Spin-trapped Adduct Research Articles

Dietary Magnesium Deficiency in Rats Enhances Free Radical Production in Skeletal Muscle

Recent studies suggest that free radicals may be involved in tissue injuries induced by magnesium deficiency. The aim of the present study was to assess the effect of magnesium deficiency on free radical production of skeletal muscle tissue. Male Wistar rats were pair-fed from weaning for 12 d either control or Mg-deficient diets containing 960 or 40 mg magnesium/kg diet, respectively. In the Mg-deficient rats, hypomagnesemia was accompanied by significantly lower magnesium and greater calcium concentrations in skeletal muscle tissue. Electron microscopy of skeletal muscle tissue revealed ultrastructural changes, including swelling mitochondria and disorganization of the sar-coplasmic reticulum network. Using the spin-trapping technique, we showed that significantly more hydroxyl radicals were generated in muscle homogenates of Mg-deficient rats. Moreover, the amount of spin trap adducts was increased in the presence of exogenous iron in both groups. In agreement with these observations, a greater concentration of thiobarbituric acid-reactive substances and a lower concentration of thiol groups were found in skeletal muscle of the Mg-deficient group compared with controls. These results strongly support the hypothesis that free radical-mediated injury could contribute to skeletal muscle lesions resulting from magnesium deficiency.

Electron Paramagnetic Resonance for the Detection of Electrochemically Generated Hydroxyl Radicals: Issues Associated with Electrochemical Oxidation of the Spin Trap.

For the detection of electrochemically produced hydroxyl radicals (HO·) from the oxidation of water on a boron-doped diamond (BDD) electrode, electron paramagnetic resonance spectroscopy (EPR) in combination with spin trap labels is a popular technique. Here, we show that quantification of the concentration of HO· from water oxidation via spin trap electrochemical (EC)-EPR is problematic. This is primarily due to the spin trap oxidizing at potentials less positive than water, resulting in the same spin trap-OH· adduct as formed from the solution reaction of OH· with the spin trap. We illustrate this through consideration of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap for OH·. DMPO oxidation on a BDD electrode in an acidic aqueous solution occurs at a peak current potential of +1.90 V vs SCE; the current for water oxidation starts to rise rapidly at ca. +2.3 V vs SCE. EC-EPR spectra show signatures due to the spin trap adduct (DMPO-OH·) at potentials lower than that predicted thermodynamically (for water/HO·) and in the region for DMPO oxidation. Increasing the potential into the water oxidation region, surprisingly, shows a lower DMPO-OH· concentration than when the potential is in the DMPO oxidation region. This behavior is attributed to further oxidation of DMPO-OH·, production of fouling products on the electrode surface, and bubble formation. Radical scavengers (ethanol) and other spin traps, here N-tert-butyl-α-phenylnitrone, α-(4-pyridyl N-oxide)-N-tert-butylnitrone, and 2-methyl-2-nitrosopropane dimer, also show electrochemical oxidation signals less positive than that of water on a BDD electrode. Such behavior also complicates their use for the intended application.

Application of chemical derivatization techniques combined with chemical ionization mass spectrometry to detect stabilized Criegee intermediates and peroxy radicals in the gas phase

