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Erythrocyte Ascorbate Recycling: Antioxidant Effects in Blood
Free Radical Biology & Medicine, Vol. 24, No. 5, pp. 789 –797, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/98 $19.00 1 .00
PII S0891-5849(97)00351-1
Original Contribution ERYTHROCYTE ASCORBATE RECYCLING: ANTIOXIDANT EFFECTS IN BLOOD SHALU MENDIRATTA, ZHI-CHAO QU,
and JAMES
M. MAY
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA (Received 6 May 1997; Revised 15 September 1997; Accepted 16 September 1997)
Abstract—Ascorbic acid is an important antioxidant in human plasma, but requires efficient recycling from its oxidized forms to avoid irreversible loss. Human erythrocytes prevented oxidation of ascorbate in autologous plasma, an effect that required recycling of ascorbate within the cells. Erythrocytes had a high capacity to take up dehydroascorbate, the two-electron oxidized product of ascorbate, and to reduce it to ascorbate. Uptake and conversion of dehydroascorbate to ascorbate was saturable, was half-maximal at 400 mM dehydroascorbate, and achieved a maximal intracellular ascorbate concentration of 1.5 mM. In the presence of 100 mM dehydroascorbate, erythrocytes had the capacity to regenerate a 35 mM ascorbate concentration in blood every 3 min. Ascorbate recycling from DHA required intracellular GSH. Depletion of erythrocyte GSH by more than 50% with diamide did not acutely affect the cellular ascorbate content, but did impair the subsequent ability of GSH-depleted cells to recycle dehydroascorbate to ascorbate. Whereas erythrocyte ascorbate recycling was coupled to GSH, an overwhelming extracellular oxidant stress depleted both ascorbate and a-tocopherol before the GSH content of cells fell appreciably. Recycled ascorbate was released from cells into plasma, but at a rate less than one tenth that of dehydroascorbate uptake and conversion to ascorbate. Nonetheless, ascorbate released from cells protected endogenous a-tocopherol in human LDL from oxidation by a water soluble free radical initiator. These results suggests that recycling of ascorbate in erythrocytes helps to maintain the antioxidant reserve of whole blood. © 1998 Elsevier Science Inc. Keywords—Antioxidants, Oxidant stress, Oxidation-reduction, Glutathione, Biological transport, Free radical
INTRODUCTION
The intracellular conversion of DHA to ascorbate is largely dependent on the GSH system,7,13 and may be enzyme-mediated. Ascorbate generated in this manner rapidly accumulates against a transmembrane concentration gradient. With time this gradient gradually dissipates, presumably due to efflux of ascorbate from the cell.5 Although release of recycled ascorbate from cells seems a plausible mechanism to protect plasma components from oxidation, this has not been demonstrated. A related question is whether and to what extent intracellular ascorbate recycling protects the erythrocyte itself from oxidant stress, especially since this cell is equipped with so many different types of defense against oxygen free radicals.16,17 Of the non-enzymatic defenses of the erythrocyte, important roles have been established for a-tocopherol in the membrane18 –20 and GSH in the cytoplasm.21,22 A role for ascorbate would be supported by demonstrating that the vitamin is depleted with or before a-tocopherol and GSH in the face of an oxidant stress. The results of this work show that recycling of
Ascorbic acid, or vitamin C, is a primary antioxidant in plasma, since it is consumed first during an oxidant stress, and since peroxidation of plasma lipids does not occur until ascorbate is depleted.1,2 The two-electron oxidation product of ascorbate, dehydroascorbate (DHA), is labile at physiologic pH and temperature (halflife 5 5–7 min).3,4 Unless reduced back to ascorbate, DHA undergoes irreversible ring-opening to 2,3-diketogulonic acid. Dehydroascorbate can be reduced or recycled to ascorbate in blood by both erythrocytes5–7 and neutrophils,8,9 but not by plasma alone.3 Quantitatively the erythrocyte is the most likely cell to fulfill this function in blood. Erythrocytes rapidly take up DHA on the glucose transporter10 –12 and reduce it to ascorbate.5–7 Address reprint request to: J. M. May, Department of Medicine, 736 Medical Research Building II, Vanderbilt University School of Medicine, Nashville, TN 37232-6303; Tel: 615-936-1653; Fax: 615-9361667; E-Mail: [email protected]. 789
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ascorbate in erythrocytes maintains both intracellular and extracellular levels of the vitamin, that extracellular ascorbate spares a-tocopherol in LDL from oxidation, and that ascorbate is in the first line of defense against an overwhelming external oxidant stress to the erythrocyte. EXPERIMENTAL PROCEDURES
Cell preparation Blood was drawn from normal volunteers by venipuncture and anticoagulated with heparin. Erythrocytes were washed three times by centrifugation in ten volumes of phosphate-buffered saline (PBS), which consisted of 154 mM NaCl and 12.5 mM Na2HPO4 in deionized water, adjusted to pH 7.4. The buffy coat of white cells was carefully removed with each wash. The 1.019 –1.063 density fraction of LDL was prepared by the method of Havel. et al.23 Measurement of plasma and intracellular ascorbate and glutathione concentrations Following incubations as noted, erythrocytes were pelleted by centrifugation and ascorbate concentrations in cells and plasma were measured by HPLC with electrochemical detection. In most experiments, plasma was cooled on ice and ultrafiltered as described below prior to assay of ascorbate. In an alternate method, 100 ml of plasma was added to 0.9 ml of ice-cold 50 mM perchloric acid with mixing. After several minutes on ice, the sample was microfuged for 1 min, and an aliquot of the supernatant was taken for HPLC assay of ascorbate. The ultrafiltration method required immediate sample processing to avoid loss of ascorbate, whereas ascorbate was stable for several hours in cold perchloric acid. For assay of intracellular ascorbate, 100 ml of packed cells was diluted with 0.3 ml of PBS, quickly frozen in dry iceacetone, and the frozen hemolysate was allowed to thaw on ice. The hemolysate was ultrafiltered as described by Iheanacho, et al.24 Briefly, the hemolysate was transferred to a Centricon-10 filter apparatus (Amicon, Inc., Beverly, MA) and centrifuged at 4°C for 30 min at 5000 (3g. This resulted in 0.3– 0.4 ml of a clear ultrafiltrate, which was diluted 10 –20-fold with mobile phase before assay. In hemolysates that were spiked with D-isoascorbate in amounts that were 10 –20% of the hemolysate ascorbate content, no losses of isoascorbate during the ultrafiltration step could be detected. It is likely that GSH present in the hemolysate helped to prevent loss of both ascorbate and added isoascorbate during preparation of the sample for analysis. Ascorbate and GSH were assayed by HPLC using the ion-pairing method of Pachla and Kissinger.25 The mo-
bile phase consisted of 80 mM sodium acetate, 1 mM tridecylamine, 1 mM EDTA, and 30% methanol, pH 5.2 before methanol addition. The mobile phase was pumped with an ESA Model 5700 pump (Bedford, MA), filtered with an ESA graphite carbon in-line filter, and preconditioned with an ESA Model 5020 guard cell set at 0.5 volts in the oxidizing mode. Samples were injected with a BioRad AS-100 injector in a volume of 100 ml. Separation was carried out on a 10 3 1 cm Waters RadiaPak C18 column (300 mm 4 m), with a 4 mm guard column of the same packing material. Detection was accomplished with an ESA Model 5100A detector using a Model 5010 analytical cell with the first electrode set at zero and the detecting electrode set at 0.4 volts. At a flow rate of 1 ml/min, ascorbate eluted at 8 –9.5 min, and GSH eluted at 10 –11.5 min, with complete separation of the peaks. The assay sensitivity was 10 pmol/sample for ascorbate and 1 nmol/sample for GSH. The concentration of oxidized GSH (GSSG) was measured using the enzymatic cycling assay of Tietze26 as previously described.13 Measurement of a-tocopherol in erythrocytes and in human LDL Cell and LDL contents of a-tocopherol were measured by HPLC with electrochemical detection according to the method of Lang, et al.27 The a-tocopherol in LDL was measured in buffer alone or in buffer from which cells had been removed by centrifugation. For assay of erythrocyte a-tocopherol, packed cells (0.2 ml) were chilled on ice, mixed with 50 ml of a 10 mg/ml solution of butylated hydroxytoluene in methanol, and lysed with 0.8 ml of ice-cold 3% (w/v) sodium dodecyl sulfate. This hemolysate was further diluted with 1 ml of ice-cold 5 mM sodium phosphate buffer, pH 7.4, that contained 10 mM ascorbic acid. The hemolysate was vortexed, treated with 2 ml of reagent alcohol (95% ethanol and 5% isopropanol, v/v), and mixed again. Following addition of 2 ml of hexane, the sample was vortexed vigorously for 1–2 min, then centrifuged to separate the phases. One ml of the hexane upper phase was taken to dryness under nitrogen, and the residue was dissolved in 0.5 ml of a mixture of methanol and reagent alcohol (1:1, v/v) for HPLC injection. Samples were chromatographed in the isocratic mode on a Waters DeltaPak C18 column (300 mm 5 m) with a 4 mm guard column of the same packing material. The mobile phase was 94% methanol and 6% water containing 100 mM sodium acetate. Pre-column conditioning of the buffer was found to be unnecessary. Tocopherol detection was carried out by a simplification of the reduction-oxidation method described by Takeda, et al.28 An ESA model 5021 conditioning cell, preceded by a graphite in-line filter, was placed just after the
