Lipid Peroxidation. Definition of Experimental Conditions for Selective Study of the Propagation and Termination Phases

To find experimental conditions to selectively study the propagation phase of lipoperoxidation we studied the lipoperoxidation, catalyzed by FeCl₂, of liposomes in a buffering condition where Fe²⁺ autoxidation and oxygen active species generation does not occur. Liposomes from egg yolk phosphatidylcholine. prepared by vortex mixing, do not oxidize Fe²⁺: on the contrary they oxidize Fe²⁺ when prepared by ultrasonic irradiation. Dimyristoyl phosphatidylcholine liposomes prepared by ultrasonic irradiation do not oxidize Fe²⁺. During sonication polyunsaturated fatty acid residues autoxidize and lipid hydroperoxides (LOOH) are generated. Only when LOOH are present in the liposimes Fe²⁺ oxidizes and its rate of oxidation depends on the amount of LOOH in the assay. The reaction results in the generation of both LOOH and thiobarbituric acid reactive material (TBAR): it is inhibited by butylated hydroxytoluene and has a acidic pH optimum; it is not inhibited by catalase and OH' scavengers. The reaction studied. thus, appears to be the chain branching and propagation phase of lipoperoxidation. When we studied the dependence of Fe²⁺ oxidation, LOOH and TBAR generation on FeCl₂ concentration, we observed that at high FeCl₂ concentrations the termination phase of lipoperoxidation was prevalent. Thus. by selecting the appropriate FeCl₂ concentration the proposed experimental system allows study of either the propagation or the termination phase of lipoperoxidation.     

Iron (III) Stimulation of Lipid Hydroperoxide-Dependent Lipid Peroxidation

In an experimental system where both Fe²⁺ autoxidation and generation of reactive oxygen species is negligible, the effect of FeCl₂ and FeCl₃ on the peroxidation of phosphatidylcholine (PC) liposomes containing different amounts of lipid hydroperoxides (LOOH) was studied; Fe²⁺ oxidation, oxygen consumption and oxidation index of the liposomes were measured. No peroxidation was observed at variable FeCl₂/FeCl₃ ratio when PC liposomes deprived of LOOH by triphenyl-phosphine treatment were utilized. By contrast, LOOH containing liposomes were peroxidized by FeCl₂. The FeCl₂ concentration at which Fe²⁺ oxidation was maximal, defined as critical Fe²⁺ concentration [Fe²⁺]*, depended on the LOOH concentration and not on the amount of PC liposomes in the assay. The LOOH-dependent lipid peroxidation was stimulated by FeCl₃, addition; the oxidized form of the metal increased the average length of radical chains, shifted to higher values the [Fe²⁺]* and shortened the latent period. The iron chelator KSCN exerted effects opposite to those exerted by FeCl₃ addition. The experimental data obtained indicate that the kinetics of LOOH-dependent lipid peroxidation depends on the Fe²⁺/Fe³⁺ ratio at each moment during the time course of lipid peroxidation. The results confirm that exogenously added FeCl₃ does not affect the LOOH-independent but the LOOH-deendent lipid peroxidation; and suggest that the Feg, endogenously generated exerts a major role in the control of the LOOH-dependent lipid peroxidation.     

Carvedilol inhibition of lipid peroxidation. A new antioxidative mechanism

To define the molecular mechanism(s) of carvedilol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process.

Carvedilol inhibits the peroxidation of sonicated phosphatidylcholine liposomes triggered by FeCl₂ addition whereas atenolol, pindolol and labetalol are ineffective. The inhibition proved not to be ascribable (a) to an effect on Fe²⁺ autoxidation and thus on the generation of oxygen derived radical initiators; (b) to the scavenging of the inorganic initiators O^·-₂ and ^·OH; (c) to an effect on the reductive cleavage of organic hydroperoxides by FeCl₂; (d) to the scavenging of organic initiators. The observations that (a) carvedilol effectiveness is inversely proportional to the concentration of FeCl₂ and lipid hydroperoxides in the assay; (b) the drug prevents the onset of lipid peroxidation stimulated by FeCl₃ addition and; (c) it can form a complex with Fe³⁺, suggest a molecular mechanism for carvedilol action. It may inhibit lipid peroxidation by binding the Fe³⁺ generated during the oxidation of Fe²⁺ by lipid hydroperoxides in the substrate. The lag time that carvedilol introduces in the peroxidative process would correspond to the time taken for carvedilol to be titrated by Fe³⁺; when the drug is consumed the Fe³⁺ accumulates to reach the critical parameter that stimulates peroxidation. According to this molecular mechanism the antioxidant potency of carvedilol can be ascribed to its ability to bind a species, Fe³⁺, that is a catalyst of the process and to its lipophilic nature that concentrates it in the membranes where Fe³⁺ is generated by a site specific mechanism.     

