Structural analysis of photonic crystal fibers by side scattering of laser light.

A side-scattering technique for investigating the inner microstructure of photonic crystal fibers (PCFs) is reported. Multiple scattering is reduced by filling the hollow PCF channels with index-matching fluid. The scattered signal is measured for fixed angles of incidence and detection while the fiber is rotated. A pattern of peaks, unique to each PCF, whether solid or hollow core, correlates closely with the symmetry planes of the PCF structure. As an example of the technique, the twist profile of a structural rocking filter is directly measured.

Effect of antioxidant protection by p-coumaric acid on low-density lipoprotein cholesterol oxidation.

Mechanisms in which p-coumaric acid (CA) acts as an antioxidant are not well understood. This study investigated whether CA can act as a direct scavenger of reactive oxygen species (ROS) and whether it minimizes the oxidation of low-density lipoprotein (LDL). Rats were administered CA in drinking water at low or high doses for 10, 21, and 30 days (uptakes were 29 and 317 mg/day, respectively). Blood levels of 8-epiprostaglandin F(2alpha) were monitored as a marker of LDL oxidation. Oral administration of CA (317 mg/day) for 30 days significantly inhibited LDL oxidation. CA also reduced LDL cholesterol levels in serum but had no effect on levels of high-density lipoprotein cholesterol. In vitro studies that used electron spin resonance in combination with spin trapping techniques were used to determine the ability of CA to scavenge ROS and alter LDL oxidation. CA effectively scavenged.OH in a dose-dependent manner. IC(50) and maximum velocity for CA scavenging of.OH were 4. 72 microM and 1.2 microM/s, respectively, with a rate constant of 1. 8 x 10(11) M(-1). s(-1). Our studies suggest that the antioxidant properties of CA may involve the direct scavenging of ROS such as.OH.

Scavenging of superoxide anion radical by chaparral.

Chaparral is considered to act as an antioxidant. However, the inhibitory effects of chaparral on specific radical species are not well understood. Using electron paramagnetic resonance (EPR) spectroscopy in combination with spin trapping techniques, we have found that chaparral scavenges superoxide anion radical (O2*-) in a dose-dependent manner. 5,5-dimethyl-lpyrroline-N-oxide (DMPO) was used as a spin trapping agent and the reaction of xanthine and xanthine oxidase as a source of O2*-. The kinetic parameters, IC50 and Vmax, for chaparral scavenging of O2*- were found to be 0.899 microg/mL and 8.4 ng/mL/sec, respectively. The rate constant for chaparral scavenging O2*- was found to be 1.22 x 10(6) g(-1) s(-1). Our studies suggest that the antioxidant properties of chaparral may involve a direct scavenging effect of the primary oxygen radical, O2*-.

Scavenging of reactive oxygen species by melatonin.

The direct effects of the neurohormone melatonin on reactive oxygen species (ROS) were investigated. Melatonin was found to inhibit DMPO-O-2 formation in a dose-dependent manner. At the level of 1. 7+/-0.07 mM, melatonin caused 50% inhibition of EPR signal intensity of DMPO-O-2 during the reaction of xanthine and xanthine oxidase. The reaction rate constant of melatonin with O2- was found to be 1.25+/-0.07x103 M-1 s-1. However, melatonin (up to 1.2 mM) did not exhibit significant effect toward OH radical, produced by the Fenton reaction. In addition, we found no evidence for the formation of the melatonin indolyl cation radical that presumably precedes conversion of melatonin to its stable N1-acetyl-N2-5-methoxykynuramine (AMK) metabolite following sequential reactions of melatonin with O2- and OH. On the other hand, melatonin was capable of scavenging H2O2 in a dose-dependent manner with an IC50=0.5+/-0.02 mM. The reaction rate constant of melatonin with H2O2 was found to be 2.52+/-0.19x105 M-1 s-1. Furthermore, melatonin was also found to inhibit 1O2-dependent 2,2,6,6-tetramethylpiperidine oxide (TEMPO) radical formation during rose bengal photodynamic reaction. The results suggest that melatonin's antioxidant properties, in part, may involve a direct effect on scavenging of ROS.

Fractionation of aqueous cigarette tar extracts: fractions that contain the tar radical cause DNA damage.

