A nondestructive technique to determine the rate of oxygen permeation into solid dosage forms.

The current study investigated the use of electron paramagnetic resonance (EPR) spectroscopy as a nondestructive method to quantify the partial pressure of oxygen (PO2) in tablets and hard shell capsules. Lithium phthalocyanine crystals (LiPC) were placed inside the dosage forms. The peak-to-peak linewidth of the first derivative of the LiPC EPR spectra was measured and, by calibration tables, the oxygen partial pressure, pO2, within the dosage form was determined. The intra-dosage form pO2 was followed as a function of time after changing the exterior gas stream composition. Results showed initial oxygen concentrations comparable to atmospheric levels in all tablets and capsules investigated. Oxygen rapidly permeated into unsealed gelatin and cellulosic hard shell capsules. Banding at the cap/body joint significantly reduced the oxygen permeation rate. Oxygen also rapidly permeated into tablet compacts, regardless of the compressional force used during tableting, while application of a polymeric film significantly decreased the rate of oxygen permeation. This EPR technique was shown to be a suitable nondestructive method to study oxygen permeation kinetics in solid dosage forms.

Oxidative stress response genes in Mycobacterium tuberculosis: role of ahpC in resistance to peroxynitrite and stage-specific survival in macrophages.

The Mycobacterium tuberculosis ahpC gene, encoding the mycobacterial orthologue of alkylhydroperoxide reductase, undergoes an unusual regulatory cycle. The levels of AhpC alternate between stages of expression silencing in virulent strains grown as aerated cultures, secondary to a natural loss of the regulatory oxyR function in all strains of the tubercle bacillus, and expression activation in static bacilli by a yet undefined mechanism. The reasons for this unorthodox regulatory cycle controlling expression of an antioxidant factor are currently not known. In this work, M. tuberculosis H37Rv and Mycobacterium smegmatis mc(2)155 ahpC knockout mutants were tested for sensitivity to reactive nitrogen intermediates, in particular peroxynitrite, a highly reactive combinatorial product of reactive nitrogen and oxygen species, and sensitivity to bactericidal mechanisms in resting and activated macrophages. Both M. tuberculosis ahpC::Km(r) and M. smegmatis ahpC::Km(r) showed increased susceptibility to peroxynitrite. In contrast, inactivation of ahpC in M. tuberculosis did not cause increased sensitivity to donors of NO alone. M. tuberculosis ahpC::Km(r) also showed decreased survival in unstimulated macrophages, but the effect was no longer detectable upon IFNgamma activation. These studies establish a specific role for ahpC in antioxidant defences involving peroxynitrite and most likely additional cidal mechanisms in macrophages, with the regulatory cycle likely contributing to survival upon coming out of the stationary phase during dormancy (latent infection) or upon transmission to a new host.

Firefly flashing is controlled by gating oxygen to light-emitting cells.

Although many aspects of firefly bioluminescence are understood, the mechanism by which adult fireflies produce light as discrete rapid flashes is not. Here we examine the most postulated theory, that flashing is controlled by gating oxygen access to the light-emitting cells (photocytes). According to this theory, the dark state represents repression of bioluminescence by limiting oxygen, which is required for bioluminescence; relief from this repression by transiently allowing oxygen access to the photocytes allows the flash. We show that normobaric hyperoxia releases the repression of light emission in the dark state of both spontaneously flashing and non-flashing fireflies, causing continual glowing, and we measure the kinetics of this process. Secondly, we determine the length of the barriers to oxygen diffusion to the photocytes in the aqueous and gas phases. Thirdly, we provide constraints upon the distance between any gas-phase gating structure(s) and the photocytes. We conclude from these data that the flash of the adult firefly is controlled by gating of oxygen to the photocytes, and demonstrate that this control mechanism is likely to act by modulating the levels of fluid in the tracheoles supplying photocytes, providing a variable barrier to oxygen diffusion.

The evolution of bioluminescent oxygen consumption as an ancient oxygen detoxification mechanism.

