Biomedical subjects

T P Ryan

Publications and source records attributed to T P Ryan.

At least 19 recordsLinked to original sources

PubMed2006

Certified reference material for radionuclides in fish flesh sample IAEA-414 (mixed fish from the Irish Sea and North Sea).

A certified reference material (CRM) for radionuclides in fish sample IAEA-414 (mixed fish from the Irish Sea and North Seas) is described and the results of the certification process are presented. Nine radionuclides (40K, 137Cs, 232Th, 234U, 235U, 238U, 238Pu, 239+240Pu and 241Am) were certified for this material. Information on massic activities with 95% confidence intervals is given for six other radionuclides (90Sr, 210Pb(210Po), 226Ra, 239Pu, 240Pu 241Pu). Less frequently reported radionuclides (99Tc, 129I, 228Th, 230Th and 237Np) and information on some activity and mass ratios are also included. The CRM can be used for quality assurance/quality control of the analysis of radionuclides in fish sample, for the development and validation of analytical methods and for training purposes. The material is available from IAEA, Vienna, in 100 g units.

Animals↗

PubMed2003

Gene expression analysis of the acute phase response using a canine microarray.

The safety of pharmaceuticals is typically assessed in the dog and rat prior to investigation in humans. As a result, a greater understanding of adverse effects in these preclinical testing species would improve safety assessment. Despite this need, there is a lack of tools to examine mechanisms and identify biomarkers in the dog. To address this issue, we developed an Affymetrix-based oligonucleotide microarray capable of monitoring the expression of thousands of canine genes in parallel. The custom canine array contains 22,774 probe sets, consisting of 13,729 canine and 9045 human-derived probe sets. To improve cross-species hybridization with human-derived probes, the detection region was moved from the variable 3' UTR to the more homologous coding region. Testing of this strategy was accomplished by comparing hybridization of naive dog liver RNA to the canine array (coding region design) and human U133A array (standard 3' design). Although raw signal intensity was greater with canine-specific probe sets, human-derived probes detected the expression of additional liver transcripts. To assess the ability of this tool to detect differential gene expression, the acute phase response was examined in beagle dogs given lipopolysaccharide (LPS). Hepatic gene expression 4 and 24 h post-LPS administration was compared to gene expression profiles of vehicle-treated dogs (n=3/group). Array data was consistent with an acute inflammatory response, with transcripts for multiple cytokines and acute phase proteins markedly induced 4 h after LPS challenge. Robust changes in the expression of transcripts involved with glucose homeostasis, biotransformation, and extracellular matrix remodeling were observed 24 h post-dose. In addition, the canine array identified several potential biomarkers of hepatic inflammation. Strong correlations were found between gene expression data and alterations in clinical chemistry parameters such as serum amyloid A (SAA), albumin, and alkaline phosphatase (ALP). In summary, this new genomic tool successfully detected basal canine gene expression and identified novel aspects of the acute phase response in dog that shed new light on mechanisms underlying inflammatory processes.

Acute-Phase Reaction↗

PubMed2002

Transuranic biokinetic parameters for marine invertebrates--a review.

A catalogue of biokinetic parameters for the transuranic elements plutonium, americium, curium, neptunium, and californium in marine invertebrates is presented. The parameters considered are: the seawater-animal concentration factor (CF); the sediment-animal concentration ratio (CR); transuranic assimilation efficiency; transuranic tissue distribution and transuranic elimination rates. With respect to the seawater-animal CF, authors differ considerably on how they define this parameter and a seven-point reporting system is suggested. Transuranic uptake from sediment by animals is characterised by low CRs. The assimilation efficiencies of transuranic elements in marine invertebrates are high compared to vertebrates and mammals in general and the distribution of transuranics within the body tissue of an animal is dependent on the uptake path. The elimination of transuranics from most species examined conformed to a standard biphasic exponential model though some examples with three elimination phases were identified.

Animals↗

PubMed2001

Technetium-99 in the Irish marine environment.

