Use Of Ascorbate Research Articles

Preparation of metal ion buffers for biological experimentation: a methods approach with emphasis on iron and zinc.

Transition metal ions are a challenge to study in physiology because of problems associated with solubility, oxidation, binding, and attaining appropriate free activities in solution. This review discusses these problems and potential ways of accommodating them. Special attention is given to iron and zinc ions, but many of the concepts can be applied for studying other transition metals. Selection of reagents appropriate for metal work (including water, salts, noncomplexing pH buffers) is briefly discussed. Calculation of the solubility product (K(sp)) for common iron and zinc precipitates is covered, as well as techniques used to solubilize Fe(3+) with organic chelates. Factors that affect Fe(2+) oxidation are mentioned, and the use of ascorbate as a reducing agent is considered. Measurement of the rate of Fe(2+) oxidation (or Fe(3+) reduction) with the Fe(2+) chromophores ferrozine and BPS is also discussed. Generation of a free metal ion activity through use of metal buffers (chelators) is discussed. Theoretical problems associated with this technique are explored, and selected shareware metal ion buffer calculators are described. Finally, techniques for measuring and minimizing nonspecific binding of iron and zinc ions to biological membranes are considered.

Cantaloupe melon peroxidase: characterization and effects of additives on activity.

Peroxidase in cantaloupe melon (Cucumis melo L. var. reticulatus Naud.), a fruit commonly fresh cut processed, was characterized to determine reaction pathway, optimal conditions for activity and effect of some additives on enzymatic action. Mn2+, CaCl2, NaNO2 and kinetin had partial inhibitory effects on enzyme activity. Activity was effectively inhibited by compounds capable of chelating peroxidase heme iron such as diethyldithiocarbamate and tiron, but unaffected by EDTA. Free radical scavenger, superoxide dismutase, also had no effect on reaction velocity. Enzymatic action was consistent with that of ascorbate peroxidase based on the relatively higher affinity for ascorbate over guaiacol. Optimum activity temperature was 50-55 degrees C. The enzyme was stable at temperatures below 40 degrees C and at 50 degrees C for up to 10 min. Over 90% of total activity was lost at 80 degrees C within 5 min. Broad pH optima, 5.5-7.5 at 50 degrees C and 6-7 at 30 degrees C, were obtained. Peroxidase activity in cantaloupe was higher than those in strawberry (Fragaria ananassa Duch.) and lettuce (Lactuca sativa L.), suggesting a relatively high oxidative stress in fresh cut cantaloupe. The potential use of ascorbate as an additive in fresh cut cantaloupe melon was demonstrated by its ability to preserve color in minimally processed fruits for 25 days at 4 degrees C, possibly as a result of an enhanced antioxidative action of the ascorbate-peroxidase complex and trace metal ion cofactors.

Use of Ascorbate in the Preparation and Maintenance of Brain Slices

Ascorbate and glutathione (GSH) are normally concentrated in brain cells at millimolar levels. However, both of these low-molecular-weight antioxidants are washed out of mammalian brain tissue during slice preparation and subsequent incubation. Ascorbate, which is not synthesized in the brain, can be added back to slices by active uptake from the incubation medium. Levels of GSH, on the other hand, are regulated by synthesis rather than uptake, and cannot be readily maintained in slices. Importantly, maintenance of brain slice ascorbate content at at least 50% of that in vivo, prevents the increase in slice water content that normally occurs during incubation. Slices with maintained ascorbate levels also have better histological characteristics than ascorbate-depleted tissue. The medium concentration of ascorbate sufficient to maintain content and inhibit edema formation is 400 μM, which is the normal concentration in brain extracellular fluid. This paper describes methods to maintain ascorbate levels in brain slices, including procedures to minimize oxidation in oxygenated incubation media. Also described is an HPLC analysis for ascorbate and GSH that is based on direct injection rather than extraction of samples.