Endogenous reductants such as hydrogen sulfide and alkylthiols provided free radical scavenging systems during the early evolution of life. The development of oxygenic photosynthesis spectacularly increased oxygen levels, and ancient life forms were obliged to develop additional antioxidative systems. We develop here the hypothesis of how "prototypical" bioluminescent reactions had a plausible role as an ancient defense against oxygen toxicity through their "futile" consumption of oxygen. As oxygen concentrations increased, sufficient light would have been emitted from such systems for detection by primitive photosensors, and evolutionary pressures could then act upon the light emitting characteristics of such systems independently of their use as futile consumers of oxygen. Finally, an example of survival of this ancient mechanism in present-day bioluminescent bacteria (in the Euprymna scolopes-Vibrio fischeri mutualism) is discussed. Once increasing ambient oxygen levels reached sufficiently high levels, the use of "futile" oxygen consumption became too bioenergetically costly, so that from this time the evolution of bioluminescence via this role was made impossible, and other mechanisms must be developed to account for the evolution of bioluminescence by a wide range of organisms that patently occurred after this (e.g., by insects).

Applications of in vivo electron paramagnetic resonance (EPR) spectroscopy: measurements of pO2 and NO in endotoxin shock.

Recent developments of EPR instrumentation that allow the use of large tissue samples or whole animals and the ability to image spatially resolved EPR signals has led to novel applications of EPR spectroscopy in vivo. Utilising a 1 GHz EPR spectrometer with a 3.4-cm birdcage resonator, it was possible to detect and measure nitric oxide and oxygen in the livers of mice with lipopolysaccharide (LPS)-induced septic shock. Nitric oxide was detected as the nitric oxide (NO) complex of Fe-diethyldithiocarbamic acid (Fe-DETC) while pO2 was measured from the EPR linewidth of the oxygen-sensitive coal material 'gloxy'. LPS treatment stimulated the production of nitric oxide in the liver and the general circulation and the oxygenation of liver tissue was decreased. Selective placement of the EPR probes allowed images of nitric oxide and oxygen to be obtained in the liver. The spectral and spatial information obtained with this technique will allow improved understanding of the pathophysiology of such diseases.

Direct determination of the kinetics of oxygen diffusion to the photocytes of a bioluminescent elaterid larva, measurement of gas- and aqueous-phase diffusional barriers and modelling of oxygen supply.

We describe the development and use of a direct kinetic technique to determine the time taken for oxygen to diffuse from the external environment into the light-producing cells (photocytes) in the prothorax of bioluminescent larvae of Pyrearinus termitilluminans. This was achieved by measuring the time course of the pseudoflash induced through sequential anoxia followed by normoxia. We have also determined the separate times taken for this oxygen diffusion in gaseous and tissue (predominantly aqueous) phases by using helium and nitrogen as the carrier gas. Of the total time taken for diffusion, that in the gas phase required 613+/-136 ms (mean +/- s.e. m., N=5) whilst that in the aqueous phase required 1313+/-187 ms. These values imply pathlengths of diffusion in the gaseous and aqueous phases of 4.80x10(-)(3)+/-0.53x10(-)(3) and 8. 89x10(-)(5)+/-0.61x10(-)(5 )m, respectively. In addition, the pathlength of gas-phase diffusion was used to derive a parameter relating to the tortuosity of the tracheal system. These values, together with those obtained upon bioluminescent oxygen consumption, have been used to model oxygen supply to the photocyte. From these studies, it would also appear that the modulation of tracheolar fluid levels might be a significant mechanism of control of tissue oxygen levels in at least some insects.

Measurement of oxygen partial pressure, its control during hypoxia and hyperoxia, and its effect upon light emission in a bioluminescent elaterid larva.