Technetium-99 activity concentrations in seawater and biota from Irish coastal waters are presented. Time series measurements of 99Tc in seawater and Fucus vesiculosus from the western Irish Sea show that activity concentrations have increased in line with the increase in discharges of 99Tc from Sellafield. The peak in activity concentrations in both seawater and Fucus vesiculosus occurred in 1997 approximately two years after the peak in 99Tc discharges. The highest activity concentration recorded in Fucus vesiculosus showed a 29-fold increase over the mean concentration for the period 1988-1993. Technetium-99 activity concentrations were measured in fish, lobsters, prawns, mussels and oysters landed at major fishing ports on the east and northeast coasts of Ireland between 1996 and 1998. Concentration factors for 99Tc in the brown seaweed Fucus vesiculosus and certain species of fish, crustaceans and molluscs from the Irish Sea were estimated. In general, these concentration factors were higher than those in the literature which were derived from laboratory studies, but agreed well with values which were based on field studies. The mean committed effective doses to Irish typical and heavy seafood consumers due to 99Tc in the period 1996-1998 were 0.061 and 0.24 microSv, respectively.

Animals↗

PubMed2001

Temporal gene expression analysis of monolayer cultured rat hepatocytes.

The use of cultured primary hepatocytes within toxicology has proven to be a valuable tool for researchers, however, questions remain with regard to functional differences observed in these hepatocytes relative to the intact liver. Cultured hepatocytes have typically been described as dedifferentiated, a classification based upon the investigation of a few key cellular processes or hepatocellular markers. In the present study, parallel expression monitoring of approximately 8700 rat genes was used to characterize mRNA changes over time in hepatocyte cultures using Affymetrix microarrays. We isolated and labeled mRNA from whole rat livers, hepatocyte-enriched cell pellets, and primary cultured hepatocytes (4, 12, 24, 48, and 72 h postplating), and hybridized these samples to microarrays. From these data, several pairwise and temporal gene expression comparisons were made. Gene expression changes were confirmed by RT/PCR and by performing replicate experiments and repeated hybridizations using a rat toxicology sub-array that contained a 900-gene subset of the 8700-gene rat genomic microarray. PCR data qualitatively reproduced the temporal patterns of gene expression observed with microarrays. Cluster analysis of time course data using self-organizing maps (SOM) revealed a classic hepatocyte dedifferentiation response. Functional grouping of genes with similar transcriptional patterns showed time-dependent regulation of phase I and phase II metabolizing enzymes. In general, cytochrome P450 mRNA expression was repressed, but expression of phase II metabolizing enzymes varied by class (upregulation of glucuronidation, downregulation of sulfation). Potential metabolic targets for toxic insult, such as glutathione metabolism, gluconeogenesis, and glycolysis, were also affected at the transcriptional level. Progressive induction of several genes associated with the cellular cytoskeleton and extracellular matrix was observed in accord with physical changes in cell shape and connectivity associated with cellular adhesion. Finally, many transcriptional changes of genes involved in critical checkpoints throughout the hepatocyte cell cycle and differentiation process were observed. In total, these data establish a more comprehensive understanding of hepatocellular dedifferentiation and reveal many novel aspects of physiological and morphological hepatocyte adaptation. An assembly of all transcripts that demonstrated differential expression in this study can be found in the Supporting Information.

Animals↗

PubMed1998

Protein oxidation: examination of potential lipid-independent mechanisms for protein carbonyl formation.

Previous data indicated that diquat-mediated protein oxidation (protein carbonyl formation) occurs through multiple pathways, one of which is lipid dependent, and the other, lipid independent. Studies reported here investigated potential mechanisms of the lipid-independent pathway in greater detail, using bovine serum albumin as the target protein. One hypothesized mechanism of protein carbonyl formation involved diquat-dependent production of H2O2, which would then react with site-specifically bound ferrous iron as proposed by Stadtman and colleagues. This hypothesis was supported by the inhibitory effect of catalase on diquat-mediated protein carbonyl formation. However, exogenous H2O2 alone did not induce protein carbonyl formation. Hydroxyl radical-generating reactions may result from the H2O2-catalyzed oxidation of ferrous iron, which normally is bound to protein in the ferric state. Therefore, the possible reduction of site-specifically bound Fe3+ to Fe2+ by the diquat cation radical (which could then react with H2O2) was also investigated. The combination of H2O2 and an iron reductant, ascorbate, however, also failed to induce significant protein carbonyl formation. In a phospholipid-containing system, an ADP:Fe2+ complex induced both lipid peroxidation and protein carbonyl formation; both indices were largely inhibitable by antioxidants. There was no substantial ADP:Fe(2+)-dependent protein carbonyl formation in the absence of phospholipid under otherwise identical conditions. Based on the lipid requirement and antioxidant sensitivity, these data suggest that ADP:Fe(2+)-dependent protein carbonyl formation occurs through reaction of BSA with aldehydic lipid peroxidation products. The precise mechanism of diquat-mediated protein carbonyl formation remains unclear, but it appears not to be a function of H2O2 generation or diquat cation radical-dependent reduction of bound Fe3+.

