High-Performance Liquid Chromatographic (HPLC) Assay Using Fluorescence Detection for the Simultaneous Determination of Gallopamil and Norgallopamil in Human Plasma

Gallopamil is a calcium-channel antagonist with reported activity in experimental animals three to five times higher than that of verapamil. An automated high-performance liquid chromatographic (HPLC) method with fluorescence detection is described for the simultaneous determination of gallopamil and its metabolite norgallopamil in plasma. Gallopamil was well resolved from norgallopamil and other metabolites, allowing simultaneous quantitation of both drugs. The detection limit for both gallopamil and norgallopamil was 0.9 ng/ml. This method has been successfully used for the determination of gallopamil and norgallopamil following the administration of 25-, 37.5-, and 50-mg oral doses of drug.     

Pharmacokinetics of Amonafide in dogs

Summary Amonafide, one of a series of imide derivatives of 1,8-naphthalic acid synthesized by Brana et al. [2] has shown significant antitumor activity against a variety of experimental tumors, including L1210 leukemia and P388 leukemia. Along with the clinical trial at our institute, we have studied the disposition of Amonafide in dogs by HPLC and fluorometry. Six dogs received Amonafide i.v. at 5 mg/kg (100 mg/m²) over 15 min; three were sacrificed at 6 h, and three at 24 h. The initial plasma t_1/2, of Amonofide was 2.4±0.4 min, the intermediate t_1/2, 26.8±3.7 min, and the terminal t_1/2, 21.7±4.0 h. the peak plasma concentration achieved was 6.3±1.7 g/ml. The average apparent volume of distribution was 12.84±0.541/kg, and the total clearance was 0.56±0.161/kg/h. In 24 h, 9.5%±0.2% of the administered dose was excreted in the urine as the parent drug, and 7.4%±1.4% in the bile in 6 h. Amonafide penetrated the CSF readily and achieved the highest concentration 20–25 min after administration, which was 30% of the concurrent plasma level. Amonafide underwent extensive metabolism to at least three major metabolites and two or more minor metabolites. The and plasma t_1/2 of the major metabolite, an N-oxide derivative, were 24.8 min and 28.6 h, respectively. The 24-h cumulative urinary excretion was 1.4% of the injected dose, and the cumulative biliary excretion was 16.7% in 6 h. At autopsy 6 h after dosing, the liver contained the highest percentage (0.23% of administered dose) of unchanged Amonafide, followed by the stomach (0.11%), lung (0.04%), kidney (0.04%), and pancreas (0.03%). The rest of the major organs retained less than 0.02% of the Amonafide dose. One day after dosing, no detectable amount of Amonafide was found in any of these tissues, indicating that Amonafide appears to be extensively metabolized and not significantly retained in the dog.     

Detection of haematoporphyrin derivative and haematoporphyrin excited states in cell environments

The photosensitiser currently used in the photodynamic therapy of cancer is haematoporphyrin derivative. Pulsed laser studies of this material and also of the "parent" molecule haematoporphyrin in polyoma-transformed fibroblast cells have now been studied. We report, for the first time, the observation of the triplet absorption of these sensitisers in cells. The corresponding triplet-triplet spectra are red shifted compared to aqueous buffer, lambda(max) 420 nm shifts to 460 nm. We also report our failure to observe singlet oxygen from the cells even though the triplet state can be seen to interact with the oxygen and even though singlet oxygen can be readily detected with the same sensitisers bound to serum albumin.     

Effect of two different types of malnutrition on the rate of elimination of ethanol in rats

Rats were fed "3% casein" or a "calorie deficient" diet, in the form of commercial pellet diet (SDS) at 50% of the amount consumed by the control group, which was fed SDS pellets ad libitum. Both of the deficient groups showed failure of weight gain in comparison with the control group. Blood levels of ethanol were measured for 3 hr after intraperitoneal injection of 1 or 1.5 g/kg at 15, 29 and 36 days after commencement of the diet. In addition the calorie deficient group was studied immediately after feeding as well as in the fasting state. Blood levels of ethanol were measured and the apparent volume of distribution and rate of removal of ethanol from the blood were calculated. A rate of ethanol metabolism/g of liver was derived. The rate of removal of ethanol was markedly decreased in the 3% casein group to less than half of control values. Three hours after injection of ethanol circulating levels were less than 50 mg/100 ml in the control and calorie deficient groups but over 200 mg/100 ml in the group fed protein deficient diets. There were no major changes in volume of distribution and the only explanation for the finding is that there is a failure of ethanol metabolism in the rats fed the low protein diet. The implication is that protein deficient human populations who often consume considerable quantities of ethanol may have a high level of tissue exposure to ethanol though the rate of metabolite formation may be low.     

