Selective Amperometric Recording of Endogenous Ascorbate

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Selective Amperometric Recording of Endogenous Ascorbate Secretion from A Single Rat Adrenal Chromaffin Cell with Pretreated Carbon Fiber Microelectrodes Kai Wang, Tongfang Xiao, Qingwei Yue, Fei Wu, Ping Yu, and Lanqun Mao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02508 • Publication Date (Web): 04 Aug 2017 Downloaded from http://pubs.acs.org on August 4, 2017

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Analytical Chemistry

Selective Amperometric Recording of Endogenous Ascorbate Secretion From A Single Rat Adrenal Chromaffin Cell with Pretreated Carbon Fiber Microelectrodes Kai Wang,†,‡ Tongfang Xiao,†,‡ Qingwei Yue,†,‡ Fei Wu,†,‡ Ping Yu,†,‡ Lanqun Mao†,‡,* †

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems,

Institute of Chemistry, the Chinese Academy of Sciences (CAS), Beijing 100190, China. ‡

University of Chinese Academy of Sciences, Beijing 100049, China

*

Corresponding Author. E-mail: [email protected], Fax: +86-10-62559373.

ABSTRACT Quantitative description of ascorbate secretion at a single cell level is of great importance in physiological studies; however, most studies on the ascorbate secretion have so far been performed through analyzing cell extracts with high performance liquid chromatography, which lacks of time resolution and analytical performance on a single cell level. This study demonstrates a single cell amperometry with carbon fiber microelectrodes (CFEs) to selectively amperometric monitoring vesicular secretion of endogenous ascorbate from a single rat adrenal chromaffin cell. The CFEs are electrochemically pretreated in a weakly basic solution (pH 9.5) and such pretreatment essentially enables the oxidation of ascorbate to occur at a relatively low potential (i.e., 0.0 V vs. Ag/AgCl), and further a high selectivity for ascorbate measurement over endogenously existing electroactive species such as epinephrine, norepinephrine and dopamine. The selectivity is ensured by much larger amperometric response at the pretreated CFEs toward ascorbate over those toward other endogenously existing electroactive species added into the solution or ejected to the electrode with a micropuffer pipette, and by the totally suppressed current response by adding ascorbate oxidase into the cell lysate. With the pretreated CFE-based single-cell amperometry developed here, exocytosis of endogenous ascorbate of rat adrenal chromaffin cells is directly observed and ensured with the calcium ion-dependent high K+-induced secretion of endogenous ascorbate from the cells. Moreover, the quantitative information of the exocytosis of endogenous ascorbate is provided.

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Introduction L-Ascorbic acid (AA, vitamin C) is a lactone (C6H8O6) and its hydroxyl groups at positions 2 and 3 ionize with pKa values of 4.17 and 11.57 in aqueous media. Although AA is relatively water soluble, it does not readily permeate lipid bilayer because of its size and presence as a negatively charged form (i.e., ascorbate) in physiological solutions.1,2 In biological systems, ascorbate functions as both an antioxidant and a cofactor of enzymes involved in biosynthetic reactions including the synthesis of catecholamines, carnitine, amino acids, cholesterol, and certain peptide hormones.1-4 In physiological systems, ascorbate acts as a neuromodulator of glutamatergic, cholinergic, dopaminergic and GABAergic transmission, and antioxidant against oxidative stress.5-10 The pathological studies of ascorbate have suggested that ascorbate has potential therapeutic roles against neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, ischemic stroke and Huntingdon’s disease.5,9-17 Most mammalian species can synthesize AA in the liver, but this ability is lost in humans, other primates, and guinea pigs.18 The requirement of AA is recognized through the role of which in preventing the pathological state known as scurvy.3 In biological system, the transport mechanisms of ascorbate have been demonstrated in a variety of cells and tissues.1,4,19 Previous attempts have suggested that ascorbate is transported into the brain and tissues via Sodium-dependent Vitamin C Transporter-2 (SVCT2), which causes accumulation of ascorbate within cells against in a concentration gradient.4 Cellular efflux of ascorbate is mediated through complex mechanisms. For example, Knoth et al. reported that cellular efflux of ascorbate was mediated through volume-sensitive osmolyte and anion channels.20 Cellular efflux of ascorbate was also suggested to be possibly mediated through homo- and hetero-exchange system associated with glutamate at the plasma membrane.21,22 Daniels et al. proposed another important mechanism, through which newly taken-up ascorbate was secreted from cultured bovine adrenal chromaffin cells coincidentally with catecholamine.23 This observation suggests that the efflux of ascorbate might undergo through an exocytotic process. So far, most studies on ascorbate exocytosis have been conducted by analyzing ascorbate in the cell extracts with high performance liquid chromatography.18,23-25 Although this method is effective for qualitatively describing the exocytosis of ascorbate, the lacking of high temporal resolution and analytical performance on a single cell level of this method make it difficult to directly observe and quantitatively describe the exocytosis of ascorbate. In this context, single-cell amperometry that utilizes a microelectrode closely positioned to a single cell to on-site real-time monitor the release of electrochemically active compounds from the cell hold a great promise; however, this method has not been successfully applied to monitor the exocytosis of ascorbate possibly because of the limitation in selectivity and sensitivity,26 although this method has prone to be particularly attractive for studying the exocytosis and intracellular cytometric analysis at a single cell level.27-36 2