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analytical column and set in the reducing mode at 20.5 volts. An ESA model 5011 analytical cell was used for detection, with the first electrode in the analytical cell set at 20.5 volts, and the second detecting electrode set at 0.6 volts. At a flow rate of 1 ml/min, b-tocopherol eluted at 5.6 –5.9 min, and a-tocopherol eluted at 6.1– 6.4 min. The assay sensitivity for a-tocopherol was 2–5 pmol. Other assays Hemolysis of erythrocytes was measured as the increase in absorbance at 540 nm of erythrocyte supernatants compared to absorbance produced by hemolysis of a known volume of erythrocytes in water.29 Protein in LDL was measured using the BCA method (Pierce Chemical Co., Rockford, IL). The amounts of ascorbate, GSH, a-tocopherol, and ferrocyanide were expressed relative to the intracellular water space of the erythrocytes,30 which was confirmed in separate studies to be 70% of the packed cell volume. Data and statistical analysis Curve-fitting was carried out by nonlinear leastsquares regression in the graphics software packaged FigP (Biosoft Inc., Cambridge, UK). Statistical significance was assessed by one- or two-way ANOVA using the statistical software package Sigmastat 2.0 (Jandel Scientific, San Rafael, CA). All results are shown as mean 6 SEM. RESULTS
Incubation of plasma in vitro exposed to air at 37°C resulted in a decrease in the plasma ascorbate concentration over 4 h (Fig. 1A). The results are expressed as a fraction of the zero-time value, since there was marked variation in the endogenous plasma and erythrocyte ascorbate contents from different donors (from 15 to over 100 mM). The decline in plasma ascorbate was completely prevented when 40% erythrocytes from the same donor were incubated with the plasma, suggesting that the cells had helped to maintain the extracellular ascorbate concentration. The initial concentration of ascorbate in plasma was 96 6 14% of that in erythrocytes from the same donor (N 5 4), so the maintenance of plasma ascorbate could not be ascribed simply to diffusion of a high initial concentration of ascorbate out of cells. Intracellular concentrations of ascorbate and GSH were unaffected by incubation of cells in plasma from the original donor (results not shown). When a stronger oxidant challenge was generated in plasma by a 1 mM concentration of the water soluble free
Fig. 1. Erythrocyte-dependent protection against ascorbate oxidation. Panel A: freshly prepared plasma was incubated at 37°C in the absence (circles) or presence (squares) of washed 40% erythrocytes derived from the same plasma. At the indicated times, plasma and plasma from cells were assayed for ascorbate, which is expressed as a fraction of the zero-time value. In 7 experiments, fractional ascorbate levels in plasma alone were different than those in plasma containing cells ( p , .02). Panel B: plasma was incubated at 37°C in the absence (circles) or presence (squares) of 40% erythrocytes derived from the same plasma and 1 mM AAPH per total volume. Data from 6 experiments are shown as a fraction of the initial value. Fractional ascorbate levels in plasma alone were different than those in plasma containing cells ( p , .05).
radical initiator 2,29-azobis(2-amidinopropane)hydrochloride (AAPH) (Wako Chemical Co., Richmond, VA), erythrocytes prevented significant loss of extracellular ascorbate, in contrast to nearly complete loss of ascorbate in plasma alone (Fig. 1B). Since AAPH should not enter cells, its effective extracellular concentration was about 70% higher in the incubations with cells, which further accentuates the differences observed. Intracellular ascorbate and GSH were again unchanged during the external challenge with AAPH (results not shown). The preservation of extracellular ascorbate by erythrocytes in these experiments may be due in part to DHA uptake and
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Fig. 2. Time course of DHA-dependent ascorbate appearance in erythrocytes and release into plasma. Washed, 40% erythrocytes were incubated at 37°C in the plasma from which they were originally derived in the presence of 100 mM DHA. At the indicated times, cells and plasma were rapidly separated in a microfuge, the cells were washed three times by centrifugation in 3 volumes of PBS, and the cells and original plasma were assayed for ascorbate. Extracellular (circles) and intracellular (squares) ascorbate concentrations are shown for 4 experiments. The increase in both curves from initial values was significant ( p , .05).
conversion to ascorbate within the cells, with subsequent leak of ascorbate across the cell membrane. This possibility was examined by measurement of ascorbate in plasma and in erythrocytes following incubation of cells with DHA. Erythrocytes rapidly internalized 100 mM DHA, which is seen as an increase in the intracellular ascorbate content (Fig. 2). Uptake resulted in a maximal intracellular ascorbate concentration of ascorbate that was more than ten-fold higher than the initial extracellular ascorbate concentration, although this level could not be sustained for more than about 60 min. The extracellular ascorbate concentration doubled in response to DHA loading, in support of the notion that it originated from intracellular ascorbate. Therefore, release of ascorbate can account for at least part of the protective effect of erythrocytes on plasma ascorbate. The intracellular concentration of GSH was unchanged during the uptake of 100 mM DHA in the presence of glucose (results not shown). Over the first 5 min of incubation, 173 nmol of ascorbate was generated per ml of erythrocyte cytoplasm, or ;54 nmol per ml of blood at a 45% hematocrit. Thus, under these conditions, a 30 –35 mM concentration of ascorbate in blood can be regenerated in about 3 min. Release of ascorbate into plasma is much slower than the recycling capacity, however. During incubation of erythrocytes with increasing concentrations of DHA for 1 h in plasma, the intracel-
Fig. 3. Uptake and reduction of increasing concentrations of DHA by erythrocytes. Washed, 40% erythrocytes were incubated at 37°C with the indicated concentrations of DHA in the plasma from which they were derived. After 60 min, ascorbate concentrations were determined in plasma and in cells washed as described in the legend to Fig. 2. Extracellular (circles) and intracellular (squares) ascorbate concentrations are shown for 6 experiments, with the smooth line for the intracellular ascorbate depicting the fit to a rectangular hyperbola (apparent Km 5 403 mM, Vmax 5 1678 nmol/(ml erythrocytes-h), r 5 0.98).
lular ascorbate concentration increased to a maximum of almost 1.5 mM (Fig. 3). When these data were fit to a rectangular hyperbola, an apparent half-maximal effect was observed at about 400 mM extracellular DHA. The extracellular ascorbate concentration rose with increasing extracellular DHA to almost tenfold that of plasma at an initial extracellular DHA concentration of 5 mM. In the presence of plasma that contained glucose, intracellular GSH and the GSSG/GSH ratio were not significantly affected, nor was hemolysis evident (results not shown). These data show that erythrocytes have a substantial, but saturable, ability to recycle DHA to ascorbate, and that the latter accumulates, at least transiently, against a concentration gradient. Loss or efflux of ascorbate from the cells is much slower than uptake and recycling, but is proportional to the intracellular ascorbate concentration. Incubation of erythrocytes with increasing concentrations of ascorbate for 3 h resulted in a linear increase in intracellular ascorbate (Fig. 4). At low initial ascorbate concentrations, intracellular ascorbate concentrations at the end of 3 h were consistently greater than those outside cells. Such accumulation of ascorbate against a gradient was much less marked than observed for DHA (Fig. 3), and likely reflects uptake and recycling of a small amount of DHA that had been generated extracellularly. The cell contents of GSH and GSSG were unchanged during the incubation with ascorbate (results not shown).
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Fig. 4. Uptake of ascorbate by erythrocytes in plasma. Washed, 40% erythrocytes were incubated at 37°C with increasing concentrations of ascorbate in the plasma from which they were derived. At 3 h, cells and plasma were separated, the cells were washed as described in the legend to Fig. 2, and plasma (circles) and cells (squares) were assayed for ascorbate Data from 5 experiments are shown.