This study investigates the respiratory physiology of bioluminescent larvae of Pyrearinus termitilluminans in relation to their tolerance to hypoxia and hyperoxia and to the supply of oxygen for bioluminescence. The partial pressure of oxygen (P(O2)) was measured within the bioluminescent prothorax by in vivo electron paramagnetic resonance (EPR) oximetry following acclimation of larvae to hypoxic, normoxic and hyperoxic (normobaric) atmospheres and during periods of bioluminescence (during normoxia). The P(O2) in the prothorax during exposure to an external P(O2) of 15.2, 160 and 760 mmHg was 10.3+/-2.6, 134+/-0.9 and 725+/-73 mmHg respectively (mean +/- s.d., N=5; 1 mmHg=0.1333 kPa). Oxygen supply to the larvae via gas exchange through the spiracles, measured by determining the rate of water loss, was also studied in the above atmospheres and was found not to be dependent upon P(O2). The data indicated that there is little to no active control of extracellular tissue P(O2) within the prothorax of these larvae. The reduction in prothorax P(O2) observed during either attack-response-provoked bioluminescence or sustained feeding-related bioluminescence in a normoxic atmosphere was variable, but fell within the range 10-25 mmHg. The effect of hypoxic atmospheres on bioluminescence was measured to estimate the intracellular P(O2) within the photocytes of the prothorax. Above a threshold value of 50-80 mmHg, bioluminescence was unaffected by P(O2). Below this threshold, an approximately linear relationship between P(O2) and bioluminescence was observed. Taken together with the extracellular P(O2) measurements, this suggests that control of P(O2) within the photocyte may occur. This work establishes that EPR oximetry is a valuable technique for long-term measurement of tissue P(O2) in insects and can provide valuable insights into their respiratory physiology. It also raises questions regarding the hypothesis that bioluminescence can have a significant antioxidative effect by reduction of prothorax P(O2 )through oxygen consumption.

Trapping of free radicals with direct in vivo EPR detection: a comparison of 5,5-dimethyl-1-pyrroline-N-oxide and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide as spin traps for HO* and SO4*-.

To spin trap hydroxyl radical (HO*) with in vivo detection of the resultant radical adducts, the use of two spin traps, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) (10 mmol/kg) has been compared. In mice treatment with 5-aminolevulinic acid and Fe3+ resulted in detection of adducts of hydroxyl radicals (HO*), but only with use of DEPMPO. Similarly, 'HO* adducts' generated via nucleophilic substitution of SO4*- adducts formed in vivo could be observed only when using DEPMPO as the spin trap. The reasons for the differences observed between DEPMPO and DMPO are likely due to different in vivo lifetimes of their hydroxyl radical adducts. These results seem to be the first direct in vivo EPR detection of hydroxyl radical adducts.

Photodynamically generated bovine serum albumin radicals: evidence for damage transfer and oxidation at cysteine and tryptophan residues.

Porphyrin-sensitized photoxidation of bovine serum albumin (BSA) results in oxidation of the protein at (at least) two different, specific sites: the Cys-34 residue giving rise to a thiyl radical (RS.); and one or both of the tryptophan residues (Trp-134 and Trp-214) resulting in the formation of tertiary carbon-centred radicals and disruption of the tryptophan ring system. In the case of porphyrins such as hematoporphyrin, which bind at specific sites on BSA, these species appear to arise via long-range transfer of damage within the protein structure, as the binding site is some distance from the ultimate site of radical formation. This transfer of damage is shown to depend on a number of factors including the conformation of the protein, the presence of blocking groups and pH. Alteration of the protein conformation results in radical formation at additional (or alternative) sites, as does blocking of the preferred loci of radical formation. The formation of these thiyl and tryptophan-derived radicals does not lead to significant aggregation or fragmentation of the protein, though it does result in a dramatic enhancement in the susceptibility of the oxidised protein to proteolytic degradation by a range of proteases. The generation of protein-derived radicals also results in an enhancement of photobleaching of the porphyrin, suggesting that protein radical generation is linked to porphyrin photooxidation.

Protein hydroperoxides and carbonyl groups generated by porphyrin-induced photo-oxidation of bovine serum albumin.