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In this study, we demonstrate a single-cell amperometry to on-site selectively monitor the exocytosis of ascorbate from a single adrenal chromaffin cell with CFEs. We find that the pretreatment of CFEs in a weakly basic solution facilitates the electrochemical oxidation of ascorbate and thus enables the selective detection of ascorbate without interference from other electroactive species co-existing in the cell. With the single-cell amperometry developed here, we directly observe the release of endogenous ascorbate from a single adrenal chromaffin cell, which is K+- and Ca2+-dependent, providing a straightforward evidence for the occurrence of exocytosis of endogenous ascorbate from primary cultured adrenal chromaffin cells. This study describes the first direct observation on the exocytosis of endogenous ascorbate from cultured adrenal chromaffin cells with single cell amperometry, which is envisaged to be of great implications in physiological and pathological investigations on the mechanisms of ascorbate efflux in the cells.

EXPERIMENTAL SECTION Reagents and Solutions. Ascorbic acid (AA), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), epinephrine (E), norepinephrine (NE), and ascorbate oxidase (AAOx, Cucurbita species, EC 1.10.3.3) were all purchased from Sigma and used as supplied. A stock solution of AA (1.0 mM) was prepared just before use. Artificial cerebrospinal fluid (aCSF) was prepared by mixing NaCl (126 mM), KCl (2.4 mM), KH2PO4 (0.5 mM), MgCl2 (0.85 mM), NaHCO3 (27.5 mM), Na2SO4 (0.5 mM), and CaCl2 (1.1 mM) into Milli-Q water, and the solution pH was adjusted pH 7.4. The Ca2+- and Mg2+-free D-Hanks solution was prepared by mixing NaCl (109.5 mM), KCl (5.7 mM), NaHCO3 (23.8 mM), NaH2PO4 (10.1 mM), Na-HEPES (7.3 mM), HEPES (17.3 mM), D-glucose (10 mM), streptomycin (250 µg/mL), and penicillin G (250 µg/mL) into Milli-Q water. The standard extracellular solution was prepared by mixing NaCl (135 mM), KCl (5 mM), MgCl2 (1 mM), CaCl2 (2 mM), HEPES (10 mM), and glucose (10 mM) into Milli-Q water. The Ca2+-free extracellular solution was prepared with almost the same procedure as that for standard extracellular solution with an exception that CaCl2 was not added and 1 mM EGTA was added. The K+-stimulating solution was prepared by mixing NaCl (78 mM), KCl (70 mM), CaCl2 (2 mM), MgCl2 (1 mM), glucose (10 mM) and HEPES (10 mM) into Milli-Q water. The Ca2+-free K+-stimulating solution was prepared with the same procedure as that for the K+-stimulating solution with an exception that CaCl2 was not added and 1 mM EGTA was added. The pH values of all solutions were adjusted to 7.4. Other chemicals were of at least analytical reagent grade and used without further purification. Aqueous solutions were prepared with Milli-Q water. Pretreatment of CFEs. CFEs were prepared by sealing carbon fibers (5 µm in diameter, ProCFE, Dagan, Minneapolis, MN) into glassy capillary as described previously.37 The CFEs were cut to leave 10 µm length of the fiber at the tip of the glass for a single cell analysis. The CFEs were electrochemically pretreated in 0.5 M H2SO4, with 3