Abstract. Short-lived highly reactive atmospheric species, such as organic peroxy radicals (RO2) and stabilized Criegee intermediates (SCIs), play an important role in controlling the oxidative removal and transformation of many natural and anthropogenic trace gases in the atmosphere. Direct speciated measurements of these components are extremely helpful for understanding their atmospheric fate and impact. We describe the development of an online method for measurements of SCIs and RO2 in laboratory experiments using chemical derivatization and spin trapping techniques combined with H3O+ and NH4+ chemical ionization mass spectrometry (CIMS). Using chemical derivatization agents with low proton affinity, such as electron-poor carbonyls, we scavenge all SCIs produced from a wide range of alkenes without depleting CIMS reagent ions. Comparison between our measurements and results from numeric modeling, using a modified version of the Master Chemical Mechanism, shows that the method can be used for the quantification of SCIs in laboratory experiments with a detection limit of 1.4×107 molecule cm−3 for an integration time of 30 s with the instrumentation used in this study. We show that spin traps are highly reactive towards atmospheric radicals and form stable adducts with them by studying the gas-phase kinetics of the reaction of spin traps with the hydroxyl radical (OH). We also demonstrate that spin trap adducts with SCIs and RO2 can be simultaneously probed and quantified under laboratory conditions with a detection limit of 1.6×108 molecule cm−3 for an integration time of 30 s for RO2 species with the instrumentation used in this study. Spin trapping prevents radical secondary reactions and cycling, ensuring that measurements are not biased by chemical interferences, and it can be implemented for detecting RO2 species in laboratory studies and potentially in the ambient atmosphere.

Heterogeneity of radiation response in mesenchymal subtype glioblastoma: molecular profiling and reactive oxygen species generation.

Radiotherapy-induced tumor death remains critical in the successful first-line management of glioblastoma, whereas resistance to radiation serves as a major factor in disease progression. Mesenchymal shift has been identified as a driver in GBM recurrence, with gene expression associated with enhanced repair of macromolecular damage caused by radiation. Using distinct mesenchymal subtype GBM cells lines, radiation response was assessed by clonogenic assay and orthotopic mouse tumor model. RNA-sequencing was performed in the setting of increasing radiation dosing while real-time assessment of ROS generation was achieved by the measurement of hydroxyl spin trap adducts via electron paramagnetic resonance. Radiation-induced cell death determined by clonogenic assay was significantly different at low dose (4-8Gy) between the resistant U3035 cells and the sensitive U3020 cells. Similar trends were present in the in vivo NSG mouse model following radiation dosing on post-implantation day 7-10, with the rate of reduction in tumor bioluminescence reversing between the U3020 and U3035 cells after the third dose of radiation. Changes in gene expression following radiation determined by RNA-sequencing indicate both U3035 and U3020 cells demonstrate a shift toward more mesenchymal profiles, with concurrent shift away from pro-neural subtype gene expression in the U3020 cells that appeared to develop resistance to radiation in vivo. Persistence of ROS generated following radiation was greater in U3020 cells shown to be more sensitive to radiation. Despite the same molecular classification, distinct GBM cell lines can demonstrate differential response to radiation and potential for mesenchymal shift associated with radiation resistance.

Garlic as Anti-oxidants and Free Radical Scavengers

The antioxidant activities of fresh garlic extracts and commercially available garlic products were evaluated using a recently developed technique for the determination of lipid hydroperoxides, initial intermediates in the lipid oxidation process. This measurement system uses reverse-phase column chromatography with chemiluminescence detection. Activity of the garlic preperations was compared to that of synthetic antioxidants. Protection against lipid oxidation provided by garlic extract was found to be concentration dependent, as was the protection provided by many synthetic antioxidants. In addition, garlic extract was also found to scavenge OH radicals, as evidenced by the reduction of the spin trap adduct in the presence of OH radicals.

Influence of phosphorous upon the formation of DMPO-[rad]OH and POBN-O2[rad]¯ spin-trapping adducts in carbon-supported P-promoted Fe-based photocatalysts

