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Rhodanese from Thiobacillus A2 was shown by proton nuclear magnetic resonance (NMR) spectroscopy to use dihydrolipoate or dihydrolipoamide as acceptor of the sulphane moiety of thiosulphate with the formation of alpha-lipoate or lipoamide respectively. Correlation is shown between assays of the enzyme activity by NMR spectroscopy and by ultraviolet spectrophotometry.
The action of the polyene antibiotic filipin on the permeability of liposomes prepared with lecithin and several plant and other steroids was studied. The effect of filipin was found to be dependent upon the type of steroid incorporated into the membrane. The interaction of filipin with steroids was found to be related both to the functional group at the 3-position and the aliphatic chain of the steroid. Complex formation of the steroid with filipin in aqueous solutions, as detected by ultraviolet spectrophotometry, correlated with the ability of filipin to increase the permeability of the liposomes.From these results it was suggested that the action of filipin could be used as a diagnostic tool to determine the presence of different steroids in plant membranes. Differences in the sensitivity of subcellular organelles to filipin may be due to differences in the steroid composition of their membranes.
The FAD analogue, N6-(6-carboxyhexyl)-FAD, carrying a hexanoic acid residue at the N6 position of the adenine moiety was synthesized. A new semi-synthetic oxidase, N6-(6-carbamoylhexyl)-FAD-D-amino acid oxidase, was prepared by reacting the succinimido ester of N6-(6-carboxyhexyl)-FAD with apo-D-amino-acid oxidase from pig kidney in the presence of benzoate. Reaction conditions and methods have been developed for preparing pure semi-synthetic and fully active N6-(6-carbamoylhexyl)-FAD-D-amino acid oxidase that contains 1 covalently bound FAD analogue/subunit, as verified by redialysis, ultraviolet spectrophotometry, electrospray ionization (ESI)-MS and peptide mapping. Presumably, the N6-(6-carbamoylhexyl)-FAD moiety of this semi-synthetic D-amino-acid oxidase (DAAO), selectively bound to Lys163, has a structurally similar position to that of the non-covalently bound FAD of the native holoenzyme, since both DAAO forms show very similar kinetic properties (semi-synthetic DAAO, Vmax(app) = 17.7 mumol min-1 mg-1; KM(app) = 4.5 mM; native holo-DAAO, Vmax = 12.2 mumol min-1 mg-1; KM = 1.8 mM). Compared with the native holo-D-amino acid oxidase. this new semi-synthetic N6-(6-carbamoylhexyl)-FAD-D-amino acid oxidase is a considerably more stable enzyme that shows meso-thermostability and withstands inactivation on dilution. Probably, the lack of dissociation of FAD and, consequently, the absence of the instable apoenzyme are responsible for these phenomena. Preliminary investigations resulted in finding convenient and reproducible crystallization conditions for N6-(6-carbamoylhexyl)-FAD-D-amino acid oxidase. The single crystals, obtained by the sitting-drop method using ammonium sulfate as precipitant, belong to the tetragonal space group I422 with cell dimensions a = 16.3 nm, c = 13.6 nm. The crystals diffract to 0.3-nm resolution, with two molecules being present in the asymmetric unit, demonstrating the two-subunit quarternary structure of this semi-synthetic D-amino-acid oxidase.
A substance, proteinaceous in nature, with an anti-agglutinic action on clumped sperm has been extracted from human semen. It appears to exist in two forms: an active reduced form and an inactive oxidised form. The protein was extracted from oxidised seminal plasma by salting with saturated ammonium sulphate. The extract was purified by preferential adsorption on a calcium carbonate column, the eluate being collected as fractions. The antagglutinin gave an extinction curve, as determined by ultraviolet spectrophotometry, similar to that as reported for the bull. Extraction recoveries, from semen stored frozen at different temperatures, indicate that the substance is possibly enzyme labile. An effect of the antagglutinin on clumped human sperm is illustrated.
