Biomedical subjects

H M Swartz

Publications and source records attributed to H M Swartz.

At least 163 records · Page 9Linked to original sources

PubMed1988

Distribution of 5-doxylstearic acid in the membranes of mammalian cells.

Concentration-dependent spin broadening of ESR spectra of the nitroxide 5-doxylstearic acid has been used to evaluate the distribution of 5-doxylstearic acid in the membranes of intact mouse thymus-bone marrow (TB) and Chinese hamster ovary (CHO) cells. TB cells, CHO cells, erythrocytes, and isolated plasma membranes from CHO cells were labelled with 5-doxylstearic acid and the peak to peak linewidths of the central line of the resulting ESR spectra were measured. The measured line widths were linearly dependent on the amount of 5-doxylstearic acid incorporated into the sample over the range of 0-0.18 mol nitroxide per mol lipid. In erythrocytes, the relationship between linewidths approximated a linear function at lower concentrations of 5-doxylstearic acid, up to 0.07 mol nitroxide per mol lipid. The amount of broadening of the central line for a given amount of 5-doxylstearic acid was far less for intact cells than for either erythrocytes or plasma membrane, indicating that the 5-doxylstearic acid samples a much larger lipid pool in the intact cells. With the broad assumption that the mobility of the 5-doxylstearic acid is similar in different membranes, the size of the lipid pool sampled by 5-doxylstearic acid is approximately equal to the total cellular lipid in intact cells. If a given concentration of 5-doxylstearic acid sampled only the plasma membrane of TB or CHO cells, we would expect to see a linewidth corresponding to a 12-20-fold greater local concentration of 5-doxylstearic acid than was observed, since the plasma membranes of CHO and TB cells represent only 5-8 percent of the total cellular lipid. Therefore, the 5-doxylstearic acid must distribute into most or all cellular membranes of intact cells and is not localized in the plasma membrane alone.

Animals↗

PubMed1988

Kinetics of enzyme-mediated reduction of lipid soluble nitroxide spin labels by living cells.

Nitroxide spin labels can be reduced to the corresponding hydroxylamines in cells. The selective action of inhibitors, and thermal and chemical inactivation demonstrate that the reduction of nitroxides in cells is an enzymatic or enzyme-mediated process. The kinetics of reduction of doxylstearates are affected by the position of the doxyl moiety along the stearic acid chain. The doxyl moiety of 5-doxylstearate is close to the membrane surface, and its reduction is first order with respect to the nitroxide, whereas the doxyl moieties of 10- and 12-doxylstearate are in the membrane hydrocarbon region and their reduction is a zero-order process. The reduction of 16-doxylstearate which usually has a mixture of first- and zero-order kinetics becomes zero order with addition of an extracellular broadening agent, potassium trioxalatochromiate(III). These results suggest that the rate of reduction of doxyl moieties is controlled by their accessibility to reducing equivalents, i.e., the rate-limiting step for the reduction of the doxyl moiety deep in the membrane is the diffusion of reducing equivalents within or into the membrane. The reduction of doxylstearates in cells is inhibited by rotenone but not antimycin A, cyanide, propyl gallate or SKF-525A. It appears that the reduction of doxylstearates takes place at the level of the ubiquinone in the respiratory chain in mitochondria in these cells.

Animals↗

PubMed1988

Oxidation of hydroxylamines to nitroxide spin labels in living cells.

In the presence of oxygen, cells can oxidize hydroxylamines, which are the products of the reduction of nitroxides in cells, back to nitroxides. Lipid-soluble hydroxylamines are oxidized much more rapidly than water-soluble ones, and most of this oxidation is inactivated by heat or trichloroacetic acid, indicating that the principal mechanism is enzyme-linked. The rates of oxidation of some lipophilic hydroxylamines are comparable to the rates of reduction of the corresponding nitroxides. Hydroxylamines formed by reduction of aqueous soluble nitroxides are not oxidized by cells, except for slight oxidation of some pyrrolidine derivatives. The latter is due to autoxidation. The kinetics of oxidation of reduced lipid-soluble nitroxides are all first-order with respect to hydroxylamines, regardless of the position of the nitroxide group along the carbon backbone, indicating that the oxidation occurs within the membrane. The oxidation of hydroxylamines in cells in inhibited by cyanide but not by antimycin A or SKF-525A. We also describe an effective method to oxidize hydroxylamines and follow this reaction; the method is based on the use of perdeuterated [15N]Tempone.