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potential-controlled amperometry at +2.0 V for 30 s, at -1.0 V for 10 s, and then with cyclic voltammetry within a potential range from 0 to +1.0 V at a scan rate of 0.1 V s-1 until a stable cyclic voltammogram was obtained. After that, the CFEs were electrochemically pretreated in 0.10 M KCl solution buffered with Borax buffer (pH 9.5) containing 10 mM sodium tetraborate with a potential-controlled amperometry at +1.3 V for 20 min. Pretreatment of CFEs was carried out on a computer-controlled Electrochemical Analyzer (CHI Instrument, Shanghai, China) with CFEs as working electrode, platinum wire as counter electrode, and a microsized Ag/AgCl electrode prepared previously38 as reference electrode. Culture of Primary Rat Adrenal Chromaffin Cells. The use and care of animals was approved and directed by the Institutional Animal Care and Use Committee of National Center for Nanoscience and Technology and the Association for Assessment and Accreditation of Laboratory Animal Care. Primary rat adrenal chromaffin cells were prepared as described previously.39 Briefly, adult rats (200-250g, Wistar) were first anaesthetized with chloral hydrate (345mg/Kg, ip) and then four adrenal glands were quickly removed from the rats and immediately immersed in ice-cold, Ca2+- and Mg2+-free D-Hanks solution. The adrenal medulla were carefully dissected from the adrenal cortex under a microscope and dissociated by incubation in 0.5 mL tissue digestion solution containing 3 mg/mL collagenase, 3 mg/mL bovine serum albumin, 0.2 mg/mL deoxyribonuclease and 2.4 mg/mL hyaluronidase for 70 min at 37 °C. After incubation, the tissue was triturated with a fire-polished, glass Pasteur pipette. Dissociated cells were re-suspended into Dulbecco’s-modified Eagle’s medium supplemented with 10% fetal bull serum, 50 µg/mL penicillin G, and 50 µg/mL streptomycin. The suspension of isolated cells was then placed onto a poly-l-lysine-coated coverslip for culture. The cells were allowed to settle and adhere on the coverslip for 30 min and then kept in the incubator at 37°C before use. All amperometric recordings were carried out within 4 hours after the cells were incubated for at least 1 hour. Single-cell Amperometric Recording. Electrochemical recordings of ascorbate secretion from a single rat adrenal chromaffin cell were performed on an inverted microscope (TS100-F, Nikon) in a Faraday cage with Patch Clamp system. A piece of coverslip containing the cultured cells was placed in the recording chamber and perfused with standard extracellular solution at approximately 1 mL/min. The tip of CFE was advanced gently to touch the surface of chromaffin cells, as judged by a slight deformation in the cell outline. The electrode was polarized at 0.0 V to on-site selectively record the ascorbate secretion from a single adrenal chromaffin cell. Oxidation currents were recorded on an Axon700B amplifier (Axon Instrument, CA, USA) and pClamp software version 10.5 (Axon Instrument, CA, USA), low-pass filtered at 0.3k Hz, and digitized at 2k Hz before storage in a computer. In the single-cell amperometric experiments, all solutions were delivered to the cells via amultiple-channel micropuffer system (RCP-2B; Inbio Inc.,

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Wuhan, China). The tip of the puffer pipette was about 100 µm far from the target cells. All experiments were performed at room temperature (25-28 °C).