Mutants of Aspergillus fennelliae (Neosartorya fennelliae) resistant to relatively high levels of amphotericin B and low levels of nystatin were obtained by successive transfers of wild type in the presence of increasing concentrations of the polyenes. The resistance of the mutants to the polyenes was accompanied by both qualitative and quantitative changes in the sterol composition of the cells. Those resistant to amphotericin B (AF5-AB1 and p-AB1) lacked ergosterol, the major sterol of the wild type, but contained a new sterol clearly distinguished by the pattern of ultraviolet spectrophotometry and thin-layer chromatography. The mutants resistant to nystatin, however, contained both ergosterol and a new sterol, but the former was produced in a much reduced amount, as compared with the wild type. Genetic analysis indicated that the lack of ergosterol is closely associated with a reduced growth rate, poor asexual reproduction, and the loss of sexual reproduction. Growth studies revealed that the addition of ergosterol to the media did not affect the growth pattern of the mutants. Mutants resistant to amphotericin B showed an increased minimal inhibitory concentration for nystatin, pimaricin, and filipin. Mutants resistant to nystatin, however, conferred increased minimal inhibitory concentration for pimaricin and filipin but not for amphotericin B.
The primary and ultimate biodegradability of phthalic acid, monobutyl phthalate, and five structurally diverse phthalic acid ester plasticizers in river water and activated sludge samples were determined via ultraviolet spectrophotometry, gas chromatography, and CO2 evolution. The compounds studied underwent rapid primary biodegradation in both unacclimated river water and acclimated activated sludge. When activated sludge acclimated to phthalic acid esters was used as the inoculum for the CO2 evolution procedure, greater than 85% of the total theoretical CO2 was evolved. These studies demonstrate that the phthalic acid ester plasticizers and intermediate degradation products readily undergo ultimate degradation in different mixed microbial systems at concentrations ranging from 1 to 83 mg/liter.
A study was conducted to determine the effects of o-nitrobenzoate, p-aminobenzoate, benzocaine (ethyl aminobenzoate), ethyl benzoate, methyl benzoate, salicylic acid (o-hydroxybenzoate), trans-cinnamic acid (beta-phenylacrylic acid), trans-cinnamaldehyde (3-phenylpropenal), ferulic acid (p-hydroxy-3-methoxycinnamic acid), aspirin (o-acetoxy benzoic acid), and anthranilic acid (o-aminobenzoic acid) upon growth and aflatoxin release in Aspergillus flavus NRRL 3145 and A. parasiticus NRRL 3240. A chemically defined medium was supplemented with various concentrations of these compounds and inoculated with spores, and the developing cultures were incubated for 4, 6, and 8 days at 27 degree C in a mechanical shaker. At the beginning of day 8 of incubation, aflatoxins were extracted from cell-free filtrates, separated by thin-layer chromatography, and quantitated by ultraviolet spectrophotometry. The structure of these aromatic compounds appeared to be critically related to their effects on mycelial growth and aflatoxin release. At concentrations of 2.5 and 5.0 mg per 25 ml of medium, methyl benzoate and ethyl benzoate were the most effective in reducing both mycelial growth and aflatoxin release by A. flavus and A. parasiticus. Inhibition of mycelial growth and aflatoxin release by various concentrations of the above-named aromatic compounds may indicate the possibility of their use as fungicides.
In a survey of Streptomyces species, methods were designed and followed that would specifically select strains capable of producing heat-stable, nonpolyenic, antifungal antibiotics. Of 500 strains grown in shaken flasks, 240 of the culture liquors contained active factors as demonstrated by paper-disc assay against Mucor ramannianus. Culture filtrates and mycelial extracts of the active strains were examined by ultraviolet spectrophotometry; 166 were nonpolyenic as determined by absorption spectra. Heat-stability tests of the nonpolyenic antibiotics over a broad pH range revealed that 15 were stable under all test conditions, 70 moderately stable, and 81 unstable. Culture liquors containing stable, nonpolyenic antifungal agents were chromatographed with eight solvent systems in an attempt to identify the antibiotics. The producing cultures were studied by cross-antagonism tests to discover similarities with producers of known antibacterial antibiotics. Two of the antibiotics produced by promising strains were identified as cycloheximide and musarin. Six antibiotics, presumably new, were detected.