Carbon-supported PO43−/Fe3+-based nanocomposites were prepared by a thermal hydrolysis method. The influence of the molar ratio PO43−/Fe3+ upon the kinetics of adsorption and photocatalytic degradation of methylene blue was evaluated. In terms of the surface reaction rate, up to 8 times higher photoactivity (99 % dye conversion) than the commercial F400 activated carbon (39 % dye conversion) was found for the sample containing a ratio PO43−/Fe3+ ca. 0.5. This composite was selected for a carefully study of spin-trapping of OH and O2‾ adduct formation. O2‾. A preferential formation of O2‾ and a strong inhibiting effect was observed in the formation of these reactive oxygen species attributed to the phosphate-based groups. In addition, a composite was prepared using the best carbon-supported P-Fe-based photocatalysts and a commercial TiO2. An inhibiting effect of the photocatalytic activity of TiO2 was observed in presence of P-containing catalysts in agreement with the decrease in the spin-trapping signals. A mechanism for the trimer aggregation of methylene blue adsorption on carbon-supported P-Fe-based nanocomposite following degradation under UV–vis irradiation is proposed.

Online Quantification of Criegee Intermediates of α-Pinene Ozonolysis by Stabilization with Spin Traps and Proton-Transfer Reaction Mass Spectrometry Detection.

Biogenic alkenes, which are among the most abundant volatile organic compounds in the atmosphere, are readily oxidized by ozone. Characterizing the reactivity and kinetics of the first-generation products of these reactions, carbonyl oxides (often named Criegee intermediates), is essential in defining the oxidation pathways of organic compounds in the atmosphere but is highly challenging due to the short lifetime of these zwitterions. Here, we report the development of a novel online method to quantify atmospherically relevant Criegee intermediates (CIs) in the gas phase by stabilization with spin traps and analysis with proton-transfer reaction mass spectrometry. Ozonolysis of α-pinene has been chosen as a proof-of-principle model system. To determine unambiguously the structure of the spin trap adducts with α-pinene CIs, the reaction was tested in solution, and reaction products were characterized with high-resolution mass spectrometry, electron paramagnetic resonance, and nuclear magnetic resonance spectroscopy. DFT calculations show that addition of the Criegee intermediate to the DMPO spin trap, leading to the formation of a six-membered ring adduct, occurs through a very favorable pathway and that the product is significantly more stable than the reactants, supporting the experimental characterization. A flow tube set up has been used to generate spin trap adducts with α-pinene CIs in the gas phase. We demonstrate that spin trap adducts with α-pinene CIs also form in the gas phase and that they are stable enough to be detected with online mass spectrometry. This new technique offers for the first time a method to characterize highly reactive and atmospherically relevant radical intermediates in situ.

Improved sensitivity for imaging spin trapped hydroxyl radical at 250 MHz.

Radicals, including hydroxyl, superoxide, and nitric oxide, play key signaling roles in vivo. Reaction of these free radicals with a spin trap affords more stable paramagnetic nitroxides, but concentrations in vivo still are so low that detection by electron paramagnetic resonance (EPR) is challenging. Three innovative enabling technologies have been combined to substantially improve sensitivity for imaging spin-trapped radicals at 250 MHz. 1) Spin-trapped adducts of BMPO have lifetimes that are long enough to make imaging by EPR at 250 MHz feasible. 2) The signal-to-noise ratio of rapid-scan EPR is substantially higher than for conventional continuous-wave EPR. 3) An improved algorithm permits image reconstruction with a spectral dimension that encompasses the full 50 G spectrum of the BMPO-OH spin adduct without requiring the wide sweeps that would be needed for filtered backprojection. A 2D spectral-spatial image is shown for a phantom containing ca. 5 μM BMPO-OH.

Catalytic Property of Ferryl (IV) Species in the Heterogeneous Thermal Fenton-Like Oxidation System

