Harold M. Swartz

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.

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.

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.

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.

Metabolism of nitroxide spin labels in subcellular fraction of rat liver: I. Reduction by microsomes

As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of seven nitroxides in microsomes obtained from rat liver. The nitroxides were chosen to provide information on the effects of the type of charge, lipophilicity and the ring on which the nitroxide group is located. Important variables that were studied included adding NADH, adding NADPH, induction of enzymes by intake of phenobarbital and the effects of oxygen. Reduction to nonparamagnetic derivatives and oxidation back to paramagnetic derivatives were measured by electron-spin resonance spectroscopy. In general, the relative rates of reduction of nitroxides were similar to those observed with intact cells, but the effects of the various variables that were studied often differed from those observed in intact cells. The rates of reduction were very slow in the absence of added NADH or NADPH. The relative effect of these two nucleotides changed when animals were fed phenobarbital, and paralleled the levels of NADPH cytochrome c reductase, cytochrome P-450, cytochrome b5 and NADH cytochrome c reductase; results with purified NADPH-cytochrome c reductase were consistent with these results. In microsomes from uninduced animals the rate of reduction was about 10-fold higher in the absence of oxygen. The products of reduction of nitroxides by microsomes were the corresponding hydroxylamines. We conclude that there are significant NADH- and NADPH-dependent paths for reduction of nitroxides by hepatic microsomes, probably involving cytochrome c reductases and not directly involving cytochrome P-450. From this, and from parallel studies now in progress in our laboratory, it seems likely that metabolism by microsomes is an important site of reduction of nitroxides. However, mitochondrial metabolism seems to play an even more important role in intact cells.

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.

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.

Cellular metabolism of proxyl nitroxides and hydroxylamines

Previous data from model systems indicated that the proxyl nitroxides should be especially resistant to bioreduction and therefore could be an effective solution to this often problematic characteristic of nitroxides. Therefore, we investigated the rate of reduction by cells and by the usual model system, ascorbate, of four proxyl nitroxides and three reference nitroxides. We found that, while the rate of reduction by ascorbate of the proxyl nitroxides was slower than the rate of a prototypic pyrrolidine nitroxide (PCA), the reverse was true for reduction by cells. We also studied the rate of oxidation of the corresponding hydroxylamines. The rate of oxidation by cells of the proxyl hydroxylamines was relatively fast, especially for the most lipophilic derivative. These results indicate that: (i) proxyl nitroxides may not be unusually resistant to bioreduction by functional biological systems; (ii) accurate knowledge of relative rates of metabolism of nitroxides and hydroxylamines in cells and tissues will require direct studies in these systems because the rates may not closely parallel those observed in model (chemical) systems; and (iii) proxyl nitroxides show potential value as agents to measure oxygen concentrations by the rates of oxidation of their corresponding hydroxylamines.

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.

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.