Previously, we have shown that aqueous cigarette tar (ACT) extracts contain a long-lived tar radical that associates with DNA in isolated rat alveolar macrophages and causes DNA damage in isolated rat thymocytes. These ACT solutions reduce oxygen to produce superoxide and, ultimately, hydrogen peroxide. In this study, we report the fractionation of ACT solutions prepared from the tar from five cigarettes using Sephadex columns. The fractions were analyzed by UV and electron paramagnetic resonance (EPR) spectroscopy and gas chromatography/mass spectrometry (GC/MS). The fractions containing polyphenolic species (principally catechol and hydroquinone, as determined by MS) caused most of the observed DNA damage in rat thymocytes. These DNA-damaging fractions produced superoxide, H2O2, and hydroxyl radicals. Stable free radicals were identified as o- and p-benzosemiquinone radicals by EPR spectroscopy. Hydroxyl radicals were detected by EPR spin-trapping with 5, 5-dimethyl-1-pyrroline N-oxide (DMPO). Catalase inhibited the EPR signal of the DMPO-OH adduct, indicating that H2O2 is the precursor of the hydroxyl radical spin adduct. The Sephadex separation resulted in a 90-fold concentration of the hydrogen peroxide-generating capacity of the fractions that contained polyphenols, relative to the unfractionated ACT solution. Another fraction, which contained nicotine, caused some DNA damage, but this damage was 28-fold less than the damage caused by the most damaging phenolic fraction. These results support our hypothesis that the tar radical system is an equilibrium mixture of semiquinones, hydroquinones, and quinones. The tar radical associates with DNA, causes DNA damage, and very likely is involved in the toxicity associated with cigarette smoking.

Simultaneous detection of carotenoids and vitamin E in human plasma.

A simplified method for analysis of the antioxidants carotenoids and vitamin E in human plasma is presented. The method is based on high-performance liquid chromatography with a single column, a flow-rate gradient, and detection at 450 and 290 nm with a diode array detector. It gives good separation of the vitamin E isomers and the major carotenoids in plasma, with a 25 min analysis time. It was found that hydrolysis of triglycerides and cholesterol esters is required to obtain good recovery of non-polar carotenoids such as lycopene, alpha-carotene and beta-carotene. Two methods were used for hydrolysis of the non-polar lipids, saponification with ethanolic KOH and digestion with an enzyme mixture of lipase and cholesterol esterase. It was found that the enzymatic digestion gave the best recoveries, better than 94% for all of the antioxidants, and preserved several carotenoids. A plasma pool is used for day to day calibration of the method, which eliminates the need for stock solutions of carotenoids that are stable for only a month due to oxidative breakdown and their tendency to crystallize when stored at -20 degrees C in organic solvents.

Absorbance changes of carotenoids in different solvents.

Carotenoids are typically measured in tissues with the high performance liquid chromatography (HPLC) and quantitation is usually done by calibrating with stock solutions in solvents. Four carotenoids including lutein, zeaxanthin, lycopene and beta-carotene were dissolved in hexane and methanol respectively, and their absorbance characteristics were compared. Lutein shows absorbance spectra that are almost independent of solvents at various concentrations. Spectra of zeaxanthin, lycopene and beta-carotene were found to be more solvent-dependent. The absorbance of zeaxanthin at lambda max is about approximately 2 times larger in methanol than in hexane at the higher concentrations, and increased non-linearly with increasing concentration in hexane. The absorbance of lycopene at lambda max in hexane is approximately 4 fold larger than in methanol, but the absorbance of the methanol sample can be recovered by re-extracting this sample in hexane. The absorbance of beta-carotene in hexane is larger than in methanol, and increased linearly with increasing concentration. But beta-carotene showed a non-linear concentration effect in methanol. There are very small variations in lambda max for all four carotenoids between hexane and methanol, due to differences in molar extinction coefficients. The non-linear concentration effects for these carotenoids are probably due to differences in solubility leading to the formation of microcrystals. Thus, care should be taken with quantitation of tissue carotenoid values, when they depend on measurement of concentrations in stock solutions.

Inhibition of acetylcholinesterase by the neurotoxicant, 1-methyl-4-phenyl-2,3-dihydropyridinium ion.