The Mechanism of Iron (III) Stimulation of Lipid Peroxidation

A study conducted on Fe²⁺ autoxidation showed that its rate was extremely slow at acidic pH values and increased by increasing the pH; it was stimulated by Fe³⁺ addition but the stimulation did not present a maximum at a Fe²⁺/Fe³⁺ ratio approaching 1:1. The species generated during Fe³⁺-catalyzed Fe²⁺ autoxidation was able to oxidize deoxyribose; the increased Fe²⁺ oxidation observed at higher pHs was paralleled by increased deoxyribose degradation. The species generated during Fe³⁺-catalyzed Fe²⁺ autoxidation could not initiate lipid peroxidation in phosphatidylcholine liposomes from which lipid hydroperoxides (LOOH) had been removed by treatment with triph-enylphosphine. Neither Fe²⁺ oxidation nor changes in the oxidation index of the liposomes due to lipid peroxidation were observed at pHs where the Fe³⁺ effect on Fe²⁺ autoxidation and on deoxyribose degradation was evident. In our experimental system, a Fe²⁺/Fe³⁺ ratio ranging from 1:3 to 2:1 was unable to initiate lipid peroxidation in LOOH-free phosphatidylcholine liposomes. By contrast Fe³⁺ stimulated the peroxidation of liposomes where increasing amounts of cumene hydroperoxide were incorporated. These results argue against the participation of Fe³⁺ in the initiation of LOOH-independent lipid peroxidation and suggest its possible involvement in LOOH-dependent lipid peroxidation.     

The oxidation of chlorophyll a in alcohols

Oxidation potentials for the chlorophyll (Chl/Chl⁺) couple, relative to that of the Fe²⁺/Fe³⁺ couple, were determined in methanol, ethanol, and isopropanol from the initial extent of reaction of chlorophyll with FeCl₃. The presence of 4,7-dimethylphenanthroline was necessary to get sufficient oxidation of chlorophyll in isopropanol. The values were +0.75 V, +0.865 V, and approx. +1.0 V, respectively, based on an assumed value of +0.77 V for the Fe²⁺/Fe³⁺ potential. It is suggested that alcohols, especially methanol, may stabilize Chl⁺ by adding to the carbonyl group or the conjugated double bond system.     

Iron (II) Ions Induced Oxidation of Ascorbic Acid and Glucose

Lipid peroxidation (LPO) of polyunsaturated fatty acids (PUFAs) is suspected to be involved in the generation of chronic diseases. A model reaction for LPO is the air oxidation of PUFAs initiated by Fe²⁺ and ascorbic acid. In the course of such model reactions glycolaldehyde (GLA) was detected as main aldehydic product. Since it is difficult to explain the generation of GLA by oxidation of PUFAs, it was suspected that GLA might be derived by oxidation of ascorbic acid. This assumption was verified by treatment of ascorbic acid with Fe²⁺.

Produced aldehydic compounds were trapped by addition of pentafluorobenzylhydroxylamine hydrochloride (PFBHA-HCl), trimethylsilylated and finally identified by gas chromatography/mass spectrometry (GC/MS). Oxidation of ascorbic acid with O₂ in presence of iron ions produced not only glycolaldehyde (GLA), but also glyceraldehyde (GA), dihydroxyacetone (DA) and formaldehyde. Glyoxal (GO) and malondialdehyde (MDA) were detected as trace compounds.