Whereas erythrocyte GSH was unaffected by DHA loading in plasma containing glucose, a role for GSH in recycling ascorbate was evident when intracellular GSH was partially oxidized with diamide. Incubation of cells with increasing amounts of diamide in the absence of glucose resulted in a decrease in intracellular GSH to about 35% of the initial value (Fig. 5A). When GSH-depleted cells were incubated for an additional 30 min in the presence of 5 mM D glucose, the diamide-induced decrease in GSH was reversed (Fig. 5A). Intracellular ascorbate tended to decrease as the concentration of diamide was increased, whether or not glucose was present (Fig. 5B). Nonetheless, the ability of GSH to maintain or recycle endogenous ascorbate was minimally compromised by oxidation of more than 50% of cell GSH. On the other hand, when cells depleted of GSH by diamide were washed several times and then incubated with 500 mM DHA, their ability to generate ascorbate was clearly impaired (Fig. 6). Diamide pretreatment decreased recycling of DHA to ascorbate by 86% in the absence of glucose, and decreased it by about 50% in cells that were subsequently exposed to glucose. Glucose was required for recycling of ascorbate from DHA, whether or not the cells had been exposed to diamide. In the absence of glucose, intracellular GSH was decreased 47% by DHA alone, and 72% by both DHA and diamide. Whereas addition of glucose to diamide-treated cells completely restored intracellular GSH to control values, DHA reduction to ascorbate did not fully recover (Fig. 6). GSH-dependent ascorbate recycling was insufficient to protect even endogenous ascorbate when cells were exposed to an overwhelming oxidant stress, even the presence of glucose. In the experiments shown in Fig. 7,
Fig. 5. Diamide oxidation of erythrocyte GSH. Erythrocytes were incubated at a 10% hematocrit in glucose-free PBS with the indicated concentration of diamide for 10 min on ice, washed three times by centrifugation in 10 volumes of ice-cold glucose-free PBS, and resuspended to a 10% hematocrit in the absence (circles) or presence (squares) of 5 mM D-glucose. Following a 10 min incubation at 37°C, cells and buffer were separated, and GSH (Panel A) and ascorbate (Panel B) contents were determined. The decrease in GSH induced by diamide significant ( p , .001) in the three experiments shown.
cells were incubated for up to 200 min with an extracellular AAPH concentration of 83 mM. This contrasts with the 1.7 mM extracellular concentration of AAPH used in the experiments shown in Fig. 1B. AAPH at this high concentration caused progressive and similar decreases in both intracellular ascorbate and membrane a-tocopherol (Fig. 7A). GSH decreased at about half the rate of ascorbate and a-tocopherol. Hemolysis also increased over the last half of the incubation. (Fig. 7B). Similar results were observed when erythrocytes were incubated in PBS with the cell-impermeant oxidant ferricyanide. PBS, rather than plasma, was used to avoid direct reaction of ferricyanide with ascorbate and other antioxidants present in plasma. Over the first 30 min of incubation, ascorbate was depleted by ferricyanide to about 50% of its initial value, despite the presence of glucose (Fig. 8). In these experiments, a-tocopherol showed a progressive, albeit slower initial decline than
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Fig. 6. DHA loading of diamide-treated erythrocytes. Control and diamide-treated erythrocytes were prepared as described in the legend to Fig. 5, and incubated at a 10% hematocrit for an additional 30 min at 37°C with 500 mM DHA in the presence or absence of 5 mM D-glucose, as indicated. Ascorbate (open bars) and GSH (hatched bars) contents are shown from three experiments, with an asterisk (*) indicating p , .05 versus corresponding samples in the preceding group of bars that were treated with glucose.
ascorbate. Intracellular GSH was unaffected by ferricyanide. These results with two different cell-impermeant agents suggest that ascorbate and a-tocopherol are more sensitive to an external oxidant stress than is GSH. That is, ascorbate can be selectively oxidized before GSH stores are significantly depleted. Whereas release of recycled ascorbate is much slower than the recycling process itself, it was nonetheless possible to show that ascorbate derived from DHA-loaded erythrocytes protects LDL form oxidation. In the presence of AAPH, the a-tocopherol content of human LDL decreased over 4 h (Fig. 9). When erythrocytes at a 40% hematocrit were included in the incubation, the rate of a-tocopherol oxidation in the LDL was significantly slowed, although there was no lag-phase. When erythrocytes were loaded with 100 mM DHA during the incubation, little loss of a-tocopherol in LDL occurred until after the second hour of incubation, indicative of a lagphase. When ascorbate oxidase was included in the incubation, the protection by DHA-loading was abolished (Fig. 9). Under these conditions, ascorbate oxidase does not significantly deplete ascorbate in DHA-loaded cells (results not shown). The failure of DHA-loading to protect extracellular LDL in the presence of ascorbate oxidase suggests that the effect was due to ascorbate that had diffused out of the cells. DISCUSSION
Since human cells cannot synthesize ascorbate, the vitamin must be delivered to them by the bloodstream.
Fig. 7. Depletion of antioxidants with high-dose AAPH. Washed, 40% erythrocytes were incubated at 37°C in the plasma from which they were derived with AAPH at an extracellular concentration of 83 mM. Panel A: at the times indicated, cells were separated from plasma, washed in PBS, and assayed for ascorbate (circles, N 5 6), GSH (squares, N 5 6), and a-tocopherol (triangles, N 5 5), which are shown as a fraction of the initial value. The decreases in ascorbate and a-tocopherol were greater than the fall in GSH ( p , .05). Panel B: hemolysis was measured in five experiments. The increase in hemolysis was significant for the last three time points sampled ( p , .05).
Ascorbate is highly susceptible to oxidation in plasma,1,2 but is protected in whole blood by erythrocytes.3,31 During their investigation of the mechanism of this protection, Borsook and colleagues found that addition of DHA to whole blood generated a reducing substance in plasma, but with the methods available at the time they were unable to confirm the substance as ascorbate.3 The present results, in agreement with those of Christine, et al.5 show directly that erythrocytes replenish plasma ascorbate by recycling DHA and slowly releasing ascorbate into plasma. The erythrocyte is well suited to the task of maintaining plasma ascorbate. First, it is the most abundant cell in blood in terms of cytoplasmic mass, second, it has a high capacity ascorbate recycling system, and third, in contrast to nucleated cells, it does not maintain a large concentration gradient of ascorbate across the plasma
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Fig. 8. Depletion of antioxidants by ferricyanide. Erythrocytes at a 20% hematocrit were incubated at 37°C in PBS containing 5 mM D-glucose and ferricyanide at an extracellular concentration of 1.1 mM. At the indicated times, cells were separated from buffer, washed in PBS, and assayed for ascorbate (circles, N 5 6), GSH (squares, N 5 6), and a-tocopherol (triangles, N 5 4). Both ascorbate and a-tocopherol differed from GSH ( p , .05).
membrane. With regard to the latter, Evans et al.32 reported plasma ascorbate concentrations that were slightly lower than erythrocyte ascorbate concentrations in normal volunteers. We found no significant difference between erythrocyte and plasma ascorbate concentrations, indicating that erythrocytes lack an uptake mechanism capable of sustaining a transmembrane concentration gradient of ascorbate. Most nucleated cells have a sodium- and energy-dependent ascorbate transporter that can maintain a 20 – 40-fold concentration gradient, depending on the cell type.11 Human erythrocytes appear to lack such a transporter, since direct transport measurements show that ascorbate enters and leaves the erythrocyte very slowly (Fig. 4), with a half-time of hours.6,7 On the other hand, erythrocytes can transiently accumulate concentrations of ascorbate as high as 1–2 mM by a coupled uptake and reduction mechanism. In this mechanism, DHA is rapidly taken up by facilitated diffusion on the GLUT1 glucose transporter,6,12,13 an abundant membrane protein in erythrocytes.33 Once inside the cells, DHA is reduced to ascorbate, and the latter is trapped within cells. This process is sufficient to generate and maintain a tenfold concentration gradient of ascorbate across the cell membrane for about 60 min from 100 mM extracellular DHA (Fig. 2). It also results in a tenfold increase in the apparent ‘‘affinity’’ for DHA uptake, from a Km of 3– 4 mM of the high capacity site of GLUT1 for DHA12 to one of about 400 mM (Fig. 3). Such coupled uptake and reduction serves as a potent scavenging mechanism for DHA generated by oxidant stress in plasma.
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Fig. 9. Protection of a-tocopherol in LDL by ascorbate released from erythrocytes. Human LDL (;0.5 mg/ml protein) was incubated at 37°C in PBS containing 5 mM glucose and 5 mM AAPH (circles). Similar incubations were carried out with LDL and washed erythrocytes at a 40% hematocrit as follows: cells alone (squares); cells plus 100 mM DHA (diamonds); and cells plus 100 mM DHA and 4 U/ml ascorbate oxidase (triangles). In the presence of cells, the extracellular concentration of AAPH was 8.3 mM. At the indicated times, LDL in aliquots of the medium or cell supernatant was assayed for a-tocopherol content. Data are shown from 7–9 experiments. Two-way ANOVA of the average fractional decrease was significant at the p , .05 for cells 1 DHA versus the three other data sets, and for LDL alone versus the three other data sets.