During the oxidative degradation of nonbiodegradable Orange IV by means of H2O2/FeIIIF2+/R oxidation method (FeIIIF2+ complex supported on cationic exchange resin as a catalyst), the binding energy of the Fe(2p3/2) region for X-ray photoelectron spectroscopy (XPS) spectra increased with the reaction time during the initial 2.5 h and then decreased continuously. The electron paramagnetic resonance (EPR) signal for hydroxyl radical (OH) spin trap adduct of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) also increased with the reaction time within a certain period of time. These results confirmed that ferryl (IV) (FeIVF=O+/R) species was generated along with the formation of OH radicals at the beginning of the reaction. The formed ferryl (IV) species enhanced the DMPO-OH signal compared with the catalyst of FeIIIF2+/R. The addition of t-butanol did not significantly reduce the reaction rate, which increased with reaction time in the process of ferryl (IV) catalytic decomposition of H2O2. This indicated that there was a more reactive species than ferryl (IV), which cannot be scavenged by t-butanol, and it was likely to be Fe (V). A possible mechanism for a resin-supported thermal Fenton-like oxidation reaction is proposed to better understand the process in the presence of iron complex catalysts.

Self-doping and surface plasmon modification induced visible light photocatalysis of BiOCl

In this study we demonstrate that self-doping and surface plasmon resonance could endow a wide-band-gap ternary semiconductor BiOCl with remarkable visible light driven photocatalytic activity on the degradation of organic pollutants and photocurrent generation properties. The self-doped BiOCl with plasmonic silver modification was synthesized by a facile one-pot nonaqueous approach and systematically characterized using X-ray diffraction, scanning electron microscopy, transmission electron microscopy, UV-visible diffuse reflectance spectra, electron spin resonance, and X-ray photoelectron spectroscopy. The photocurrent enhancement was found to be intimately dependent on the irradiation wavelength and matched well with the intensity of the absorption of the Ag nanoparticles. Reactive species trapping experiments and electron spin resonance spectroscopy with 5,5-dimethyl-1-pyrroline-N-oxide spin-trapping adducts confirmed that more oxidative species could be generated from the photogenerated electrons due to the plasmon-excitation of the metallic Ag in the self-doped BiOCl with plasmonic silver modification, which is responsible for the great enhancement of photocatalytic activity and photocurrent. Surface photovoltage spectroscopy and time-resolved photoluminescence spectroscopy results revealed the transfer of plasmon-band-induced electrons from Ag nanoparticles into BiOCl and the acceleration effect of surface plasmon resonance-induced intense oscillating electric fields on this electron transfer. This study would not only provide direct evidence of plasmonic photocatalysis, but also shed light on the design of highly efficient metal-semiconductor composite photocatalysts.

Effect of phosphorylation of the maize starch on thermal generation of stable and short‐living radicals

AbstractEffect of hydrolysis, phosphorylation and treatment with high hydrostatic pressure on thermal generation of stable and short‐living radicals in maize starch was studied by X‐ray diffraction (XRD), electron paramagnetic resonance (EPR) spectroscopy, differential scanning calorimetry and polarized light microscopy. Phosphorus was introduced into maize starch as mono‐ and distarch phosphates. XRD indicated localization of phosphate groups in amorphous part of the granule whereas calorimetric data suggested some cross‐linking of the distarch phosphates. Stable and short‐living radicals with unpaired electron localized at carbon atom were generated in all investigated samples in the temperature range commonly used for processing food. The number of detected short‐living radicals, stabilized by a spin trap, is of two orders of magnitude greater than that of the stable radical species. Hydrolysis and phosphorylation strongly increase the number of stable radicals while pretreatment of the starch with high hydrostatic pressure diminishes their amount. The EPR spectra of stable radicals consist of two components, single line and another one with hyperfine structure, indicating interaction of unpaired electron with neighboring hydrogen atom. The EPR spectra of the spin trap adducts with short‐living radicals contain three components from species differing in their dynamic properties depending on localization in zones of various degree of crystallinity.