The effect of the neurotoxicant 1-methyl-4-phenyl-2,3-dihydropyridinium ion (MPDP+) on acetylcholinesterase (AchE) activity was investigated. The MPDP+ was found to inhibit the enzyme in a dose-dependent manner. The kinetic parameter, Km, for the substrate acetylthiocholine was found to be 0.22 mM, and the Kis and Kii for MPDP+ inhibition of AChE were found to be 0.265 and 0.578 mM, respectively. It was found that MPDP+ is neither a substrate of AChE nor a time-dependent inactivator. The studies of reaction kinetics indicate the inhibition of AChE to be a noncompetitive inhibition. The inhibition of AchE by MPDP+ was virtually reversed by either dilution or gel exclusion chromatography. These data suggest that once MPDP+ enters the basal ganglia of the brain, it can inhibit the AChE and thereby increase the acetylcholine level in the basal ganglia, leading to potential cell dysfunction. It appears likely that the nigrostriatal toxicity by MPDP+ leading to Parkinson's disease-like syndrome may, at least in part, be mediated via the AChE inhibition.

EPR-spin trapping kinetic studies of superoxide radicals produced by photosensitized hypocrellin A. A photodynamic therapeutic agent.

Kinetics of superoxide radicals generation by a photodynamic therapeutic agent, Hypocrellin A has been studied by EPR-spin trapping technique. The rate constant (k(g)) for superoxide radicals generation by Hypocrellin A was found to be 5.0 x 10(-3) s-1. The first-order rate constant (k1) and the half-life time (t0.5) for DMPO-O2- decay were 2.86 x 10(-3) s-1 and 242.3 s, respectively. It appears that oxyradicals, generated during the photodynamic process, may be associated with the treatment of various skin diseases by photodynamic therapy of Hypocrellin A.

Elimination of n-butylated hydroxytoluene methylation during fatty acid analysis by gas chromatography.

An improved method for fatty acids analysis with optimum recovery of highly polyunsaturated fatty acids methyl esters in biological systems is presented. The method is based on transesterification of phospholipid and triacylglycerols to fatty acid methyl esters using a commercially available reagent, Methyl-Prep II. Without proper precautions, as much as 50% of n-butylated hydroxytoluene (BHT) added to prevent oxidation of polyunsaturated fatty acids, could be methylated during the transesterification step. Methylated BHT elutes close to 14:0 (myristic acid) and no longer functions as an antioxidant, but the modified conditions virtually eliminate the methylation of BHT. Sample extraction and methylation was completed in 30 min at room temperature. A chelator (diethylenetriamine-pentaacetic acid; DTPA) is also added to prevent peroxidation of metal catalyzed free radical chain reactions. The standard deviations of the major fatty acids from multiple human plasma samples prepared on different days were less than 5%. The recovery of arachidonic acid, 20:4, from plasma was improved using the new method, and the recovery for docosahexaenoic acid, 22:6, spiked to human plasma was found to be 99%.

EPR studies on the kinetics of quenching singlet oxygen.

The specific reaction between singlet oxygen (1O2) and 2,2,6,6-tetra-methyl-piperidine (TEMP) was utilized to investigate the kinetics of singlet oxygen quenching. Rose bengal was used for the generation of singlet oxygen (1O2). The deuterium isotope effect (rho) was found to be 22.1. The rate constant (kT) of the reaction of TEMP with 1O2 in H2O, D2O and ethanol were found to be 9.0 x 10(5) M-1s-1, 1.5 x 10(6) M-1s-1 and 7.3 x 10(5) M-1s-1, respectively. The intercept of the plot of [TEMPO]-1 at different H2O/D2O ratios was found to be constant, indicating the lifetime of 3Sens is independent of solvent deuteration. The quenching of 1O2 by azide and the sensitizer itself were confirmed by the constant intercept on the kinetic plots. Thus, the quenching rate constants for azide anion in ethanol solution (96%) and for ground state rose bengal in 0.05 M phosphate buffer (pH 7.4), were found to be 8.36 x 10(8) M-1s-1 and 1.72 x 10(4) M-1s-1, respectively. These studies not only verify the specificity of the reaction between TEMP and 1O2 but also provide a sensitive and specific assay for 1O2.

EPR studies of spin-trapped free radicals in paraquat-treated lung microsomes.