Porphyrin-sensitized photo-oxidation of bovine serum albumin results in oxidation at specific sites to produce protein radical species: at the Cys-34 residue (to give a thiyl radical) and at one or both tryptophan residues (Trp-134 and Trp-214) to give tertiary carbon-centered radicals and cause disruption of the indole ring system. This study shows that these photo-oxidation processes also consume oxygen and give rise to hydrogen peroxide, protein hydroperoxides, and carbonyl functions. The yield of hydrogen peroxide, protein hydroperoxides, and carbonyl functions is shown to be dependent on illumination time, the nature of the sensitizer, and the concentration of oxygen; the yield of hydroperoxides can also be markedly diminished by the presence of a spin trap which reacts with the initial protein radicals. The mechanism of formation of the protein hydroperoxides is suggested to be primarily through type I processes (i.e., independent of singlet oxygen), while type II (singlet oxygen) mechanisms may play a significant role in protein carbonyl formation. Reaction of the protein hydroperoxide species with metal ion complexes is shown to produce further protein-derived radicals which are predominantly present on amino acid side chains.

Lipid peroxidation-dependent chemiluminescence from the cyclization of alkylperoxyl radicals to dioxetane radical intermediates.

This work reveals a novel mechanism for triplet carbonyl formation (and hence chemiluminescence) during lipid peroxidation, whose chemiluminescence has been attributed to both triplet carbonyls and singlet oxygen. As a model for polyunsaturated fatty acid hydroperoxides, we have synthesized 3-hydroperoxy-2,3-dimethyl-1-butene by photooxygenation of tetramethylethylene. One-electron oxidation of this hydroperoxide with heme proteins and peroxynitrite to the corresponding alkylperoxyl radical results in chemiluminescence, both direct and 9,10-dibromoanthracene-2-sulfonate-sensitized, the latter attributed to the formation of triplet acetone. It is postulated that triplet acetone results from the cyclization of the alkylperoxyl radical to a dioxetane radical intermediate followed by its thermolysis. This is supported by EPR spin-trapping experiments in which discrimination between carbon-centered radicals derived from the alkyloxyl and alkylperoxyl radicals is achieved through the use of one-electron oxidants and reductants, e.g., FeII- and TiIII.

Use of isotopically labelled spin-traps to determine definitively the presence or absence of non-radical addition artefacts in EPR spin-trapping systems.

EPR spin-trapping, although a powerful, sensitive technique for the study of free radicals, can be susceptible to artefacts; one of the most intractable to determine has been the non-radical addition of a substrate to a spin-trap followed by oxidation of the product to an EPR-detectable nitroxide. This work details how differentially isotopically labelled spin-traps (either nitroso or nitrone) may be used to determine the presence (or absence) of such artefacts, and provide a semi-quantitative measure of the extent of their contribution to the total EPR spectra in spin-trapping reactions. Artefactual 'ene' addition of the nitroso spin-trap 3,5-dibromo-4-nitroso-benzenesulphonic acid (DBNBS) to tryptophan followed by oxidation to EPR-detectable products has been confirmed, as has its nucleophilic addition to the thiol of glutathione to give non-EPR detectable products. The nitrone α-phenyl-N-tert-butylnitrone (PBN) exhibited no such reactivity.

A study of photochemically-generated protein radical spin adducts on bovine serum albumin: the detection of genuine spin-trapping and artefactual, non-radical addition in the same molecule.

Photo-oxidation of bovine serum albumin (BSA) by porphyrins produces protein-centred radicals that can be spin trapped by 3, 5-dibromo-4-nitrosobenzenesulphonic acid (DBNBS) and 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO). In the case of DMPO, a thiyl radical from the Cys-34 residue is trapped, whereas with DBNBS signals from both this thiyl and tertiary carbon-centred species are observed. However, specific chemical modification of the Cys-34 residue, in combination with dual-isotope spin-trapping techniques, shows that the signal assigned to the Cys-34 thiyl adduct with DBNBS is a nitroxide artefact resulting from sequential (non-radical) nucleophilic addition and oxidation. In contrast, both the Cys-34 thiyl DMPO adduct and the tertiary carbon-centred DBNBS adducts result from genuine spintrapping. This study shows that such artefacts can be detected-even with anisotropic EPR spectra-through the use of appropriately substituted spin-traps, and that nitroso spin-traps need to be employed with great care in systems containing free thiol groups.

The synthesis and use of a (15)N and (2)H isotopically-labelled derivative of the spin-trap 3, 5-dibromo-4-nitrosobenzenesulphonic acid.

