Determination of dopamine in the presence of ascorbic acid using poly(3,5-dihydroxy benzoic acid) film modified electrode

A polymerized film of 3,5-dihydroxy benzoic acid (DBA) was prepared on the surface of a glassy carbon electrode (GCE) in neutral solution by cyclic voltammetry (CV). The poly(DBA) film-coated GCE exhibited excellent electrocatalytic activity toward the oxidation of dopamine (DA). A linear range of 1.0 × 10⁻⁷ to 1.0 × 10⁻⁴ M and a detection limit of 6.0 × 10⁻⁸ M were observed in pH 7.4 phosphate buffer solutions. Moreover, the interference of ascorbic acid (AA) was effectively eliminated. This work provides a simple and easy approach to selective detection of DA in the presence of AA.     

Chemometric-assisted kinetic-spectrophotometric method for simultaneous determination of ascorbic acid, uric acid, and dopamine

A chemometric-assisted kinetic spectrophotometric method has been developed for simultaneous determination of ascorbic acid (AA), uric acid (UA), and dopamine (DA). This method relies on the difference in the kinetic rates of the reactions of analytes with a common oxidizing agent, tris(1,10-phenanthroline) and iron(III) complex (ferritin, [Fe(phen)₃]³⁺) at pH 4.4. The changes in absorbance were monitored spectrophotometrically. The data obtained from the experiments were processed by chemometric methods of artificial neural network (ANN) and partial least squares (PLS). Acceptable techniques of prediction set, randomization t test, cross-validation, and Y randomization were applied for the selection of the best chemometric method. The results showed that feedforward artificial neural network (FFANN) is more efficient than the other chemometric methods. The parameters affecting the experimental conditions were optimized, and it was found that under optimal conditions Beer’s law is followed in the concentration ranges of 4.3–74.1, 4.3–78.3, and 2.0–33.0 μM for AA, UA, and DA, respectively. The proposed method was successfully applied to the determination of analytes in serum and urine samples.     

Au-nanoclusters incorporated 3-amino-5-mercapto-1,2,4-triazole film modified electrode for the simultaneous determination of ascorbic acid, dopamine, uric acid and nitrite

A novel biosensor has been constructed by the electrodeposition of Au-nanoclusters (nano-Au) on poly(3-amino-5-mercapto-1,2,4-triazole) (p-TA) film modified glassy carbon electrode (GCE) and employed for the simultaneous determination of dopamine (DA), ascorbic acid (AA), uric acid (UA) and nitrite (NO₂⁻). NH₂ and SH groups exposed to the p-TA layer are helpful for the electrodeposition of nano-Au. The combination of nano-Au and p-TA endow the biosensor with large surface area, good biological compatibility, electricity and stability, high selectivity and sensitivity and flexible and controllable electrodeposition process. In the fourfold co-existence system, the linear calibration plots for AA, DA, UA and NO₂⁻ were obtained over the range of 2.1–50.1 μM, 0.6–340.0 μM, 1.6–110.0 μM and 15.9–277.0 μM with detection limits of 1.1 × 10⁻⁶ M, 5.0 × 10⁻⁸ M, 8.0 × 10⁻⁸ M and 8.9 × 10⁻⁷ M, respectively. In addition, the modified biosensor was applied to the determination of AA, DA, UA and NO₂⁻ in urine and serum samples by using standard adding method with satisfactory results.     

Simultaneous determination of dopamine, ascorbic acid, and uric acid using carbon ionic liquid electrode

A recently constructed carbon composite electrode using room temperature ionic liquid as pasting binder was employed as a novel electrode for sensitive, simultaneous determination of dopamine (DA), ascorbic acid (AA), and uric acid (UA). The apparent reversibility and kinetics of the electrochemical reaction for DA, AA, and UA found were improved significantly compared to those obtained using a conventional carbon paste electrode. The results show that carbon ionic liquid electrode (CILE) reduces the overpotential of DA, AA, and UA oxidation, without showing any fouling effect due to the deposition of their oxidized products. In the case of DA, the oxidation and reduction peak potentials appear at 210 and 135mV (vs Ag/AgCl, KCl, 3.0M), respectively, and the CILE shows a significantly better reversibility for dopamine. The oxidation peak due to the oxidation of AA occurs at about 60mV. For UA, a sharp oxidation peak at 340mV and a small reduction peak at 250mV are obtained at CILE. Differential pulse voltammetry was used for the simultaneous determination of ternary mixtures of DA, AA, and UA. Relative standard deviation for DA, AA, and UA determinations were less than 3.0% and DA, AA, and UA can be determined in the ranges of 2.0x10(-6)-1.5x10(-3), 5.0x10(-5)-7.4x10(-3), and 2.0x10(-6)-2.2x10(-4)M, respectively. The method was applied to the determination of DA, AA, and UA in human blood serum and urine samples.     

