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H M Swartz

Publications and source records attributed to H M Swartz.

At least 145 records · Page 8Linked to original sources
PubMed1990

Interactions of nitroxides with plasma and blood: effect on 1/T1 of water protons.

Nitroxide stable free radicals (nitroxides) have potential utility as MRI contrast-enhancing agents with the additional capability of reflecting redox metabolism. In order to gain a better understanding of their potential interactions in vivo, we have studied the longitudinal NMRD profiles (1/T1 as a function of field strength) and ESR spectra for lipophilic and aqueous-soluble nitroxides in blood, plasma, and plasma components. Typical water-soluble nitroxides do not interact appreciably with blood, plasma, or plasma proteins. Fatty acid nitroxides do interact physically with blood, predominantly by intercalation within red blood cell membranes and binding to albumin. The latter interaction results in significantly enhanced relaxivity for the nitroxide/HSA complex. Relaxation of water protons in this case is dominated by inner sphere processes, ostensibly due to water molecules hydrogen bonded to nitroxide moieties. The rotational reorientation time for the complex, the electronic relaxation time, and the exchange time for the water molecule reversably bound to the nitroxide, all appear significantly to influence the correlation time (approximately 16 ns) for this inner sphere contribution.

Blood Proteins↗
PubMed1990

Measurement of time-resolved oxygen concentration changes in photosynthetic systems by nitroxide-based EPR oximetry.

The application of recent developments of EPR oximetry to photosynthetic systems is described and used to study rapid processes in isolated thylakoid membranes from spinach and in intact photoautotrophic soybean cells. Using the peak heights of 15N perdeuterated Tempone and two microwave power levels oxygen evolution and consumption were measured. The method measured time-resolved oxygen concentration changes in the micromolar range. Oxygen evolution was linearly proportionate to the chlorophyl concentration of thylakoid membrane over the range studied (0-2 mg/ml). Oxygen evolution associated with single turnover light pulses was consistent with the four state model. The time (t1/2) to reach equilibrium of oxygen concentrations after a single turnover pulse was 0.4-0.5 ms, indicating that the evolution of oxygen coupled to the S4-S0 transition may be shorter than reported previously. The time for equilibrium of oxygen after single turnover pulses in soybean cells was relatively long (400 ms), which suggests that there are significant barriers to the free diffusion of oxygen in this system. The method also was used to study oxygen consumption by the electron transport chain of photosystem I and photosystem II. We conclude that EPR oximetry can provide quantitative and time-resolved data on oxygen concentrations with a sensitivity that is useful for studies of such systems.

Cyclic N-Oxides↗
PubMed1990

Principles of the metabolism of nitroxides and their implications for spin trapping.

A review of the principal interactions of nitroxides with cells suggests that if these same phenomena occur with spin adducts the result could be considerable experimental confusion and error. In particular, these could lead to differential rates of loss of spin adducts, thereby potentially invalidating conclusions on the amounts or even the types of free radicals that are trapped. In addition, shuttling of electrons between nitroxides and hydroxylamines also very significantly could alter the amounts and types of spin adducts that are observed.

Cells↗
PubMed1989

Development of nitroxides for selective localization inside cells.

The use of nitroxides to measure intracellular phenomena, especially oxygen concentrations, is a new and potentially important approach to a number of physiological and pathophysiological studies. This study provides data indicating the feasibility of developing nitroxides that localize selectively in the intracellular compartment; it is based on the use of readily hydrolysed ester linkages, such that the nitroxides become converted intracellularly to ionic derivatives that do not cross cell membranes readily. Up to 120-fold increased concentrations of intracellular nitroxides (and their one electron reduction product, the hydroxylamines) were obtained. The ESR spectra of the intracellular nitroxides were consistent with their conversion to the ionic species. Preliminary studies indicate that these nitroxides have the properties needed for their use as probes of intracellular concentrations of oxygen and that it should be feasible to synthesize nitroxides that will be even more effective for this purpose.

Animals↗
PubMed1989

Oximetry in cells and tissues using a nitroxide-liposome system.

In order to avoid the complication of reduction of nitroxides in biological media during oxygen measurements, liposomes containing a water-soluble nitroxide, 2,2,6,6-tetramethyl-piperidine-N-oxyl-4-trimethylammonium (Cat1), were used in studies of oxygen consumption by thymus-bone-marrow cells. The superhyperfine structure of Cat1 contained in liposomes was found to be sensitive to oxygen concentration in a fashion similar to that of free Cat1. Measurements of cellular respiration using Cat1 contained in liposomes agreed well with the results obtained using free Cat1. Using this nitroxide-liposome system, the respiration of liver slices was measured successfully, whereas such measurements using free cat1 were complicated by rapid reduction of the nitroxide. This nitroxide-liposome system also could be used in conjunction with a membrane permeable nitroxide and an extracellular broadening agent to measure intracellular and extracellular oxygen concentrations simultaneously.