Affinity Labels↗

PubMed1988

Modulation of water proton relaxation rates by liposomes containing paramagnetic materials.

Liposomes containing paramagnetic chemicals have significant potential for enhancing the effectiveness and selectivity of contrast agents in magnetic resonance imaging. We have studied the interactions of water, liposomes, and contrast agents using NMR and ESR. We find that the permeability of liposomes to water is of major importance in determining the effects of these systems on water proton relaxation. Key factors include (1) liposome composition, which determines the phospholipid membrane phase behavior as a function of temperature; (2) liposome structure (unilamellar or multilamellar); (3) location of the contrast agent in the liposome (aqueous space or lipid membrane); and (4) binding of contrast agents by liposomes. By appropriate selection of these factors it is possible to obtain effective proton relaxation enhancement in a manner suitable for particular experimental or clinical needs for contrast in MRI. For maximum relaxivity using lipophilic nitroxides, the optimum system appears to be 5-doxyl stearate in multilamellar vesicles with a membrane composition that gives a phase transition temperature slightly above body temperature.

Contrast Media↗

PubMed1988

Delivery of nitroxide spin label to cultured cells by liposomes.

The positively charged nitroxide spin label, 2,2,6,6-tetramethyl-piperidine-N-oxyl-4-trimethylammonium (Cat1), was encapsulated in two types of liposomes, phosphatidylserine/phosphatidylcholine (PS/PC) and phosphatidylserine/distearoylphosphatidylcholine/dipalmitoylphosphatidyl choline (PS/DSPC/DPPC). The liposomes were incubated with mouse thymus-bone marrow (TB) cells to study the uptake and metabolism of nitroxides entrapped in liposomes. The effects of temperature, metabolic inhibitors, and fixation of cells were investigated. The results indicate that different mechanisms are involved in the uptake of these two types of liposomes. PS/PC liposomes interact predominantely with the plasma membrane of TB cells and release Cat1 continuously, whereas the majority of PS/DSPC/DPPC liposomes are taken into the cells intact via endocytosis. These findings suggest that it may be possible to deliver nitroxides selectively, either to the membrane of cells or to their interior by manipulating the lipid composition of the liposomes. This study also found that the rate of reduction of Cat1 delivered using liposomes was increased under hypoxic conditions. Thus, the use of liposomes for in vivo delivery of nitroxides has the potential to provide NMR contrast that reflects different metabolic conditions.

Animals↗

PubMed1988

Electron spin resonance imaging of tissues with lipid-rich areas.

Electron spin resonance imaging and spectroscopy of lipid-rich samples were studied at 9 Ghz. Using both lipid- and aqueous-soluble nitroxides good delineation of fat- and muscle-rich regions of tissue was obtained. Several different principles can be used to differentiate between these types of tissues and potentially between different types of lipids. These principles include lipophilicity of nitroxides, viscosity (which is higher in lipids), oxygen solubility (oxygen is four times more soluble in lipids), and the distribution of paramagnetic metal ions. These techniques have potential applicability for directly studying metabolism and oxygen concentration in tissues by ESR techniques and for investigating potential contrast agents for in vivo NMR techniques.

Adipose Tissue↗

PubMed1988

The effect of melanin on iron associated decomposition of hydrogen peroxide.