RESULTS AND DISCUSSION Electrochemical Oxidation of Ascorbate at the Pretreated CFEs. Figure 1 shows cyclic voltammograms (CVs) obtained at pristine (i.e., without electrochemical pretreatment in a weakly basic solution) (A) and pretreated (B) CFEs in aCSF (pH 7.4) before (red) and after (black) the addition of ascorbate into the solution. At pristine CFE, ascorbate oxidation occurs at ca. +0.30 V with tailed current response under the experimental conditions employed. Compared with that at the pristine CFE, the oxidation of ascorbate occurs at a more negative potential of -0.10 V at the pretreated CFE and the current quickly reaches a well-defined steady state at +0.20 V. This comparison demonstrates that the electrochemical pretreatment in a weakly basic solution applied to the CFEs essentially accelerates the kinetics of ascorbate oxidation at the as-pretreated CFEs. As documented previously,40 ascorbate is one of the inner-sphere redox species with electron transfer kinetics sensitive to the chemical structure of the electrode. Although the mechanism underlying this kinetics enhancement at the pretreated CFEs remains unclear at the present stage and necessitates more in-depth future studies mainly because of the structure complexity of carbons, in particular those of the carbons subjected to various pretreatments,41,42 the change in the surface chemistry and hydrophilicity as well as density of electronic state of CFEs caused by the pretreatment in a weakly basic solution would constitute a main consequence for the kinetics enhancement. The kinetics enhancement virtually enables the oxidation of ascorbate to commence at a much negative potential (-0.10 V) than those for the oxidation of DA (ca. 0.0 V), E (+0.01 V), NE (+0.03 V) and DOPAC (+0.05 V) (Figure S1), suggesting little interference from these electroactive species toward the detection of ascorbate. As shown in Figure 1C and D, when the pretreated CFEs were polarized at 0.0 V, the addition of DA, E, NE, or DOPAC into the solution did not produce obvious current response compared with that of ascorbate. To validate the single-cell amperometry with the pretreated CFEs for on-site real-time monitoring the exocytosis of ascorbate from a single rat adrenal chromaffin cell, we further studied the selectivity and sensitivity of the method in the cell culture coverslip containing pure aCSF (i.e., without rat adrenal chromaffin cells) that is then used for a single-cell analysis, in which the pretreated CFE and a micropuffer pipette were positioned adjacently. In this case, solutions were ejected from the micropuffer pipette and detected at the adjacent CFEs at 0.0 V. As shown in Figure 2A, the ejection of E, NE, or DA from the micropuffer pipette did not produce current response at the CFE, while the ejection of ascorbate produced an obvious current response, demonstrating the selectivity of this method for ascorbate detection. Moreover, the selectivity of the method was further studied with the chromaffin cell lysates. To do this, 1 mL sample of chromaffin

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I / nA


4 2 0 -0.2

0.0 0.2 0.4 E / V vs. Ag/AgCI

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DOPAC AA

Figure 1. (A, B) Typical CVs at pristine (A) and pretreated (B) CFEs in aCSF (pH 7.4) before (red) and after (black) addition of 200 µM ascorbate into the solution. Scan rate, 50 mVs-1. (C) Typical amperometric current-time response obtained with the pretreated CFE for DA (10 µM), E (10 µM), NE (10 µM), DOPAC (20 µM), and ascorbate (200 µM) as indicated in the figure. The electrode was polarized at 0.0 V vs. Ag/AgCl. (D) Histograms the current responses obtained at the pretreated CFE toward different compounds.

cells was first subjected to homogenization by ultrasonication and then filtered by 0.22 µm microfilter. The cell lysates were immediately used in the experiments. The cell lysates before and after AAOx (120 units mL-1) treatment were separately ejected to the CFE (Figure 2B). As shown in Figure 2B, the ejection of pure chromaffin cell lysates (i.e., without AAOx treatment) yields a clear current response that was ascribed to the oxidation of endogenous ascorbate in chromaffin cells (top). On the contrary, the ejection of the same cell lysates pretreated with AAOx did not produce obvious current response at the electrode (bottom). These results again demonstrate the selectivity of the method toward ascorbate with the CFEs pretreated in the weakly basic solution. In addition to its high selectivity, the method with the pretreated CFEs shows a good linearity for the measurement of ascorbate. The currents recorded in the system mentioned above increase with increasing the concentration of 6