Adenosine Diphosphate↗

PubMed1997

Quantitative assessment of variables that influence soft-tissue electrovaporization in a fluid environment.

OBJECTIVES: To evaluate the process of soft-tissue electrovaporization and to study variables that affect tissue clearance rates in a laboratory setting, in order to identify parameters that can optimize transurethral electrovaporization of the prostate. METHODS: Fresh bovine skeletal muscle, equivalent in impedance and surface properties to the human prostate, was submerged in 3.3% sorbitol solution and electrovaporized with a grooved monopolar electrode attached to the weighted arm of a linear actuator. The effects of excursion rate, applied mechanical load, power setting, electrode configuration, and generator performance on the volume of tissue removed, were assessed. RESULTS: Tissue removal increased significantly when electrode excursion rate was slowed from 25 to 15 mm/s (P < 0.05) and then to 10 mm/s (P < 0.05); when the load was increased from 20 to 50 g (P < 0.005); and when dial power was increased from 120 to 150 W (P < 0.01). Tissue removal was generator dependent. There was no significant difference between the Force 40 and the Force 2 (P > 0.4), but a new computer-controlled constant power output generator (Force FX) did significantly improve tissue vaporization at an equivalent power setting (P < 0.005 and P < 0.01, respectively). Tissue removal was also dependent upon electrode configuration, with the VaporTrode-Grooved Bar removing significantly more tissue than either an ungrooved roller bar of equivalent size or 2-mm smooth roller ball, respectively, both after a single pass (P < 0.001 and P < 0.05) and after five repeated passes (P < 0.05 and P < 0.005). The histologic depth of tissue thermal effect was less than 1 mm, but it was 38% greater for the VaporTrode-Grooved Bar (0.68 mm) than for the standard cutting loop (0.5 mm, P < 0.01). CONCLUSIONS: Using a novel method to quantify tissue removal, we have demonstrated that electrode configuration, excursion rate, applied load, power setting, and generator performance are interdependent factors that influence the efficacy of the electrovaporization process in a fluid environment.

Animals↗

PubMed1997

Pulmonary ferritin: differential effects of hyperoxic lung injury on subunit mRNA levels.

Ferritin is an iron storage protein that is regulated at the transcriptional and transcriptional levels, resulting in a complex mixture of tissue- and condition-specific isoforms. The protein shell of ferritin is composed of 24 subunits of two types (heavy or light), which are encoded by two distinct and independently regulated genes. In the present studies, the isoform profile for lung ferritin differed from other tissues (liver, spleen, and heart) as determined by isoelectric focusing (IEF) and polyacrylamide gel electrophoresis (PAGE). Lung ferritin was composed of equal amounts of heavy and light subunits. Differences in isoform profiles were the result of tissue-specific differential expression of the ferritin subunit genes as demonstrated by Northern blot analyses. Like heart ferritin, lung ferritin exhibited a low iron content that did not increase extensively in response to iron challenge, which contrasts with ferritins isolated from liver or spleen. When animals were exposed to hyperoxic conditions (95% oxygen for up to 60 h), ferritin heavy subunit mRNA levels did not markedly change at any of the investigated time points. In contrast, ferritin light subunit mRNA increased severalfold in response to hyperoxic exposure. Investigation of the cytoplasmic distribution of ferritin mRNA showed that a substantial portion was associated with the ribonucleoprotein (RNP) fraction of the cytosol, suggesting that a pool of untranslated ferritin mRNA exists in the lung. Upon hyperoxic insult, all ferritin light subunit mRNA pools (RNP, monosomal, polysomal) were elevated, although a specific shift from RNP to polysomal pools was not evident. Therefore, the increase in translatable ferritin mRNA in response to hyperoxia resulted from transcriptional rather than specific translational activation. The observed pattern of light chain-specific transcriptional induction of ferritin is consistent with the hypothesis that hyperoxic lung injury is at least partially iron mediated.