Animals↗
PubMed1989

Metabolism in rat liver microsomes of the nitroxide spin probe tempol.

Paramagnetic nitroxide spin labels have been extensively used to probe various biophysical and biochemical properties of the cellular environment. Recently nitroxides have been proposed as contrast enhancing agents in proton magnetic resonance imaging and contrast enhancement has been demonstrated in animal studies. Nitroxides, possessing a stable unpaired electron, increases the relaxation rates of protons, providing an enhancement of contrast. Nitroxides are metabolized intracellularly principally via reversible reduction to hydroxylamines. Rates of reduction depend on the physical characteristics of the nitroxides, in general 5-membered pyrrolidine ring are reduced more slowly than those with a 6-membered piperidine ring. Oxidation back to the nitroxide is relevant for lipid soluble hydroxylamines, while is low for water soluble ones. It is known that nitroxides are metabolized by subcellular fractions (cytosol, mitochondria, microsomes), though the enzymatic and non-enzymatic systems involved are poorly characterized. In the present study, the first of the necessary steps toward a systematic study of the metabolism of nitroxides by subcellular organelles, we have chosen to study the metabolism of 4-hydroxy 2,2,6,6-tetramethylpiperidine-N-oxyl in isolated rat liver microsomes. Microsomes were able to reduce Tempol slowly without any substrate addition; when NADPH was added, the reduction rate substantially increased. In phenobarbitone induced rats the reduction rate was significantly higher than in not-induced microsomes. NADPH-dependent reduction rate was inhibited by thallium chloride (an inhibitor of the flavin-centered cytochrome P-450 reductase), superoxide dismutase, and by N-ethylmaleimide; menadione increased it. The Tempol reduction rate was not significantly affected by various cytochrome P-450 inhibitors with the sole exception of metyrapone. A solution containing purified cytochrome P-450 reductase and NADPH readily reduced Tempol. Microsomes fortified with NADPH were able to reduce Tempol at an appreciable rate. In order to distinguish between reduction of nitroxides to hydroxylamine or destruction of nitroxides following nitroxide reduction, microsomal suspensions were treated with a mild oxidant (ferricyanide 0.5-10 mM). The recovery varied from 40 to 60%, indicating a process of probe destruction leading to as yet unknown metabolites. The present study clearly indicates that, in this model system, cytochrome c (P-450) reductase and not cytochrome P-450 is responsible for the observed Tempol metabolism; along with hydroxylamine formation, other Tempol derived metabolites are formed during the process.

Animals↗
PubMed1989

The products of the reduction of doxyl stearates in cells are hydroxylamines as shown by oxidation by 15N-perdeuterated Tempone.

The use of nitroxides in functional biological systems has increased greatly as it has become evident that such studies can provide valuable biophysical and metabolic data. This has led to a need to understand the nature of the metabolism of nitroxides and their products. This paper presents data indicating the value of 15N-perdeuterated Tempone specifically to indicate the amount of hydroxylamines that are present in a cellular system. Using this technique, we found that in the mammalian cells that we studied the principal or only products of reduction of doxyl stearates were the corresponding hydroxylamines.

Animals↗
PubMed1989

Detection of bone marrow involvement in patients with cancer.

Current methods for the study of bone marrow to evaluate possible primary or metastatic cancers are reviewed. Bone marrow biopsy, radionuclide scan, computed tomography and magnetic resonance imaging (MRI) are analyzed with regard to their clinical usefulness at the time of diagnosis and during the course of the disease. Bone marrow biopsy is still the examination of choice not only in hematologic malignancies but also for tumors that metastasize into the marrow. Radionuclide scans are indicated for screening for skeletal metastases, except for those from thyroid carcinoma and multiple myeloma. Computed tomography is useful for cortical bone evaluation. MRI shows a high sensitivity in finding occult sites of disease in the marrow but its use has been restricted by high cost and limited availability. However, the future of MRI in bone marrow evaluation seems assured. MRI is already the method of choice for diagnosis of multiple myeloma, when radiography is negative, and for quantitative evaluation of lymphoma when a crucial therapeutic decision (i.e. bone marrow transplantation) must be made. Finally, methods are being developed that will enhance the sensitivity and specificity of MRI studies of bone marrow.

Adult↗
PubMed1989

Metabolism of aqueous soluble nitroxides in hepatocytes: effects of cell integrity, oxygen, and structure of nitroxides.