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ascorbate ejected from the micropuffer pipette and are linear with the concentration within the range from 0.05 to 2 mM ( I / pA = 25.04 C /mM - 1.78, γ = 0.9892). Taken together, these results substantially validate the single-cell amperometry developed here with the CFE pretreated in a weakly basic solution for on-site recording the secretion of ascorbate from a single adrenal chromaffin cell. 40 pA

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SE

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E Cell Lysates AA NE

Cell Lysates + AAOx

DA Figure 2. (A) Amperometric response of the CFE at 0.0 V toward a 10 s puffer application of standard extracellular solution (SE), 10 µM E, 200 µM ascorbate (AA), 10 µM NE, and 10 µM DA. (B) Amperometric response of the CFE at 0.0 V toward a 10 s puffer application of cell lysate (top) and cell lysate + AAOx (bottom).

Ascorbate Secretion from A Single Adrenal Chromaffin Cell. As reported previously,18,26,43 isolated chromaffin cells lose endogenous ascorbate rapidly in culture and, as a consequence, the cells must be replenished with ascorbate prior to subsequent study of ascorbate secretion. However, the subcellular distribution of newly transported ascorbate in ascorbate-supplemented chromaffin cells has previously shown to be predominantly extragranular following mechanical disruption and thereby it has remained unresolved whether the distribution of newly transported ascorbate reflects the true distribution of ascorbate in the original tissue.18,20,44,45 The use of single-cell amperometry developed here essentially makes it possible to immediately on-site monitor the secretion of endogenous ascorbate from acutely dissociated rat adrenal chromaffin cells. As typically depicted in Figure 3A, puff application of K+-stimulating solution resulted in well-defined amperometric spikes recorded with the CFE closed attached to the cell (left), reflecting vesicular release from the cell.30-36 This result suggests the successful recording of the vesicular release of endogenous ascorbate from a single rat adrenal chromaffin cell with the single-cell amperometry. To further confirm the vesicular release of ascorbate observed in this study, we investigated the dependency of the high K+-induced endogenous ascorbate release on calcium pathway since the process of vesicular release is known to be calcium ion dependent.26,39,46 To do this, we replaced the 7



standard extracellular solution (i.e., containing 2 mM Ca2+) with Ca2+-free solution containing 1 mM EGTA for at least 3 min, puff applied Ca2+-free K+-stimulating solution to the cell, and recorded the ascorbate release with the same method as that in standard extracellular solution with normal K+-stimulating solution applied to the cell. As shown in Figure 3A (right), the removal of Ca2+ from both the extracellular solution and the K+-stimulating solution well suppressed the high K+-induced secretion of ascorbate. To confirm that the amperometric spikes recorded in Figure 3A (left) induced by high K+ were due to the release of endogenous ascorbate, we measured the chromaffin cells’ response to high K+ with the pretreated CFEs at different working potentials. We found that, when the electrode was held at -0. 20 V, at which the oxidation of ascorbate does not occur, 70 mM KCl induced no amperometric spikes (Figure 3B, right). Whereas, the same puffer application of 70 mM KCl induced spikes when the electrode was hold at 0.0 V (Figure 3B, left). These

Extracellular Solution Containing no Ca2+

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K+-stimulating Solution

5s

K+-stimulating Solution

Figure 3 (A) (Left) Amperometric spikes recorded with the pretreated CFE closely attached to a single rat adrenal chromaffin cell that was cultured in the standard extracellular solution (i.e., containing 2 mM Ca2+) and stimulated by K+-stimulating solution. (Right) Current-time trace recorded with the pretreated CFE closely attached to a single rat adrenal chromaffin cell that was cultured in the Ca2+-free extracellular solution and stimulated by Ca2+-free K+-stimulating solution. Inset, schematic illustration of the single cell amperometry with the pretreated CFE. (B) High K+ evoked amperometric spikes recorded with the pretreated CFE closely attached to a single rat adrenal chromaffin cell cultured in the standard extracellular solution (i.e., containing 2 mM Ca2+) at different holding potentials of 0.0 V (left) and -0.20 V (right). Inset, a typical amperogram spike for ascorbate secretion.