Animals↗

PubMed1997

Prostate heating patterns comparing electrosurgical transurethral resection and vaporization: a prospective randomized study.

PURPOSE: A prospective study was performed to determine if transurethral electrosurgical vaporization of the prostate is associated with unseen heat damage to vital periprostatic structures compared to conventional loop resection. In addition, energy consumption and its relationship to observed tissue temperature at the prostate periphery were evaluated for each treatment. MATERIALS AND METHODS: Patients with moderate to severe symptoms of benign prostatic bladder outflow obstruction and objective evidence of diminished flow or acute urinary retention were randomized to undergo either transurethral loop resection or electrosurgical vaporization after stratification for gland volume. Instrumentation was standardized for both groups except for the monopolar electrode used. The radiofrequency power source in the study was a new computer controlled generator with a constant power delivery feature. Regional tissue heating patterns were evaluated with optical fiber probes in real time. Four stationary sites were chosen for temperature measurements, namely the lateral lobe of the prostate, neurovascular bundle beside the prostatic apex at the level of the external sphincter, and anterior rectal wall at the level of the prostatic base and apex. A pull back technique was used to search for hot points in the long axis of the probe (3-dimensional temperature mapping) in 2 patients from each group. Incident generator panel power settings for the electrosurgical vaporization treatments were equivalent to those commonly used for loop resection (150 watts) and were adjusted up or down as needed. RESULTS: Prostate electrosurgical vaporization was possible at generator panel settings that were nearly equivalent to those for transurethral resection of the prostate (130 to 190 watts). No significant rectal or sphincteric heating was detected with either procedure. Conductive cooling of the neurovascular bundles was observed in 2 patients in each group toward the end of the operation. More energy was used per minute of treatment during electrosurgical vaporization than with regular loop resection (p < 0.004) but this was not associated with unwanted tissue heating. CONCLUSIONS: Neither conventional loop resection nor electrosurgical vaporization of the prostate appeared to be unsafe treatments with respect to unseen deep heating effects to vital periprostatic structures when performed at equivalent low incident power. The extra energy used during electrosurgical vaporization provided the benefit of improved coagulative hemostasis concurrently with shallow tissue vaporization using pure cutting current alone, without compromising treatment safety.

Aged↗

PubMed1996

Factors influencing electrovaporization in the treatment of benign prostatic hyperplasia.

Electrovaporization refers to the process of vaporizing tissue using electrical energy. Proposed as a new treatment method for benign prostatic hyperplasia (BPH), this technique allows removal of prostatic tissue with simultaneous coagulation, thereby minimizing blood loss. The recent increase in popularity of electrovaporization in the treatment of BPH warrants a quantitative assessment of the process, including an objective evaluation of its influencing factors. In this study, the effects of power, mechanical loading, and excursion rate on tissue removal by electrovaporization were examined in fresh skeletal muscle. A monopolar electrode was attached to the weighted arm of a linear motion system and rolled across the tissue surface while activated by a radio-frequency generator. The tissue samples were frozen and cut longitudinally to allow visualization and measurement of the vaporized groove using an optical imaging technique. The volume of tissue removed increased significantly when power was increased from 120 to 150 W (46 to 119 mm3, p = 0.006), when the load was increased from 20 to 50 gm (20 to 119 mm3, p = 0.002), and when the excursion rate was decreased from 25 to 15 mm/s (29 to 69 mm3, p < 0.05) and from 15 to 10 mm/s (69 to 137 mm3, p < 0.05). There was no significant gain in volume removed when power was increased to 180 W or when the load was increased to 70 gm, indicating that these factors are constrained with regard to optimal tissue removal. Using a novel method to quantitatively assess tissue removal by electrovaporization, this study has demonstrated that greater tissue removal can be achieved by increasing power, increasing the load, or decreasing the excursion rate, but only within limits.

Animals↗

PubMed1996

Tissue impedance as a function of temperature and time.