The effects of melanin on the iron-catalyzed decomposition of hydrogen peroxide to hydroxyl radicals and hydroxyl ions have been studied using electron spin resonance, spin trapping and visible light spectrophotometry. Melanin altered these reactions by several different mechanisms and consequently, depending on conditions, can significantly increase or decrease the yield of reactive products, including hydroxyl radicals. For low concentrations of ferrous ions, melanin decreased the yield of hydroxyl radicals due to binding of ferrous ions by melanin; ferrous ions bound to melanin did not decompose H2O2 efficiently. Melanins increased the rate of hydroxyl radical production if the predominant form of iron was ferric, due to the ability of melanin to reduce ferric to ferrous iron. Hydroxyl radical production in the presence of a strong chelator (e.g. EDTA) and melanin was greater than in the presence of a weak chelator (e.g. ADP) and melanin. Melanin also increased the rate of destruction of the DMPO-OH adduct.

Adenosine Diphosphate↗

PubMed1987

Effects of nitroxides on the magnetic field and temperature dependence of 1/T1 of solvent water protons.

We report a study of the longitudinal NMRD profiles (proton longitudinal relaxation rates as a function of field strength) over a broad range of magnetic field (0.01 to 50 MHz proton Larmor frequency) and temperature (-9.6 to 37 degrees C) for aqueous solutions of (i) a fatty acid-nitroxide/albumin complex and (ii) 10 low molecular weight nitroxides. Analysis of the NMRD profile for the fatty acid-nitroxide/albumin complex provides a lower bound estimate for the rotational correlation time of the complex, which permits the calculation of an upper bound on the inner sphere contribution to relaxation of the uncomplexed nitroxides. Inner sphere processes, ostensibly due to water molecules hydrogen bonded to the nitroxide moiety, dominate the relaxation effects of the slowly rotating macromolecular nitroxide/albumin complex. By extrapolation, the contribution of these inner sphere processes are negligible for rapidly tumbling nitroxides free in solution, which affect solvent proton relaxation almost entirely through outer sphere processes (i.e., translational diffusion). A comparison of the relaxation data for aqueous solutions of the uncomplexed nitroxides with the theory of outer sphere relaxation of J.H. Freed (J. Chem. Phys. 68, 4034 (1978] yields values for the distance of closest approach of the water and nitroxide molecules, as well as for their relative diffusion constants, at five different temperatures. Our results indicate that the rather modest relaxivities of aqueous solutions of nitroxides increase substantially with increased solvent viscosity and with protein binding, supporting the potential utility of nitroxides for enhancement of contrast in nuclear magnetic resonance images.

Cyclic N-Oxides↗

PubMed1987

Oxygen-dependent metabolism of potential magnetic resonance contrast agents.

Contrast agents have been investigated in an effort to increase the differences between the proton relaxation times of normal and pathologic tissue, and thus improve the ability to detect regions of pathology. Data on the oxygen-dependent metabolism of three nitroxides that are potential MRI contrast agents are presented. The amount of active, paramagnetic nitroxide remaining in suspensions of mammalian cells was followed as a function of time using electron-spin resonance spectroscopy. Data on the rates of nitroxide reduction over a wide range of oxygen concentrations are presented and show these rates as a function of the intracellular oxygen concentration. When intracellular oxygen concentrations were measured, only cells that were severely hypoxic reduced these potential contrast agents at increased rates. Because regions of some types of pathology are often hypoxic, the oxygen-dependent metabolism of nitroxides should allow contrast to be achieved on the basis of differences in metabolism between normal and diseased tissues. Further, the data suggest that future studies of nitroxide metabolism need to take into account the O2 concentration in order to understand the observed reduction rates and to predict the rates under other conditions.

Animals↗

PubMed1987

Modification of relaxation of lipid protons by molecular oxygen and nitroxides.

Measurement of proton 1/T1 in model lipids from 0.00023 to 1.3 Tesla, at 5 degrees C and 37 degrees C, shows that both oxygen and nitroxides effectively enhance the relaxation rates of the protons of lipids. Equilibration with 1 atm of oxygen has a sixfold greater effect on lipid protons than on water protons, primarily because of the high lipid solubility of oxygen. The maximum relative relaxivity of oxygen for lipid protons occurs at 0.12-0.8 Tesla. Lipid soluble nitroxides have a four- to eightfold greater effect on lipid protons than on water protons. This high relaxivity, combined with the high lipid solubility possible with nitroxides, could lead to significant contrast in vivo in lipid environments.