The capacity of the erythrocyte to regenerate ascorbate from DHA is substantial. Ascorbate recycling can be measured indirectly as ascorbate-dependent reduction of extracellular ferricyanide.30,34 From the initial rate of ascorbate-dependent extracellular ferricyanide reduction, we have calculated that erythrocytes can regenerate the amount of ascorbate typically present in blood every 3 min.35 A similar estimate was derived in this work by measuring the initial rate of uptake and reduction of 100 mM DHA by 40% erythrocytes incubated in plasma from the same donor. The source of reducing equivalents for this high capacity recycling derives ultimately from glucose, but directly from GSH. We recently found that GSH depletion decreases the ability of erythrocytes to reduce ferricyanide.13 In the present work, we show directly that recycling of DHA to ascorbate is impaired in cells partially depleted of GSH with diamide, and that recycling is almost absent without glucose (Fig. 6). Sasaki, et al.36 have also shown in canine lens epithelial cells that glucose utilization in the hexose monophosphate shunt is required for GSH-dependent recycling of ascorbate. The mechanism of GSH-dependent ascorbate recycling is controversial.37 Direct reduction of DHA by GSH clearly plays a role, but it is difficult to explain the rapid (Fig. 2) and saturable (Fig. 3) ascorbate accumulation in erythrocytes without positing one or more enzymatic components, as well. In this regard, a GSH-
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dependent DHA reductase activity has been purified from erythrocytes,15 which may be a member of the glutaredoxin family.14 Ascorbate recycling may also serve to protect the erythrocyte against a transmembrane oxidant stress. In this work an overwhelming oxidant stress was generated outside erythrocytes using either AAPH (Fig. 7) or ferricyanide (Fig. 8). The free radical species generated outside cells by AAPH should attack a-tocopherol in the cell membrane before reaching intracellular defenses. Whereas the cell-impermeant ferricyanide may selectively oxidize intracellular ascorbate via a transmembrane oxidoreductase,38,39 it also depletes a-tocopherol in the membrane.40 At relatively high concentrations, both agents progressively oxidized intracellular ascorbate and membrane a-tocopherol, with little or no effect on GSH. The concerted decrease in ascorbate and a-tocopherol suggests that both antioxidants serve in the initial defense of the erythrocyte against oxidant stress originating outside the cell. The failure of GSH to fall under this degree of stress likely reflects its millimolar concentration in erythrocytes, and the ability of cells provided with glucose to recycle GSSG to GSH via glutathione reductase and the hexose monophosphate shunt.41 In this regard, an 83 mM extracellular AAPH concentration in plasma decreased erythrocyte ascorbate by only about 50% over 3 h (Fig. 7), in comparison to nearly complete oxidation of plasma ascorbate by 1 mM AAPH over 2 h (Fig. 1B). Despite the presence of other ascorbate-sparing antioxidants in plasma, including uric acid and albumin,42,43 this large difference attests to the high capacity of GSH-dependent ascorbate recycling in the cells. It has been shown that ascorbate can recycle a-tocopherol in LDL in the face of an oxidant stress.44,45 In this work we found that a-tocopherol in LDL was protected against oxidation by AAPH in the presence of erythrocytes (Fig. 9). Part of this protection was due to consumption by erythrocytes of oxygen free radical species generated from AAPH. However, loading erythrocytes with DHA provided even greater protection against loss of a-tocopherol in LDL than erythrocytes alone. Since this protection was reversed when ascorbate oxidase was present outside the cells, we conclude that it was due to ascorbate that had left the cells, and not to any antioxidant effect of DHA,46 or to transfer of a-tocopherol from the cells to LDL.47,48 Thus, recycling of ascorbate within erythrocytes helps to maintain ascorbate in plasma, which in turn can protect or recycle a-tocopherol in LDL. Acknowledgements—Supported by NIH grant DK50435 and a Research Grant from the American Diabetes Association. We thank Ms. Kathy Carter in the laboratories of Drs. Sergio Fazio and MacRae Linton for the preparation of the human LDL.
REFERENCES 1. Frei, B.; Stocker, R.; Ames, B. N. Antioxidant defenses and lipid peroxidation in human blood plasma. Proc. Natl. Acad. Sci. USA 85:9748 –52; 1988. 2. Frei, B.; England, L.; Ames, B. N. Ascorbate is an outstanding antioxidant in human blood plasma. Proc. Natl. Acad. Sci. USA 86:6377– 6381; 1989. 3. Borsook, H.; Davenport, H. W.; Jeffreys, C. E. P.; Warner, R. C. The oxidization of ascorbic acid and its reduction in vitro and in vivo. J. Biol. Chem. 117:237–279; 1937. 4. Dhariwal, K. R.; Washko, P. W.; Levine, M. Determination of dehydroascorbic acid using high-performance liquid chromatography with coulometric electrochemical detection. Anal. Biochem. 189:18 –23; 1990. 5. Christine, L.; Thomson, G.; Iggo, B.; Brownie, A. C.; Stewart, C. P. The reduction of dehydroascorbic acid by human erythrocytes. Clin. Chim. Acta 1:557–569; 1956. 6. Hughes, R. E.; Maton, S. C. The passage of vitamin C across the erythrocyte membrane. Br. J. Haematol. 14:247–53; 1968. 7. Okamura, M. Uptake of L-ascorbic acid and L-dehydroascorbic acid by human erythrocytes and HeLa cells. J. Nutr. Sci. Vitaminol. 25:269 –279; 1979. 8. Washko, P. W.; Wang, Y.; Levine, M. Ascorbic acid recycling in human neutrophils. J. Biol. Chem. 268:15531–15535; 1993. 9. Washko, P.; Rotrosen, D.; Levine, M. Ascorbic acid transport and accumulation in human neutrophils. J. Biol. Chem. 264:18996 – 19002; 1989. 10. Mann, G. V.; Newton, P. The membrane transport of ascorbic acid. Ann. NY Acad. Sci. 258:243–252; 1975. 11. Rose, R. C. Transport of ascorbic acid and other water-soluble vitamins. Biochim. Biophys. Acta 947:335–366; 1988. 12. Vera, J. C.; Rivas, C. I.; Fischbarg, J.; Golde, D. W. Mammalian facilitative hexose transporters mediate the transport of dehydroascorbic acid. Nature 364:79 – 82; 1993. 13. May, J. M.; Qu, Z. C.; Whitesell, R. R.; Cobb, C. E. Ascorbate recycling in human erythrocytes: Role of GSH in reducing dehydroascorbate. Free Radic. Biol. Med. 20:543–551; 1996. 14. Wells, W. W.; Xu, D. P.; Yang, Y. F.; Rocque, P. A. Mammalian thioltransferase (glutaredoxin) and protein disulfide isomerase have dehydroascorbate reductase activity. J. Biol. Chem. 265: 15361–15364; 1990. 15. Mieyal, J. J.; Starke, D. W.; Gravina, S. A.; Dothey, C.; Chung, J. S. Thioltransferase in human red blood cells: purification and properties. Biochemistry 30:6088 – 6097; 1991. 16. Winterbourn, C. C.; Stern, A. Human red cells scavenge extracellular hydrogen peroxide and inhibit formation of hypochlorous acid and hydroxyl radical. J. Clin. Invest. 80:1486 –1491; 1987. 17. Agar, N. S.; Sadrzadeh, S. M. H.; Hallaway, P. E.; Eaton, J. W. Erythrocyte catalase. A somatic oxidant defense? J. Clin. Invest. 319 –321; 1986. 18. Mino, M.; Nishida, Y.; Murata, K.; Takegawa, M.; Katsui, G.; Yuguchi, Y. Studies on the factors influencing the hydrogen peroxide hemolysis test. J. Nutr. Sci Vitaminol. 24:383–395; 1978. 19. Yamamoto, Y.; Niki, E.; Eguchi, J.; Kamiya, Y.; Shimasaki, H. Oxidation of biological membranes and its inhibition. Free radical chain oxidation of erythrocyte ghost membranes by oxygen. Biochim. Biophys. Acta 819:29 –36; 1985. 20. Tamai, H.; Miki, M.; Mino, M. Hemolysis and membrane lipid changes induced by xanthine oxidase in vitamin E deficient red cells. J. Free Rad. Biol. Med. 2:49 –56; 1986. 21. Trotta, R. J.; Sullivan, S. G.; Stern, A. Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxdie. The relative roles of haem- and glutathione-dependent decomposition of t-butyl hydroperoxide and membrane lipid hydroperoxides in lipid peroxidation and haemolysis. Biochem. J. 212:759 –772; 1983. 22. Meister, A. Glutathione-ascorbic acid antioxidant system in animals. J. Biol. Chem. 269:9397–9400; 1994.