Tissue impedance dependence on temperature has been measured for six tissue types. The information was gathered using an automated laboratory under computer control. This information is needed to be able to input these values into a computer model that predicts temperature distribution produced by delivery of radio frequency energy. Due to the thermal dose of time and temperature, tissue properties change and no published data are available that document this. Since there are no theoretical predictions, empirical data were measured to supply this information. Using an aluminum cylindrical cavity of volume 1.69 cm3, muscle, liver, brain, and fat tissue impedance were measured at 500 kHz over a range of temperatures. All tests began at room temperature where baseline measurements were made. The cylinder was then placed in a constant temperature water bath at between 30 and 90 degrees C. The tests were run for a period of 10 to 30 minutes. Temperature homogeneity was carefully studied throughout the volume of the cylinder. It was found that thermal equilibrium occurred within four minutes. Special care was taken with the tissue sample in regard to optimize moisture and freshness, and minimize fat content. Grain orientation was also taken into consideration depending on the test. For all tissue types, resistivity decreased initially as the sample temperature equilibrated with the bath temperature. For temperatures less than 75 degrees C, resistivity values remained approximately constant over time.

Adipose Tissue↗

PubMed1995

Diquat-dependent protein carbonyl formation. Identification of lipid-dependent and lipid-independent pathways.

In a previous report on diquat-dependent oxidative damage in rat hepatic microsomes, protein oxidation, as measured by protein carbonyl (PC) formation, was observed in addition to lipid peroxidation (LP). Both phenomena were antioxidant sensitive. Inhibition of PC formation was somewhat surprising given the proposed mechanism of metal-catalyzed protein oxidation. Studies reported here examined diquat-dependent PC formation in greater detail. In rat hepatic microsomes, diquat-dependent thiobarbituric acid-reactive substances (TBARS) and PC formation were time and concentration dependent. In this system, LP was inhibited completely by U-74006F or U-78517G, whereas PC formation was inhibited only partially by these antioxidants. In an essentially lipid-free system consisting of purified rat hepatic cytochrome P450 reductase, BSA and an NADPH-generating system, PC formation was also observed, but was not antioxidant-sensitive. Under these conditions, minimal diquat-dependent TBARS formation was observed. The observation of relative antioxidant insensitivity is consistent with H2O2 (generated during the diquat redox cycle) catalyzing protein oxidation via a site-specific, metal-catalyzed mechanism. Thus, different pathways would appear to be involved in diquat-dependent PC formation in lipid-containing and lipid-free systems. Carbon tetrachloride induces LP following reductive activation to the trichloromethyl free radical, a pathway not directly involving H2O2 generation. In the microsomal system, CCl4 induced TBARS and PC formation, both of which were completely inhibitable by antioxidants. Taken together, these data suggest that diquat induces PC formation by lipid-dependent (antioxidant-sensitive) and lipid-independent (antioxidant-insensitive) pathways. In microsomes, both pathways contribute to diquat-dependent PC formation. Data for the lipid-independent pathway are consistent with the mechanism of metal-catalyzed protein oxidation proposed by Stadtman and colleagues (reviewed in Free Radic Biol Med 9: 315-325, 1990), while the lipid-dependent pathway is likely secondary to LP itself--via a Michael-type addition reaction between hydroxyalkenals and protein sulfhydryl groups, amino groups or other protein nucleophiles. The latter pathway is also responsible for carbon tetrachloride-dependent PC formation. Additional studies are in progress to further characterize the lipid-independent mechanism.

Animals↗

PubMed1994

Brain hyperthermia: I. Interstitial microwave antenna array techniques--the Dartmouth experience.

PURPOSE: Microwave antennas of various designs were inserted into arrays of nylon catheters implanted in brain tumors with the goal of raising temperatures throughout the target volume to 43.0 degrees C. METHODS AND MATERIALS: All antennas were flexible, and included dipole, choke dipole, modified dipole, and helical designs driven at 915 or 2450 MHz. Antennas were tested in brain-equivalent phantom in arrays. Phase shifting and phase rotation techniques were incorporated into the treatment system to steer power in the tumor, assisted by a treatment planning computer that predicted power deposition patterns and temperature distributions. Choke antennas were designed and tested to reduce a dependence of the central power location on depth of insertion into tissue. Temperature data analysis used only central and orthogonal axes mapping data measured at 2.0 mm intervals. RESULTS: A total of 23 patients were treated, using from one to six microwave antennas. Minimum tumor temperatures, averaged over the 60 min treatment, ranged from 37.2-44.3 degrees C (mean 40.0 degrees C) and maximum average tumor temperatures ranged from 46.5-60.1 degrees C (mean 49.1 degrees C). The percentage of all measured temperatures reaching therapeutic levels (> or = 43.0 degrees C) was 70.9. T90, the temperature at which 90% of all measured temperatures equaled or exceeded, was 40.8 degrees C, and T50 was 44.2 degrees C. CONCLUSION: Patient data analysis showed that the array of four dipole antennas spaced 2.0 cm apart were capable of heating a volume of 5.9 cm (along the central array axis) x 2.8 cm x 2.8 cm.

