bedside drainage of a cervical esophagogastric anastomosis leak should undergo cervical reexploration in the operating room; major gastric ischemia or necrosis, or both, may warrant takedown of the anastomosis and intrathoracic stomach.     

Hyperthermia following MDMA administration in rats: effects of ambient temperature, water consumption, and chronic dosing

In two experiments it was found that the hyperthermia which follows MDMA ("Ecstasy") results from an interaction of direct pharmacological effect of the drug and the prevailing environmental conditions in which it is administered. In Experiment 1, rats given fixed doses of either 2.5, 5.0 or 7.5 mg/kg MDMA or saline were injected on different days at ambient temperatures (Ta's) of 11, 24, and 30 degrees C. At each Ta drinking water was freely available following dosing on one session and temporarily unavailable on a second. The hyperthermic and hyperkinetic responses were monitored using remote biotelemetry. Experiment 2 used a between-subject design in which each group of rats received a standard 7.5 mg/kg dose of MDMA administered at only one of the three levels of Ta(24 degrees C) and at only one level of the water-availability factor. Dosing in some groups was continued for a further 13 days to test for tolerance or sensitization effects. Ambient temperature significantly affected the magnitude of the hyperthermia but not the hyperkinesis. Water deprivation during the drugged period significantly augmented the hyperthermia, but only in the high Ta (30 degrees C.) condition. Chronic dosing produced sensitization of both hyperthermic and hyperkinetic responses. The findings indicate that ambient temperature, water consumption and frequency of drug use affect the hyperthermia which follows MDMA administration.     

Hypoxaemia and release of endothelin-1

BACKGROUND: Secretion of the vasoconstrictor peptide endothelin-1 from vascular endothelium is increased by various stimuli. Whether hypoxaemia affects plasma levels of endothelin-1 in humans is unknown, but this may be important in the haemodynamic response to hypoxaemia. The plasma endothelin-1 concentrations in hypoxaemic humans has therefore been measured. METHODS: Plasma levels of endothelin-1 were measured by specific radioimmunoassay in 10 control subjects at rest and following 30 minutes of acute hypoxaemia (SaO2 75-80%) induced by breathing a nitrogen/oxygen mixture, and in 10 patients with hypoxaemic cor pulmonale. RESULTS: The plasma endothelin-1 concentration in control subjects was increased from a mean (SE) of 0.90 (0.11) pmol/l at baseline to 2.34 (0.34) pmol/l during hypoxaemia. In patients with cor pulmonale the plasma endothelin-1 concentration was 2.96 (0.34) pmol/l, raised in comparison with control subjects at rest but similar to levels in controls during hypoxaemia. CONCLUSIONS: Plasma levels of endothelin-1 were increased by hypoxaemia in humans. The raised levels observed in patients with cor pulmonale may largely be attributable to the effects of hypoxaemia, although the pathophysiological significance of these observations remains to be established.