Cyclic N-Oxides↗

PubMed1987

Further evidence that the pigment in the Dubin-Johnson syndrome is not melanin.

The pigment in the Dubin-Johnson syndrome (DJS) is shown unequivocally not to be a typical melanin or closely related polymer. In electron spin resonance (ESR) studies of DJS pigment from a hepatoma, it is shown that, unlike true melanins, the pigment associated with the DJS syndrome has no free radical in the absence of light. Exposure to even low levels of visible light over a broad frequency range induces a free radical in the DJS pigment. Previous studies did not appreciate the sensitivity to light of this pigment and therefore erroneously concluded that the DJS pigment had a permanent free radical. The light induced ESR signal in DJS tissue has spectroscopic properties that differ significantly from any known melanins. The pigment is not extracted by lipophilic solvents and is centrifuged down at 50,000g, but not at 5,000g.

Carcinoma, Hepatocellular↗

PubMed1986

Cellular metabolism of water-soluble nitroxides: effect on rate of reduction of cell/nitroxide ratio, oxygen concentrations and permeability of nitroxides.

In order to interpret more accurately studies that have used nitroxides and to improve the efficacy of the use of nitroxides in both basic studies of cells and as contrast agents for in vivo NMR, we have initiated a systematic study of the distribution and metabolism of nitroxides in biological systems. Overall, the results provide a reasonably coherent picture of some aspects of the interactions between nitroxides and cells. Reduction of the nitroxides appears to be an intracellular process, so that one of the principal variables that affects the rate of reduction is the ability of a nitroxide to enter cells. The entrance of nitroxides into cells shows considerable variability and ranges from essentially no penetration (e.g., 2,2,6,6-tetramethylpiperidine-N-oxyl-4-trimethylamine), through rates that are comparable to rates of reduction (e.g., 2,2,5,5-tetramethyl-pyrrolidine-N-oxyl-3-carboxylic acid), to rates that are so fast that there is complete equilibrium between intracellular and extracellular compartments (e.g., Tempone). The presence of a charged group on the nitroxide appears to be the important variable that affects their ability to enter cells. Once a nitroxides enters the cell, the structure of the nitroxide, e.g., piperidine vs. pyrrolidine ring, is major factor that affects the rate of reduction. The rates of reduction increase with increasing concentrations of nitroxides. This indicates that the principal mechanism(s) of reduction do not saturate in the concentration range we studied. We observed no abrupt changes in the rates of reduction over the entire concentration range of cells and nitroxides that we studied, which suggests that the mechanism(s) of nitroxide reduction did not change. The presence of oxygen decreased the observed rate of reduction of many of the nitroxides and this effect was independent of the concentration of nitroxide.

Animals↗

PubMed1986

Hypoxia-sensitive NMR contrast agents.

The rate of reduction of nitroxides is shown to be more rapid in hypoxic cells. The rate of reduction and the effect of hypoxia on the reduction rate vary for different nitroxides. These findings indicate that it may be feasible to develop in vivo NMR contrast agents that selectively will indicate areas of hypoxia and thereby aid in the detection of disease processes such as neoplasia, ischemia, and inflammation.

Animals↗

PubMed1986

Liposome delivery of NMR contrast agents for improved tissue imaging.

Large unilamellar liposomes containing the paramagnetic contrast agent manganese chloride were shown to selectively enhance the intensity of the proton NMR signal from the livers of experimental mice. The kinetics of the observed effect indicate that the liposomes are rapidly removed from the circulation and deposited in high concentration within the liver. This process is known to involve the phagocytic activity of macrophages. These results show that liposomes could provide a useful and efficient vehicle for delivering NMR contrast agents to organs like the liver, spleen, and bone marrow. It may also be possible to use liposomes to selectively deliver contrast agents to tissue regions of developing pathology that are rich in macrophage activity.

Animals↗

PubMed1985

Measurement of intracellular oxygen concentration using the spin label TEMPOL.