The optimum use of nitroxides in viable biological systems, including live animals, requires knowledge of the metabolism of nitroxides by major organ systems, especially the liver. We report here details of the metabolism of several prototypic aqueous soluble nitroxides in suspensions of freshly isolated hepatocytes. The general patterns of metabolism were similar to those observed in other types of cells (previous studies have been done principally in cells from tissue culture, such as CHO cells) including the primary initial reaction being reduction to the hydroxylamine, an increased rate of metabolism of some nitroxides in hypoxic cells, faster rates of reduction of nitroxides on six-membered piperidine rings compared to five-membered pyrrolidine rings, and most metabolism being intracellular. Metabolism in hepatocytes differed from other cell lines in having (1) significant reduction in the extracellular medium due to ascorbate that was released from damaged hepatocytes; (2) decreased rates of metabolism in freeze-thawed cells due to damage to subcellular organelles. These results provide much of the data needed to understand the role of the liver in the metabolism of nitroxides by intact animals and explain some previously puzzling results which indicated an apparent unusually high rate of metabolism of a charged nitroxide (Cat1) by hepatocytes. Our results also indicate that the use of freshly isolated cells or tissue homogenates may introduce experimental artifacts in the study of the metabolism of nitroxides.

Air↗
PubMed1989

Effects of oxygen on the metabolism of nitroxide spin labels in cells.

The products of the reduction of nitroxides in cells are the corresponding hydroxylamines, which cells can oxidize back to the nitroxides in the presence of oxygen. Both the reduction of nitroxides and the oxidation of hydroxylamines are enzyme-mediated processes. For lipid-soluble nitroxides, the rates of reduction are strongly dependent on the intracellular concentration of oxygen; severely hypoxic cells reduce nitroxides more rapidly than cells supplied with oxygen. In contrast, the rates of oxidation of hydroxylamines increase smoothly with increasing intracellular oxygen concentration up to 150 microM. In order to separate the effects on the rates of metabolism of nitroxides due directly to oxygen from effects due to the redox state of enzymes, we studied the cells under conditions in which each of these variables could be changed independently. Oxygen affects the metabolism of these nitroxides primarily by interacting with cytochrome c oxidase to change the redox state of the enzymes in the respiratory chain. Our results are consistent with the conclusions that in these cells reduction of lipophilic nitroxides occurs at the level of ubiquinone in the respiratory chain in mitochondria, and oxidation of the corresponding hydroxylamines occurs at the level of cytochrome c oxidase.

Animals↗
PubMed1989

Oxygen gradients in CHO cells: measurement and characterization by electron spin resonance.

The concentration of oxygen within cells is important in many physiological and pathological processes, but such oxygen-dependent processes are generally studied as a function of the concentration of extracellular oxygen, due to a lack of suitable methods. Using a newly developed technique based on ESR spectroscopy, we show that respiration stimulation of a cell suspension can result in a significant difference between average intracellular and extracellular concentrations of oxygen. These results indicate that studies of oxygen-dependent phenomena in cells may require measurement of intracellular oxygen concentrations and imply that there are mechanisms in cells that restrict the free diffusion of oxygen.

Animals↗
PubMed1989

In vivo localized ESR spectroscopy reflecting metabolism.

Localized electron spin resonance spectroscopy in live mice was performed using a surface coil operating at 1.1 GHz with sufficient sensitivity and stability to measure quantitatively the time course of the distribution, uptake, and reduction of nitroxides in selected organs/regions (liver, bladder, head) of mice. The ability to measure regional concentrations of nitroxides in vivo could be used for pharmacokinetic analysis of drugs labeled with nitroxides and for measurement of oxygen concentrations and redox metabolism.

Animals↗
PubMed1989

Exchange and shuttling of electrons by nitroxide spin labels.

The ability of nitroxide spin labels to act as oxidizers of reduced nitroxides (hydroxylamines) in biological and model systems was demonstrated. All of the nitroxides tested were able to act as oxidizing agents with respect to hydroxylamine derivatives of nitroxides. The rates of these reactions were first order with respect to nitroxide concentration and with respect to hydroxylamine concentration, making the reaction second order overall. The second-order rate constants are reported for a number of these reactions. These reactions proceeded to an equilibrium state and the equilibrium constants for several combinations of reactants are presented. Both the rate constants and the equilibrium constants were found to be dependent on the ring structure of the nitroxide and hydroxylamine, with piperidines being reduced more easily and pyrrolidines and oxazolidines being oxidized more easily. All of the hydroxylamine derivatives were oxidized by air to their respective nitroxides, with the rate of this oxidation greater for pyrrolidines than for piperidines. Furthermore, hydroxylamines that are permeable to lipid bilayers were able to act as shuttles of reducing equivalents to liposome-encapsulated nitroxides that were otherwise inaccessible to reducing agents. This mechanism of shuttling of electrons was able to explain the relatively rapid reduction by cells of a nonpermeable nitroxide in the presence of a permeable nitroxide.