Reaction of thymidine and ascorbic acid induced by UV in the presence of salicylic acid

When a neutral solution of thymidine and ascorbic acid was irradiated with UV light of wavelength longer than 300 nm in the presence of salicylic acid as a photosensitizer, six product peaks appeared in an HPLC chromatogram in addition to small amounts of thymidine dimers. The six products were identified as three pairs of diastereomers of 5-(2-deoxy-2-l-ascorbyl)-5,6-dihydrothymidine, 5-(2-l-ascorbyl)-5,6-dihydrothymidine, and 5,6-dihydrothymidine. These results suggest that novel DNA damage may be generated by ascorbic acid with salicylic acid induced by sunlight.     

Determination of ascorbic acid by polypyrrole potentiometric detector in ion chromatography

A potentiometric detector, based on electrodeposition of polypyrrole onto Pt electrode, was investigated in this study. The chromatographic performance of the PPy detector was investigated in ion chromatography. The resulting PPy detector exhibited good performance for AA determination with a wide linear range (1.0 × 10⁻⁶ to 1.0 × 10⁻² M), a highly reproducible response (R.S.D. of 2.45%), without any interference and long-term stability. The calculated detection limit was 7.0 × 10⁻⁵ mM at 3σ. The calibration results showed good Nernstian behavior and also satisfactory low detection limit. In order to verify the reliability of the PPy detector, it was applied to the determination of AA in pharmaceutical samples. The results were satisfactory and agreed closely with the manufacturers’ stated contents. The PPy electrode can be used as an alternative novel potentiometric detector material for determination of AA in standards and pharmaceutical samples.     

Highly sensitive and selective uric acid biosensor based on a three-dimensional graphene foam/indium tin oxide glass electrode

A three-dimensional (3D) continuous and interconnected network graphene foam (GF) was synthesized by chemical vapor deposition using nickel foam as a template. The morphologies of the GF were observed by scanning electron microscopy. X-ray diffraction and Raman spectroscopy were used to investigate the structure of GF. The graphene with few layers and defect free was closely coated on the backbone of the 3D nickel foam. After etching nickel, the GF was transferred onto indium tin oxide (ITO) glass, which acted as an electrode to detect uric acid using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The GF/ITO electrode showed a high sensitivity for the detection of uric acid: approximately 9.44 mA mM⁻¹ in the range of 25 nM–0.1 μM and 1.85 mA mM⁻¹ in the range of 0.1–60 μM. The limit of detection of GF/ITO electrode for uric acid is 3 nM. The GF/ITO electrode also showed a high selectivity for the detection of uric acid in the presence of ascorbic acid. This electrode will have a wide range of potential application prospects in electrochemical detection.     

Uric acid determination in the presence of ascorbic acid using self-assembled submonolayer of dimercaptothiadiazole-modified gold electrodes

This article reports the determination of uric acid (UA) in the presence of ascorbic acid (AA) using a self-assembled submonolayer of heteroaromatic dithiol, 2,5-dimercapto-1,3,4-thiadiazole (DMcT), on gold (Au) electrode. Submonolayer to multilayers of DMcT can be prepared on Au electrode by varying the soaking time of Au electrode in 1mM aqueous solution of DMcT. The formation of submonolayer, monolayer, and multilayers of DMcT on Au electrode was confirmed from its reductive desorption measurements and electrochemical blocking behavior toward ferricyanide. Interestingly, submonolayer of DMcT separates the voltammetric signal of UA from AA by 210 mV, whereas monolayer and multilayers of DMcT fail to separate them. The voltammetric signals of AA and UA are highly stable and reproducible at submonolayer of DMcT. Fast electron transfer, weak hydrogen bonding interactions with AA and UA, and prevention of fouling effect caused by oxidized product of AA can be achieved at submonolayer of DMcT, and thus it successfully separates the voltammetric signals of AA and UA. The practical application of the current system is demonstrated by measuring the concentration of UA in human urine samples without any treatment.     