Erythrocyte ascorbate recycling 23. Havel, R. J.; Eder, H. A.; Bragdon, J. H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. Clin. Invest. 34:1345–1353; 1955. 24. Iheanacho, E. N.; Hunt, N. H.; Stocker, R. Vitamin C redox reactions in blood of normal and malaria-infected mice studied with isoascorbate as a nonisotopic marker. Free Radic. Biol. Med. 18:543–552; 1995. 25. Pachla, L. A.; Kissinger, P. T. Analysis of ascorbic acid by liquid chromatography with amperometric detection. Methods Enzymol. 62:15–24; 1979. 26. Tietze, F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: Applications to mammalian blood and other tissues. Anal. Biochem. 27:502–522; 1969. 27. Lang, J. K.; Gohil, K.; Packer, L. Simultaneous determination of tocopherols, ubiquinols, and ubiquinones in blood, plasma, tissue homogenates, and subcellular fractions. Anal. Biochem. 157:106 –116; 1986. 28. Takeda, H.; Shibuya, T.; Yanagawa, K.; Kanoh, H.; Takasaki, M. Simultaneous determination of a-tocopherol and a-tocopherolquinone by high-performance liquid chromatography and coulometric detection in the redox mode. J Chromatogr. 722: 287–294; 1996. 29. Sato, Y.; Kamo, S.; Takahashi, T.; Suzuki, Y. Mechanism of free radical-induced hemolysis of human erythrocytes: hemolysis by water-soluble radical initiator. Biochemistry 34:8940 – 8949; 1995. 30. Orringer, E. P.; Roer, M. E. An ascorbate-mediated transmembrane-reducing system of the human erythrocyte. J. Clin. Invest. 63:53–58; 1979. 31. Kellie, A. E.; Zilva, S. S. CXXVII. The catalytic oxidation of ascorbic acid. Biochem. J. 29:1028 –1035; 1935. 32. Evans, R. M.; Currie, L.; Campbell, A. The distribution of ascorbic acid between various cellular components of blood, in normal individuals, and its relation to the plasma concentration. Br. J. Nutr. 47:473– 482; 1982. 33. Baldwin, S. A.; Baldwin, J. M.; Lienhard, G. E. Monosaccharide transporter of the human erythrocyte. Characterization of an improved preparation. Biochemistry 21:3836 –3842; 1982. 34. Schipfer, W.; Neophytou, B.; Trobisch, R.; Groiss, O.; Goldenberg, H. Reduction of extracellular potassium ferricyanide by transmembrane NADH:(acceptor)oxidoreductase of human erythrocytes. Int. J. Biochem. 17:819 – 823; 1985. 35. May, J. M.; Qu, Z. -C.; Whitesell, R. R. Ascorbic acid recycling enhances the antioxidant reserve of human erythrocytes. Biochemistry 34:12721–12728; 1995. 36. Sasaki, H.; Giblin, F. J.; Winkler, B. S.; Chakrapani, B.; Leverenz, V.; Shu, C. C. A protective role for glutathione-dependent
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reduction of dehydroascorbic acid in lens epithelium. Invest. Opthalmol. Vis. Sci. 36:1804 –1817; 1995. Winkler, B. S.; Orselli, S. M.; Rex, T. S. The redox couple between glutathione and ascorbic acid: A chemical and physiological perspective. Free Radic. Biol. Med. 17:333–349; 1994. Zamudio, I.; Cellino, M.; Canessa-Fischer, M. The relation between membrane structure and NADH: (Acceptor) Oxidoreductase activity of erythrocyte ghosts. Arch. Biochem. Biophys. 129: 336 –345; 1969. Crane, F. L.; Lo¨w, H.; Clark, M. G. Plasma Membrane Redox Enzymes. In: Martonosi, A. N., ed. The Enzymes of Biological Membranes, Second Edition. New York: Plenum Press; 1985; 465–510. May, J. M.; Qu, Z. C.; Morrow, J. D. Interaction of ascorbate and a-tocopherol in resealed human erythrocyte ghosts—Transmembrane electron transfer and protection from lipid peroxidation. J. Biol. Chem. 271:10577–10582; 1996. Meister, A. Glutathione metabolism. Methods Enzymol. 251:3–7; 1995. Sevanian, A.; Davies, K. J.; Hochstein, P. Serum urate as an antioxidant for ascorbic acid. Am. J. Clin. Nutr. 54:1129S–1134S; 1991. Halliwell, B.; Gutteridge, J. M. C. The antioxidants of human extracellular fluids. Arch. Biochem. Biophys. 280:1– 8; 1990. Jialal, I.; Grundy, S. M. Preservation of the endogenous antioxidants in low density lipoprotein by ascorbate but not probucol during oxidative modification. J. Clin. Invest. 87:597– 601; 1991. Jialal, I.; Vega, G. L.; Grundy, S. M. Physiologic levels of ascorbate inhibit the oxidative modification of low density lipoprotein. Atherosclerosis 82:185–191; 1990. Retsky, K. L.; Freeman, M. W.; Frei, B. Ascorbic acid oxidation product(s) protect human low density lipoprotein against atherogenic modification. Anti- rather than prooxidant activity of vitamin C in the presence of transition metal ions J. Biol. Chem. 268:1304 –1309; 1993. Bieri, J. G.; Evarts, R. P.; Thorp, S. Factors affecting the exchange of tocopherol between red blood cells and plasma. Am. J. Clin. Nutr. 30:686 – 690; 1977. Bjornson, L. K.; Gniewkowski, C.; Kayden, H. J. Comparison of exchange of alpha-tocopherol and free cholesterol between rat plasma lipoproteins and erythrocytes. J. Lipid Res. 16:39–53; 1975.
ABBREVIATIONS
AAPH 2,29-azobis(2-amidinopropane)hydrochloride DHA dehydroascorbate PBS phosphate-buffered saline
PII S0891-5849(97)00351-1
Original Contribution ERYTHROCYTE ASCORBATE RECYCLING: ANTIOXIDANT EFFECTS IN BLOOD SHALU MENDIRATTA, ZHI-CHAO QU,
and JAMES
M. MAY
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA (Received 6 May 1997; Revised 15 September 1997; Accepted 16 September 1997)
Abstract—Ascorbic acid is an important antioxidant in human plasma, but requires efficient recycling from its oxidized forms to avoid irreversible loss. Human erythrocytes prevented oxidation of ascorbate in autologous plasma, an effect that required recycling of ascorbate within the cells. Erythrocytes had a high capacity to take up dehydroascorbate, the two-electron oxidized product of ascorbate, and to reduce it to ascorbate. Uptake and conversion of dehydroascorbate to ascorbate was saturable, was half-maximal at 400 mM dehydroascorbate, and achieved a maximal intracellular ascorbate concentration of 1.5 mM. In the presence of 100 mM dehydroascorbate, erythrocytes had the capacity to regenerate a 35 mM ascorbate concentration in blood every 3 min. Ascorbate recycling from DHA required intracellular GSH. Depletion of erythrocyte GSH by more than 50% with diamide did not acutely affect the cellular ascorbate content, but did impair the subsequent ability of GSH-depleted cells to recycle dehydroascorbate to ascorbate. Whereas erythrocyte ascorbate recycling was coupled to GSH, an overwhelming extracellular oxidant stress depleted both ascorbate and a-tocopherol before the GSH content of cells fell appreciably. Recycled ascorbate was released from cells into plasma, but at a rate less than one tenth that of dehydroascorbate uptake and conversion to ascorbate. Nonetheless, ascorbate released from cells protected endogenous a-tocopherol in human LDL from oxidation by a water soluble free radical initiator. These results suggests that recycling of ascorbate in erythrocytes helps to maintain the antioxidant reserve of whole blood. © 1998 Elsevier Science Inc. Keywords—Antioxidants, Oxidant stress, Oxidation-reduction, Glutathione, Biological transport, Free radical
INTRODUCTION
The intracellular conversion of DHA to ascorbate is largely dependent on the GSH system,7,13 and may be enzyme-mediated. Ascorbate generated in this manner rapidly accumulates against a transmembrane concentration gradient. With time this gradient gradually dissipates, presumably due to efflux of ascorbate from the cell.5 Although release of recycled ascorbate from cells seems a plausible mechanism to protect plasma components from oxidation, this has not been demonstrated. A related question is whether and to what extent intracellular ascorbate recycling protects the erythrocyte itself from oxidant stress, especially since this cell is equipped with so many different types of defense against oxygen free radicals.16,17 Of the non-enzymatic defenses of the erythrocyte, important roles have been established for a-tocopherol in the membrane18 –20 and GSH in the cytoplasm.21,22 A role for ascorbate would be supported by demonstrating that the vitamin is depleted with or before a-tocopherol and GSH in the face of an oxidant stress. The results of this work show that recycling of
Ascorbic acid, or vitamin C, is a primary antioxidant in plasma, since it is consumed first during an oxidant stress, and since peroxidation of plasma lipids does not occur until ascorbate is depleted.1,2 The two-electron oxidation product of ascorbate, dehydroascorbate (DHA), is labile at physiologic pH and temperature (halflife 5 5–7 min).3,4 Unless reduced back to ascorbate, DHA undergoes irreversible ring-opening to 2,3-diketogulonic acid. Dehydroascorbate can be reduced or recycled to ascorbate in blood by both erythrocytes5–7 and neutrophils,8,9 but not by plasma alone.3 Quantitatively the erythrocyte is the most likely cell to fulfill this function in blood. Erythrocytes rapidly take up DHA on the glucose transporter10 –12 and reduce it to ascorbate.5–7 Address reprint request to: J. M. May, Department of Medicine, 736 Medical Research Building II, Vanderbilt University School of Medicine, Nashville, TN 37232-6303; Tel: 615-936-1653; Fax: 615-9361667; E-Mail: [email protected]. 789