The comparative effects of ketoconazole, itraconazole, and fluconazole on the lipids of five Sporothrix schenckii strains were investigated. Quantitative analysis of lipids and sterols was completed, as well as qualitative analysis of sterols, by thin-layer chromatography and ultraviolet spectrophotometry. Growth of the S. schenckii isolates in the presence of azole derivative concentrations below the minimum inhibitory concentration (MIC) resulted in significant alterations in the lipid and sterol contents as compared with the control values. Furthermore, lanosterol was detected in these azole-treated cells. These results were in complete agreement with the proposed mechanism of action of azoles, which act by inhibiting ergosterol biosynthesis with a consequent accumulation of lanosterol. Concerning the MIC values, fluconazole was found to be the least effective drug. On the other hand, as determined from a comparison of the effects of the three azoles on the sterol content of the strains studied, no significant differences in efficacy were found among the tested drugs.
The model hydrogen peroxide-myeloperoxidase-chloride system is capable of generating the powerful oxidant hypochlorous acid, which can be quantitated by trapping the generated species with the beta-amino acid, taurine. The resultant stable product, taurine chloramine, can be quantitated by its ability to oxidize the sulfhydryl compound, 5-thio-2-nitro-benzoic acid to the disulfide, 5,5'-dithiobis(2-nitroben-zoic acid) or to oxidize iodide to iodine. Using this system, purified myeloperoxidase in the presence of chloride and taurine converted stoichiometric quantities of hydrogen peroxide to taurine chloramine. Chloramine generation was absolutely dependent on hydrogen peroxide, myeloperoxidase, and chloride and could be inhibited by catalase, myeloperoxidase inhibitors, or chloride-free conditions. In the presence of taurine, intact human neutrophils stimulated with either phorbol myristate acetate or opsonized zymosan particles generated a stable species capable of oxidizing 5-thio-2-nitrobenzoic acid or iodide. Resting cells did not form this species. The oxidant formed by the stimulated neutrophils was identified as taurine chloramine by both ultraviolet spectrophotometry and electrophoresis. Taurine chloramine formation by the neutrophil was dependent on the taurine concentration, time, and cell number. Neutrophil-dependent chloramine generation was inhibited by catalase, the myeloperoxidase inhibitors, azide, cyanide, or aminotriazole and by chloride-free conditions, but not by superoxide dismutase or hydroxyl radical scavengers. Thus, it appears that stimulated human neutrophils can utilize the hydrogen peroxide-myeloperoxidase-chloride system to generate taurine chloramine. Based on the demonstrated ability of the myeloperoxidase system to generate free hypochlorous acid we conclude that neutrophils chlorinate taurine by producing this powerful oxidant. The biologic reactivity and cytotoxic potential of hypochlorous acid and its chloramine derivatives suggest that these oxidants play an important role in the inflammatory response and host defense.
We have investigated whether fluorimetric determination of the lecithin/sphingomyelin ratio is feasible after HPLC. We found that the saturated phospholipid, dipalmitoyl lecithin, which is secreted by the fetal lung, and which cannot be detected by ultraviolet spectrophotometry, can be determined fluorimetrically. For this purpose we have added the fluorescence-enhancing substance, 1,6-diphenyl-1,3,5-hexatriene to the effluent. With the aid of a continuous-flow system we showed that the method can be used for a rapid assay of the ratio of these phospholipids in amniotic fluid.
