Impairment of ascorbic acid's anti-oxidant properties in confined media: inter and intramolecular reactions with air and vanadate at acidic pH

The anti-oxidant properties of L-ascorbic acid were investigated in the confined medium produced by a sodium bis(2-ethylhexyl)sulfosuccinate (aerosol-OT, AOT) self-assembled reverse micelle. Using 1H-1H NOESY (proton-proton 2D nuclear overhauser enhancement correlation spectroscopy) NMR spectroscopy, the location of ascorbic acid was investigated and found to be at the AOT-interface in contrast to earlier studies where the ascorbate was assumed to be in the water pool in these microemulsions. The reaction of ascorbic acid with oxygen was investigated using EPR spectroscopy. A delocalized monoanionic ascorbate radical was observed in microemulsions prepared from pH 5.6 stock solutions. This is in contrast to studies carried out in aqueous media where no radical formation was observed. The oxidation of ascorbic acid by aqueous V(V) was investigated in reverse micelles. Modest changes in the kinetic parameters were observed for this system compared to that in water. Details of these reactions were examined and can be summarized as the microemulsion solvating and stabilizing reactive intermediates via rate inhibition or enhancement. The inhibition of the oxidation is due to solvation stabilization of ascorbic acid in microemulsion media. Since ascorbate is a valuable marker of oxidative stress, our results suggest that compartmentization can modify the stabilization of the ascorbate radical and the changes in properties could be important in biological systems.     

Impairment of ascorbic acid’s anti-oxidant properties in confined media: Inter and intramolecular reactions with air and vanadate at acidic pH

The anti-oxidant properties of l-ascorbic acid were investigated in the confined medium produced by a sodium bis(2-ethylhexyl)sulfosuccinate (aerosol-OT, AOT) self-assembled reverse micelle. Using ¹H–¹H NOESY (proton-proton 2D nuclear overhauser enhancement correlation spectroscopy) NMR spectroscopy, the location of ascorbic acid was investigated and found to be at the AOT-interface in contrast to earlier studies where the ascorbate was assumed to be in the water pool in these microemulsions. The reaction of ascorbic acid with oxygen was investigated using EPR spectroscopy. A delocalized monoanionic ascorbate radical was observed in microemulsions prepared from pH 5.6 stock solutions. This is in contrast to studies carried out in aqueous media where no radical formation was observed. The oxidation of ascorbic acid by aqueous V(V) was investigated in reverse micelles. Modest changes in the kinetic parameters were observed for this system compared to that in water. Details of these reactions were examined and can be summarized as the microemulsion solvating and stabilizing reactive intermediates via rate inhibition or enhancement. The inhibition of the oxidation is due to solvation stabilization of ascorbic acid in microemulsion media. Since ascorbate is a valuable marker of oxidative stress, our results suggest that compartmentization can modify the stabilization of the ascorbate radical and the changes in properties could be important in biological systems.     

Involvement of ascorbic acid and ab-type cytochrome in plant plasma membrane redox reactions

Summary Higher plant plasma membranes contain ab-type cytochrome that is rapidly reduced by ascorbic acid. The affinity towards ascorbate is 0.37 mM and is very similar to that of the chromaffin granule cytochromeb ₅₆₁. High levels of cytochromeb reduction are reached when ascorbic acid is added either on the cytoplasmic or cell wall side of purified plasma membrane vesicles. This result points to a transmembrane organisation of the heme protein or alternatively indicates the presence of an effective ascorbate transport system. Plasma membrane vesicles loaded by ascorbic acid are capable of reducing extravesicular ferricyanide. Addition of ascorbate oxidase or washing of the vesicles does not eliminate this reaction, indicating the involvement of the intravesicular electron donor. Absorbance changes of the cytochromeb -band suggest the electron transfer is mediated by this redox component. Electron transport to ferricyanide also results in the generation of a membrane potential gradient as was demonstrated by using the charge-sensitive optical probe oxonol VI. Addition of ascorbate oxidase and ascorbate to the vesicles loaded with ascorbate results in the oxidation and subsequent re-reduction of the cytochromeb. It is therefore suggested that ascorbate free radical (AFR) could potentially act as an electron acceptor to the cytochrome-mediated electron transport reaction. A working model on the action of the cytochrome as an electron carrier between cytoplasmic and apoplastic ascorbate is discussed.Abbreviations AFR ascorbate free radical - AO ascorbate oxidase - DTT dithiothreitol - FCCP carbonylcyanidep-trifluorome-thoxyphenylhydrazon - Hepes N-(2-hydroxyethyl)-piperazine-N-(2-ethanesulfonic acid) - Oxonol VI bis(3-propyl-5-oxoisoxazol-4-yl) penthamethine oxonol - PMSF phenylmethylsulfluoride     