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ascorbate in erythrocytes maintains both intracellular and extracellular levels of the vitamin, that extracellular ascorbate spares a-tocopherol in LDL from oxidation, and that ascorbate is in the first line of defense against an overwhelming external oxidant stress to the erythrocyte. EXPERIMENTAL PROCEDURES
Cell preparation Blood was drawn from normal volunteers by venipuncture and anticoagulated with heparin. Erythrocytes were washed three times by centrifugation in ten volumes of phosphate-buffered saline (PBS), which consisted of 154 mM NaCl and 12.5 mM Na2HPO4 in deionized water, adjusted to pH 7.4. The buffy coat of white cells was carefully removed with each wash. The 1.019 –1.063 density fraction of LDL was prepared by the method of Havel. et al.23 Measurement of plasma and intracellular ascorbate and glutathione concentrations Following incubations as noted, erythrocytes were pelleted by centrifugation and ascorbate concentrations in cells and plasma were measured by HPLC with electrochemical detection. In most experiments, plasma was cooled on ice and ultrafiltered as described below prior to assay of ascorbate. In an alternate method, 100 ml of plasma was added to 0.9 ml of ice-cold 50 mM perchloric acid with mixing. After several minutes on ice, the sample was microfuged for 1 min, and an aliquot of the supernatant was taken for HPLC assay of ascorbate. The ultrafiltration method required immediate sample processing to avoid loss of ascorbate, whereas ascorbate was stable for several hours in cold perchloric acid. For assay of intracellular ascorbate, 100 ml of packed cells was diluted with 0.3 ml of PBS, quickly frozen in dry iceacetone, and the frozen hemolysate was allowed to thaw on ice. The hemolysate was ultrafiltered as described by Iheanacho, et al.24 Briefly, the hemolysate was transferred to a Centricon-10 filter apparatus (Amicon, Inc., Beverly, MA) and centrifuged at 4°C for 30 min at 5000 (3g. This resulted in 0.3– 0.4 ml of a clear ultrafiltrate, which was diluted 10 –20-fold with mobile phase before assay. In hemolysates that were spiked with D-isoascorbate in amounts that were 10 –20% of the hemolysate ascorbate content, no losses of isoascorbate during the ultrafiltration step could be detected. It is likely that GSH present in the hemolysate helped to prevent loss of both ascorbate and added isoascorbate during preparation of the sample for analysis. Ascorbate and GSH were assayed by HPLC using the ion-pairing method of Pachla and Kissinger.25 The mo-
bile phase consisted of 80 mM sodium acetate, 1 mM tridecylamine, 1 mM EDTA, and 30% methanol, pH 5.2 before methanol addition. The mobile phase was pumped with an ESA Model 5700 pump (Bedford, MA), filtered with an ESA graphite carbon in-line filter, and preconditioned with an ESA Model 5020 guard cell set at 0.5 volts in the oxidizing mode. Samples were injected with a BioRad AS-100 injector in a volume of 100 ml. Separation was carried out on a 10 3 1 cm Waters RadiaPak C18 column (300 mm 4 m), with a 4 mm guard column of the same packing material. Detection was accomplished with an ESA Model 5100A detector using a Model 5010 analytical cell with the first electrode set at zero and the detecting electrode set at 0.4 volts. At a flow rate of 1 ml/min, ascorbate eluted at 8 –9.5 min, and GSH eluted at 10 –11.5 min, with complete separation of the peaks. The assay sensitivity was 10 pmol/sample for ascorbate and 1 nmol/sample for GSH. The concentration of oxidized GSH (GSSG) was measured using the enzymatic cycling assay of Tietze26 as previously described.13 Measurement of a-tocopherol in erythrocytes and in human LDL Cell and LDL contents of a-tocopherol were measured by HPLC with electrochemical detection according to the method of Lang, et al.27 The a-tocopherol in LDL was measured in buffer alone or in buffer from which cells had been removed by centrifugation. For assay of erythrocyte a-tocopherol, packed cells (0.2 ml) were chilled on ice, mixed with 50 ml of a 10 mg/ml solution of butylated hydroxytoluene in methanol, and lysed with 0.8 ml of ice-cold 3% (w/v) sodium dodecyl sulfate. This hemolysate was further diluted with 1 ml of ice-cold 5 mM sodium phosphate buffer, pH 7.4, that contained 10 mM ascorbic acid. The hemolysate was vortexed, treated with 2 ml of reagent alcohol (95% ethanol and 5% isopropanol, v/v), and mixed again. Following addition of 2 ml of hexane, the sample was vortexed vigorously for 1–2 min, then centrifuged to separate the phases. One ml of the hexane upper phase was taken to dryness under nitrogen, and the residue was dissolved in 0.5 ml of a mixture of methanol and reagent alcohol (1:1, v/v) for HPLC injection. Samples were chromatographed in the isocratic mode on a Waters DeltaPak C18 column (300 mm 5 m) with a 4 mm guard column of the same packing material. The mobile phase was 94% methanol and 6% water containing 100 mM sodium acetate. Pre-column conditioning of the buffer was found to be unnecessary. Tocopherol detection was carried out by a simplification of the reduction-oxidation method described by Takeda, et al.28 An ESA model 5021 conditioning cell, preceded by a graphite in-line filter, was placed just after the
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analytical column and set in the reducing mode at 20.5 volts. An ESA model 5011 analytical cell was used for detection, with the first electrode in the analytical cell set at 20.5 volts, and the second detecting electrode set at 0.6 volts. At a flow rate of 1 ml/min, b-tocopherol eluted at 5.6 –5.9 min, and a-tocopherol eluted at 6.1– 6.4 min. The assay sensitivity for a-tocopherol was 2–5 pmol. Other assays Hemolysis of erythrocytes was measured as the increase in absorbance at 540 nm of erythrocyte supernatants compared to absorbance produced by hemolysis of a known volume of erythrocytes in water.29 Protein in LDL was measured using the BCA method (Pierce Chemical Co., Rockford, IL). The amounts of ascorbate, GSH, a-tocopherol, and ferrocyanide were expressed relative to the intracellular water space of the erythrocytes,30 which was confirmed in separate studies to be 70% of the packed cell volume. Data and statistical analysis Curve-fitting was carried out by nonlinear leastsquares regression in the graphics software packaged FigP (Biosoft Inc., Cambridge, UK). Statistical significance was assessed by one- or two-way ANOVA using the statistical software package Sigmastat 2.0 (Jandel Scientific, San Rafael, CA). All results are shown as mean 6 SEM. RESULTS
Incubation of plasma in vitro exposed to air at 37°C resulted in a decrease in the plasma ascorbate concentration over 4 h (Fig. 1A). The results are expressed as a fraction of the zero-time value, since there was marked variation in the endogenous plasma and erythrocyte ascorbate contents from different donors (from 15 to over 100 mM). The decline in plasma ascorbate was completely prevented when 40% erythrocytes from the same donor were incubated with the plasma, suggesting that the cells had helped to maintain the extracellular ascorbate concentration. The initial concentration of ascorbate in plasma was 96 6 14% of that in erythrocytes from the same donor (N 5 4), so the maintenance of plasma ascorbate could not be ascribed simply to diffusion of a high initial concentration of ascorbate out of cells. Intracellular concentrations of ascorbate and GSH were unaffected by incubation of cells in plasma from the original donor (results not shown). When a stronger oxidant challenge was generated in plasma by a 1 mM concentration of the water soluble free
Fig. 1. Erythrocyte-dependent protection against ascorbate oxidation. Panel A: freshly prepared plasma was incubated at 37°C in the absence (circles) or presence (squares) of washed 40% erythrocytes derived from the same plasma. At the indicated times, plasma and plasma from cells were assayed for ascorbate, which is expressed as a fraction of the zero-time value. In 7 experiments, fractional ascorbate levels in plasma alone were different than those in plasma containing cells ( p , .02). Panel B: plasma was incubated at 37°C in the absence (circles) or presence (squares) of 40% erythrocytes derived from the same plasma and 1 mM AAPH per total volume. Data from 6 experiments are shown as a fraction of the initial value. Fractional ascorbate levels in plasma alone were different than those in plasma containing cells ( p , .05).