The yield of the aldehydic compounds was increased by addition of lipid hydroperoxides (LOOH) or H₂O₂. The buffer influenced the reaction considerably: Iron ions react with Tris buffer by producing dihydroxyace-tone (DA). Since ascorbic acid is present in biological systems and Fe²⁺ ions are obviously generated by cell damaging processes, the production of GLA and other aldehydic components might add to the damaging effects of LPO.

Glucose suffers also oxidation to short-chain aldehydic compounds in aqueous solution, but this reaction requires addition of equimolar amounts of Fe²⁺ together with equimolar amounts of H₂O₂ or 13-hydroperoxy-9-cis-11-trans-octadecadienoic acid (13-HPODE). Therefore this reaction, also influenced by the buffer system, seems to be not of biological relevance.     

Binding of Ferric Ions to Nuclei and Other Tissue Sites in Sections Stained for Sulfated Mucosubstances by the High-Iron Diamine Method

Binding of Fe³⁺ occurred in nuclei and several other sites when tissue sections, after a prior staining by the high-iron diamine (HID) method for sulfomucins, were immersed for 1 hr in 0.06 N HCl containing 1% potassium ferrocyanide (Prussian blue reaction). Apparently Fe³⁺, which is derived from FeCl₃ present in the HID dye bath, unites directly with these tissue components, although one cannot exclude the possibility that iron is first bound to colorless diamine complexes and then to tissues. The visualization of Fe³⁺ by ferrocyanide provides a simple way of obtaining a suitable nuclear stain combined with general counters taming for the HID method.     

Mechanisms of Saccharomyces Cerevisiae PMA1 H+-ATPase inactivation by Fe2+, H2O2 and Fenton reagents

Although considerably more oxidation-resistant than other P-type ATPases, the yeast PMA1 H⁺-ATPase of Saccharomyces cerevisiae SY4 secretory vesicles was inactivated by H₂O₂, Fe²⁺, Fe- and Cu-Fenton reagents. Inactivation by Fe²⁺ required the presence of oxygen and hence involved auto-oxidation of Fe²⁺ to Fe³⁺. The highest Fe^2- (100 μM) and H₂O₂ (100 mM) concentrations used produced about the same effect. Inactivation by the Fenton reagent depended more on Fe²⁺ content than on H₂O₂ concentration, occurred only when Fe²⁺ was added to the vesicles first and was only slightly reduced by scavengers (mannitol, Tris, NaN₃, DMSO) and by chelators (EDTA, EGTA, DTPA, BPDs, bipyridine, 1, 10-phenanthroline). Inactivation by Fe- and Cu- Fenton reagent was the same; the identical inactivation pattern found for both reagents under anaerobic conditions showed that both reagents act via OH^·. The lipid peroxidation blocker BHT prevented Fenton-induced rise in lipid peroxidation in both whole cells and in isolated membrane lipids but did not protect the H⁺-ATPase in secretory vesicles against inactivation. ATP partially protected the enzyme against peroxide and the Fenton reagent in a way resembling the protection it afforded against SH-specific agents. The results indicate that Fe²⁺ and the Fenton reagent act via metal-catalyzed oxidation at specific metal-binding sites, very probably SH-containing amino acid residues. Deferrioxamine, which prevents the redox cycling of Fe²⁺, blocked H⁺-ATPase inactivation by Fe²⁺ and the Fenton reagent but not that caused by H₂O₂, which therefore seems to involve a direct non-radical attack. Fe-Fenton reagent caused fragmentation of the H⁺-ATPase molecule, which, in Western blots, did not give rise to defined fragments bands but merely to smears.     

DNA Single Strand Breakage by H2O2 and Ferric or Cupric Ions: Its Modulation by Histidine

The role of histidine on DNA breakage induced by hydrogen peroxide (H₂O₂) and ferric ions or by H₂O₂ and cupric ions was studied on purified DNA. L-histidine slightly reduced DNA breakage by H₂O₂ and Fe³⁺ but greatly inhibited DNA breakage by H₂O₂ and Cu²⁺. However, only when histidine was present, the addition of EDTA to H₂O₂ and Fe³⁺ exhibited a bimodal dose response curve depending on the chelator metal ratio. The enhancing effect of histidine on the rate of DNA degradation by H₂O₂ was maximal at a chelator metal ratio between 0.2 and 0.5, and was specific for iron. When D-histidine replaced L-histidine, the same pattern of EDTA dose response curve was observed. Superoxide dismutase greatly inhibited the rate of DNA degradation induced by H₂O₂, Fe³⁺, EDTA and L-histidine involving the superoxide radical.