Free radical mediated interaction of ascorbic acid and ascorbate/Cu(II) with viral and plasmid DNAs

Previous studies indicate that ascorbic acid, when combined with copper or iron cleaves several viral DNA. ln this study, we generated the ascorbate radical anion electrochemically in a simple chemical environment without the participation of a metal ion. This solution possesses viral DNA scission activity. Ohe absence of catalytic metal ions [Fe (III) and Cu(II)] in the incubation medium was evidenced by metal chelating agents such as desferrioxamine and EDTA. Ohe radical quenching at high EDTA concentration was attributed to ionic strength of EDTA rather than metal chelation. Ohe effects of antioxidants, radical scavangers, catalase, superoxide dismutase and some proteins on DNA cleavage have been tested. Cleavage may not arise directly from ascorbate free radical but the reaction of the radical form of ascorbate with oxygen may produce the actual reactive species. Aerobic oxidation of ascorbate itself strictly requires transition metal catalysts, however electrochemically produced ascorbyl radical avoided the kinetic barrier that prevented direct oxidation of ascorbic acid with oxygen and eliminated the need for the transition metal ion catalysts.     

Inhibition of human leukocyte 3-hydroxy-3-methylglutaryl coenzyme A reductase activity by ascorbic acid. An effect mediated by the free radical monodehydroascorbate

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in microsomes isolated from cultured lymphoid (IM-9) cells or freshly isolated human leukocytes was markedly decreased by either ascorbic acid or its oxidized derivative, dehydroascorbate. Inhibition of IM-9 leukocyte HMG-CoA reductase activity was log linear between 0.01 and 10 mM ascorbic acid (25 and 81% inhibition, respectively) and 0.1 and 10 mM dehydroascorbate (5 and 75% inhibition, respectively). Inhibition was noncompetitive with respect to HMG-CoA (Km = 10.2 microM (RS); ascorbic acid, Ki = 6.4 mM; dehydroascorbate, Ki = 15 mM) and competitive with respect to NADPH (Km = 16.3 microM; acetic acid, Ki = 6.3 mM; dehydroascorbate, Ki = 3.1 mM). Ascorbic acid and dehydroascorbate are interconverted through the free radical intermediate monodehydroascorbate. Reducing agents are required to convert dehydroascorbate to monodehydroascorbate, but prevent formation of the free radical from ascorbate. In microsomes from IM-9 cells, the reducing agent, dithiothreitol, abolished HMG-CoA reductase inhibition by ascorbate but enhanced inhibition by dehydroascorbate. In addition, the concentration of monodehydroascorbate present in ascorbate solutions was directly proportional to the degree of HMG-CoA reductase inhibition by 1.0 mM ascorbate. Fifty per cent inhibition of enzyme activity occurred at a monodehydroascorbate concentration of 14 microM. These data indicate that monodehydroascorbate mediates inhibition of HMG-CoA reductase by both ascorbate and dehydroascorbate. This effect does not appear to be due to free radical-induced membrane lipid modification, however, since both ascorbate and dehydroascorbate inhibited the protease-solubilized, partially purified human liver enzyme. Since inhibition of HMG-CoA reductase occurs at physiological concentrations of ascorbic acid in the human leukocyte (0.2-1.72 mM), this vitamin may be important in the regulation of endogenous cholesterol synthesis in man.     

Adventitious chemistry at reduced water activities: free radicals and polyhydroxy agents

Free radicals have been associated with loss of viability of lyophilized bacteria exposed to oxygen. Free radical concentration was proportional to the log of the oxygen pressure in the sample. Sugars, such as lactose or sucrose, preserved viability and inhibited free radical production. Lyophilized tissue, particularly liver and spleen, also reacted with oxygen to produce free radicals, which appear to be associated with ascorbic acid in the tissues. Pure ascorbic acid in air does not produce free radicals, but when mixed with protein before lyophilization it reacts with oxygen in air. When a mixture of sodium ascorbate and phenylalanine or tryptophan is lyophilized, free radicals identical to those observed in tissue are obtained. Propyl gallate and di- or trihydroxybenzoates also react with oxygen when lyophilized with phenylalanine, but the g value of the free radical is significantly less than that obtained with ascorbate. A number of amino acids and similar nitrogenous compounds act as catalysts to form propyl gallate free radicals. As with the bacterial or tissue preparations, various sugars or similar carbohydrates inhibited free radical production by either ascorbate or gallate. In the absence of water the free radicals produced by the action of oxygen on lyophilized samples are stable for years. The rate of free radical production is increased by small amounts of moisture (exposure to moist air), but at humidities over 30% rh the radicals are unstable.     