Characteristics of the spin-trapping reaction of a free radical derived from AAPH: further development of the ORAC-ESR assay

The characteristics of the spin-trapping reaction in the oxygen radical absorbance capacity (ORAC)-electron spin resonance (ESR) assay were examined, focusing on the kind of spin traps. 2,2-Azobis(2-amidinopropane) dihydrochloride (AAPH) was used as a free radical initiator. The spin adducts of the AAPH-derived free radical were assigned as those of the alkoxyl radical, RO· (R=H(2)N(HN)C-C(CH(3))(2)). Among the spin traps tested, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5,5-dimethyl-4-phenyl-1-pyrroline N-oxide (4PDMPO), 5-(2,2-dimethyl-1,3-propoxycyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO), and 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) were applicable to the ORAC-ESR assay. Optimal formation of spin-trapped radical adduct was observed with 1 mM AAPH, 10 mM spin trap, and 5 s UV irradiation. The calibration curve (the Stern-Volmer's plot) for each spin trap showed good linearity, and their slopes, k (SB)/k (ST), were estimated to be 87.7±2.3, 267±15, 228±9, and 213±16 for DMPO, 4PDMPO, CYPMPO, and DEPMPO, respectively. Though the k (SB)/k (ST) values for selected biosubstances varied with various spin traps, their ratios to Trolox (the relative ORAC values) were almost the same for all spin traps tested. The ORAC-ESR assay also had a very good reproducibility. The ORAC-ESR assay was conducted under stoichiometric experimental conditions. The present results demonstrate the superiority of the ORAC-ESR assay.

Electron and Spin-Density Analysis of Tirapazamine Reduction Chemistry

Tirapazamine (TPZ, 1, 3-amino-1,2,4-benzotriazine 1,4-N,N-dioxide), the radical anion 2 formed by one-electron reduction of 1, and neutral radicals 3 and 4 formed by protonation of 2 at O(N4) or O(N1), respectively, and their N-OH homolyses 3 → 5 + ·OH and 4 → 6 + ·OH have been studied with configuration interaction theory, perturbation theory, and density functional theory. A comprehensive comparative analysis is presented of structures and electronic structures and with focus on the development of an understanding of the spin-density distributions of the radical species. The skeletons of radicals 3 and 4 are distinctly nonplanar, several stereoisomeric structures are discussed, and there exists an intrinsic preference for 3 over 4. The N-oxides 1, 5, and 6 have closed-shell singlet ground states and low-lying, singlet biradical (SP-1, SP-6) or biradicaloid (SP-5) excited states. The doublet radicals 2, 3, and 4 are heavily spin-polarized. Most of the spin density of the doublet radicals 2, 3, and 4 is located in one (N,O)-region, and in particular, 3 and 4 are not C3-centered radicals. Significant amounts of spin density occur in both rings in the singlet biradical(oid) excited states of 1, 5, and 6. The dipole moment of the N2-C3(X) bond is large, and the nature of X provides a powerful handle to modulate the N2-C3 bond polarity with opposite effects on the two NO regions. Our studies show very low proton affinities of radical anion 2 and suggest that the pK(a) of radical [2+H] might be lower than 6. Implications are discussed regarding the formation of hydroxyl from 3 and/or 4, regarding the ability of 5 and 6 to react with carbon-centered radicals in a manner that ultimately leads to oxygen transfer, and regarding the interpretation of the EPR spectra of reduced TPZ species and of their spin-trap adducts.

Thermal generation of stable spin trap adducts with super-hyperfine structure in their EPR spectra: An alternative EPR spin trapping assay for radical scavenging capacity determination in dimethylsulphoxide

Thermal decomposition of potassium persulphate (K2S2O8) was studied in detail by the EPR spin trapping technique in dimethylsulphoxide (DMSO), employing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 5-ethoxycarbonyl-5-methyl-1-pyrroline-N-oxide (EMPO) and 5-(diisopropoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DIPPMPO) as spin traps. DMPO and/or its derivatives exclusively trapped the primary formed SO4• − radical anions producing stable spin adducts with half-lives exceeding 2 h at room temperature. High-resolution EPR spectra of these adducts showed unusually rich hyperfine structure due to the interaction of the unpaired electron with all magnetically active nuclei of the spin trap moiety. In contrast to aprotic DMSO solvent, •DMPO-OH adducts dominated in mixed DMSO/water solutions with water content higher than 50%. The thermal decomposition of K2S2O8 in DMSO represents an effective source of free radicals for the radical scavenging capacity (RSC) determination assay, applicable to hydrophilic as well as hydrophobic antioxidants. Efficiency of the assay is demonstrated with a series of cereal samples.