Electron Spin Resonance and Spin Trapping techniques were used to demonstrate the generation of free radicals during incubation of paraquat with lung microsomes. Aerobic incubation of paraquat resulted in the production of superoxide radical (.O2-) which was trapped by 5, 5-dimethyl-pyrroline-N-oxide or phenyl-tert-butyl-nitrone. The formation of .O2- and hydroxyl radical (.OH) by paraquat during microsomal incubation was also confirmed by the inhibition of the EPR spectra of DMPO-O2- and DMPO-OH spin adducts by superoxide dismutase and dimethyl sulfoxide, respectively. Our results provide direct evidence for the generation of reactive oxygen species during redox cycling of paraquat in microsomes. The formation of DMPO-H spin adduct during incubation of paraquat in the microsomal system is a strong indication of the involvement of hydrogen atom transfer mechanisms in paraquat-induced generations of reactive oxygen species in the lung.

The specificity and product of quenching singlet oxygen by 2,2,6,6-tetramethylpiperidine.

The specificity of 2,2,6,6-tetramethylpiperidine to singlet oxygen was shown using Rose Bengal as a singlet oxygen generator, and Xanthine-Xanthine Oxidase and KO2 as the sources for the superoxide radical. The highest concentration of produced-singlet oxygen occurred at 25% of O2 by Rose Bengal photosensitization. The linewidth of the EPR signal for photosensitized nitroxyl radical, increasing solvent polarity. Deuterated solvents enlarge the EPR signal intensity in a dose-dependent manner. No EPR signal increase was observed in xanthine-xanthine oxidase reaction or KO2 systems, indicating that TEMP does not react with the superoxide anion. Thus, reaction of TEMP with 1O2 is highly specific.

Evidence for superoxide radical production in peroxynitrite decomposition.

Peroxynitrite decomposition was investigated by ESR spin trapping. The spin trap used was 5,5-dimethyl-1-pyrroline N-oxide (DMPO). A mixture of peroxynitrite and DMPO generated predominantly DMPO-O2- adduct. A combination of SOD and catalase suppressed the formation of DMPO-O2-. The DMPO-O2- signal reached its maximum at pH lower than 7 and decreased as pH increased. The DMPO-O2- signal also depended on peroxynitrite concentration with maximum signal intensity appearing at 4.2 mM. The results demonstrate that peroxynitrite decomposition generates O2.-. Since reaction of H2O2 with NO2- generates peroxynitrite, the results point out a pathway for conversion of H2O2 to O2.- via peroxynitrite as an intermediate.

Detection of free radicals in aqueous extracts of cigarette tar by electron spin resonance.

Aqueous extracts of cigarette tar (ACT) autooxidize to produce semiquinone, hydroxyl, and superoxide radicals in air-saturated buffered aqueous solutions. The semiquinone species were detected by direct electron spin resonance (ESR) measurements and identified as o- and p-benzosemiquinone radicals by comparison with the ESR signals of catechol and hydroquinone radicals under similar conditions. The rate of formation of these radicals was dependent on pH. Hydroxyl and superoxide radicals were detected as 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) spin adducts by ESR spin trapping. Superoxide dismutase (SOD) (20 units/ml) inhibited the formation of the superoxide spin adduct of DMPO completely. Addition of Fe2+ to this system increased the ESR signal intensity of hydroxyl radical spin adduct of DMPO three to five times. These results indicate that superoxide and hydroxyl radicals are produced during the autooxidation of hydroquinone- and catechol-related species in ACT.

The ESR properties, DNA nicking, and DNA association of aged solutions of catechol versus aqueous extracts of tar from cigarette smoke.

Previous studies in our laboratories have shown that extracts from mainstream or sidestream tobacco smoke nick DNA. These solutions contain the tar semiquinone free radical, and the tar radical becomes associated with cellular DNA. Aged solutions of catechol contain a semiquinone species that has ESR properties similar to those of the radical in cigarette tar extracts, and we have used these catechol solutions as a model for the tar radical. Both the radical in aged catechol solutions and the cigarette tar radical become associated with the DNA in mammalian cells and also nick DNA. The nicking of DNA caused by both tar and aged catechol solutions follows saturation kinetics. Aged catechol solutions thus allow the study of a model for the radical present in cigarette tar, without interference from the other toxic constituents in tar extracts.

Acetylcholinesterase inhibition by 1-methyl-4-phenylpyridinium ion, a bioactivated metabolite of MPTP.