Animals↗
PubMed1989

Rapid assessment of liposomal stability in blood by an aqueous nitroxide spin label.

An electron spin resonance method using an aqueous nitroxide spin label, 2,2,6,-tetramethyl-piperidine-N-oxyl-4-trimethyl-ammonium, for rapid assessment of liposome stability in blood is presented. The retention of the nitroxide in liposomes is measured by its electron spin resonance signal intensity, a procedure which does not require separation of the sample from the blood. Any nitroxide that is released from the liposomes is reduced by external ascorbic acid which is added to the sample. The method permits kinetic studies on the integrity of liposomes in the presence of destabilizing factors such as detergent, blood, or alteration in temperature.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1989

Evaluation of dibromonitrosobenzene sulfonate as a spin trap in biological systems.

In the present study dibromonitrosobenzene sulfonate (DBNBS) was examined for its suitability for spin trapping for ESR detection of superoxide radicals in biological systems. This nitroso spin trap recently has been reported to yield very persistent spin adducts with O2. as well as with various carbon-centered radicals. In the present work the possible toxicity of DBNBS, the partitioning of its spin adducts into cells, and the stability of the adducts and the parent compound inside cells were studied. No significant toxicity was found. In cellular systems, however, DBNBS did not produce detectable adducts with O2.; it also did not detectably trap superoxide generated in the xanthine/xanthine oxidase system. Both DBNBS and a DBNBS adduct performed extracellularly and then added to cell suspensions were rapidly metabolized by cells. Intracellular spin adducts were not detected under any condition. Evidently, in spite of its promising features, DBNBS will not be useful for spin trapping radicals in cellular systems or for detecting superoxide radicals in any biological system.

Benzenesulfonates↗
PubMed1989

The cellular-induced decay of DMPO spin adducts of .OH and .O2.

In a recent report, it was concluded that DMPO, often considered the spin trap of choice for detection of superoxide and hydroxyl radical adducts in biological systems, may be unsuitable for many biological uses because of its instability in cellular systems. It was demonstrated in red blood cells and in hamster V79 cells that the DMPO spin adducts of .O2- and .OH are metabolized very rapidly so that even if formed, they may not be detected in many experiments with cells. Because of the potential importance of these findings to experiments already reported on the occurrence of oxygen radicals in cellular systems, and the implications of these findings for future experiments, we have extended the studies on DMPO to other cellular, systems. We have also investigated the role of oxygen in this system because it has been shown recently that very hypoxic cells reduce some nitroxides much more rapidly than oxic cells and therefore it seemed possible that the rapid loss of radical adducts of DMPO was due to the hypoxic conditions under which the previous experiments were carried out. The results of the present experiments indicate that the loss of the DMPO spin adducts occurs in other cell systems as well, that the decomposition rate is independent of the concentration of oxygen, and that the final products of cellular metabolism of DMPO adducts are different from those of most nitroxides. There is no evidence that intracellular DMPO-spin adducts of oxygen radicals can be observed under conditions similar to those used in this study. We conclude that DMPO is not likely to be a suitable agent for studying intracellular oxygen radicals.

Animals↗
PubMed1989

Sources of the increased longitudinal relaxation rates observed in melanotic melanoma. An in vitro study of synthetic melanins.

Researchers have suggested that the increased longitudinal relaxation rates (1/T1) of solvent water protons often found in melanoma result either from the paramagnetism of stable free radicals occurring in melanin or from that of methemoglobin in nonacute hemorrhagic regions of the tumor. However, field-cycling relaxometry and model solutions of synthetic melanin produced data which show that free radicals in melanin do not contribute significantly to 1/T1; instead, aggregation of melanin into macromolecular particles and binding of biologically-common paramagnetic metal ions (ie Fe3+, Mn2+, and Cu2+) to melanin effectively do increase 1/T1. These data have been combined with published histochemical data on melanin-containing tissues, while disregarding any additional effect related to hemorrhage. The result indicates that in melanoma the expected contribution of melanin-bound Fe3+ to 1/T1, at typical imaging fields, predominates under estimated in vivo conditions; furthermore, the total contribution from all sources, specifically due to the presence of melanin, is sufficient to account for reported measurements of 1/T1 in melanoma. Comparing the latter results with published data on T1 relaxation in model solutions of methemoglobin suggests that co-existing regions of nonacute microhemorrhage also may contribute significantly to 1/T1 under certain conditions. Finally, the implications for 1/T2 of melanin occurring in vivo within discrete melanosomes is discussed.

Chemical Phenomena↗