Catalytic Metals, Ascorbate and Free Radicals: Combinations to Avoid

Trace levels of transition metals can participate in the metal-catalyzed Haber-Weiss reaction (superoxide-driven Fenton reaction) as well as catalyze the oxidation of ascorbate. Generally ascorbate is thought of as an excellent reducing agent; it is able to serve as a donor antioxidant in free radical-mediated oxidation processes. However, as a reducing agent it is also able to reduce redox-active metals such as copper and iron, thereby increasing the pro-oxidant chemistry of these metals. Thus ascorbate can serve as both a pro-oxidant and an antioxidant. In general, at low ascorbate concentrations, ascorbate is prone to be a pro-oxidant, and at high concentrations, it will tend to be an antioxidant. Hence there is a crossover effect. We propose that the "position" of this crossover effect is a function of the catalytic metal concentration. In this presentation, we discuss: (1) the role of catalytic metals in free radical-mediated oxidations; (2) ascorbate as both a pro-oxidant and an antioxidant; (3) catalytic metal catalysis of ascorbate oxidation; (4) use of ascorbate to determine adventitious catalytic metal concentrations; (5) use of ascorbate radical as a marker of oxidative stress; and (6) use of ascorbate and iron as free radical pro-oxidants in photodynamic therapy of cancer.

On-Site Monitoring of Total Copper by Anodic Stripping Voltammetry, During Algicide Dosing of a Reservoir

Abstract The use of on-site anodic stripping voltammetry (ASV) to monitor and evaluate the effectiveness of copper-based algicide dosing of a reservoir is presented. An initial comparison of the copper levels determined by anodic stripping voltammetry (ASV) and inductively coupled plasma-atomic emission spectrometry (ICP-AES) was performed in the laboratory using 46 water samples collected during an algal control treatment of a reservoir. Good correlation was observed at the 95% confidence limit. Use of ascorbate as oxygen scavenger in chloride media was found effective for copper determination in the field by ASV. The ascorbate-chloride electrolyte removes the need for inconvenient nitrogen or argon bubbling for oxygen removal. ASV monitoring was used to monitor algicide dosing operations by boat and aircraft. Data during the first 60 minutes of dosing is presented at surface, 5 and 10 metre depths.

Iron speciation at physiological pH in media containing ascorbate and oxygen.

The stability of iron ascorbate solutions was investigated, under both anaerobic and aerobic conditions, with the Fe2+ and Fe3+ indicators, respectively ferrozine and mimosine, at different pH values. The species present under the differing conditions were investigated by electron paramagnetic resonance (EPR) and Mössbauer spectroscopy and by gel-filtration chromatography. At physiological pH (6.8-7.4) iron ascorbate solutions rapidly form mononuclear chelatable Fe3+ species as reflected by indicator studies and EPR. Mössbauer spectroscopy fails to detect any Fe2+ species. EPR studies show a time-dependent decrease in rhombic Fe3+, particularly in oxygenated buffers, consistent with a conversion to polynuclear Fe. O2 uptake studies show that the conversion of Fe2+ to Fe3+ in Fe-ascorbate solutions at pH > 7.0 was accompanied by rapid O2 consumption but preceded depletion of ascorbate. Addition of high concentrations of mannitol (50-200 mM) reduces the O2 consumption and partly stabilizes the rapidly chelatable Fe form. Gel filtration studies show that the oxidation of Fe-ascorbate solutions at pH 7.4 is accompanied by an increase in the apparent relative molecular mass of the Fe, presumably due to Fe polymer formation. These studies indicate that inherent instability of Fe-ascorbate solutions above neutral pH and clearly have important implications in the use of ascorbate in studies of Fe physiology.

Use of Ascorbate for the Treatment of Refractory Idiopathic Thrombocytopenic Purpura in Children

Use of Ascorbate for the Treatment of Refractory Idiopathic Thrombocytopenic Purpura in Children

Treatment of idiopathic thrombocytopenic purpura with ascorbate.

The treatment of idiopathic thrombocytopenic purpura (ITP) includes corticosteroids, danazol, splenectomy and various immunosuppressives. Treatment can be difficult for those patients refractory to these modalities and/or those patients intolerant of the secondary effects. In this paper we report on the use of ascorbate in the treatment of ITP and its successful use in seven of 11 patients studied. We found that therapy with ascorbate appeared to improve the platelet count and the intravascular survival of platelets. Because of excellent patient compliance and its lack of toxicity, it may be an alternative for the treatment of ITP. The exact role of ascorbate in the treatment of ITP, as well as its mechanism of action, await further study.