Synergistic interaction between the probucol phenoxyl radical and ascorbic acid in inhibiting the oxidation of low density lipoprotein.

Chain-breaking antioxidants such as butylated hydroxytoluene, alpha-tocopherol, and probucol have been shown to decrease markedly the oxidative modification of low density lipoprotein (LDL). Their mechanism of action appears to involve scavenging of LDL-lipid peroxyl radicals. The purpose of this study was to investigate the occurrence of radical reactions produced during oxidation of LDL and LDL-containing probucol initiated by lipoxygenase or copper. In addition, we have investigated the possibility of a synergistic interaction between ascorbate and probucol in inhibiting the oxidation of LDL. Incubation of LDL-containing probucol and lipoxygenase produced a composite electron spin resonance (ESR) spectrum due to the endogenous alpha-tocopheroxyl radical and probucol-derived phenoxyl radical. The spectral assignment was further verified by chemical oxidation of alpha-tocopherol and probucol. In the presence of ascorbic acid, these radicals in the LDL particle were reduced to their parent compounds with concomitant formation of the ascorbate radical. In both the peroxidation of linoleic acid and the copper-initiated peroxidation of LDL, the antioxidant activity of probucol was significantly increased by low (3-6 microM) concentrations of ascorbate. The probucol-dependent inhibition of LDL oxidation was enhanced in the presence of ascorbic acid. We conclude that the reaction between the phenoxyl radical of probucol and ascorbate results in a synergistic enhancement of the antioxidant capacity of these two compounds and speculate that such reactions could play a role in maintaining the antioxidant status of LDL during oxidative stress in vivo.     

Rooting hastened in onions by ascorbate and ascorbate free radical

Treatment of onion bulbs with ascorbate or its free radical hastened root emergence on the basal plate in relation to treatments with water or dehydroascorbate. This stimulation was accompanied by a significant increase of DNA synthesis per primordium. After a 24-h imbibition, ascorbate and ascorbate free radical also increased cell length. Ascorbate and ascorbate free radical apparently activated the onset of cell proliferation in root primordia, resulting in a shortening in G₁-S transition. The possible action of the ascorbate system at the plasma membrane level is discussed.Abbreviations ASC ascorbic acid - AFR ascorbate free radical - DHA dehydroascorbate     

Optimization of ascorbic and uric acid separation in human plasma by free zone capillary electrophoresis ultraviolet detection

In this paper we propose a new fast free zone capillary electrophoresis method for the simultaneous determination of ascorbic acid (AA) and uric acid (UA) in human plasma. We investigated the effect of analytical parameters, such as concentration and pH of borate running buffer, cartridge temperature, and sample treatment, on resolution, migration times, corrected peak areas, and efficiency. A good separation was achieved using a 60.2-cmx75-microm uncoated silica capillary and 100 mmol/L sodium borate buffer, pH 8, when metaphosphoric acid was employed as protein precipitant, in less than 4 min. These conditions gave a good reproducibility of migration times (CV 0.35 and 0.34%) and peak areas (CV 3.2 and 3.1%) for ascorbate and urate, respectively. The limit of detection was 0.5mg/L for both analytes when the detection was performed at 254 nm for AA and at 292 nm for UA. We compared the present method with a validated capillary electrophoresis assay by measuring plasma urate and ascorbate in 32 normal subjects and the obtained data were analyzed by the Passing and Bablok regression.     

Ascorbic acid is an endogenous cytosolic inhibitor of ATP-supported rat liver mitochondrial calcium transport

ATP-supported but not Site I or Site II respiratory chain-linked 45Ca2+ transport into isolated rat liver mitochondria is profoundly inhibited by a small molecule present in the cytosolic fraction. This inhibitor was purified and shown to be identical with ascorbic acid in a number of chemical properties, cytosolic abundance, susceptibility to ascorbate oxidase, and to agents that otherwise block the effect of authentic ascorbic acid. Experiments with a variety of free radical scavengers and glutathione indicated that ascorbate inhibition of calcium transport is mediated through a 1-electron-free radical mechanism rather than a conventional 2-electron reaction. Calcium transport mechanisms may, therefore, be a target in the pathophysiology of disease processes that influence the intracellular ratios and levels of ascorbate and physiological radical scavengers.     

