Comparison of biochemical markers of bone turnover in Paget disease treated with pamidronate and a proposed model for the relationships between measurements of the different forms of pyridinoline cross-links

We have compared the use of new markers of bone turnover in the assessment and treatment of Paget disease and made observations on the mechanisms of bone resorption. Urine hydroxyproline (Hyp) as a bone resorption marker and serum alkaline phosphatase (ALP) as a bone formation marker have traditionally been used to biochemically assess and monitor treatment of Paget disease. Hyp and total ALP were compared with total urine pyridinoline (Pyd) and deoxypyridinoline (Dpd), free urine Pyd and Dpd, urine type I collagen N-terminal cross-linked telopeptide (NTX), type I collagen C-terminal propeptide (PICP), serum osteocalcin, and bone ALP in Paget patients treated with pamidronate. Patients were divided into three biochemical severity-based treatment groups by their fasting urine hydroxyprolline excretion (HypE) levels (Le., group 1, HypE < 5.0 mumol/l of glomerular filtrate [GF]; group 2, HypE of 5.0-9.9 mumol/l of GF; group 3, HypE > 10 mumol/l of GF). Group 1 received one 60 mg intravenous infusion of pamidronate, and groups 2 and 3 received four and six 60 mg infusions at weekly intervals, respectively. Fasting serum and morning urine specimens were taken before and at 2, 6, 13, and 26 weeks after starting treatment. Baseline Z scores were used to compare separation of patient results from normal, and the difference in Z scores from baseline to 13 weeks was used to compare response to treatment. Baseline discrimination and response to treatment at all disease activity levels was greatest for NTX and was poor for osteocalcin, PICP, and C-terminal cross-linked telopeptide of type I collagen (ICTP). The other markers showed good discrimination and response at medium and high levels of disease activity. NTX, total Pyd and Dpd, free Pyd and Dpd, and ICTP are all pyridinoline cross-link-based markers, but discrimination and response by NTX was generally much greater than for the others. Determination of the mechanism of the difference between NTX and other cross-link measures is necessary for appropriate use of the markers and may also lead to a better understanding of the bone resorption process. It has been proposed that the greater sensitivity and discrimination of NTX is because it is more bone-specific than the other cross-link markers with significant amounts of free Pyd and Dpd coming from nonbone sources. We propose another model where the proportion of peptide-bound cross-links such as NTX may be increased in high bone turnover states partly due to a rate-limiting step in their degradation to free cross-links. Conditions with high bone resorption rates would have high levels of NTX that would decline rapidly when resorption rates fall to a level where the capacity to degrade NTX matches the rate of production.     

Diagnosis and pathophysiology of paradoxical embolism

A review of the experience with 134 consecutive patients with germinal cell testicular neoplasia indicates that definitively accurate staging of the malignancy at presentation is the single most important prognostic factor. Nearly two-thirds of the patients with all types of germinal malignancies survived or died of other causes and the highest survival rates were seen among patients with earlier stages of seminoma. In recent years patients with all types of germinal malignancies of the testis have been treated by radical retroperitoneal lymphadenectomy with enhancement of survivals. Patients with seminoma and non-seminomatous tumors exhibited increased survival rates with node dissection. In 66 consecutive lymphadenectomies the complication rate was less than 14 per cent, with only 1 death related to the operation. Retroperitoneal lymphadenectomy not only affords therapeutic advantage but also provides an opportunity for accurate surgical staging of disease and allows for rational decision relative to additional treatment, radiation therapy or chemotherapy.     

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.     

The surface-active compounds of a Pseudomonas sp. S-27 culture

Pseudomonas species S-27 was grown on various substrates. It was established that the Pseudomonas species S-27 strain can produce biosurfactants of ramnolipid nature decreasing the surface and interfacial tension to 29.2 and 0.05 mN/m. respectively, as well as a biopolymer stabilizing the emulsions with hydrocarbons and oils. The biosurfactant and bioemulsifier synthesis is shown to depend of the substrate nature.     

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.     

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.