We have developed a noninvasive method with general applicability for measuring intracellular oxygen using the spin label TEMPOL (2,2,6,6,-tetramethypiperidine-N-oxyl-4-ol) which has superhyperfine structure in its electron spin resonance spectra that is broadened in the presence of oxygen. This broadening is linear over a range of 1 to 6 ppm oxygen which covers the important physiological range of oxygen concentration. Viscosity, TEMPOL concentration, and instrument modulation intensity also can affect superhyperfine structure but the contributions from these effects can be determined. The TEMPOL distributes equally into the intra- and extracellular compartments but its intracellular signal can be studied selectively by addition of transition metal ions such as potassium ferricyanide and potassium tris(oxalato)chromiate, which broaden away the signal from extracellular TEMPOL and do not cross the cell membrane to affect the intracellular TEMPOL. Results with a cell culture line (mouse thymus-bone marrow) indicate that under our experimental conditions these cells may maintain an average intracellular oxygen concentration lower than the extracellular oxygen concentration, and that there is not a constant relationship between extracellular and intracellular oxygen concentrations.

Animals↗

PubMed1985

Effect of melanin on radiation response of CHO cells.

The effect of the presence of melanin on the response of mammalian cells to ionizing radiation was investigated in a model system utilizing the ability of Chinese hamster ovary cells to incorporate melanin by endocytosis. Cells were incubated in monolayer cultures from 2 to 20 hours with melanin prepared from 'beef eye' or synthesized by air oxidation of 3,4-dihydroxyphenylalanine. For asynchronous cultures, the survival curve parameters for cells incubated with both types of melanin were indistinguishable from those of the same cells without added melanin. The radiation response to fractionated doses of 6 Gy separated by various periods did not indicate any effect of melanin on the extent or kinetics of repair of sublethal damage. Likewise, the repair of potentially lethal damage in plateau phase cultures was unaffected by the presence of melanin. Thus the explanation for the clinical radiation resistance of melanomas in the absence of a direct radiation effect might more likely be found in consideration of other factors such as the role of melanin in oxygen consumption or in differentiation.

Animals↗

PubMed1984

Studies on human uterine cervix and rat uterus using S-, X- and Q-band electron-spin-resonance spectroscopy.

In previous studies we have reported on the detection of a strong e.s.r. signal in samples of normal human cervix; the signal is much reduced or absent in samples of invasive cancer of the cervix. In order to identify the species responsible for the strong signal, we have used X-, S- and Q-band e.s.r. spectroscopy. The major signal that is detectable in ground-up samples of cervix preserved at -196 degrees C has features consistent with the presence of a peroxy free radical. Good agreement with the experimental findings was obtained by computer simulation, using values for the g-tensor of gx = 2.002, gy = 2.005 and gz = 2.036. The peroxy radical is produced on grinding the normal cervix samples to a powder under liquid N2, and appears to be formed by modification of a pre-existing oxygen-containing complex. Control experiments eliminated the possibility that the strong signals seen in frozen powders prepared from normal cervix were artefacts only of the grinding procedure. Experiments with rats in vivo and with cervix samples in vitro are consistent with the conclusion that the peroxy radical is formed by disturbing the cyclo-oxygenase system that is involved in prostaglandin synthesis.

Animals↗

PubMed1984

Identification by electron spin resonance of free radicals formed during the oxidation of 4-hydroxyanisole catalyzed by tyrosinase.

We have observed the formation of free radicals during the oxidation of the melanocytotoxic agent 4-hydroxyanisole with the enzyme tyrosinase as a catalyst. The first free radical to form is identified as the 4-methoxy-1,2-benzosemiquinone radical anion. The peak concentration of this radical increases with tyrosinase concentration; a minimum concentration of 50 micrograms/ml of tyrosinase was needed to observe this radical. The peak concentration of this radical is independent of 4-hydroxyanisole concentration. This radical is produced by reverse dismutation of the primary product, 4-methoxy-1,2-benzoquinone and 4-methoxycatechol produced indirectly.

Anisoles↗