Diels—Alder/Thiol—Olefin Co‐oxygenation Approach to Antimalarials Incorporating the 2,3‐Dioxabicyclo[3.3.1]nonane Pharmacophore.

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Detection of reactive free radicals derived from nucleosides by liquid chromatography coupled to tandem mass spectrometry of DMPO spin trapping adducts

In this study, reactive free radicals derived from several nucleosides were spin trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and then detected by high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). This method provides a specific detection of spin trapping adducts derived from nucleosides with a very high sensitivity: quantities as low as 0.5 picomoles of spin trapping adducts corresponding to concentrations of 2.5 10(-8) mol . L(-1) were detected. Different spin trapping adducts were characterized by HPLC/ESI-MS/MS in three well-known systems producing free radicals photochemically: the photolysis of 5-halo-2'-deoxyuridines, the photolysis of 5-thiophenylmethyl-2'-deoxyuridine and the photolysis of thymidine with menadione bisulfite as a photosensitizer. A new radical photoreactivity of uridine derivatives was also detected by this method both at the nucleoside and at the RNA level, showing that the method is also relevant for studying spin trapping adducts derived from DNA and RNA strands.

Diels–Alder/thiol–olefin co-oxygenation approach to antimalarials incorporating the 2,3-dioxabicyclo[3.3.1]nonane pharmacophore

A Diels–Alder/thiol–olefin co-oxygenation approach to the synthesis of novel bicyclic endoperoxides 17a– 22b is reported. Some of these endoperoxides (e.g., 17b, 19b, 22a and 22b) have potent nanomolar in vitro antimalarial activity equivalent to that of the synthetic antimalarial agent arteflene. Iron(II)-mediated degradation of sulfone-endoperoxide 19b and spin-trapping with TEMPO provide a spin-trapped adduct 25 indicative of the formation of a secondary carbon centered radical species 24. Reactive C-radical intermediates of this type may be involved in the expression of the antimalarial effect of these bicyclic endoperoxides.

The line asymmetry of electron spin resonance spectra as a tool to determine the cis: trans ratio for spin-trapping adducts of chiral pyrrolines N-oxides: The mechanism of formation of hydroxyl radical adducts of EMPO, DEPMPO, and DIPPMPO in the ischemic–reperfused rat liver

Nonstereospecific addition of free radicals to chiral nitrones yields cis/ trans diastereoisomeric nitroxides often displaying different electron spin resonance (ESR) characteristics. Glutathione peroxidase–glutathione (GPx-GSH) reaction was applied to reduce the superoxide adducts (nitrone/ OOH) to the corresponding hydroxyl radical (HO ) adducts (nitrone/ OH) of two nitrones increasingly used in biological spin trapping, namely 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide, and of 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DIPPMPO), a sterically hindered DEPMPO analogue. The method offered improved conditions to record highly resolved ESR spectra and by accurate simulation of line asymmetry we obtained clear evidence for the existence of previously unrecognized isomer pairs of cis- and trans-[DEPMPO/ OH] and [DIPPMPO/ OH]. Additional nitrone/ OH generation methods were used, i.e. photolysis of hydrogen peroxide and the Fenton reaction. We developed a kinetic model involving first- and second-order decay and a secondary conversion of trans to cis isomer to fully account for the strongly configuration-dependent behavior of nitrone/ OH. In the reductive system and, to a lower extent, in the Fenton or photolytic systems cis-nitrone/ OH was the more stable diastereoisomer. In various biologically relevant milieu, we found that the cis: trans-nitrone/ OH ratio determined right after the spin adduct formation significantly differed upon the GPx-GSH vs (Fenton or photolytic) systems of formation. This new mechanistic ESR index consistently showed for all nitrones that nitrone/ OH signals detected in the postischemic effluents of ischemic isolated rat livers are the reduction products of primary nitrone/ OOH. Thus, ESR deconvolution of cis/ trans diastereoisomers is of great interest in the study of HO formation in biological systems.