Effects of radioligand oxidation and ascorbate-induced lipid peroxidation on serotonin-1 receptor assay: use of ascorbate and ethylenediamine tetraacetic acid buffers to prevent (3H)-5-HT binding artifacts.

The effects of ascorbic acid and ascorbate-induced lipid peroxidation upon (3H)-5-HT binding were examined in total membrane fractions prepared from mouse cortex and hippocampus. Low concentrations of ascorbic acid promoted lipid peroxidation of membranes as assessed by malondialdehyde production relative to intermediate concentrations of ascorbate. The actual concentration of ascorbate required for expression of pro-oxidative and antioxidative properties was dependent upon assay conditions such as temperature and ionic constituents. Ascorbate-induced lipid peroxidation was completely prevented by addition of 1 mM ethylenediamine tetraacetic acid (EDTA) and enhanced by GTP at concentrations typically used in binding assays. Marked lipid peroxidation was associated with loss of (3H)-5-HT binding sites with little effect upon affinity of the receptor for the ligand. In contrast, mild lipid peroxidation occurring during the binding assay with the ligand present was associated with a decreased affinity for (3H)-5-HT and the appearance of a curvilinear Scatchard plot. Under assay conditions that prevented ascorbate-induced lipid peroxidation, Scatchard analysis indicated only a single high-affinity binding site for (3H)-5-HT even when assayed with an expanded range of ligand concentrations. Preparation of ligand and assay of (3H)-5-HT binding in the absence of ascorbate resulted in shallow, markedly curvilinear Scatchard plots. These data support the continued use of ascorbate in (3H)-5-HT binding assays to prevent ligand degradation and the addition of 1 mM EDTA to prevent lipid peroxidation from occurring during the binding assay.

Simultaneous determination of 3-methoxy-4-hydroxyphenylglycol, 5-hydroxyindoleacetic acid, and homovanillic acid in cerebrospinal fluid with high-performance liquid chromatography using electrochemical detection

An improved high-performance liquid chromatographic method with electrochemical detection (HPLC-EC) for the simultaneous determination of 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA) in cerebrospinal fluid (CSF) of humans and nonhuman primates is described. Quantitation is based on the use of an internal standard, 5-fluoro-HVA. Sample preparation consists of mixing an aliquot of CSF with a solution of the internal standard followed by ultrafiltration. The precision of the method is high, with within-run and between-run coefficients of variation of 2–6% and less than 10%, respectively, in the concentration ranges of the metabolites encountered in human lumbar CSF. Accuracy was tested by comparing the present HPLC method with specific gas chromatographicmass spectrometric (GS-MS) assays for MHPG and HVA and a GC-MS-validated HPLC assay for 5-HIAA: the correlations obtained were 0.968 for MHPG, 0.989 for 5-HIAA, and 0.999 for HVA, with no systematic bias between the methods. The use of ascorbate as a preserving agent for monoamine metabolites in CSF was not found to be necessary when proper care was exercised in sample handling and storage. The analysis of samples with up to 2% ascorbic acid was possible as well, but MHPG had to be assayed separately using an extraction procedure and an alternative internal standard, 3-ethoxy-4-hydroxyphenylglycol.

Megadoses of vitamin C prevent the development of tolerance and physical dependence on morphine in mice

In mice ascorbate, when co-administered with morphine, suppresses the development of tolerance and physical dependence on the drug, without significantly affecting its analgesic properties as inferred from unaltered ED50 values. The duration of morphine-induced analgesia, however, is progressively reduced with an increase in the amounts of ascorbate. Ascorbate at 1g/kg body weight does not alter the pH of blood, and has no effect on the levels of lipid-neroxides in blood and brain. Studies presented in this paper suggest the potential use of ascorbate in the prevention of development of tolerance in therapeutic applications of narcotics as analgesics. Cultured Neuroblastoma x Glioma hybrid cells (NG 108-15) respond to opiates in two different ways. The rapid receptor mediated inhibition of adenylate cyclase is followed by a long-lived compensatory increase in its activity (1–4). In a recent report (5) we have shown that ascorbate suppresses the delayed etorphine-induced compensatory increase in cAMP levels in NG 108-15 cells without affecting the short-term inhibitory response of cells to the drug. It has been suggested that while the former may be the basis of narcotic dependence and tolerance, the latter is responsible for the analgesic effect. These observations, based on a model system, prompted us to examine the effect of ascorbate on the pharmacological properties of morphine at the organismal level.