Photosensitized formation of ascorbate radicals by chloroaluminum phthalocyanine tetrasulfonate: an electron spin resonance study.

The chloroaluminum phthalocyanine tetrasulfonate sensitized photooxidation of ascorbic acid to ascorbate radical (A.-) was followed by electron spin resonance (ESR) spectroscopy. In air saturated aqueous media, steady-state amounts of A.- are rapidly established upon irradiation. The ESR signal disappears within a few seconds after the light is extinguished--more slowly under constant irradiation as oxygen is depleted. No photooxidation was observed in deaerated media. The effect of added superoxide dismutase, catalase, desferrioxamine, and singlet oxygen scavengers (NaN3 and tryptophan) was studied, as was replacement of water by D2O and saturation with O2. The results are indicative of free radical production by direct reaction between ascorbate ion and sensitized phthalocyanine (a Type I mechanism) in competition with the (Type II) reaction of HA- with singlet oxygen, a reaction which does not produce ascorbate radical intermediates.     

Structure of ascorbic acid and its biological function. Determination of the conformation of ascorbic acid and isoascorbic acid by infrared and ultraviolet investigations

The four O-H bands of ascorbic acid could be assigned by means of infrared investigations. It could be shown by electron spin resonance and nuclear magnetic resonance measurements that the radical sodium ascorbate is formed by a cyclic side-chain structure resulting in a loss of C(6)-OH and C(3)-OH. The C(2) = C(3) double bond is still maintained as could be shown by infrared and ultraviolet absorption spectroscopy. In the case of complete oxidation of ascorbic acid to dehydroascorbic acid, C(6)-OH is reestablished (indicating the reopening of the furanoid ring), while C(2)-OH as well as the C(2) = C(3) double bond have disappeared due to the deprotonation of C(2)-OH and C(3)-OH. In the case of isoascorbic acid and its radical potassium isoascorbate similar results are obtained with one distinct difference: in the case of isoascorbic acid, C(2)-OH does not appear while C(3)-OH exhibits a shoulder.     

Reduction of cytochrome C by ascorbic free radical.

The rate of "in vivo" reduction of cytochrome c by ascorbic acid (AA) increases from 69 nmoles of cytochrome c for minute, to 202 nanomoles when ascorbate oxidase is added. Since the AA oxidation by AA oxidase is a system to generate ascorbic free radical (AFR), data suggest that AFR is a better reducing compound than ascorbate in cytochrome c reduction. Since the addition of oxidized glutathione and human immunoglobulins (-S-S- bridge containing compounds) in the medium produces a remarkable decrease in cytochrome c reduction, it is suggested that AFR could also reduce -S-S- groups.     

Ascorbate activates soluble guanylate cyclase via H2O2-metabolism by catalase

Conditions necessary for the activation by ascorbic acid of soluble guanylate cyclase purified from bovine lung have been examined. Ascorbic acid (0.1-10 mM) did not directly activate the enzyme, nonetheless, pronounced activation by ascorbate (3-10 mM) was observed in incubation mixtures containing 1 microM bovine liver catalase. Superoxide dismutase (SOD) and mannitol did not affect the catalase-dependent activation of guanylate cyclase elicited by ascorbate, suggesting that superoxide anion and hydroxyl radical were not mediating the activation of the enzyme. However, SOD enhanced the relatively low level activation of the enzyme elicited by catalase in the absence of added ascorbate. Pronounced inhibition (both with and without added ascorbate) was observed of catalase-dependent activation of guanylate cyclase by either ethanol (100 mM) or a fungal catalase preparation. Neither ethanol nor fungal catalase inhibited activation of guanylate cyclase by S-nitrosyl-N-acetyl-penicillamine (SNAP), a source of the nitric oxide free radical. These observations indicate that autoxidation of ascorbic acid or thiols present with the guanylate cyclase preparation leads to generation of H2O2, and its metabolism by bovine liver catalase mediates the concomitant activation of guanylate cyclase. The mechanism of activation appears to be associated with the presence of Compound I of catalase and to be inhibited by superoxide anion.     