radical initiator 2,29-azobis(2-amidinopropane)hydrochloride (AAPH) (Wako Chemical Co., Richmond, VA), erythrocytes prevented significant loss of extracellular ascorbate, in contrast to nearly complete loss of ascorbate in plasma alone (Fig. 1B). Since AAPH should not enter cells, its effective extracellular concentration was about 70% higher in the incubations with cells, which further accentuates the differences observed. Intracellular ascorbate and GSH were again unchanged during the external challenge with AAPH (results not shown). The preservation of extracellular ascorbate by erythrocytes in these experiments may be due in part to DHA uptake and
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Fig. 2. Time course of DHA-dependent ascorbate appearance in erythrocytes and release into plasma. Washed, 40% erythrocytes were incubated at 37°C in the plasma from which they were originally derived in the presence of 100 mM DHA. At the indicated times, cells and plasma were rapidly separated in a microfuge, the cells were washed three times by centrifugation in 3 volumes of PBS, and the cells and original plasma were assayed for ascorbate. Extracellular (circles) and intracellular (squares) ascorbate concentrations are shown for 4 experiments. The increase in both curves from initial values was significant ( p , .05).
conversion to ascorbate within the cells, with subsequent leak of ascorbate across the cell membrane. This possibility was examined by measurement of ascorbate in plasma and in erythrocytes following incubation of cells with DHA. Erythrocytes rapidly internalized 100 mM DHA, which is seen as an increase in the intracellular ascorbate content (Fig. 2). Uptake resulted in a maximal intracellular ascorbate concentration of ascorbate that was more than ten-fold higher than the initial extracellular ascorbate concentration, although this level could not be sustained for more than about 60 min. The extracellular ascorbate concentration doubled in response to DHA loading, in support of the notion that it originated from intracellular ascorbate. Therefore, release of ascorbate can account for at least part of the protective effect of erythrocytes on plasma ascorbate. The intracellular concentration of GSH was unchanged during the uptake of 100 mM DHA in the presence of glucose (results not shown). Over the first 5 min of incubation, 173 nmol of ascorbate was generated per ml of erythrocyte cytoplasm, or ;54 nmol per ml of blood at a 45% hematocrit. Thus, under these conditions, a 30 –35 mM concentration of ascorbate in blood can be regenerated in about 3 min. Release of ascorbate into plasma is much slower than the recycling capacity, however. During incubation of erythrocytes with increasing concentrations of DHA for 1 h in plasma, the intracel-
Fig. 3. Uptake and reduction of increasing concentrations of DHA by erythrocytes. Washed, 40% erythrocytes were incubated at 37°C with the indicated concentrations of DHA in the plasma from which they were derived. After 60 min, ascorbate concentrations were determined in plasma and in cells washed as described in the legend to Fig. 2. Extracellular (circles) and intracellular (squares) ascorbate concentrations are shown for 6 experiments, with the smooth line for the intracellular ascorbate depicting the fit to a rectangular hyperbola (apparent Km 5 403 mM, Vmax 5 1678 nmol/(ml erythrocytes-h), r 5 0.98).
lular ascorbate concentration increased to a maximum of almost 1.5 mM (Fig. 3). When these data were fit to a rectangular hyperbola, an apparent half-maximal effect was observed at about 400 mM extracellular DHA. The extracellular ascorbate concentration rose with increasing extracellular DHA to almost tenfold that of plasma at an initial extracellular DHA concentration of 5 mM. In the presence of plasma that contained glucose, intracellular GSH and the GSSG/GSH ratio were not significantly affected, nor was hemolysis evident (results not shown). These data show that erythrocytes have a substantial, but saturable, ability to recycle DHA to ascorbate, and that the latter accumulates, at least transiently, against a concentration gradient. Loss or efflux of ascorbate from the cells is much slower than uptake and recycling, but is proportional to the intracellular ascorbate concentration. Incubation of erythrocytes with increasing concentrations of ascorbate for 3 h resulted in a linear increase in intracellular ascorbate (Fig. 4). At low initial ascorbate concentrations, intracellular ascorbate concentrations at the end of 3 h were consistently greater than those outside cells. Such accumulation of ascorbate against a gradient was much less marked than observed for DHA (Fig. 3), and likely reflects uptake and recycling of a small amount of DHA that had been generated extracellularly. The cell contents of GSH and GSSG were unchanged during the incubation with ascorbate (results not shown).
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Fig. 4. Uptake of ascorbate by erythrocytes in plasma. Washed, 40% erythrocytes were incubated at 37°C with increasing concentrations of ascorbate in the plasma from which they were derived. At 3 h, cells and plasma were separated, the cells were washed as described in the legend to Fig. 2, and plasma (circles) and cells (squares) were assayed for ascorbate Data from 5 experiments are shown.
Whereas erythrocyte GSH was unaffected by DHA loading in plasma containing glucose, a role for GSH in recycling ascorbate was evident when intracellular GSH was partially oxidized with diamide. Incubation of cells with increasing amounts of diamide in the absence of glucose resulted in a decrease in intracellular GSH to about 35% of the initial value (Fig. 5A). When GSH-depleted cells were incubated for an additional 30 min in the presence of 5 mM D glucose, the diamide-induced decrease in GSH was reversed (Fig. 5A). Intracellular ascorbate tended to decrease as the concentration of diamide was increased, whether or not glucose was present (Fig. 5B). Nonetheless, the ability of GSH to maintain or recycle endogenous ascorbate was minimally compromised by oxidation of more than 50% of cell GSH. On the other hand, when cells depleted of GSH by diamide were washed several times and then incubated with 500 mM DHA, their ability to generate ascorbate was clearly impaired (Fig. 6). Diamide pretreatment decreased recycling of DHA to ascorbate by 86% in the absence of glucose, and decreased it by about 50% in cells that were subsequently exposed to glucose. Glucose was required for recycling of ascorbate from DHA, whether or not the cells had been exposed to diamide. In the absence of glucose, intracellular GSH was decreased 47% by DHA alone, and 72% by both DHA and diamide. Whereas addition of glucose to diamide-treated cells completely restored intracellular GSH to control values, DHA reduction to ascorbate did not fully recover (Fig. 6). GSH-dependent ascorbate recycling was insufficient to protect even endogenous ascorbate when cells were exposed to an overwhelming oxidant stress, even the presence of glucose. In the experiments shown in Fig. 7,
Fig. 5. Diamide oxidation of erythrocyte GSH. Erythrocytes were incubated at a 10% hematocrit in glucose-free PBS with the indicated concentration of diamide for 10 min on ice, washed three times by centrifugation in 10 volumes of ice-cold glucose-free PBS, and resuspended to a 10% hematocrit in the absence (circles) or presence (squares) of 5 mM D-glucose. Following a 10 min incubation at 37°C, cells and buffer were separated, and GSH (Panel A) and ascorbate (Panel B) contents were determined. The decrease in GSH induced by diamide significant ( p , .001) in the three experiments shown.
cells were incubated for up to 200 min with an extracellular AAPH concentration of 83 mM. This contrasts with the 1.7 mM extracellular concentration of AAPH used in the experiments shown in Fig. 1B. AAPH at this high concentration caused progressive and similar decreases in both intracellular ascorbate and membrane a-tocopherol (Fig. 7A). GSH decreased at about half the rate of ascorbate and a-tocopherol. Hemolysis also increased over the last half of the incubation. (Fig. 7B). Similar results were observed when erythrocytes were incubated in PBS with the cell-impermeant oxidant ferricyanide. PBS, rather than plasma, was used to avoid direct reaction of ferricyanide with ascorbate and other antioxidants present in plasma. Over the first 30 min of incubation, ascorbate was depleted by ferricyanide to about 50% of its initial value, despite the presence of glucose (Fig. 8). In these experiments, a-tocopherol showed a progressive, albeit slower initial decline than
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Fig. 6. DHA loading of diamide-treated erythrocytes. Control and diamide-treated erythrocytes were prepared as described in the legend to Fig. 5, and incubated at a 10% hematocrit for an additional 30 min at 37°C with 500 mM DHA in the presence or absence of 5 mM D-glucose, as indicated. Ascorbate (open bars) and GSH (hatched bars) contents are shown from three experiments, with an asterisk (*) indicating p , .05 versus corresponding samples in the preceding group of bars that were treated with glucose.
ascorbate. Intracellular GSH was unaffected by ferricyanide. These results with two different cell-impermeant agents suggest that ascorbate and a-tocopherol are more sensitive to an external oxidant stress than is GSH. That is, ascorbate can be selectively oxidized before GSH stores are significantly depleted. Whereas release of recycled ascorbate is much slower than the recycling process itself, it was nonetheless possible to show that ascorbate derived from DHA-loaded erythrocytes protects LDL form oxidation. In the presence of AAPH, the a-tocopherol content of human LDL decreased over 4 h (Fig. 9). When erythrocytes at a 40% hematocrit were included in the incubation, the rate of a-tocopherol oxidation in the LDL was significantly slowed, although there was no lag-phase. When erythrocytes were loaded with 100 mM DHA during the incubation, little loss of a-tocopherol in LDL occurred until after the second hour of incubation, indicative of a lagphase. When ascorbate oxidase was included in the incubation, the protection by DHA-loading was abolished (Fig. 9). Under these conditions, ascorbate oxidase does not significantly deplete ascorbate in DHA-loaded cells (results not shown). The failure of DHA-loading to protect extracellular LDL in the presence of ascorbate oxidase suggests that the effect was due to ascorbate that had diffused out of the cells. DISCUSSION
Since human cells cannot synthesize ascorbate, the vitamin must be delivered to them by the bloodstream.