Discrimination of ascorbate-dependent nonenzymatic and enzymatic, membrane-bound reduction of nitric oxide in denitrifying Pseudomonas perfectomarinus

The marine nitrite-respiring (denitrifying) bacterium, Pseudomonas perfectomarinus, catalyzes by a membrane-bound enzyme the reduction of nitric oxide to nitrous oxide with ascorbate-reduced phenazine methosulfate as electron donor. The entire nitric oxide-reducing capability of a cell-free system was membrane bound and this process was studied with respect to pH and substrate dependency. The enzymatic process was perturbed by an identical nonenzymatic reduction by iron(II) ascorbate in neutral to alkaline aqueous solution. 2 mol nitric oxide and 1 mol ascorbate were consumed per mol nitrous oxide formed. Enzymatic and nonenzymatic processes were discriminated by their differential behavior towards pH and metal-chelating agents. The pH optimum for the enzymatic and nonenzymatic reaction was 5.2 and greater than 7.0, respectively. EDTA (10 mM) inhibited the nonenzymatic reduction completely without interfering with the membrane-bound activity. The nonenzymatic system mimics the reaction of nitric oxide reductase and could serve as a model to study the formation of the N-N bond in denitrification. Enzymatic generation of nitric oxide by cytochrome cd and subsequent nonenzymatic reduction to nitrous oxide simulate an overall quasi-enzymatic nitrous oxide formation by cytochrome cd. The nonenzymatic reduction of nitric oxide might have occurred in previous work due to the ubiquitous use of ascorbate in studies on nitrite respiration and the likelihood of adventitious iron in biological samples.

Dopamine receptor mediated inhibition by pergolide of electrically-evoked 3H-dopamine release from striatal slices of cat and rat: slight effect of ascorbate.

The dopamine receptor agonist pergolide inhibited the calcium-dependent, electrically evoked overflow of tritium from slices of the striatum of cat or rat prelabelled with 3H-dopamine. This inhibition of tritium overflow by nanomolar concentrations of pergolide was antagonized by the benzamide neuroleptic S-sulpiride (0.1 microM). In millimolar concentrations, L- ascorbate had slight or no effects on this dopamine receptor mediated inhibition, in striatal slices of either the cat or the rat. Since these same concentrations of ascorbate have been reported to completely block the specific binding of 3H-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN) and of 3H-apomorphine to presumed dopamine receptors, the present results suggest a dissociation between the characteristics of 3H-ADTN and 3H-apomorphine binding and the dopamine autoreceptor. Previous contradictory results concerning the existence of inhibitory dopamine receptors which modulate depolarization-evoked overflow of dopamine from the striatum of the rat are thus apparently not due to a species difference nor to the use of ascorbate, but rather to differences in experimental conditions.

The use of ascorbate as a probe of opioid receptor structure: evidence for two independent mechanisms of receptor destruction by ascorbate.

Ascorbate (20mM) pretreatment of brain membrane suspensions at 37 degrees produced a rapid irreversible loss of specific opioid binding. There was no reduction in specific 3H-halo-peridol binding. Ascorbate induced loss of opioid binding under these experimental conditions was not blocked by low concentrations of EDTA or MN++. In contrast, the slowly developing loss of opioid binding during exposure to 1 mM ascorbate at 23 degrees was completely inhibited by 10(-5)M EDTA or Mn++. At 37 degrees, D-isoascorbate, and several other reducing agents (glutathione, dithiothreitol, cysteine) produced a loss of opioid binding similar to that seen with ascorbate. It is concluded that 1 mM ascorbate at 23 degrees, and 20 mM ascorbate at 37 degrees, destroy opioid binding sites by two independent mechanisms. Lipid peroxidation is implicated at low ascorbate concentrations; a reductive process appears to be responsible for the ascorbate induced loss of binding at higher concentrations.

