Standardization and potential use of HPLC for detection of cellular placental alkaline phosphatase using established tumour cell lines and fresh tumour biopsies

Placement alkaline phosphatase (PLAP) is one of the cellular phosphatases (ALP) expressed in patients with testis cancers, particularly in seminomas. Using various techniques including Western blot and high-performance liquid chromatography (HPLC) systems and ATC2, a newly developed specific anti-PLAP monoclonal antibody (Mab), the presence of active form of PLAP in lysates prepared from testis tumour fragment and tumour cell lines, was studied. This was carried out following isolation of PLAP from biological samples using CNBr Sepharose-conjugated ATC2 beads. The results showed that: (1) The target for the newly developed Mab ATC2 was PLAP. (2) The ATC2-conjugated bead system was an efficient method for isolating pure PLAP. (3) Diethylamine (DEA), in contrast to urea and glycine, was the most efficient for separation of PLAP from ATC2-conjugated beads, as the isolated molecule did not lose any phosphatase activity and there was very little uncoupling of the ATC2 Mab from the beads. (4) ATC2-conjugated CNBr beads could pick up PLAP from a solution containing standard PLAP and lysates prepared from tumour cell lines or testis tissue fragments positive for the PLAP. (5) HPLC profile of testis tumour lines and testis tumours showed two distinct peaks with ALP activity, one at retention time 7-8 min (corresponding to 95 kDa molecule) and one at 12-13 min corresponding to 70 kDa molecule). These data demonstrated the potential use of various biochemical methods in combination with HPLC for isolation of the fully functional molecules with ALP activity from different samples including lysates prepared from patients with testis cancer. The nature of ALP activity at 95 kDa is being investigated as no such molecule has been reported previously. These techniques might have an important implication for an early detection of germ cell tumours, particularly in patients with equivocal ultrasound.     

Reproducibility of lumbar paraspinal surface electromyogram power spectra

OBJECTIVE: To establish the reproducibility of surface recordings of electromyograms from lumbar erector spinae muscles. DESIGN: Repeated measurements in 10 male volunteers under controlled conditions. METHODS: While isometric load was held constant at (2/3) and (1/3) of maximum voluntary contraction, surface electromyograms were recorded from the 4th/5th lumbar interspace on three occasions separated by at least a day. Fresh surface electrodes were applied on each test occasion. The raw electromyographic signal was filtered and digitized and the signal processed by fast Fourier transformation to give median frequency and total amplitude spectra. Linear regression lines of median frequency and amplitude against time were calculated. The 'spectral halfwidth" was defined as the full width at half maximum of the composite amplitude spectrum. Reproducibility was assessed by calculation of the intraclass correlation coefficient. RESULTS: Reproducibility of initial median frequency and spectral halfwidth were found to be excellent at both (2/3) (ICCC 0.91 and 0.88) and (1/3) (ICCC 0.84 and 0.83) maximum voluntary contraction. Initial power and regression slopes of power and median frequency were not reproducible. CONCLUSIONS: The reproducibility of two electromyographic variables, the initial median frequency and the spectral halfwidth, were found to be satisfactory.     

The 11th Meeting of the European Society for Paediatric Infectious Diseases (ESPID) 26–28 May 1993 Helsinki,Finland

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The 11th Meeting of the European Society for Paediatric Infectious Diseases (ESPID) 26–28 May 1993 Helsinki, Finland     

The effects of clofibrate on plasma glucose,lipoproteins, fibrinogen,and other biochemical and haematological variables in patients with mature onset diabetes mellitus

Summary We have studied the effects of clofibrate treatment on glucose tolerance and plasma insulin, plasma triglyceride, cholesterol and non-esterified fatty acid (NEFA) levels, and on various haematological variables (including plasma fibrinogen level, red cell flexibility, whole blood viscosity, and plasma -thromboglobulin level) in patients with mature-onset diabetes. Twenty-two patients (11 men and 11 women) were randomly allotted to treatment with clofibrate, 1 g twice daily, or a corn-oil placebo for 12 weeks, and then changed to the alternate medication for another 12 weeks. Half the patients took clofibrate in the first 12 weeks of the study, and half took the placebo. The patients stayed on their usual diet, and 13 also took tolbutamide before and during the trial. The trial was double-blind. At the beginning, middle and end of the trial fasting measurements were made, and plasma glucose, insulin, triglyceride, and NEFA concentrations were then measured repeatedly during the next 8 h (from 8.00 a. m. to 4 p. m.), to allow calculation of the mean 8-h concentration of these substances. In general, plasma concentrations of glucose, triglyceride, cholesterol, NEFA and fibrinogen were lower when the patients were taking clofibrate then when they were taking the corn-oil placebo, but higher when taking the placebo than at entry to the trial. We favour the explanation that clofibrate has lowered these concentrations, when compared with the placebo. The alternative interpretation, that 2 g per day of the placebo increases plasma concentrations of glucose, triglyceride, cholesterol, NEFA and fibrinogen, and that clofibrate has little effect, seems unlikely. The first interpretation, that clofibrate has a positive effect when compared with an inert placebo, has been adopted when interpreting the results. Clofibrate treatment led to a 15% lower fasting blood glucose level, and 11% lower mean 8-h glucose concentration than did placebo (p<0.01) but it did not significantly change plasma insulin concentration. The fasting and mean 8-hour concentrations of plasma triglyceride and fasting plasma cholesterol concentrations were reduced by clofibrate (by 44%, 33% and 10% respectively, p<0.05). Clofibrate decreased the fasting plasma NEFA level by 27% (p<0.01), and the mean 8-h plasma NEFA concentration by 23% (p<0.05). A weak relationship between the mean 8-h levels of plasma NEFA and plasma glucose (r=0.49, p<0.05) was consistent with the suggestion that the change in plasma glucose could, in part, be due to a change in NEFA concentration. The mean plasma fibrinogen concentration was decreased 23% by clofibrate (p<0.01). There was a positive correlation between the observed decrease during treatment and the baseline fibrinogen concentration (r=0.80, p<0.001), i. e. the greatest decrease occurred in those subjects with the highest plasma fibrinogen concentrations. Whole blood viscosity fell slightly, but erythrocyte flexibility was not significantly changed by clofibrate. The mean haemoglobin concentration and leucocyte count fell slightly during clofibrate treatment and the platelet count rose. -thromboglobulin was not affected. Clofibrate treatment was associated with rises in plasma albumin, urea, creatine kinase and aspartate aminotransferase, and falls in plasma bilirubin, -glutamyl-transpeptidase and alkaline phosphatase. Most of these changes occurred within the reference range.     

