Measurements of pO2 in vivo, including human subjects, by electron paramagnetic resonance.
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Animals↗
Biomedical subjects
H M Swartz
Publications and source records attributed to H M Swartz.
Measurements of intracellular concentrations of oxygen: experimental results and conceptual implications of an observed gradient between intracellular and extracellular concentrations of oxygen.
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Animals↗
The roles of neuromelanin, binding of metal ions, and oxidative cytotoxicity in the pathogenesis of Parkinson's disease: a hypothesis.
A characteristic feature of both Parkinson's disease (idiopathic paralysis agitans) and normal aging is loss of pigmented neurons in the substantia nigra. This has been found to correlate with the accumulation of neuromelanin and with oxidative stress in this brain region, but a clear association between these factors has not been established. Based on our recent demonstration that neuromelanin is a true melanin, containing bound metal ions in situ, we present a general model for its accumulation in vivo and the hypotheses (1) that it has a cytoprotective function in the sequestration of redox-active metal ions under normal conditions but (2) that it has a cytotoxic role in the pathogenesis of Parkinson's disease. Thus, neuromelanin accumulates normally through the autooxidation of catecholamines and serves tightly to bind redox-active metal ions, processes which would accelerate under conditions of intracellular or extracellular oxidative stress. Based on the known properties of melanin, however, neuromelanin also has the potential for exacerbating oxidative stress, eg by generating H2O2 when it is intact or by releasing redox-active metal ions if it loses its integrity; these reactions also would modulate the reactivity of the neuromelanin. By overwhelming intracellular antioxidative defense mechanisms, such a positive-feedback cycle could turn a condition of chronic or repeated oxidative stress in vulnerable neurons into an acute crisis, leading to cellular death. If the cumulative stress in duration and/or degree is severe enough, neuronal depletion could be sufficient to cause Parkinson's disease during life. One possible trigger for this cascade is suggested by the increased nigral iron contents in postmortem parkinsonian brains and the correlation of this disease with urban living where exposure to heavy metal ions is high: the saturation of neuromelanin with redox-active metal ions. Parkinson's disease therefore may be a form of accelerated aging in the substantia nigra associated with environmental toxins in which neuromelanin has a central, active role.
Aging↗
Reduction of lipid-soluble nitroxides in CHO cells and macrophage tumor cells.
There is a need to understand the metabolism of nitroxides because of their usefulness in measurements in living cells of complex phenomena, such as biophysical properties, redox metabolism, and the concentration of oxygen at specific sites. As part of a systematic study of the metabolism of nitroxides in cells, the authors studied Chinese hamster ovary (CHO) cells and mouse macrophage tumor (M5076) cells, using a set of lipophilic nitroxides based on 5 doxyl stearate: the free acid, the methyl ester of the acid, and a phosphatidylcholine with two doxyl stearates esterified to the glycerol. The rates of metabolism of these nitroxides under anoxia differed significantly as a function of both the type of cell and the type of nitroxide. The rates of reduction of the three lipophilic nitroxides depended on their localization. The rates of reduction were first order for all three lipophilic nitroxides, and the only products detected were the respective hydroxylamines. Effects of freeze-thawing and incubation temperature differed in the two cell lines. The authors conclude that the metabolism of nitroxides in different cell lines can be quite different. This may be especially important in understanding studies using nitroxides in living cells, functional organs, and in vivo.
Animals↗
In vivo measurement of oxygen concentration using sonochemically synthesized microspheres.
Proteinaceous microspheres filled with nitroxides dissolved in an organic liquid have been synthesized for the first time using high intensity ultrasound; these were used to measure oxygen concentrations in living biological systems. The microspheres have an average size of 2.5 microns, and the proteinaceous shell is permeable to oxygen. Encapsulation of the nitroxides into the microsphere greatly increased the sensitivity of the electron paramagnetic resonance signal line width to oxygen because of the higher solubility of oxygen in organic solvents. The encapsulation also protected the nitroxide from bioreduction. No decrease in intensity of the electron paramagnetic resonance signal was observed during 70 min after intravenous injection of the microspheres into a mouse. Measurement of the changes in oxygen concentration in vivo by means of restriction of blood flow, anesthesia, and change of oxygen content in the respired gas were made using these microspheres.
Anesthetics, General↗
Biophysics of melanin and informal group on the characterization of neuromelanin.
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Biophysics↗
Cardiac hypertrophy alters myocardial response to ischaemia and reperfusion in vivo.