Quantitative relationship between photosystem I electron transport and ATP formation

The Photosystem I-dependent transport of electrons from diaminodurene to methylviologen is linear with reaction time and supports a constant rate of phosphorylation. However, if the diaminodurene is not kept fully reduced by the presence of excess ascorbate, the oxidized diaminodurene accumulates and begins to compete with the methylviologen as the electron acceptor. Thus, although the rate of ATP formation remains unchanged, an increasing proportion of the electron transport becomes cyclic and hence unmeasured. This leads to a rapid increase in the apparent efficiency of phosphorylation which is misleading. In contrast, it is known that the oxidized form of 3,3′-diaminobenzidine polymerizes to form an insoluble substance which should not be available to serve as an electron acceptor. However, 3,3′-diaminobenzidine is not a satisfactory donor of electrons in Photosystem I reactions for two reasons: the rate of electron transport quickly falls with reaction time and the oxidized form of 3,3′-diaminobenzidine seems to be an exceptionally efficient electron acceptor near the beginning of the period of illumination when it is presumably not yet polymerized. Thus in the first 2–3 sec of illumination when the reaction is still rapid much of the electron transport is cyclic and therefore unmeasured, especially in the absence of excess ascorbate. This cycling of electrons, which leads to an inflated apparent efficiency ( P e 2 > 2 ), is particularly pronounced at low donor concentrations. When cyclic electron transport is avoided by the use of ascorbate or by the selection of appropriate reaction times, both diaminodurene and 3,3′-diaminobenzidine support phosphorylation with an efficiency which is approximately half of the efficiency exhibited by the overall Hill reaction. The same is true when 2,5-diaminotoluene, tetrachlorohydroquinone, 4,5-dimethyl- o-phenylenediamine, and reduced 2,6-dichloroindophenol serve as electron donors. With these six substances, the phosphorylation efficiences were 0.57 ± 0.1 molecules of ATP formed for each pair of electrons transferred ( P e 2 ). In the same chloroplasts preparations, the transport of electrons from water to methylviologen-supported phosphorylation with a P e 2 of 1.2.

Ion Transport by Heart Mitochondria: VII. ACTIVATION OF THE ENERGY-LINKED ACCUMULATION OF Mg++ BY Zn++ AND OTHER CATIONS

Abstract The effect of a number of heavy metal ions on the energy-linked accumulation of Mg++ by isolated heart mitochondria has been studied with the use of ascorbate and tetramethylphenylenediamine as the energy source. With this substrate Mg++ uptake is activated strongly by Zn++ and Cd++, and to a lesser degree by Cu++, Hg++, and Ag+. In contrast to this activation of Mg++ accumulation, these reagents depress oxidative phosphorylation and the energy-linked accumulation of Ca++. The cation-dependent increase in Mg++ accumulation requires phosphate or arsenate and is inhibited by inhibitors of respiration and uncouplers of phosphorylation. It is strongly inhibited by adenosine diphosphate and by sulfhydryl group reagents, but is insensitive to oligomycin. It is also depressed by Na+ and K+. The increased ion uptake in the presence of Zn++ is accompanied by increased respiration. This observation is consistent with a greater demand for free energy to support the increased ion accumulation. Zn++ has little effect on the loss of accumulated ions to the suspending medium, and appears to act by stimulating the rate of uptake of Mg++. A large portion of the available Zn++ is taken up by the mitochondria in a reaction which does not require the expenditure of free energy. Mitochondria which are tightly coupled accumulate Mg++ rather slowly compared to preparations which show poorer respiratory control. Mg++ accumulation in tightly coupled mitochondria is activated to a greater degree by Zn++ and Cd++ than is the corresponding reaction in more loosely coupled preparations. It is suggested that specific membrane lesions which favor the energy-linked uptake of Mg++ can be induced by the addition of Zn++ or Cd++, and perhaps by other procedures. The relationship of the Zn++-dependent uptake of Mg++ to other induced ion transport systems of the mitochondrion is discussed.