The synthesis and aqueous superoxide anion scavenging of water-dispersible lutein esters

Xanthophyll carotenoids of the C40 series, which includes commercially important compounds such as lutein, zeaxanthin, and astaxanthin, have poor aqueous solubility in the native state. Hawaii Biotech, Inc. (HBI) and others have shown that the aqueous dispersibility of derivatized carotenoids can be increased by varying the chemical structure of the esterified moieties. In the current study, the published series of novel, highly water-dispersible C40 carotenoid derivatives has been extended to include (3 R,3′ R,6′ R)-lutein (β,ε-carotene-3,3′-diol) derivatives. Two novel derivatives were synthesized by esterification with inorganic phosphate and succinic acid, respectively, and subsequently converted to the sodium salts. Red-orange, clear, aqueous suspensions were obtained after addition of these novel derivatives to USP-purified water. Aqueous dispersibility of the disuccinate sodium salt of lutein was 2.85 mg/mL; the diphosphate salt demonstrated a >10-fold increase in dispersibility at 29.27 mg/mL. As reported previously, these aqueous suspensions were obtained without the addition of heat, detergents, co-solvents, or other additives. The direct aqueous superoxide scavenging abilities of these novel derivatives were subsequently evaluated by electron paramagnetic resonance (EPR) spectroscopy in a well-characterized in vitro isolated human neutrophil assay. The novel derivatives were nearly identical aqueous-phase scavengers, demonstrating dose-dependent suppression of the superoxide anion signal (as detected by spin-trap adducts of DEPMPO) in the millimolar range. These lutein-based soft drugs will likely find utility in those commercial and clinical applications for which aqueous-phase singlet oxygen quenching and direct radical scavenging may be required.

Free Radical-Dependent Dysfunction of Small-for-Size Rat Liver Grafts: Prevention by Plant Polyphenols

The mechanisms by which small-for-size liver grafts decrease survival remain unclear. This study investigated the role of free radicals in injury to small-for-size grafts. Rat liver explants were reduced in size ex vivo and transplanted into recipients of the same or greater body weight, resulting in a graft weight and standard liver weight of approximately 50% and 25%, respectively. A polyphenol extract from Camellia sinenesis (20 microg/mL) or an equivalent concentration of epicatechin was added to the storage solution and the lactated Ringer poststorage rinse solution. Serum alanine aminotransferase release increased from approximately 60 U/L before implantation to 750, 1410, and 2520 U/L after full-size, half-size, and quarter-size transplantation, respectively. Total bilirubin increased slightly after transplantation of full-size and half-size grafts but increased 104-fold in quarter-size grafts. In quarter-size grafts, histological changes included necrosis, leukocyte infiltration, and eosinophilic inclusion body formation. Polyphenol treatment ameliorated these effects by > or =67%. Survival was 30% after transplantation of small-for-size grafts. After polyphenol treatment, survival increased to 70%. Free radicals in bile assessed by spin trapping and 4-hydroxynonenal adducts measured by immunohistochemistry were also greater in reduced-size grafts, an effect ameliorated by polyphenols. Epicatechin, a major polyphenol from Camellia sinenesis, also improved graft function and decreased enzyme release, histopathologic changes, and free radical formation. Increased formation of free radicals occurs after transplantation of reduced-size livers, which contributes to graft dysfunction and failure. Plant polyphenols decrease liver graft injury and increase survival of small-for-size liver grafts, most likely by scavenging free radicals.