A comparison of the spin labels MAL-3 and TEMPAMINE for measuring the internal microviscosity of human erythrocytes.

In a recent paper, Daveloose et al. (Daveloose, D., Fabre, G., Berleur, F., Testylier, G. and Letterrier, F. (1983) Biochim. Biophys. Acta 763, 41-49) described a technique to measure the internal microviscosity of erythrocytes using the spin label MAL-3 (2,2,5,5-tetramethyl-3-malimidopyrrolidinyl-N-oxyl) to supercede the use of TEMPAMINE (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amine)ferricyanide. They cite the long time (12 h) required for TEMPAMINE to enter the erythrocyte interior and the 'unphysiological' nature of the potassium ferricyanide required to isolate the TEMPAMINE signal inside the erythrocytes as reasons for developing this technique. In the present work, the penetration of TEMPAMINE into the erythrocyte interior is found to be pH-dependent and on the order of seconds, not hours. The slow penetration of TEMPAMINE described by Daveloose et al. probably is a result of their using trimethylTEMPAMINE which is membrane-impermeable. High concentrations of ferricyanide do affect red cell morphology in a reproducible fashion. The internal microviscosity as measured by TEMPAMINE reflects this. MAL-3 does not. Sample preparation time using TEMPAMINE-ferricyanide is about 1 min compared to about 40 min for the MAL-3 technique. In the presence of ferricyanide or ascorbate, MAL-3-labeled cells show slow loss of signal intensity which indicates that MAL-3 leaks out of the cells. Both the TEMPAMINE-ferricyanide and MAL-3 techniques have advantages and disadvantages.

Direct measurement of the internal viscosity of sickle erythrocytes as a function of cell density.

The rotational dynamics of TEMPAMINE can be used to study directly the intracellular environment. The extracellular signal from TEMPAMINE is broadened away by the use of potassium ferricyanide which does not enter the cell. The EPR signal which results when 1 mM TEMPAMINE, 120 mM ferricyanide, and erythrocytes are mixed together arises from TEMPAMINE only in the intracellular aqueous space. The relative viscosity measured by the motion of TEMPAMINE in various control environments is: water at 37 degrees C = 1; human plasma at 37 degrees C = 1.1; internal aqueous environment of washed erythrocytes or whole blood at 37 degrees C = 4.92 +/- 0.32. Erythrocytes can be fractionated by density. In sickle-cell anemia (SS), the percentage of cells we find with density greater than 1.128 g/ml is 15-40%, in normals (AA) and sickle trait (AS) 1%. By direct spin-label measurements with TEMPAMINE we show, for the first time, that the relative internal viscosity (eta mu) of these dense erythrocytes is markedly elevated and density-dependent. Our results show that (1) eta mu increases with increasing cell density; (2) eta mu obtained from sickle cells is higher than eta mu obtained from normal cells at a given density, and this effect is greater at 37 degrees C than at 20 degrees C; (3) eta mu is proportional to MCHC, but eta mu in erythrocytes is higher than eta mu obtained from in vitro preparations of hemoglobin S at equivalent concentrations. We conclude that the relative internal viscosity of erythrocytes is affected by three factors: the state of cell hydration, the amount of hemoglobin polymer present, and the potential interactions of the cell membrane with intracellular hemoglobin.

Rapid measurement of drug release from temperature-sensitive liposomes by electron paramagnetic resonance and radioisotope techniques.

We have developed two new methods for quantifying drug release from temperature-sensitive liposomes. Large unilamellar vesicles were made by the reverse phase evaporation process. They contained a water-soluble electron paramagnetic resonance probe, trimethyl-4-amino-2,2,6,6-tetramethyl piperidine N-oxyl and the radioisotope cytosine-[3H]1-beta-D-arabinofuranoside in their aqueous compartment. Release of the electron paramagnetic resonance probe was measured by placing the liposomes in a solution of a spin label quenching agent, potassium ferricyanide, and monitoring the reduction in signal strength. The measurement of radioisotope released involved rapid ultracentrifugation of the liposomes after which the supernatant was tested for the presence of radioactivity. Both methods were found to be rapid and convenient ways of measuring drug release from temperature-sensitive liposomes and both methods gave comparable results. The radioisotope assay provides a direct measurement of drug leakage, whereas the electron spin resonance assay provides a continuous marker for liposome stability as a function of temperature.

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.

A spin-label study of plasma membranes of adrenal chromaffin cells.