Brain Neoplasms↗

PubMed1994

Theoretical and experimental analysis of air cooling for intracavitary microwave hyperthermia applicators.

An intracavitary microwave antenna array system has been developed and tested for the hyperthermia treatment of prostate cancer at Thayer School of Engineering and Dartmouth-Hitchcock Medical Center. The antenna array consists of a choked dipole antenna inserted into the urethra and a choked dipole antenna eccentrically embedded in a Teflon obturator inserted into the rectum. To prevent unnecessary heating of the healthy tissue that surrounds each applicator, an air cooling system has been incorporated into the rectal applicator. The air cooling system was designed and modeled theoretically using a numerical solution of heat and momentum equations within the applicator, and an analytical solution of the Pennes bioheat equation in tissue surrounding the applicator. The 3-D temperature distribution produced by the air-cooled rectal applicator was measured in a perfused canine prostate.

Animals↗

PubMed1993

Inhibition of in vitro lipid peroxidation by 21-aminosteroids. Evidence for differential mechanisms.

In a previous report (Ryan and Petry, Arch Biochem Biophys 300: 699-704, 1993), the effects of two 21-aminosteroids (U-74500A and U-74006F) on the oxidation and reduction of iron in a buffer/organic solvent system were investigated. In those studies, U-74500A was found to be an efficient iron reductant and potential iron chelator, whereas U-74006F had little effect on iron redox chemistry. As an extension of those studies, we now report the effects of U-74006F and U-74500A on lipid peroxidation in systems that are dependent upon iron oxidation/reduction. In liposomes, U-74500A inhibited ADP:Fe(II)-dependent lipid peroxidation in a concentration-dependent manner, whereas U-74006F was minimally effective in this system. The mechanism of U-74500A-dependent inhibition probably involved interactions with iron, as iron oxidation was inhibited in the presence of this compound. No effects on iron oxidation were observed in the presence of U-74006F. Addition of Ferrozine to liposomal incubation mixtures indicated that at least two iron pools were present in samples containing U-74500A, one immediately bound by Ferrozine, and another that was bound more slowly. Furthermore, ADP:Fe(III)/ascorbate-dependent lipid peroxidation was blocked completely by U-74500A, presumably by formation of a redox inert complex upon reduction of the iron. U-74500A partially protected ADP:Fe(II) from oxidation by H2O2 and lipid hydroperoxides, indicating that the U-74500A:iron complex was stable in the presence of biologically relevant oxidants. U-74006F did not markedly affect iron oxidation or reduction when incorporated into phospholipid liposomes. In microsomal lipid peroxidation systems containing ADP:Fe(III) and NADPH, both U-74500A and U-74006F inhibited lipid peroxidation. U-74006F-dependent inhibition of microsomal lipid peroxidation was dependent on both NADPH and Fe(III). Further, it was enhanced when U-74006F was allowed to preincubate in this system prior to iron addition. Preincubation of U-74006F with microsomes, NADPH, and ADP:Fe(III) produced several metabolites detectable by HPLC. These results suggest that U-74500A inhibits lipid peroxidation by directly affecting iron redox chemistry, whereas U-74006F-mediated inhibition is enhanced by preincubation with a metabolically competent microsomal system.

Animals↗

PubMed1993

The effects of 21-aminosteroids on the redox status of iron in solution.