Selective detection of uric acid in the presence of ascorbic acid at physiological pH by using a beta-cyclodextrin modified copolymer of sulfanilic acid and N-acetylaniline

A beta-cyclodextrin (CD) modified copolymer membrane of sulfanilic acid (p-ASA) and N-acetylaniline (SPNAANI) on glassy carbon electrode (GCE) was prepared and used to determine uric acid (UA) in the presence of a large excess of ascorbic acid (AA) by differential pulse voltammetry (DPV). The properties of the copolymer were characterized by X-ray photoelectron spectra (XPS) and Raman spectroscopy. The oxidation peaks of AA and UA were well separated at the composite membrane modified electrode in phosphate buffer solution (PBS, pH 7.4). A linear relationship between the peak current and the concentration of UA was obtained in the range from 1.0 x 10(-5) to 3.5 x 10(-4)mol L(-1), and the detection limit was 2.7 x 10(-6)mol L(-1) at a signal-to-noise ratio of 3. Two hundred and fifty-fold excess of AA did not interfere with the determination of UA. The application of the prepared electrode was demonstrated by measuring UA in human serum samples without any pretreatment, and the results were comparatively in agreement with the spectrometric clinical assay method.     

Effects of ascorbic acid and ascorbic acid 2-phosphate, a long-acting vitamin C derivative, on the proliferation and differentiation of human osteoblast-like cells

In order to investigate the effect of ascorbic acid (AsA) and ascorbic acid 2-phosphate (Asc 2-P), a long-acting vitamin C derivative, on the growth and differentiation of human osteoblast-like cells, we supplemented the culture medium of MG-63 cells with various concentrations (0.25 to 1 mM) of these factors. Asc 2-P significantly stimulated nascent cell growth at all concentrations in the presence of fetal bovine serum (FBS). On the other hand, AsA showed a growth repressive effect depending on its concentration, and that of FBS. Asc 2-P also increased expression of osteoblast differentiation markers, such as collagen synthesis and alkaline phosphatase (ALP) activity. These stimulative activities of Asc 2-P were attenuated by inhibitors of collagen synthesis, indicating that these effects were dependent on collagen synthesis. Electron micrographs of the cells showed the formation of a three-dimensional tissue-like structure endowed with a mature extracellular matrix in the presence of Asc 2-P.     

Mitochondrial ascorbic acid transport is mediated by a low-affinity form of the sodium-coupled ascorbic acid transporter-2

Despite the fundamental importance of the redox metabolism of mitochondria under normal and pathological conditions, our knowledge regarding the transport of vitamin C across mitochondrial membranes remains far from complete. We report here that human HEK-293 cells express a mitochondrial low-affinity ascorbic acid transporter that molecularly corresponds to SVCT2, a member of the sodium-coupled ascorbic acid transporter family 2. The transporter SVCT1 is absent from HEK-293 cells. Confocal colocalization experiments with anti-SVCT2 and anti-organelle protein markers revealed that most of the SVCT2 immunoreactivity was associated with mitochondria, with minor colocalization at the endoplasmic reticulum and very low immunoreactivity at the plasma membrane. Immunoblotting of proteins extracted from highly purified mitochondrial fractions confirmed that SVCT2 protein was associated with mitochondria, and transport analysis revealed a sigmoidal ascorbic acid concentration curve with an apparent ascorbic acid transport K_m of 0.6 mM. Use of SVCT2 siRNA for silencing SVCT2 expression produced a major decrease in mitochondrial SVCT2 immunoreactivity, and immunoblotting revealed decreased SVCT2 protein expression by approximately 75%. Most importantly, the decreased protein expression was accompanied by a concomitant decrease in the mitochondrial ascorbic acid transport rate. Further studies using HEK-293 cells overexpressing SVCT2 at the plasma membrane revealed that the altered kinetic properties of mitochondrial SVCT2 are due to the ionic intracellular microenvironment (low in sodium and high in potassium), with potassium acting as a concentration-dependent inhibitor of SVCT2. We discarded the participation of two glucose transporters previously described as mitochondrial dehydroascorbic acid transporters; GLUT1 is absent from mitochondria and GLUT10 is not expressed in HEK-293 cells. Overall, our data indicate that intracellular SVCT2 is localized in mitochondria, is sensitive to an intracellular microenvironment low in sodium and high in potassium, and functions as a low-affinity ascorbic acid transporter. We propose that the mitochondrial localization of SVCT2 is a property shared across cells, tissues, and species.     