Chromaffin-cell membranes were labeled with two nitroxide spin labels, one probing the interior of the membrane and one probing the interfacial region. Both spin labels indicate that the membrane undergoes a phase transition at about 26 degrees C. An Arrhenius plot of acetylcholinesterase activity exhibits a discontinuity at 26 degrees C, consistent with the existence of a phase transition at that temperature. Acetylcholine, which stimulates chromaffin cells to secrete catecholamines, and hexamethonium, a cholinergic blocker, do not affect the rotational correlation times of the spin labels. These results argue that cholinergic stimulation does not affect the fluidity of the chromaffin-cell membrane.

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.

Analysis of the reduction of nitroxides by flavin mononucleotide.

This article describes a simple method to prepare hydroxylamines from nitroxides by photo-activated flavin mononucleotide. The half-time of reduction varied from 2 to 38.4 s for a series of nitroxides. For most nitroxides short exposures to light (min) were sufficient to produce significant amounts of hydroxylamine; longer periods of exposure increased the yields of other products. Proxyl (2,2,5-trimethyl-5-alkylpyrrolidine-N-oxyl) nitroxides were unusually reactive with a much higher yield of products which could not be reoxidized by ferricyanide to the nitroxides. Optimum conditions for reversible reduction depend on the nitroxide and the amounts of other reducible substances such as oxygen and ferricyanide that may be present.

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.

Heterocyclic amines and genotype of N-acetyltransferases as risk factors for prostate cancer.

A variety of carcinogenic heterocyclic amines are produced during the cooking of meat at high temperatures. These carcinogens are metabolized by N-acetyltransferases (NAT), which are polymorphic in the population. This study examined associations between prostate cancer (PCa) and the consumption of different kinds of meat, heterocyclic amine intake and NAT genotypes. PCa patients and controls were recruited in the Syracuse, NY area. Levels of meat and heterocyclic amine intakes were determined from validated surveys and NAT genotypes were determined by the sequences of PCR-amplified DNA from buccal swabs. A total of 152 cases and 161 controls were eligible for analysis. There was an association between PCa and history of PCa in the first-degree blood relatives (OR = 4.59, 95% CI 2.21-9.70), and family history of bladder cancer (P < 0.02). However, there was no association with the history of other cancers. There was no association between PCa and either 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) intake, or NAT1 and NAT2 genotypes. However, there was a trend of association with MeIQx and with rapid NAT2 and NAT1*10 in combination with PhIP. A new NAT1 allele with a frequency of one out of 544 chromosomes was found in the Caucasian subjects.

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.

The effect of cimetidine on the formation of sulfamethoxazole hydroxylamine in patients with human immunodeficiency virus.

Hypersensitivity reactions from trimethoprim/sulfamethoxazole are likely caused by a reactive nitroso intermediate formed from sulfamethoxazole hydroxylamine. This pilot study tested whether cimetidine inhibits the urinary excretion of sulfamethoxazole hydroxylamine. Ten outpatients infected with human immunodeficiency virus (HIV) and currently receiving trimethoprim/sulfamethoxazole prophylaxis were randomly selected from 59 eligible patients. Five received cimetidine 800 mg twice daily for 1 week and five served as controls. Two spot urine samples one week apart were obtained after a trimethoprim/sulfamethoxazole dose for all patients. Patients taking cimetidine had a significant decrease in excretion of sulfamethoxazole hydroxylamine relative to total excreted drug in the two urine samples compared with control patients. Cimetidine likely caused this decrease in sulfamethoxazole hydroxylamine excretion through inhibition of CYP3A4. Because of potential differences between HIV-infected patients and healthy subjects in oxidative metabolism, future studies of inhibitors of sulfamethoxazole hydroxylamine formation should be conducted in the HIV population.

Localization of the membrane perturber adamantylsulfate in sarcoplasmic reticular vesicles.

The properties of a new membrane perturbing agent, adamantyl sulfate (A-1-S) are described. Lobster sarcoplasmic reticular vesicles (SRV) were used as a test membrane system and the activity of the Ca2+--Mg2+-dependent ATPase in conjunction with spin label data was used to determine the location of A-1-S in the membrane. The results suggest that A-1-S perturbs only the polar region of the SRV and that this causes loss of ATPase function. Thus, ATPase activity requires integrity of the membrane polar region.

Adamantyl nitroxide: a spin label for probing membrane surfaces.