The effect of the neurotoxicant, 1-methyl-4-phenylpyridinium ion (MPP+) on acetylcholinesterase (AchE) activity was investigated. The MPP+ was found to inactivate the enzyme in a dose dependent manner. The kinetic parameter, Km for the substrate (acetylthiocholine), was found to be 0.216 mM and Ki for MPP+ for the inactivation of AChE was found to be 0.197 mM. It was found that MPP+ is neither a substrate of AChE nor the time-dependent inactivator. The studies of reaction kinetics indicate the inactivation of AChE to be a linear mixed-type inhibition. The inactivation of AChE by MPP+ was partially recovered by either dilution or gel exclusion chromatography. These data suggest that once MPP+ enters the basal ganglia of the brain, it can inactivate the AChE and thereby increase the acetylcholine level in the basal ganglia, leading to potential cell dysfunction. It appears likely that the nigrostriatal toxicity by MPP+ leading to Parkinson's disease-like syndrome may, in part, be mediated via the AChE inactivation.

Generation of reactive oxygen species during the monoamine oxidase-catalyzed oxidation of the neurotoxicant, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

The neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to generate reactive oxygen species during its interaction with monoamine oxidase type B (MAO-B). The kinetic parameters, Km and Vmax, for MAO-B-catalyzed oxidation of MPTP to the corresponding species MPDP+ were found to be 0.194 mM and 0.335 microM/min, respectively. The generation of superoxide (.O2-) and hydroxyl (.OH) radicals was detected as the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) spin adduct by spin-trapping in combination with EPR techniques. Addition of Fe2+ (10 microM) to this system caused a 5-fold enhancement in EPR signal intensity of the DMPO-OH adduct. Catalase, a scavenger of hydrogen peroxide (H2O2), inhibited the DMPO-OH spin adduct formation in a dose-dependent manner, indicating that H2O2 is produced in the MAO-B catalyzed oxidation of MPTP. Ethanol, a well known scavenger of hydroxyl radical, rapidly produced an alpha-hydroxyethyl radical signal. Superoxide dismutase inhibited the formation of DMPO-O2- and DMPO-OH spin adducts in a dose-dependent fashion. These data suggest that superoxide radicals are produced during the oxidation of MPTP by MAO-B and that the generation of H2O2 and .OH was secondary to the production of .O2-. It appears likely that the nigrostriatal toxicity of MPTP leading to Parkinson's disease-like syndrome may in part be mediated via these reactive oxygen species.

EPR kinetic studies of superoxide radicals generated during the autoxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium, a bioactivated intermediate of parkinsonian-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

1-Methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), a metabolic product of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been shown to generate superoxide radicals during its autoxidation process. The generation of superoxide radicals was detected as a 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).O2- spin adduct by spin trapping in combination with EPR techniques. The rate of formation of spin adduct was dependent not only on the concentrations of MPDP+ and oxygen but also on the pH of the system. Superoxide dismutase inhibited the spin adduct formation in a dose-dependent manner. The ability of DMPO to trap superoxide radicals, generated during the autoxidation of MPDP+, and of superoxide dismutase to effectively compete with this reaction for the available O2-, has been used as a convenient competition reaction to quantitatively determine various kinetic parameters. Thus, using this technique the rate constant for scavenging of superoxide radical by superoxide dismutase was found to be 7.56 x 10(9) M-1 s-1. The maximum rate of superoxide generation at a fixed spin trap concentration using different amounts of MPDP+ was found to be 4.48 x 10(-10) M s-1. The rate constant (K1) for MPDP+ making superoxide radical was found to be 3.97 x 10(-6) s-1. The secondary order rate constant (KDMPO) for DMPO-trapping superoxide radicals was found to be 10.2 M-1 s-1. The lifetime of superoxide radical at pH 10.0 was calculated to be 1.25 s. These values are in close agreement to the published values obtained using different experimental techniques. These results indicate that superoxide radicals are produced during spontaneous oxidation of MPDP+ and that EPR spin trapping can be used to determine the rate constants and lifetime of free radicals generated in aqueous solutions. It appears likely that the nigrostriatal toxicity of MPTP/MPDP+ leading to Parkinson's disease may largely be due to the reactivity of these radicals.