The impact of cardiac hypertrophy on myocardial biochemical and physiological responses to ischaemia-reperfusion (I-R) was investigated in vivo. Hypertrophy was produced by aortic constriction (PH) or swimming training (TH). Open-chest rat hearts in PH, TH and a sedentary control group (SC) were subjected: (1) to ischaemia, by surgical occlusion of the main descending branch of the left coronary artery for 30 min; (2) to I-R, by releasing the occluded blood vessel for 15 min; or (3) to a sham operation. Ischaemia per se had little effect on heart oxidative and antioxidant status, or lipid peroxidation. However, I-R significantly decreased glutathione (GSH) content, increased glutathione disulfide (GSSG) content, and reduced GSH/GSSG ratio in the SC hearts. These alterations were associated with decreased activities of GSH peroxidase and GSSG reductase, and an increase in lipid peroxidation. Myocardial ATP, total adenine nucleotide content and energy charge in SC were significantly decreased after ischaemia, whereas levels of purine nucleotide derivatives, particularly adenosine, were elevated. No significant alteration of GSH status of adenine nucleotide metabolism occurred after ischaemia or I-R in hypertrophied hearts. In both PH and TH, glutathione content was significantly higher than in SC, whereas activities of GSH peroxidase and GSSG reductases were lower. TH rats maintained a higher heart rate (HR), peak systolic pressure, and energy charge during I-R. These data indicate that hypertrophied but well-functioned hearts may be more resistant to I-R induced disturbances of myocardial oxidative and antioxidant functions.
Adenine Nucleotides↗
Sonochemical activation of hematoporphyrin: an ESR study.
The production of 2,2,6,6-tetramethyl-4-piperidone-N-oxyl by reaction of 2,2,6,6-tetramethyl-4-piperidone (TMPone) with ultrasonically generated active species in oxygenated solutions of hematoporphyrin (Hp) was studied by electron spin resonance spectroscopy. The nitroxide production rate in air-saturated TMPone solutions in phosphate-buffered saline of pH 9.0 was significantly higher in the presence of Hp than in its absence. The enhancement of nitroxide production by Hp was significantly inhibited in the presence of sodium azide or histidine in the solution. The production rate with Hp was doubled by substitution of deuterium oxide, while the rate without Hp increased only modestly. These results suggest that a substantial amount of active oxygen can be generated by ultrasound in aqueous solutions of Hp. Since the production rate was not reduced by mannitol and no nitroxide was produced in nitrogen-saturated solutions, it appears that hydroxyl radicals do not account for a major portion of the active oxygen species which reacted with TMPone to yield a nitroxide.
Electron Spin Resonance Spectroscopy↗
Lithium phthalocyanine: a probe for electron paramagnetic resonance oximetry in viable biological systems.
Lithium phthalocyanine (LiPc) is a prototype of another generation of synthetic, metallic-organic, paramagnetic crystallites that appear very useful for in vitro and in vivo electron paramagnetic resonance oximetry. The peak-to-peak line width of the electron paramagnetic resonance spectrum of LiPc is a linear function of the partial pressure of oxygen (pO2); this linear relation is independent of the medium surrounding the LiPc. It has an extremely exchange-narrowed spectrum (peak-to-peak line width = 14 mG in the absence of O2). Physicochemically LiPc is very stable; its response to pO2 does not change with conditions and environments (e.g., pH, temperature, redox conditions) likely to occur in viable biological systems. These characteristics provide the sensitivity, accuracy, and range to measure physiologically and pathologically pertinent O2 tensions (0.1-50 mmHg; 1 mmHg = 133 Pa). The application of LiPc in biological systems is demonstrated in measurements of pO2 in vivo in the heart, brain, and kidney of rats.
Animals↗
Simultaneous measurement of intracellular and extracellular oxygen concentrations using a nitroxide-liposome system.
The concentration of oxygen within cells is important in many physiological and pathological processes, but such oxygen-dependent phenomena are generally studied as a function of the concentration of extracellular oxygen, due to a lack of suitable methods. Electron parmamagnetic resonance (EPR) oximetric techniques offer an attractive alternative to make such measurements. Previous EPR oximetric studies of extracellular-intracellular oxygen gradients have been hindered, however, by the fact that separate samples, prepared in slightly different ways, were required for individual measurements of extracellular and intracellular oxygen concentrations. In this study we demonstrate a technique that allows simultaneous measurement of intracellular and extracellular oxygen concentrations in a single sample: extracellular measurement is achieved using positively charged nitroxides encapsulated in liposomes, while intracellular oxygen is determined using a membrane-permeable nitroxide along with an extracellular broadening agent. Application of this system to the measurement of oxygen concentrations in suspensions of rat myoblast cells gave results which are consistent with non-simultaneous measurements and which show substantial extracellular-intracellular oxygen gradients in these rapidly respiring cells.
Animals↗
Simultaneous multi-site EPR spectroscopy in vivo.