The details of a fatal self-poisoning with the anti-arrhythmic drug tocainide are described. The authors are unaware of another such case. The physiological and toxicological results are post mortem are presented and discussed, with emphasis on the method of analysis of the drug in the body fluids and tissues. Stomach content was analysed by direct solvent extraction followed by ultraviolet spectrophotometry and thin-layer chromatography. Blood, urine and liver concentrations were obtained by high-pressure liquid chromatography, blood and liver samples being extracted under basic conditions and urine analysed directly. Two methods are compared for the liver analysis; an alkaline extraction of an enzymic digestion yielded lower levels of drug than did a mechanical maceration followed by alkaline extraction and 'salting out'. The latter is an adaptation of the method used for analysis of blood. Details of therapeutic and toxic blood/plasma concentrations are taken from the literature and compared with the analytical data, leading to the conclusion that the only logical cause of death was tocainide poisoning.
Ten healthy, male volunteers (aged 19-27 yr; weight 62-67 kg) were randomly distributed into control and test groups of five subjects each in a controlled study on the effect of ranitidine on chloroquine disposition. The control group subjects received two tablets of chloroquine sulfate (300-mg base) only. The test group subjects received ranitidine 250 mg hs for four days prior to the administration of chloroquine sulfate and throughout the sample collection period. Blood samples (5 ml) were collected from the time of the chloroquine administration to the seventh day after drug administration. The samples were analyzed for chloroquine content by a combination of thin-layer chromatography and ultraviolet spectrophotometry. The Wilcoxon test at 0.05 significance level was used to compare the disposition parameters between control and test groups. Ranitidine therapy was associated with no significant alterations in chloroquine oral clearance rate, elimination rate constant, and apparent volume of distribution. Unlike cimetidine, ranitidine does not interact pharmacokinetically with chloroquine. Ranitidine, therefore, may be the H2-receptor antagonist of choice for ulcer patients receiving chloroquine.
OBJECTIVE: This study investigated a new, nonimmunologic, enzymatic-based theophylline assay for the possible interference of a clinically achievable serum theophylline concentration by four xanthine-related compounds: pentoxifylline, 1-methylxanthine, allopurinol, and oxypurinol. RATIONALE: The enzyme theophylline oxidase is used in this assay kit to convert theophylline to 1,3 dimethyluric acid in the presence of cytochrome C. Cytochrome C is reduced to ferrocyte C, which is then measured by ultraviolet spectrophotometry at one minute and again at five minutes. The rate of appearance of ferrocyte C over this time is then related to a one-point 20-micrograms/mL (111-mumol/L) theophylline standard provided with the kit and the concentration of theophylline is calculated. Xanthine-related compounds have the potential to interact with theophylline oxidase via: (1) structural similarity, and (2) inhibition of xanthine oxidase, which increases theophylline metabolite concentrations. DESIGN: Human serum was spiked with known, therapeutically achievable concentrations of pentoxifylline, 1-methylxanthine, allopurinol, and oxypurinol, both alone and in combination with theophylline in a concentration of 15 micrograms/mL (83.3 mumol/L). Two controls were used: human serum and human serum spiked with theophylline. Data were analyzed statistically with ANOVA, and clinically with +/- 10 percent control criteria. RESULTS: Means (+/- SEM) for the control samples were 2.0 (0.2) micrograms/mL for serum alone and 15.3 (0.3) micrograms/mL for theophylline-spiked serum. For the test specimens, the results ranged from 2.3 to 2.8 micrograms/mL and from 13.9 to 16.6 micrograms/mL when xanthine-related compounds were added to serum without and with theophylline, respectively. CONCLUSIONS: The results were not statistically or clinically significant for the four xanthine-related compounds at the concentrations studied.
OBJECTIVE: A significant practical problem in the standardization of dissolution testing is addressed. In vitro releasing characteristics of hydroxyzine hydrochloride tablets are presented to further the documentation of bioequivalency criteria. DESIGN: The assessment model compares the official United States Pharmacopeia disintegration approach for dissolution analysis with that of the Food and Drug Administration's recommended rotating paddle technique for inducing aqueous disruption of the solid oral dosage form. RESULTS: The rationale and significance of the study focuses attention on the variation in release of the active ingredient observed relative to the four formulation strengths. With differences in the extent of dissolution noted and official standards in mind, emphasis is placed on the development of an alternate test protocol. CONCLUSIONS: Dissolution data derived via ultraviolet spectrophotometry revealed statistically significant differences in the amount of hydroxyzine hydrochloride being released from its coated structure, the extent of which was found to be dependent on the acid nature of the simulated gastric dissolution medium used and intensity of mixing action employed.