We report the synthesis and use of d2-(15)N isotopically-labelled 3, 5-dibromo-4-nitrosobenzenesulphonic acid (DBNBS-d2-(15)N, as its sodium salt), a spin-trap possessing several advantages over non-labelled DBNBS. The simplification in the electron paramagnetic resonance spectra of radical adducts of DBNBS-d2-(15)N compared with those of DBNBS not only results in increased sensitivity, but also facilitates the assignment and analysis of complex spectra. An example of this simplification is given.

EPR studies on the effects of complexation of heme by hemopexin upon its reactions with organic peroxides.

Hemopexin, a heme-binding serum glycoprotein, is thought to play an important role in the prevention of oxidative damage that may be catalysed by free heme. Through the use of EPR techniques, the generation of free radicals from organic hydroperoxides by heme and heme-hemopexin complexes, and the concomitant formation of high oxidation-state iron species has been studied; these species are implicated as causative agents in processes such as cardiovascular disease and carcinogenesis. From the rates of production of these species from both n-alkyl and branched hydroperoxides, it has been inferred that the dramatic reduction in the yield of oxidising species generated by heme upon its complexation with hemopexin arises from steric hindrance of the access of hydroperoxide to the bound heme.

Conformational changes induced in bovine serum albumin by the photodynamic action of haematoporphyrin.

The photodynamic action of haematoporphyrin upon bovine serum albumin, spin-labelled at the cysteine-34 residue, has been shown to: (i) increase its susceptibility to proteolysis by chymotrypsin and trypsin, and (ii) increase its susceptibility to denaturation by urea. This is thought to be the result of conformational changes caused by the formation of protein radicals, although contributions from subsequent radical reactions of, for example, amino acids, may also take place. Such species have previously been shown, by EPR spin-trapping, to be formed in this system. Increased proteolytic susceptibility of non spin-labelled protein is also observed upon photolysis with haematoporphyrin, indicating that the changes observed in the spin-labelled protein also occur in the native form, and are not artefactual in nature. The significance of these photochemically-induced conformational changes within proteins in the photodynamic therapy of tumours, and other protein-radical systems is discussed.

An EPR spin trapping study of albumin protein radicals formed by the photodynamic action of haematoporphyrin.

The photodynamic action of haematoporphyrin (8,13-bis (1-hydroxyethyl)-3,7,12,17-tetramethyl-21H,23H-porphine-2,18 -dipropionic acid) has been shown to generate radical species upon isolated bovine and human serum albumins by the detection, using EPR spectroscopy, of adducts of the protein-derived radicals to spin traps. Similar radicals can also observed with fresh human serum, suggesting that similar species might be generated during in vivo photodynamic therapy. These radicals are believed to arise as a result of processes both dependent and independent of singlet oxygen formation (Type II and Type I reactions respectively). The formation of these protein radicals can be inhibited by physiologically relevant antioxidants such as glutathione and ascorbate in a dose-dependent manner; the observation of similar species with fresh serum does however suggest that these defensive processes can be easily overwhelmed. The relevance of these findings to the photodynamic therapy of tumours is discussed.

Free radical formation in murine skin treated with tumour promoting organic peroxides.

The generation of free radicals from tumour-promoting organic peroxides applied to intact murine skin samples has been studied by EPR spectroscopy using two techniques: first direct observation of ascorbyl radicals produced from reactions of peroxide-related radicals with ascorbate, an important endogenous antioxidant, and secondly, observation of radical adducts produced by spin-trapping. Free radical generation from tumour-promoting organic peroxides can be seen to occur in intact skin tissue through a one-electron reductive pathway, and takes place at sites including the viable cells of the epidermis and/or dermis. This radical generation is dependent upon the penetration of the skin by the peroxides, with the stratum corneum representing a major diffusional barrier to their penetration of skin. The technique of using ascorbyl radical measurement by EPR spectroscopy as a means of studying and quantifying radical production in intact tissues, developed in this work, may prove of much use in the study of many free radicals and their reactions in a wide range of biological systems, particularly skin. When combined with appropriate spin-trapping techniques, which enable identification of radical species and elucidation of their mechanisms of production, this enables the direct, real-time observation of radical reactions and mechanisms not previously possible in intact tissue samples.