In an effort to understand more about the perturbing properties of adamantane-like molecules on biological membranes, the spin probe adamantyl nitroxide (2,2'-dimethyl-5-adamantyl oxazolidine-N-oxyl) was synthesized, purified and characterized. Electron paramagnetic resonance (EPR) spectra were then obtained from 1:50 and 1:200 mixtures of adamantyl nitroxide with dipalmitoyl and dipalmityl phosphatidylcholine multibilayers. Above the phase transition temperature of these lipids (41 degrees C for dipalmitoyl phosphatidylcholine and 43 degrees C for dipalmityl phosphatidylcholine) the spectra of adamantyl nitroxide are similar to control spectra obtained in liquid oleic acid. Below the phase transition temperatures, however, spectral differences were observed depending on: (1) the concentration of the spin probe in the lipid; (2) the linkage between the polar head group and the hydrocarbon tails of the phospholipid; (3) the temperature of the sample. Partitioning of adamantyl nitroxide between the aqueous and hydrocarbon phases of the sample is most prominent at probe-to-lipid ratios of 1:200 and at temperatures below the pre-transition temperature of the lipid (around 33 degrees C). Computer simulations of the above results, as well as additional experiments performed at 35 GHz, show that the results arise from true partitioning and not from asymmetric probe motion. Two conclusive results of these experiments are that spectra of adamantyl nitroxide in phospholipid multibilayers are sensitive to probe concentration and to the physical characteristics of the phospholipid which they probe. The spectral differences which arise when adamantyl nitroxide is used with ether- and ester-linked phospholipids indicate that it is a sensitive probe of membrane surfaces. Employment of this molecule in membrane research should prove to be useful in obtaining additional information about membrane surface events.

New developments in the use of cytokines for cancer therapy.

Cytokines, the pleiotropic immune regulatory proteins, are involved in the regulation of cell growth, differentiation and functional activation. Novel cytokines have been enumerated in rapid succession and entered the clinical arena. IL-2 is well recognized for its antitumor effects and is accepted therapy for numerous malignancies. IL-l and IL-11 are important as thrombopoetic factors while IL-6 has been introduced in clinical trials as a platelet growth factor and as an antitumor agent. IL-4 has shown growth inhibitory effects against many solid tumor cell lines in vitro, but its direct effect on human tumors in vivo remains to be explored. IL-7 may be an important addition to the current strategies of adoptive immunotherapy. IL-12 plays a fundamental role in activating antitumor cellular immunity. When given with tumor associated antigens, IL-12 has proven effective against many forms of metastatic solid tumors. Immunotoxins appear to be promising, though the antigenicity of the molecule and antibody development aspects remain to be resolved. The current review will focus on the clinical use of novel cytokines for the treatment of cancer.

Kinetics of distribution of 5-doxylstearic acid in mouse lymphocytes.

The rate at which stearic acid nitroxide spin labels distribute through cells affects the interpretation of data obtained from these nitroxides. We used photoreduction of 5-doxylstearic acid (5DS) to determine the rate at which 5DS arrives at the outer half of the plasma membrane of mouse lymphocytes and macrophages. Our results show that 5DS is in equilibrium with the outer half of the plasma membrane of mouse lymphocytes over a time frame of 2 minutes or less. Thus, spectra data obtained from 5DS-labelled cells clearly reflects the spectrally averaged environment of all the cell membranes in lymphocytes and potentially other cells as well. This clarifies the observation that the spectral information obtained from doxylstearic acid nitroxides is relatively insensitive to environmental changes which would be expected to involve only the plasma membrane of the cell.

Viral transformation of baby hamster kidney cells affects the oxygen sensitivity of nitroxide reduction.

In order to explore and expand the potential applications of nitroxides, it is important to understand how nitroxides are metabolized by living systems. We studied the oxygen sensitivity of a BHK (baby hamster kidney) cell line and BHK cells that had been transformed by adenovirus to determine if the process of adenoviral transformation affects nitroxide metabolism. When the BHK cells are transformed by adenovirus, they reduce nitroxides essentially independently of oxygen concentration. In addition, the rate of reduction of the nitroxides decreases by a factor of 10 compared to the rate of reduction in BHK cells. These data suggest to us that the process of viral transformation has affected dramatically the metabolism of these cells; one possible interpretation is that the viral transformed cells lost the ability to perform oxidative phosphorylation. We believe that this is a metabolic event which could be exploited by magnetic resonance imaging.