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results suggest that endogenous ascorbate in the adrenal chromaffin cells efflux from the cell cytoplasm as a result of the vesicular exocytosis. As mentioned above, single-cell amperometry is a very attractive electrochemical method to record the exocytosis of neurotransmitters in various types of cells because of its high temporal resolution and capability to perform at a single cell level. To investigate the kinetics of the high K+-induced exocytosis of ascorbate, we analyzed four parameters of amperometric spikes of ascorbate release (Figure 4, top), i.e., rise time (RT) and half height duration (HHD) that reflect the fusion kinetics; and peak height (PH) and peak area (PA) that reflect the vesicle content released in each spike. N was evaluated with Faraday’s law (N = Q / nF), where N is in moles, Q is charge equal to the peak area, n is the number of electrons in the oxidation reaction (2 for ascorbate), and F is Faraday’s constant. All kinetic results are presented as mean ± SEM and summarized (Figure 4, bottom). Taken together, the combination of high selectivity of the pretreated CFEs toward ascorbate oxidation with the high temporal resolution of the single-cell amperometry essentially enable us to on-site record the secretion of ascorbate and thereby to understand the kinetics of the secretion. As far as we know, this is the first quantitative description on the exocytosis of the endogenous ascorbate with a single-cell amperometry, which is envisaged to be useful for further understanding of the mechanism and the possible pathways involved in ascorbate secretion.

HHD

PH

PA RT

RT (ms) 70 mM K+

1.6 ± 0.1

HHD (ms) 13.8 ± 0.5

PH (pA) 64 ± 3.2

6 molec)

PA (pC)

N (10

1.3 ± 0.08

3.7 ± 0.04

Figure 4 (Top) Scheme showing the four parameters used for the single peak analysis in this work. (Bottom) Kinetic parameters results of ascorbate release from adrenal chromaffin cells (n =46 spikes, 4 cells). The data are shown as mean ± SEM.

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CONCLUSIONS In summary, we have demonstrated a single-cell amperometry to on-site directly monitor the exocytosis of endogenous ascorbate from a single adrenal chromaffin cell with CFEs pretreated in a weakly basic solution. The pretreatment of the CFEs essentially accelerates the electrochemical oxidation of ascorbate and thus enables the selective detection of ascorbate without interference from other electroactive species co-existing in the cell. With the single cell amperometry developed here, we provide the first description on the kinetics of the exocytosis of endogenous ascorbate from a single adrenal chromaffin cell. This study essentially offers a straightforward evidence that endogenous ascorbate appears to locate in a vesicular compartment and efflux from the chromaffin cells through an exocytotic mechanism, which is quite similar to the exocytosis of catecholamine that have been demonstrated to occur for chromaffin vesicles. Moreover, the use of the single cell amperometry essentially provides the quantitative information on the exocytosis of endogenous ascorbate, which is believed to be useful for understanding the kinetics transport mechanism of ascorbate in physiological and pathological investigations.

ASSOCIATED CONTENT Supporting Information Cyclic voltammograms of DA, E, NE and DOPAC. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMANTION Corresponding Author *

E-mail: [email protected]. Fax: (+86)-10-62559373.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We acknowledge financial support from the National Natural Science Foundation of China (21435007, 21321003, 21210007, 91413117 for L.M., 21475138, 91132708 for P.Y.), National Basic Research Program of China (2016YFA0200104, 2013CB933704), and Chinese Academy of Sciences.

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For TOC only

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Selective Amperometric Recording of Endogenous Ascorbate

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