Human erythrocyte recycling of ascorbic acid: relative contributions from the ascorbate free radical and dehydroascorbic acid

Recycling of ascorbic acid from its oxidized forms helps to maintain the vitamin in human erythrocytes. To determine the relative contributions of recycling from the ascorbate radical and dehydroascorbic acid, we studied erythrocytes exposed to a trans-membrane oxidant stress from ferricyanide. Ferricyanide was used both to induce oxidant stress across the cell membrane and to quantify ascorbate recycling. Erythrocytes reduced ferricyanide with generation of intracellular ascorbate radical, the concentrations of which saturated with increasing intracellular ascorbate and which were sustained over time in cells incubated with glucose. Ferricyanide also generated dehydroascorbic acid that accumulated in the cells and incubation medium to concentrations much higher than those of the radical, especially in the absence of glucose. Ferricyanide-stimulated ascorbate recycling from dehydroascorbic acid depended on intracellular GSH but was well maintained at the expense of intracellular ascorbate when GSH was severely depleted by diethylmaleate. This likely reflects continued radical reduction, which is not dependent on GSH. Erythrocyte hemolysates showed both NAD- and NADPH-dependent ascorbate radical reduction. The latter was partially due to thioredoxin reductase. GSH-dependent dehydroascorbate reduction in hemolysates, which was both direct and enzyme-dependent, was greater than that of the radical reductase activity but of lower apparent affinity. Together, these results suggest an efficient two-tiered system in which high affinity reduction of the ascorbate radical is sufficient to remove low concentrations of the radical that might be encountered by cells not under oxidant stress, with back-up by a high capacity system for reducing dehydroascorbate under conditions of more severe oxidant stress.     

Changes in ascorbate, glutathione, and related enzyme activities during thidiazuron-induced bud break of apple

The changes of ascorbic acid, dehydroascorbic acid, and glutathione content and related enzyme activities were studied in apple buds during dormancy and thidiazuron-induced bud break. An increase in ascorbic acid, reduced form of glutathione (GSH), total glutathione, total non-protein thiol (NPSH) and non-glutathione thiol (RSH) occurred as a result of induction by thidiazuron during bud break, whereas dehydroascorbic acid and oxidized glutathione (GSSG) decreased during the same period. Thidiazuron also enhanced the ratio of GSH/GSSG, and activities of ascorbate free radical reductase (AFR; EC 1.6.5.4), ascorbate peroxidase (EC 1.11.1.11). dehydroascorbate reductase (DHAR; EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2). The ascorbic acid content and the activities of AFR, ascorbate peroxidase, and DHAR peaked when buds were in the side green or green tip stage just prior to the start of rapid expansion, and declined thereafter. The GSH, NPSH, RSH, ratio of GSH/GSSG, and activities of GR increased steadily during bud development.     

Changes in the Ascorbate System during Seed Development of Vicia faba L

Large changes occur in the ascorbate system during the development of Vicia faba seed and these appear closely related to what are generally considered to be the three stages of embryogenesis. During the first stage, characterized by embryonic cells with high mitotic activity, the ascorbic acid/dehydroascorbic acid ratio is about 7, whereas in the following stage, characterized by rapid cell elongation (stage 2), it is lower than 1. The different ascorbic/dehydroascorbic ratio may be correlated with the level of ascorbate free radical reductase activity, which is high in stage 1 and lower in stage 2. Ascorbate peroxidase activity is high and remains constant throughout stages 1 and 2, but it decreases when the water content of the seed begins to decline (stage 3). In the dry seed, the enzyme disappears together with ascorbic acid. Ascorbate peroxidase activity is observed to be 10 times higher than that of catalase, suggesting that ascorbate peroxidase, rather than catalase, is utilized in scavenging the H₂O₂ produced in the cell metabolism. There is no ascorbate oxidase in the seed of V. faba. V. faba seeds acquire the capability to synthesize ascorbic acid only after 30 days from anthesis, i.e. shortly before the onset of seed desiccation. This suggests that (a) the young seed is furnished with ascorbic acid by the parent plant throughout the period of intense growth, and (b) it is necessary for the seed to be endowed with the ascorbic acid biosynthetic system before entering the resting state so that the seed can promptly synthesize the ascorbic acid needed to reestablish metabolic activity when germination starts.     