Mechanism of enhanced bioavailability and diuretic effect of azosemide by ascorbic acid in rats

To increase the extent of comparative oral bioavailability (F) value and the diuretic and natriuretic effects of orally administered azosemide, ascorbic acid was coadministered to rats. The rationales for this study are that ascorbic acid might inhibit intestinal first-pass effect of azosemide and might increase the unionized fraction of azosemide at the receptor sites. After oral administration of azosemide (20 mg/kg) with 100 mg of ascorbic acid, the F value (138% vs. 100%), 8-h urinary excretion of azosemide (5.18% vs. 1.32% of oral dose), 8-h urine output (41.3 vs. 23.0 ml), and 8-h urinary excretion of sodium (24.6 vs. 15.3 mmol/kg) were greater than controls (without ascorbic acid). The amount of spiked azosemide remaining after 30 min incubation of 50 mug of azosemide with the 9000 g supernatant fraction of rat small intestine was significantly greater by 100 microg of ascorbic acid (45.3 vs. 40.9 microg) than controls (without ascorbic acid). After oral administration of azosemide with NH4Cl, the urine pH decreased by 0.5 U, and 8-h urine output (25.8 vs. 11.0 ml) and 8-h urinary excretion of sodium (13.3 vs. 6.89 mmol/kg) were significantly greater than controls (without NH4Cl). The increase in F value and diuretic and natriuretic effects of azosemide with coadministration of ascorbic acid seemed to be due to reduced intestinal first-pass metabolism of azosemide, increased urinary excretion of azosemide, and increased unionized fraction of azosemide at the renal tubular receptor sites.     

The presence of glutathione and glutathione reductase in chloroplasts: A proposed role in ascorbic acid metabolism

Both glutathione and an NADPH-dependent glutathione reductase are present in spinach (Spinacia oleracea L.) chloroplasts. It is proposed that glutathione functions to stabilise enzymes of the Calvin cycle, and it may also act to keep ascorbic acid in chloroplasts in the reduced form.Abbreviations GSH tripeptide glutathione - GSH reduced form of glutathione - GSSG oxidised form of glutathione     

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.     

Relative survivability of human osteoblasts is enhanced by 39 °C and ascorbic acid after exposure to photopolymerization ingredients

Photopolymerizable hydrogels offer great potential in cartilage tissue engineering due to their ability to conform to irregular defect shapes and be applied in a potentially minimally invasive manner. An important process requirement in the use of photopolymerizable hydrogels is the ability of the suspended cells to withstand low intensity ultraviolet light (UV) exposure (4–5 mW/cm²) and photoinitiator concentrations. For cartilage integration with underlying subchondral bone tissue, robust localized osteoblast activity is necessary. Yet, while it is known that osteoblasts do not respond well to UV light, limited work has been conducted to improve their survivability. In this study, we evaluated the cellular cytotoxicity of five different human cell sources at different UV exposure times, with and without a commercially used photoinitiator. We were able to confirm that human osteoblasts were the least tolerant to varying UV exposure times in comparison to bone marrow stem cell, periodontal ligament cell, smooth muscle and endothelial cell lineages. Moreover osteoblasts cultured at 39 °C did not deteriorate in terms of alkaline phosphatase expression or calcium deposition within the extracellular matrix (ECM), but did reduce cell proliferation. We believe however that the lower proliferation diminished osteoblast sensitivity to UV and the photoinitiator. In fact, the relative survivability of osteoblasts was found to be augmented by the combination of a biochemical factor and an elevated incubation temperature; specifically, the use of 50 mg/l of the anti-oxidant, ascorbic acid significantly (P < 0.05) increased the survivability of osteoblasts when cultured at 39 °C. We conclude that ascorbic acid at an incubation temperature of 39 °C can be included in in vitro protocols used to assess cartilage integration with bone ECM. Such inclusion will enhance conditions of the engineered tissue model system in recapitulating in vivo osteoblast activity.     