Respiratory distress syndrome in second-born versus first-born twins. A matched case-control analysis

The well-known increased risk of the respiratory distress syndrome in a twin born second as compared with the twin born first is usually attributed to the second twin's predisposition to depression at birth ("asphyxia"). We analyzed the etiologic roles of birth order, presentation, and depression at birth in the development of the respiratory distress syndrome in matched case-control populations drawn from 221 preterm twin pairs. Among the 39 twin pairs discordant for respiratory distress syndrome, the second twin was the affected member in 31 pairs. Second birth order was the only independent risk factor, but only in vaginal deliveries (matched odds ratio, 14.2; 95 percent confidence interval, 2.5 to 81.1). Second twins delivered abdominally did not have an increased risk relative to first twins (odds ratio, 0.9; confidence interval, 0 to 17.8). When depression at birth was evaluated as an outcome variable, malpresentation, rather than birth order, was the major risk factor (independent matched odds ratios of 2.7 [confidence interval, 1.0 to 7.5] and 1.3 [0.7 to 2.5], respectively). Thus, second twins' increased risk of respiratory distress syndrome cannot be explained by a predisposition to depression at birth; a more important factor may be that second twins do not benefit from the salutary effects of labor to the same extent as first twins.     

Molecular genetic characterization of XRCC4 function

XRCC4 is a generally expressed protein of 334 amino acids that is involved in the repair of DNA double-strand breaks and in V(D)J recombination, but its function is unknown. In this study, we have used a mutational approach and the yeast two-hybrid method to perform an initial characterization of this protein. We show that the XRCC4 protein is located in the nucleus. We also demonstrate that several potential phosphorylation sites are not required for XRCC4 function in a transient V(D)J recombination assay. In addition, we show that XRCC4 forms a homodimer in vivo with the homodimerization domain being located within amino acids 115-204. Finally, we define a core domain of XRCC4 that functions in V(D)J recombination and comprises amino acids 18-204. Potential functions of XRCC4 are discussed.      

Advantages of branched peptides in serodiagnosis. Detection of HIV-specific antibodies and the use of glycine spacers to increase sensitivity.

The reactivities of antibodies with branched and monomeric peptides were compared in ELISA assays. We found that lower amounts of antibodies could be detected with branched peptides than with monomeric peptides. This was observed with a monoclonal antibody and with antibodies in the sera of various HIV-positive individuals. To investigate the physical aspects of branched peptides important for the observed increase in sensitivity, glycine spacers of different lengths were introduced between the branched lysine core and the epitope reacting with the monoclonal antibody. The effect of the number of glycine residues, both on the sensitivity of antibody detection and on the amount of branched peptide needed to produce a given signal, was studied and the optimum was found at 4-5 residues. We discuss the basis for these findings and conclude that the routine use of branched peptides for serodiagnosis will give both greater sensitivity and appreciable cost savings.     

Serologic analysis of white-tailed deer sera for antibodies to Borrelia burgdorferi by enzyme-linked immunosorbent assay and western immunoblotting.

White-tailed deer serum samples were collected in the Minneapolis-St. Paul, Minn., metropolitan area during the fall and winter months from 1989 to 1992 and analyzed for antibodies to Borrelia burgdorferi, the etiologic agent of Lyme borreliosis. Ninety-eight percent of the serum samples were collected from regions where currently the vector tick, Ixodes dammini, is nonexistent. Antibodies to B. burgdorferi were detected in 2.2% of 508 samples by enzyme-linked immunosorbent assay, and their presence was confirmed by Western immunoblot analysis. Western immunoblotting yielded mean numbers of reactive bands of 0.1 and 6.0 for samples that were negative and positive for antibodies by enzyme-linked immunosorbent assay, respectively. The molecular weights of the antigens in many of the reactive bands from positive samples were similar to the molecular weights of antigens reactive with samples from humans with Lyme borreliosis. An antibody response to the major outer surface proteins A and B was not detected. Serologic analysis of deer sera may provide a valuable method for surveillance programs designed to monitor the spread of B. burgdorferi in nature.     

Effect of histamine and methacholine on nasal airway resistance in atopic and nonatopic subjects: Comparison with bronchial challenge and skin test responses

Serial nasal, intracutaneous, or bronchial challenges were carried out with solutions containing 2- or 3-fold increments in histamine (H) or methacholine (Meth) concentration until nasal airway resistance (NAR) increased by more than 100%, a large intracutaneous reaction was elicited, or FEV₁ decreased by 20% or more. Thirty nonatopic and 48 asymptomatic atopic subjects were studied, the latter group divided into rhinitic patients with and without asthma. Several types of data analysis demonstrated there was no significant difference in the nasal or cutaneous effects of H or Meth between the atopic and nonatopic groups. Comparable results were obtained in a subgroup of 39 subjects (13 normal, 13 atopic, and 13 atopic with asthma) who underwent all six test sequences (i.e., nasal, cutaneous, and bronchial with both drugs). As expected, the asthmatics showed significantly increased bronchial reactivity to both agents. In comparison with Meth, H had a much greater effect on the nasal mucosa and skin than on the bronchi. It is concluded that, contrary to bronchial responses, but in accord with cutaneous reactivity, the nasal responses of nonatopic subjects, atopic persons with allergic rhinitis alone, and subjects with both allergic rhinitis and asthma show no intergroup differences on testing with H or Meth.