Liquid chromatographic behavior of ascorbate on amine columns

Quantitation of ascorbate at concentrations normally found in biological samples and foods has previously been shown to be possible by HPLC analysis. Prefilled amine columns from three manufacturers were presently used to evaluate their potential for separating low concentrations of [14C]ascorbic acid from its degradation products, [14C]dehydroascorbic acid and [14C]diketogulonic acid. A successful separation was achieved on some columns with as little as 200 cpm (30 pmol) of total ascorbate injected. On other columns, injection of 30-500 pmol of ascorbate resulted in as much as 80% of [14C]ascorbic acid eluting with an unpredictable retention time. In these instances the inclusion of nonlabeled ascorbic acid (0.5 mg/ml) to the sample resulted in most of the [14C]ascorbic acid activity eluting at the expected retention time of ascorbic acid. The inclusion of ascorbic acid in samples injected onto the column also resulted in a more discrete peak in the elution of dehydroascorbic acid, and more complete recovery of the total [14C]activity (ascorbic acid, dehydroascorbic acid, and diketogulonic acid) injected onto the column.     

Exercise-induced endotoxemia: the effect of ascorbic acid supplementation

Strenuous, long-duration aerobic exercise results in endotoxemia due to increased plasma levels of lipopolysaccharide (LPS) leading to cytokine release, oxidative stress, and altered gastrointestinal function. However, the effect of short-term strenuous aerobic exercise either with or without antioxidant supplementation on exercise-induced endotoxemia is unknown. A significant increase in the concentration of bacterial LPS (endotoxin) was noted in the venous circulation of healthy volunteers following maximal acute aerobic exercise (0.14(-1) pre-exercise vs. 0.24(-1) postexercise, p <0.01). Plasma nitrite concentration also increased with exercise (0.09 +/- 0.05 nM x ml(-1) vs. 0.14 +/- 0.01 nM x ml(-1), p <0.05) as did ascorbate free radical levels (0.02 +/- 0.001 vs. 0.03 +/- 0.002 arbitrary units, p <0.05). Oral ascorbic acid supplementation (1000 mg) significantly increased plasma ascorbic acid concentration (29.45 mM x l(-1) to 121.22 mM x l(-1), p <0.05), and was associated with a decrease in plasma LPS and nitrite concentration before and after exercise (LPS: 0.01(-1); nitrite: 0.02 +/- 0.02 nM x ml(-1) vs. 0.02 +/- 0.03 nM x ml(-1)). Ascorbic acid supplementation led to a significant increase in ascorbate free radical levels both before (0.04 +/- 0.01 arbitrary units) and after exercise (0.06 +/- 0.02 arbitrary units, p <0.05). In conclusion, strenuous short-term aerobic exercise results in significant increases in plasma LPS levels (endotoxemia) together with increases in markers of oxidative stress. Supplementation with ascorbic acid, however, abolished the increase in LPS and nitrite but led to a significant increase in the ascorbate radical in plasma. The amelioration of exercise-induced endotoxemia by antioxidant pretreatment implies that it is a free radical-mediated process while the use of the ascorbate radical as a marker of oxidative stress in supplemented systems is limited.     

Lucigenin chemiluminescence in human seminal plasma

Seminal plasma protects spermatozoa from the detrimental effects of reactive oxygen species such as hydrogen peroxide. We investigated the lucigenin-dependent chemiluminescence in cell-free seminal plasma from andrological patients. The seminal plasma was separated from cells by centrifugation. In all seminal plasmas studied lucigenin-dependent chemiluminescence (LCL) was detected. The LCL showed a strong pH-dependence. The signal was stable if samples were stored at +4°C for up to 4 days or up to 8 days at -80°C. Filtration of the samples (0.45 and 0.22 μm pore size) did not lower their luminescence. The addition of superoxide dismutase (SOD) and ascorbic acid oxidase (AAO) lowered LCL nearly to baseline values while trolox and desferal showed moderate effect, whereas allopurinol had no effect. Electron paramagnetic resonance spectroscopy demonstrated ascorbyl radicals in seminal plasma. Physiological concentrations of ascorbic acid yielded SOD-inhibitable lucigenin-chemiluminescence. The nitroblue-tetrazolium assay showed that ascorbic acid in buffer solution produced formazan. Superoxide-anion radicals were not detected in seminal plasma by the spin-trap DEPMPO due to their low steady state concentration. It is concluded that in seminal plasma ascorbate reacts with molecular oxygen yielding ascorbyl radicals and superoxide anion. If lucigenin is added to seminal plasma, reducing substances present, such as ascorbate, reduce lucigenin to the corresponding radical; this radical reacts with molecular oxygen and also forms O₂^-2.. So LCL in human seminal plasma results from the autoxidation of ascorbate and the oxidation of the reduced lucigenin. While the physiological relevance of the former mechanism is unknown, the latter is an artifact.