These studies suggest that the enhancing effect of histidine on the rate of DNA degradation by H₂O₂ and Fe³⁺ is mediated by an oxidant which could be a ferrous-dioxygen-ferric chelate complex or a chelate-ferryl ion.     

The lactate-dependent enhancement of hydroxyl radical generation by the Fenton reaction

The effect of lactic acid (lactate) on Fenton based hydroxyl radical (^·OH) production was studied by spin trapping, ESR, and fluorescence methods using DMPO and coumarin-3-carboxylic acid (3-CCA) as the ^·OH traps respectively. The ^·OH adduct formation was inhibited by lactate up to 0.4mM (lactate/iron stoichiometry = 2) in both experiments, but markedly enhanced with increasing concentrations of lactate above this critical concentration. When the H₂O₂ dependence was examined, the DMPO-OH signal was increased linearly with H₂O₂ concentration up to 1 mM and then saturated in the absence of lactate. In the presence of lactate, however, the DMPO-OH signal was increased further with higher H₂O₂ concentration than 1 mM, and the saturation level was also increased dependent on lactate concentration. Spectroscopic studies revealed that lactate forms a stable colored complex with Fe³⁺ at lactate/Fe³⁺ stoichiometry of 2, and the complex formation was strictly related to the DMPO-OH formation. The complex formation did not promote the H₂O₂ mediated Fe³⁺ reduction. When the Fe³⁺-lactate (1:2) complex was reacted with H₂O₂, the initial rate of hydroxylated 3-CCA formation was linearly increased with H₂O₂ concentrations. All the data obtained in the present experiments suggested that the Fe³⁺-lactate (1:2) complex formed in the Fenton reaction system reacts directly with H₂O₂ to produce additional ^·OH in the Fenton reaction by other mechanisms than lactate or lactate/Fe³⁺ mediated promotion of Fe³⁺/Fe²⁺ redox cycling.     

Oxygen Toxicity. The Influence of Adenine-Nucleotides and Phosphate on Fe2+ Autoxidation

Fecl₂, in Na phosphate buffer autoxidizes forming active oxygen species which damage deoxyribose. Di-and triphosphate adenine-nucleotides inhibit both Fe²⁺ autoxidation and deoxyribose damage in Na phosphate buffer pH 7.4. The inhibition is related to the number of charges of the adenine-nucleotide molecule: ATP at pH 7.4 is a better inhibitor than ADP; at a pH (6.5) close to the pK's of the third and fourth charge of ADP and ATP, ADP inhibition is greatly decreased whereas ATP inhibition is slightly affected. The extent of ATP inhibition of Fe²⁺ autoxidation depends both on ATP/Mg²⁺ and ATP/Fe²⁺ ratios in the reaction mixture. Formation of a Fe²⁺ -nucleotide complex appears to be the mechanism through which ATP and ADP inhibit autoxidation and thus the generation of active oxygen species. These findings are discussed in relation to physiological and pathological fluctuations of nucleotide concentrations.     

Aluminum ions stimulate the oxidizability of low density lipoprotein by Fe2+: implication in hemodialysis mediated atherogenic LDL modification

Objective: Al³⁺ stimulates Fe²⁺ induced lipid oxidation in liposomal and cellular systems. Low-density lipoprotein (LDL) oxidation may render the particle atherogenic. As elevated levels of Al³⁺ and increased lipid oxidation of LDL are found in sera of hemodialysis patients, we investigated the influence of Al³⁺ on LDL oxidation.