Determination of glomerular size-selectivity in the normal rat with Ficoll.

Diffusion studies in vitro indicate that Ficoll behaves more like an ideal spherical molecule than does dextran, suggesting that Ficoll would be a better probe of glomerular pore size than the commonly used dextran. To examine the differences between these macromolecules in vivo, the fractional clearances of tritiated Ficoll and dextran were measured over a wide range of molecular sizes (Stokes-Einstein radius, rs, from 19 to 65 A) in normal euvolemic Munich-Wistar rats. Whole-kidney and single-nephron hemodynamic conditions were characterized through a combination of clearance and micropuncture measurements. The fractional clearance, or sieving coefficient (theta), for dextran significantly exceeded that of Ficoll at all molecular sizes examined, theta for dextran being approximately 10 times that for Ficoll for rs greater than 30 A. Thus, the results with Ficoll imply a more size-restrictive barrier than do the results with dextran. The values of theta for Ficoll approximated previously reported values for uncharged globular proteins. Although theta for Ficoll at rs = 35 A was much smaller than the corresponding value for dextran, it was still approximately 30 times greater than typical values of the filtrate-to-plasma concentration ratio reported for serum albumin (a polyanion) in the rat, in agreement with the concept that glomerular charge-selectivity normally plays an important role in the prevention of albuminuria. Three membrane-pore models were compared in their ability to represent the dextran and Ficoll sieving data. A lognormal pore-size distribution in parallel with a nonselective "shunt" pathway was found to be more effective than either an isoporous membrane with a shunt or a purely lognormal distribution. On the basis of these laboratory results and computations, Ficoll may be preferred over dextran in future studies of glomerular size-selectivity.     

Radical Resection of Periampullary Tumors in the Elderly: Evaluation of Long-term Results

Increasingly, patients of advanced age are coming for evaluation of periampullary tumors. Although several studies have demonstrated the safety of resecting periampullary tumors in older patients, few long-term survival data have been reported. Between 1983 and 1992 various periampullary masses were resected in 70 patients over age 65 (range 65–87 years). Total pancreatectomy was performed in 11 patients, and 59 patients underwent pancreaticoduodenectomy. The mean duration of hospitalization was 17 ± 15 days. Major complications occurred in 27 patients (39%), and operative mortality rate was 8.5%. Overall median survival was 24 months; and 5-year survival was 25%. Perioperative outcome was compared in patients aged 65 to 74 years and in patients ≥75 years old. The older age group required longer periods in the surgical intensive care unit postoperatively, but the long-term survival was similar in the two age groups. Radical resection with the intent to cure periampullary tumors is safe in selected patients of advanced age, and long-term survival is in the range of expected survival for younger patients with the same tumors.     

Predictors of blood transfusions in spinal instrumentation and fusion surgery

STUDY DESIGN: A retrospective review of 244 adult spine instrumentation and fusion surgery cases (1994-1995) from one institution. OBJECTIVES: To ascertain the predictors of blood transfusions for adult patients undergoing different types of multilevel spine surgery. SUMMARY OF BACKGROUND DATA: Blood loss and transfusion requirements during and after multilevel spine surgeries have always been perceived as great. Identifying the predictors of blood transfusion with this type of surgery may aid in reducing the amount of blood loss and the transfusion requirements. METHODS: The charts of 244 adult patients who underwent multilevel spine surgery from January 1994 to July 1995 were retrospectively reviewed. RESULTS: A large percentage of patients required blood transfusion. The significant determinants for increased amounts of allogeneic red blood cell units transfused on the day of surgery using linear multiple regression modeling were low preoperative hemoglobin concentration, tumor surgery, increased number of posterior levels surgically fused, history of pulmonary disease, decreased amount of autologous blood available, and no use of the Jackson table (R2 = 0. 63). The significant determinants for an increased amount of autologous red blood cell units transfused on the day of surgery using linear multiple regression modeling were increased autologous red blood cells available, low preoperative hemoglobin concentration, and increased number of posterior levels surgically fused (R2 = 0. 60). CONCLUSION: The need for transfusion is associated with multiple factors, suggesting that a multifaceted, integrated approach may be necessary to reduce this risk.