The effects of two 21-aminosteroids (U-74500A and U-74006F) on the oxidation and reduction of iron were investigated. U-74500A completely prevented ADP: Fe(II) autoxidation whereas U-74006F had only a slight inhibitory effect. The inhibition of Fe(II) oxidation by U-74500A was concentration dependent, with 100% inhibition occurring at concentrations equal to or greater than 25 microM in systems containing 50 microM Fe(II). When the Fe(II)-specific chelator Ferrozine was added to incubations containing U-74500A and ADP:Fe(II), formation of the Ferrozine:Fe(II) chromophore was slow, suggesting that U-74500A chelates Fe(II) with substantial affinity. Temporally, 20 min were required for complete formation of the Ferrozine-Fe(II) chromophore in the presence of U-74500A, whereas complexation in its absence was instantaneous. This phenomenon was not observed with U-74006F, Desferal, or ascorbate. In a system containing 25 microM ADP:Fe(II), U-74500A (25 microM) and U-74006F (25 microM) acted as iron reductants, reducing the iron at rates of approximately 2, and 0.1 microM/min, respectively. In addition, U-74500A fluorescence was quenched in a concentration-dependent manner upon the addition of Fe(III), further demonstrating interactions between this compound and iron. The substructures of U-74500A consist of a steroid (U-76911) and a complex amine (U-82902E). When these compounds were assayed individually, it was found that U-82902E exhibited activities similar to those of U-74500A, whereas the free steroid had no effect. Studies employing cyclic voltammetry revealed that U-74500A had a relatively low oxidation potential (E = 228 mV), whereas U-74006F was much less susceptible to oxidation (E = 810 mV). Taken together, these data suggest that subtle effects on iron redox chemistry, which would in turn inhibit or eliminate the initiation of undesired oxidative reactions, may contribute to the potent antioxidant activities of U-74500A and U-74006F.

Adenosine Diphosphate↗

PubMed1993

The role of metals in the enzymatic and nonenzymatic oxidation of epinephrine.

The effects of transition metals on nonenzymatic and ceruloplasmin catalyzed epinephrine oxidation were investigated by studying rates of epinephrine oxidation in purified buffers and in the presence of metal chelating agents. We found that epinephrine does not "autoxidize" in sodium chloride solutions prepared with deionized water that was further purified by chromatography over Chelex 100 resin prior to use. Epinephrine was oxidized rapidly in sodium chloride prepared with tap water (1.20 +/- 0.12 nmoles/min) or in deionized water (0.40 +/- 0.80 nmoles/min), but this oxidation was prevented by the addition of Desferal, a potent metal chelating agent. Epinephrine oxidation was enhanced upon the addition of ceruloplasmin, and this oxidation rate could be slowed, but not eliminated, by the addition of Desferal. If epinephrine solutions were preincubated for 72 hours with Desferal prior to ceruloplasmin addition, however, no oxidation was observed. Epinephrine was shown to form colored complexes with both iron and copper at pH 7.0. The Fe(III)-epinephrine complex was much more stable than was the Cu(II)-epinephrine complex. Oxygen consumption studies of ceruloplasmin catalyzed epinephrine oxidation showed that copper was a better promoter of epinephrine oxidation than was iron, suggesting that ceruloplasmin-catalyzed epinephrine oxidation results from adventitious copper bound to the purified enzyme. In light of these results, the physiological relevance of ceruloplasmin catalyzed oxidation of biogenic amines may be minor.

Biogenic Monoamines↗

PubMed1992

Rat ceruloplasmin: resistance to proteolysis and kinetic comparison with human ceruloplasmin.

Rat ceruloplasmin was purified from serum using fast protein liquid chromatography and compared to human ceruloplasmin isolated in the same manner. Rat ceruloplasmin was found to be more resistant to plasmin-mediated proteolysis than was human ceruloplasmin. Although both proteins were cleaved initially to products with apparent molecular weights of 116,000 and 20,000 Da, rat ceruloplasmin was resistant to further proteolysis, whereas the human enzyme was cleaved to smaller fragments. Primary structure differences could account for the different relative stabilities between the two enzymes. Kinetic analysis of rat ceruloplasmin produced a biphasic v vs v/s plot with apparent Km's of 40 and 1.5 microM for iron. When compared with the human enzyme, rat ceruloplasmin showed about one-fourth the ferroxidase activity and had a much broader pH profile than that of human ceruloplasmin. Rates of p-phenylenediamine oxidation by rat ceruloplasmin were about one-half those obtained with human ceruloplasmin, with maximal p-phenylenediamine oxidase activity at pH 5.0 for both enzymes.

Amino Acid Sequence↗