Materials and methods: Using different LDL modifying systems (Fe²⁺, Cu²⁺, free radical generating compounds, human endothelial cells, hemin/H₂O₂ and HOCl), the influence of Al³⁺ on LDL lipid and apoprotein alteration was investigated by altered electrophoretic mobility, lipid hydroperoxide-, conjugated diene- and TBARS formation.

Results: Al³⁺ could stimulate the oxidizability of LDL by Fe²⁺, but not in the other systems tested. Al³⁺ and Fe²⁺ were found to bind to LDL and Al³⁺could compete with Fe²⁺ binding to the lipoprotein. Fluorescence polarization data indicated that Al³⁺ does not affect the phospholipid compartment of LDL.

Conclusions:The results indicate that increased LDL oxidation by Fe²⁺ in presence of Al³⁺ might be due to blockage of Fe²⁺ binding sites on LDL making more free Fe²⁺ available for lipid oxidation.     

Self-reduction of the iron(III)-doxorubicin complex

The Fe³⁺-doxorubicin complex undergoes reactions that suggest that the complex self-reduces to a ferrous oxidized-doxorubicin free radical species. The Fe³⁺-doxorubicin system is observed to reduce ferricytochrome c, consume O₂ and react with 2,2′-bipyridine. Bipyridine acts as a “ferrous ion scavenger” as it reacts with the ferrous ion produced by Fe³⁺-doxorubicin self-reduction. In the absence of O₂, a ferrous doxorubicin complex accumulates. In the presence of oxygen, Fe²⁺ recycles back to Fe³⁺. The rates of these reactions were measured and the Fe³⁺-doxorubicin self-reduction was determined to be the rate-determining step. The Fe³⁺-doxorubicin induced inactivation of cytochrome c oxidase and NADH cytochrome c reductase on beef heart submitochondrial particles occurs at a rate similar to Fe³⁺-doxorubicin self-reduction. Thus the rate at which damage to these mitochondrial enzymes occurs may be controlled by a nonezymatic Fe³⁺-doxorubicin self-reduction.     

Control of the Ferric Ion Concentration in Iron-Aceto-Carmine Staining

The Fe⁺⁺⁺ concentration is controlled by adjusting the FeCl₃ normality of the iron-aceto-carmine staining solutions. Two stock mordant solutions are prepared by dissolving ferric chloride (FeCl₃·6H₂O; m. w. =270.31) in 45% glacial acetic acid, the normality of one being N/1, and of the other N/10. By combining aceto-carmine (preferably prepared from Merck's carmine No. 40 N. F., or from the Coleman and Bell product) and one or the other of the stock mordant solutions, a series of iron-aceto-carmine solutions is made up, each solution being of different normality for FeCl₃, depending on the proportions combined. Trial series: N/50, N/100, N/500, N/1000 and 0 N.

Tissue (spermatogenic) from one specimen is fixed in Carnoy-2, divided equally among the five iron-aceto-carmine solutions for staining, then squashed, dehydrated and mounted as usual. Subsequently the trial series may be retained or adjusted. Advantages of the method: 1) discloses quickly the optimum stain for a particular tissue type; 2) automatically gives an optimum stain to cells in different maturational stages; 3) results are reproducible in subsequent operations.

Tables and equations are provided for a number of other normalities and quantities of stain.     

Degradation of Hyaluronic Acid by Photosensitized Riboflavin In Vitro. Modulation of the Effect by Transition Metals, Radical Quenchers, and Metal Chelators

The effect of photoexcited riboflavin (RF) on the viscosity of hyaluronic acid (HA) solutions has been investigated. UV irradiation of RF causes under aerobic conditions fragmentation of HA and a decrease in the viscosity of its solutions. A decrease of HA viscosity occurs in PO₄-buffered solutions and is accelerated by high pH, Fe²⁺ (but much less so by Fe³⁺), certain metal chelators, and horseradish peroxidase (HRP); it is partially inhibited by catalase and less so by superoxide dismutase (SOD). The reactivity of the system was completely blocked by Tris, ethanol, aspirin, d-manitol, dimethylthiourea (DMTU), dimethylsulfoxide (DMSO), and sodium azide. These results indicate that the most likely chemical species involved in the reaction is the hydroxyl radical. Singlet oxygen (¹0₂) generation is suggested by the ability of NaN₃ and DMSO to completely inhibit the reactivity of the system. These two agents, however, may also interact with OH^√ radical, as well and suppress the reactivity of the system. H₂O₂ and seem also to be produced in significant amounts, because catalase and SOD partially block the reactivity of the system. The effect of HRP may be due to hydrogen subtraction from HA and H₂O₂ reduction to water. Photoexcitation of RF may potentially occur in vitro and in vivo in the organs and tissues that are permeable to light, such as the eye or skin, and damage HA and other cell-matrix components causing inflammation and accelerating aging.  © 1997 Elsevier Science Inc.     