Attempts to improve drug delivery from conventional jet nebulizers have included the use of storage systems to reduce drug wastage during exhalation. Venturi nebulizers enhance drug delivery during inhalation, reducing treatment times and drug wastage. This study investigated the effect of age on inhaled dose from a conventional jet nebulizer (Acorn) used both with and without a storage chamber (Mizer), compared to two Venturi nebulizers (Ventstream and Pari LC). Filters were attached to the four nebulizer systems, containing salbutamol, and 18 children with cystic fibrosis (3-16 yrs) inhaled through these devices. The quantity of drug collected on the filter was assessed using ultraviolet spectrophotometry. The particle size distribution of the aerosol from each nebulizer system was measured using laser diffraction. Inspiratory filter deposition using the Acorn was lower than the Acorn with Mizer, and both Venturi nebulizers. Filter deposition using the Acorn with Mizer was lower than the Pari LC. No trend with age, height or weight was noted using any nebulizer. Aerosol particle size using the Ventstream was lower than the other nebulizer systems. Drug output from both Venturi nebulizers was more efficient than from the jet nebulizer, used with and without the storage chamber, during inhalation by children with cystic fibrosis. The inhaled dose did not change with the patient's age or size using both types of nebulizer.
The effects of atropine on serum concentrations and pharmacokinetics of theophylline were studied in six rabbits (2.07 +/- 0.11 kg). Theophylline serum concentration was determined by ultraviolet spectrophotometry. After i.v. administration of aminophylline 12.5 mg/kg, i.m. atropine 1 mg/kg decreased significantly the maximal serum concentration of theophylline from 23.6 +/- 1.1 to 19.6 +/- 1.1 mg/l, and increased that of theophylline at the time of 6 and 8 hours after injection. After i.v. aminophylline administration without or with atropine, the pharmacokinetic parameters of theophylline calculated using a one compartment open model were: K (elimination rate constant) = 0.23 +/- 0.03, 0.19 +/- 0.02/h; t1/2 (half life) = 3.04 +/- 0.40, 3.66 +/- 0.40 h; Cl (clearance) = 0.26 +/- 0.04, 0.23 +/- 0.03 l/kg/h, respectively (P less than 0.01). But there was no significant variation in modification of volume of distribution (Vd). The results suggested that there is a significant drug interaction between atropine and theophylline.
PURPOSE: To analyze alteplase solutions following delivery through the AngioJet Xpeedior rheolytic thrombectomy device to characterize the viability of proteins exposed to high shear stress. METHODS: Alteplase solutions were prepared by reconstitution in sterile water to obtain a targeted concentration of 1.0 mg/mL. A baseline control sample was obtained. The AngioJet system was modified by occluding the return line to allow collection of the dispersed fluid volume. Alteplase solutions (n=5) were delivered through the 6-F, 120-cm Xpeedior catheter and collected. All samples were assayed and compared to baseline using qualitative visual inspection, ultraviolet spectrophotometry, size exclusion chromatography, and in vitro clot lysis assays. RESULTS: Analysis of the test solutions demonstrated a mean protein recovery of 98.0%+/-3.5% of targeted concentration; the collected protein was fully active, as determined by clot lysis assays (specific activity > or =100%). All samples were clear and colorless. No significant fragmentation or aggregation of protein was observed. CONCLUSIONS: Alteplase solutions, when delivered through the AngioJet Xpeedior rheolytic thrombectomy device, remain stable and biologically active in vitro. Further clinical investigation is warranted using this method as a novel approach for pharmacomechanical thrombolysis.