A gradient technique to measure electron paramagnetic resonance spectra simultaneously at several different locations is described. The technique is based on the use of point probes containing paramagnetic centers. The value of the magnetic field gradient is chosen to be sufficient to separate the EPR signals from the different paramagnetic probes yet at the same time small enough to change only minimally the shape of individual signals. The conditions to apply this technique are considered in detail. When experimental data have a high signal-to-noise ratio, the lineshape distortion induced by the gradient can be corrected with the aid of a known distribution function of paramagnetic centers within the probe. The maximum entropy deconvolution algorithm is successfully applied for the correction of significantly distorted lines. The technique is experimentally tested and applied to measure the concentration of oxygen in hypertrophied rat myocardium and normal rat kidney in vivo by low frequency EPR (L-band, 1.2 GHz). Two types of EPR oxygen-sensitive probes--lithium phthalocyanine crystals and synthetic carbohydrate chars--were used.
Animals↗
Oxygen tension in a murine tumor: a combined EPR and MRI study.
The effects of fusinite, a new agent for the measurement of the concentration of oxygen in vivo by EPR, on MRI images have been studied. There was little effect on spin-echo T1-weighted images, but the fusinite resulted in large effects on T2-weighted images. Especially large effects could be observed when using spoiled gradient echo sequences (SPGR). The observed measurements of oxygen by EPR corresponded to the relative vascularity at the site of the fusinite both histologically and by MRI studies of vascularity using Gd-DTPA as a contrast agent. We conclude that by using the effects of fusinite on magnetic susceptibility, it can be located accurately and noninvasively with MRI and thereby the value of the use of fusinite to measure concentration of oxygen in vivo is enhanced.
Animals↗
Total and paramagnetic metals in human substantia nigra and its neuromelanin.
A number of hypotheses on the etiology of Parkinson's disease and other CNS disorders postulate a role of metal ions and/or neuromelanin. As part of an investigation of the interactions between neuromelanin and metal ions, we have studied the amount and type of metal ions in human neuromelanin in intact substantia nigra and in isolated neuromelanin using electron paramagnetic resonance (EPR), which selectively measures metal ions which are in valence states that have unpaired electrons and total reflection X-ray fluorescence (TXRF), which measures total metals. EPR also is a principal technique for studying the biophysics of melanins by analysis of its free radicals. The studies of substantia nigra with TXRF indicated the presence of substantial amounts of iron, zinc, lead, copper, manganese, and titanium at concentrations up to 4 times greater than those of non-pigmented brain tissue (basis pedunculi). The concentrations of metal ions in isolated neuromelanin were 5-260 times higher than in substantia nigra. The studies with EPR indicated that there were substantial amounts of paramagnetic metals ions, especially iron, bound to neuromelanin in intact substantia nigra, and the presence of these metal ions modified the EPR spectra of the free radicals of neuromelanin. We conclude: 1. Compared to other regions of the mid-brain, the substantia nigra contains increased amounts of many different metal ions; 2. Many of these metal ions are in paramagnetic valence states; 3. There are high concentrations of paramagnetic metal ions bound to neuromelanin. These results are consistent with the hypotheses that postulate a role of metal ions in promoting oxidative reactions in pigmented neurons.
Aged↗
Purified human neuromelanin, synthetic dopamine melanin as a potential model pigment, and the normal human substantia nigra: characterization by electron paramagnetic resonance spectroscopy.
Neuromelanin is a poorly understood pigment that accumulates in catecholaminergic neurons during normal aging. Electron paramagnetic resonance spectroscopy, an especially effective technique for investigating melanins, is used in the present study to show unambiguously that neuromelanin is a melanin; however, it is not well modeled by synthetic dopamine melanin and thus is an atypical melanin. Some of the unusual features of neuromelanin can be explained by postulating two distinct sources for its free radicals, the dominant one possibly derived from a precursor containing sulfur. Examination of human substantia nigra by electron paramagnetic resonance spectroscopy during the purification of neuromelanin also demonstrates, contrary to some other studies, that a portion of the paramagnetic metal ions in this tissue are bound to the pigment in situ. Combined with previous histochemical data, these observations have implications for the mechanism through which neuromelanin accumulates in vivo and are consistent with its having a cytoprotective function under normal conditions, but a cytotoxic role at advanced ages and in patients with Parkinson's disease. Other results of this study show that homogenizing tissues during the purification of any natural pigment may cause contamination of the pigment by extraneous metal ions and that subsequent incubation in hot acid, though most effective in removing metal ions and hydrolyzing proteins, leads to degradation of melanin. A purification procedure using incubation in acid at room temperature, however, is well suited for identifying and characterizing unknown natural pigments by electron paramagnetic resonance spectroscopy.