An investigation into thee mechanism of citrate---FE-dependent lipid peroxidation

Chelation by citrate was found to promote the autoxidation of Fe²⁺, measured as the disapperance of 1,10-phenanthroline-chelatable Fe²⁺. The autoxidation of citrate---²⁺ could in turn promote the peroxidation of microsomal phospholipid liposomes, as judged by malondialdehyde formation. At low citrate---Fe²⁺ ratios the autoxidation of Fe²⁺ was slow and the formation of malondialdehyde was preceded by a lag phase. The lag phase evidence of this, linear initial rates of lipid peroxidation were obtained via the combination of citrate---Fe²⁺ and citrate---Fe³⁺, optimum activity occurring at a Fe³⁺---Fe²⁺ ratio of 1:1. Evidence is also presented to suggest that the superoxide and the hydrogen peroxide that are formed during the autoxidation of citrate---Fe²⁺ can either stimulate or inhibit lipid peroxidation by affecting the yield of citrate---Fe³⁺ from citrate---Fe²⁺. No evidence was obtained for the participation of the hydroxyl radical in the initiation of lipid peroxidation by citrate---Fe²⁺.     

Direct NAD(P)H hydrolysis into ADP-ribose(P) and nicotinamide induced by reactive oxygen species: a new mechanism of oxygen radical toxicity

The effect of different oxygen radical-generating systems on NAD(P)H was determined by incubating the reduced forms of the pyridine coenzymes with either Fe²⁺-H₂O₂ or Fe³⁺-ascorbate and by analyzing the reaction mixtures using a HPLC separation of adenine nucleotide derivatives. The effect of the azo-initiator 2,2'-azobis(2-methylpropionamidine)dihydrochloride was also tested. Results showed that, whilst all the three free radical-producing systems induced, with different extent, the oxidation of NAD(P)H to NAD(P)⁺, only Fe²⁺-H₂O₂ also caused the formation of equimolar amounts of ADP-ribose(P) and nicotinamide. Dose-dependent experiments, with increasing Fe²⁺ iron (concentration range 3-180 μM) or H₂O₂ (concentration range 50-1000 μM), were carried out at pH 6.5 in 50 mM ammonium acetate. NAD(P)⁺, ADP-ribose(P) and nicotinamide formation increased by increasing the amount of hydroxyl radicals produced in the medium. Under such incubation conditions NAD(P)⁺/ADP-ribose(P) ratio was about 4 at any Fe²⁺ or H₂O₂ concentration. By varying pH to 2.0, 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0 and 7.4, NAD(P)⁺/ADP-ribose(P) ratio changed to 5.5, 3.2, 1.8, 1.6, 2.0, 2.5, 3.0, 5.4 and 6.5, respectively. Kinetic experiments indicated that 90-95% of all compounds were generated within 5s from the beginning of the Fenton reaction. Inhibition of ADP-ribose(P), nicotinamide and NAD(P)⁺ production of Fe²⁺-H₂O₂-treated NAD(P)H samples, was achieved by adding mannitol (10-50 mM) to the reaction mixture. Differently, selective and total inhibition of ADP-ribose(P) and nicotinamide formation was obtained by performing the Fenton reaction in an almost completely anhydrous medium, i.e. in HPLC-grade methanol. Experiments carried out in isolated postischemic rat hearts perfused with 50 mM mannitol, showed that, with respect to values of control hearts, this hydroxyl radical scavenger prevented reperfusion-associated pyridine coenzyme depletion and ADP-ribose formation. On the basis of these results, a possible mechanism of action of ADP-ribose(P) and nicotinamide generation through the interaction between NAD(P)H and hydroxyl radical (which does not involve the C-center where “conventional” oxidation occurs) is presented. The implication of this phenomenon in the pyridine coenzyme depletion observed in postischemic tissues is also discussed.     