Changes in the ascorbic acid levels of peritoneal lymphocytes and macrophages of mice with endotoxin-induced oxidative stress

Ascorbic acid (AA) is an important cytoplasmic antioxidant that mice synthesize in the liver, the intracellular levels of which decrease in an oxidative stress situation such as endotoxic shock. The present work deals with the changes in AA levels, that modulate the immune function, in the two main immune cells, namely macrophages and lymphocytes, from female BALB/c mice suffering endotoxic shock caused by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) (100 mg/kg). The intake by cells of this antioxidant present in vitro at different concentrations was also studied. The animals show an oxidative stress, standardized in previous studies, that causes mortality at 30h after LPS injection. The cells were obtained from the peritoneum at 2, 4, 12 and 24h after LPS or PBS (control) injections and were incubated without or with AA at 0.01, 0.1 and 1 mM for 10, 30, 60, 120 or 180 min. The hepatic AA levels were also studied at 0, 2, 4, 12 and 24h after LPS injection. The peritoneal cells obtained from animals injected with LPS showed increased AA levels in relation to the control cells at all times after LPS injection, with maximal effect at 12h. The AA levels decreased after this time, in agreement with changes in the AA hepatic levels. The increase was due to the AA of lymphocytes since macrophages showed a decrease in AA at different times after LPS injection. Both cells showed an increase in the intracellular levels of AA when this antioxidant was added in vitro. This takes place mainly at 30–60 min of incubation in cells from controls and at 10 min in cells from treated mice 12–24 h after LPS injection. The incorporation decreased at these times of endotoxic shock, a few hours before death. In all cases AA levels were higher in lymphocytes than in macrophages, and 1 mM was the most effective concentration. These results suggest that the immune cells need appropriate levels of antioxidants, such as AA, under oxidative stress conditions, and that while lymphocytes take and accumulate AA, macrophages use it.     

CV and NMR study on the reaction of Mo(VI) with 3,4-dihydroxybenzoic acid and ascorbic acid in aqueous solution

The gradual release of the ligand 3,4-dihydroxybenzoic acid (3,4-DHBA) from its molybdenum complex in the presence of ascorbic acid (AscA) in a weakly acidic aqueous solution (pH ∼ 3.5) is described. We observed that the formation of the 3,4-DHBA-semiquinone oxidation state and the semidehydroascorbate is a pre-requisite for the release of the 3,4-DHBA ligand. The interaction of these radicals leads at the same time to the further degradation of AscA resulting in, among other compounds, threonic acid which participates in the reaction with molybdenum. The comparison of the complexing ability indicated that threonic acid competes with protocatechuate, while ascorbic acid is a less good ligand for the Mo(VI). Solution studies on the reaction mechanism were performed by cyclic voltammetry, NMR spectroscopy and UV-Vis spectroscopy. Isolated precipitates were investigated by NMR spectroscopy. The antioxidant properties of 3,4-DHBA and AscA were also compared using the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH).     

The oxidative damage of plasmid DNA by ascorbic acid derivatives in vitro: the first research on the relationship between the structure of ascorbic acid and the oxidative damage of plasmid DNA

To study the structure-function relationship of the oxidative-damage effect of ascorbic acid, we have focused on the interaction between plasmid DNA pUC19 and a series of ascorbic acid derivatives modified on different OH groups in the presence of transition metal ions. Some ascorbic acid derivatives can selectively cleave plasmid DNA from Form I to Form II in the presence of low concentration of Cu2+ just like ascorbic acid itself, while other derivatives oxidatively damage plasmid DNA slightly. We found that those derivatives with unattached 2-OH and 3-OH groups retain the ability to cleave the plasmid DNA. The derivatives that have been methylated on 2-OH or 3-OH can only cleave plasmid DNA softly, and those derivatives that have been protected on both 2-OH and 3-OH can hardly exert an oxidative damage on plasmid DNA under the same condition. Form these results, we can draw the conclusion that 2-OH and 3-OH groups of the ascorbic acid molecule contribute most to this biological activity.     

Effects of 4-hydroxynonenal, a lipid peroxidation product, on dopamine transport and Na+/K+ ATPase in rat striatal synaptosomes

Incubation of rat striatal synaptosomes in ascorbic acid induced the production of thiobarbituric acid reactive substances, a marker of lipid peroxidation, and 4-hydroxynonenal (4-HNE), a lipid peroxidation aldehydic product. Incubations with 4-HNE, used at a range of concentrations comparable to those obtained during peroxidation, induced a simultaneous, dose-dependent decrease of dopamine (DA) uptake and Na⁺/K⁺ ATPase activity and a loss of sulfhydryl (SH) groups. Similar results were observed in a previous study when lipid peroxidation was induced after incubation of synaptosomes in ascorbic acid. Taken together, these data suggest that 4-HNE is an important mediator of oxidative stress and may alter DA uptake after binding to SH groups of the DA transporter and to Na⁺/K⁺ ATPase. These toxic events may contribute to the onset and progression of Parkinsons disease.