Animals↗
A standardized test for the identification and characterization of melanins using electron paramagnetic resonance (EPR) spectroscopy.
Melanins are complex, incompletely understood polymeric pigments that historically have been difficult to investigate with common chemical, histochemical, and physicochemical techniques. Because these pigments uniquely contain a stable population of organic free radicals, electron paramagnetic resonance (EPR) spectroscopy is a particularly effective method for studying them, and a set of qualitative EPR criteria has been established for their identification. However, a number of practical problems have arisen in applying these criteria to identify and characterize unknown pigments in relatively scarce pathological specimens, indicating that a standardized approach is needed. As reported here, a standardized EPR test for melanin based on the EPR criteria has been developed, guided by the requirements that it be sensitive, accurate, simple, and easy to interpret. It has been evaluated using the well-characterized synthetic melanin prepared by alkaline autooxidation of 5,6-dihydroxyphenylalanine (Dopa) and initially applied to the identification and characterization of an unknown pigment purified from an unusual malignant lung tumor.
Chemistry Techniques, Analytical↗
The minocycline-induced thyroid pigment and several synthetic models: identification and characterization by electron paramagnetic resonance spectroscopy.
A distinctive side-effect of exposure to minocycline is black pigmentation of the thyroid gland. Previous studies have identified an association between this side-effect and the ability of minocycline to competitively inhibit thyroid peroxidase, but extensive histochemical analyses have resulted in ambiguous definitions of the pigment. Electron paramagnetic resonance spectroscopy, an especially effective technique for investigating melanins, is used in the present study to show that the thyroid pigment is well-modeled by synthetic pigments generated by oxidation of minocycline in vitro, thus indicating that it is a polymeric product due to oxidation of minocycline in vivo by thyroid peroxidase. The results demonstrate that pigments derived from minocycline comprise a novel class of pigments with redox and paramagnetic characteristics which are melanin-like in most respects yet are fundamentally unique. They also indicate that the thyroid pigment contains a large quantity of Fe bound tightly in situ, a finding with important pathophysiological implications in view of the redox properties of the pigment. The binding of Fe , however, also may provide a basis for noninvasively detecting the presence of pigmentation by magnetic resonance imaging of the thyroid. Other results of this study show that homogenizing tissues during the purification of any natural pigment can cause contamination of the pigment by extraneous metal ions and that subsequent incubation in hot acid, although most effective in removing metal ions and hydrolyzing tissue proteins, leads to degradation of melanin. By comparison, a purification procedure utilizing incubation in acid at room temperature generally is well-suited for identifying and characterizing natural melanins by electron paramagnetic resonance spectroscopy, but is inadequate for the thyroid pigment.
Animals↗
Simultaneous measurements of the intra- and extra-cellular oxygen concentration in viable cells.
An EPR method that can measure the intra- and extra-cellular oxygen concentration [O2] simultaneously in vitro has been developed using specially designed nitroxides. In the presence of Fe(CN)6(3-) in the medium, intracellular [O2] is measured by a neutral 15N-nitroxide and extracellular [O2] is measured by a negatively charged 14N-nitroxide, since charged species do not enter cells and the EPR spectrum of a 15N-nitroxide does not overlap with that of a 14N-nitroxide. The method is based in part on the minimal broadening of negatively charged nitroxides by Fe(CN)6(3-) and the very effective broadening of neutral nitroxides by the same paramagnetic ions. Results with this method confirm the existence of gradients in [O2] between the extracellular and intracellular compartments in CHO cells and M5076 tumor cells, even without stimulation of cellular respiration by CCCP. The nature of the barrier that needs to be involved to account for the experimental results raises some significant questions.
Animals↗
Modulation by neuromelanin of the availability and reactivity of metal ions.
Presence of neuromelanin is likely to alter significantly the amount, distribution, reactivity, and consequences of reactivity of metal ions in those parts of the brain that contain neuromelanin. The effects are complex and can be predicted only with detailed knowledge of the system because (1) melanins are strong binders of metal ions and many organic molecules; (2) the effect of binding, depending on the circumstances, can increase or decrease reactivity of metal ions; and (3) melanins can generate and react with oxidation-reduction-active species such as hydrogen peroxide, hydroxyl radicals, superoxide anions, and singlet oxygen. Neuromelanin has some significant differences from most other natural melanins because of its mode of formation (by autooxidation rather than enzymatically) and its composition (it probably is a copolymer derived from dopamine and glutathione). It probably is not possible to understand fully the role of metal ions in oxidative damage in the brain without having an adequate understanding of the structure and reactivity of neuromelanin.
Humans↗