Studies in bioluminescence IV. Properties of luciferin from Pholas dactylus

Light could be obtained by the addition of Fe²⁺ to purified luciferin from Pholas dactylus in the absence of luciferase. The total light emitted was proportional to the concentration of luciferin used. The characteristics of this nonenzymic emission correspond to those of the fast reaction previously described. It may have a physiological importance since iron is present in the luciferin. The injection of Fe²⁺ alone was not sufficient; the presence of a complexing agent such as phosphate or CN⁻ or EDTA was also necessary. Light emission could also be obtained by the addition of H₂O₂, in the presence of Fe²⁺, to luciferin. It has been demonstrated that, for a given amount of luciferin, the total light emitted by the action of varying ratios of Fe²⁺ and luciferase is constant.     

Iron and Sulfur Mineral Analysis Methods for Natural Attenuation Assessments

Based on electron acceptor abundance, Fe³⁺ and SO₄^2- reduction by bacteria may play a dominant role in intrinsic bioremediation of some organic contaminants in the subsurface. Both Fe³⁺ and SO₄^2- reduction processes involve mineral phases and may not be properly understood by evaluating only groundwater concentrations. Fe and S mineral analyses should be incorporated in natural attenuation studies; however, inherent problems with sample collection and analysis have discouraged such efforts. Methods are presented here for (1) sediment collection and anoxic preservation, (2) evaluation of biologically available Fe³⁺ and biogenically produced Fe²⁺ minerals, and (3) a simplified extended mineral sulfide analysis for ∼FeS and S°+FeS₂. These techniques are demonstrated to evaluate Fe³⁺ and SO₄^2- reduction at three sites where the soil or aquifer matrix had been contaminated with gasoline fuel, methane gas, or landfill leachate. It is expected that these techniques will permit Fe and S mineral analyses to become a routine part of natural attenuation assessments.     

Kinetics of the Competitive Degradation of Deoxyribose and Other Molecules by Hydroxyl Radicals Produced by the Fenton Reaction in the Presence of Ascorbic Acid

The competition method in which the Fenton reaction is employed as an OH radical generator and deoxyribose as a detecting molecule, has been used to determine the rate constants for reactions of the OH radical with its scavengers. Nonlinear competition plots were obtained for those scavengers which reacted with the Fenton reagents (Fe²⁺ or H₂O₂). Ascorbic acid is believed to overcome this problem. We have investigated the kinetics of deoxyribose degradation by -OH radicals generated by the Fenton reaction in the presence of ascorbic acid, and observed that the inclusion of ascorbic acid in the Fenton system greatly increased the rate of OH radical generation. As a result, the interaction between some scavengers and the Fenton reagents became negligeable and linear competition plots of A7A vs scavenger concentrations were obtained. The effects of experimental conditions such as, the concentrations of ascorbic acid, deoxyribose, H₂O₂ and Fe²⁺-EDTA, the EDTA/Fe²⁺ ratio as well as the incubation time, on the deoxyribose degradation and the determination of the rate constant for mercaptoethanol chosen as a reference compound were studied. The small standard error, (6.76± 0.21) ±' 10⁹M^-1s^-1 observed for the rate constant values for mercaptoethanol determined under 13 different experimental conditions, indicates the latter did not influence the rate constant determination. This is in fact assured by introducing a term, k_x, into the kinetic equation. This term represents the rate of-OH reactions with other reagents such as ascorbic acid, Fe²⁺-EDTA, H₂O₂ etc. The agreement of the rate constants obtained in this work with that determined by pulse radiolysis techniques for cysteine, thiourea and many other scavengers, suggests that this simple competition method is applicable to a wide range of compounds, including those which react with the Fenton reagents and those whose solubility in water is low.