Fig. 7. Depletion of antioxidants with high-dose AAPH. Washed, 40% erythrocytes were incubated at 37°C in the plasma from which they were derived with AAPH at an extracellular concentration of 83 mM. Panel A: at the times indicated, cells were separated from plasma, washed in PBS, and assayed for ascorbate (circles, N 5 6), GSH (squares, N 5 6), and a-tocopherol (triangles, N 5 5), which are shown as a fraction of the initial value. The decreases in ascorbate and a-tocopherol were greater than the fall in GSH ( p , .05). Panel B: hemolysis was measured in five experiments. The increase in hemolysis was significant for the last three time points sampled ( p , .05).
Ascorbate is highly susceptible to oxidation in plasma,1,2 but is protected in whole blood by erythrocytes.3,31 During their investigation of the mechanism of this protection, Borsook and colleagues found that addition of DHA to whole blood generated a reducing substance in plasma, but with the methods available at the time they were unable to confirm the substance as ascorbate.3 The present results, in agreement with those of Christine, et al.5 show directly that erythrocytes replenish plasma ascorbate by recycling DHA and slowly releasing ascorbate into plasma. The erythrocyte is well suited to the task of maintaining plasma ascorbate. First, it is the most abundant cell in blood in terms of cytoplasmic mass, second, it has a high capacity ascorbate recycling system, and third, in contrast to nucleated cells, it does not maintain a large concentration gradient of ascorbate across the plasma
Erythrocyte ascorbate recycling
Fig. 8. Depletion of antioxidants by ferricyanide. Erythrocytes at a 20% hematocrit were incubated at 37°C in PBS containing 5 mM D-glucose and ferricyanide at an extracellular concentration of 1.1 mM. At the indicated times, cells were separated from buffer, washed in PBS, and assayed for ascorbate (circles, N 5 6), GSH (squares, N 5 6), and a-tocopherol (triangles, N 5 4). Both ascorbate and a-tocopherol differed from GSH ( p , .05).
membrane. With regard to the latter, Evans et al.32 reported plasma ascorbate concentrations that were slightly lower than erythrocyte ascorbate concentrations in normal volunteers. We found no significant difference between erythrocyte and plasma ascorbate concentrations, indicating that erythrocytes lack an uptake mechanism capable of sustaining a transmembrane concentration gradient of ascorbate. Most nucleated cells have a sodium- and energy-dependent ascorbate transporter that can maintain a 20 – 40-fold concentration gradient, depending on the cell type.11 Human erythrocytes appear to lack such a transporter, since direct transport measurements show that ascorbate enters and leaves the erythrocyte very slowly (Fig. 4), with a half-time of hours.6,7 On the other hand, erythrocytes can transiently accumulate concentrations of ascorbate as high as 1–2 mM by a coupled uptake and reduction mechanism. In this mechanism, DHA is rapidly taken up by facilitated diffusion on the GLUT1 glucose transporter,6,12,13 an abundant membrane protein in erythrocytes.33 Once inside the cells, DHA is reduced to ascorbate, and the latter is trapped within cells. This process is sufficient to generate and maintain a tenfold concentration gradient of ascorbate across the cell membrane for about 60 min from 100 mM extracellular DHA (Fig. 2). It also results in a tenfold increase in the apparent ‘‘affinity’’ for DHA uptake, from a Km of 3– 4 mM of the high capacity site of GLUT1 for DHA12 to one of about 400 mM (Fig. 3). Such coupled uptake and reduction serves as a potent scavenging mechanism for DHA generated by oxidant stress in plasma.
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Fig. 9. Protection of a-tocopherol in LDL by ascorbate released from erythrocytes. Human LDL (;0.5 mg/ml protein) was incubated at 37°C in PBS containing 5 mM glucose and 5 mM AAPH (circles). Similar incubations were carried out with LDL and washed erythrocytes at a 40% hematocrit as follows: cells alone (squares); cells plus 100 mM DHA (diamonds); and cells plus 100 mM DHA and 4 U/ml ascorbate oxidase (triangles). In the presence of cells, the extracellular concentration of AAPH was 8.3 mM. At the indicated times, LDL in aliquots of the medium or cell supernatant was assayed for a-tocopherol content. Data are shown from 7–9 experiments. Two-way ANOVA of the average fractional decrease was significant at the p , .05 for cells 1 DHA versus the three other data sets, and for LDL alone versus the three other data sets.
The capacity of the erythrocyte to regenerate ascorbate from DHA is substantial. Ascorbate recycling can be measured indirectly as ascorbate-dependent reduction of extracellular ferricyanide.30,34 From the initial rate of ascorbate-dependent extracellular ferricyanide reduction, we have calculated that erythrocytes can regenerate the amount of ascorbate typically present in blood every 3 min.35 A similar estimate was derived in this work by measuring the initial rate of uptake and reduction of 100 mM DHA by 40% erythrocytes incubated in plasma from the same donor. The source of reducing equivalents for this high capacity recycling derives ultimately from glucose, but directly from GSH. We recently found that GSH depletion decreases the ability of erythrocytes to reduce ferricyanide.13 In the present work, we show directly that recycling of DHA to ascorbate is impaired in cells partially depleted of GSH with diamide, and that recycling is almost absent without glucose (Fig. 6). Sasaki, et al.36 have also shown in canine lens epithelial cells that glucose utilization in the hexose monophosphate shunt is required for GSH-dependent recycling of ascorbate. The mechanism of GSH-dependent ascorbate recycling is controversial.37 Direct reduction of DHA by GSH clearly plays a role, but it is difficult to explain the rapid (Fig. 2) and saturable (Fig. 3) ascorbate accumulation in erythrocytes without positing one or more enzymatic components, as well. In this regard, a GSH-
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dependent DHA reductase activity has been purified from erythrocytes,15 which may be a member of the glutaredoxin family.14 Ascorbate recycling may also serve to protect the erythrocyte against a transmembrane oxidant stress. In this work an overwhelming oxidant stress was generated outside erythrocytes using either AAPH (Fig. 7) or ferricyanide (Fig. 8). The free radical species generated outside cells by AAPH should attack a-tocopherol in the cell membrane before reaching intracellular defenses. Whereas the cell-impermeant ferricyanide may selectively oxidize intracellular ascorbate via a transmembrane oxidoreductase,38,39 it also depletes a-tocopherol in the membrane.40 At relatively high concentrations, both agents progressively oxidized intracellular ascorbate and membrane a-tocopherol, with little or no effect on GSH. The concerted decrease in ascorbate and a-tocopherol suggests that both antioxidants serve in the initial defense of the erythrocyte against oxidant stress originating outside the cell. The failure of GSH to fall under this degree of stress likely reflects its millimolar concentration in erythrocytes, and the ability of cells provided with glucose to recycle GSSG to GSH via glutathione reductase and the hexose monophosphate shunt.41 In this regard, an 83 mM extracellular AAPH concentration in plasma decreased erythrocyte ascorbate by only about 50% over 3 h (Fig. 7), in comparison to nearly complete oxidation of plasma ascorbate by 1 mM AAPH over 2 h (Fig. 1B). Despite the presence of other ascorbate-sparing antioxidants in plasma, including uric acid and albumin,42,43 this large difference attests to the high capacity of GSH-dependent ascorbate recycling in the cells. It has been shown that ascorbate can recycle a-tocopherol in LDL in the face of an oxidant stress.44,45 In this work we found that a-tocopherol in LDL was protected against oxidation by AAPH in the presence of erythrocytes (Fig. 9). Part of this protection was due to consumption by erythrocytes of oxygen free radical species generated from AAPH. However, loading erythrocytes with DHA provided even greater protection against loss of a-tocopherol in LDL than erythrocytes alone. Since this protection was reversed when ascorbate oxidase was present outside the cells, we conclude that it was due to ascorbate that had left the cells, and not to any antioxidant effect of DHA,46 or to transfer of a-tocopherol from the cells to LDL.47,48 Thus, recycling of ascorbate within erythrocytes helps to maintain ascorbate in plasma, which in turn can protect or recycle a-tocopherol in LDL. Acknowledgements—Supported by NIH grant DK50435 and a Research Grant from the American Diabetes Association. We thank Ms. Kathy Carter in the laboratories of Drs. Sergio Fazio and MacRae Linton for the preparation of the human LDL.
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ABBREVIATIONS
AAPH 2,29-azobis(2-amidinopropane)hydrochloride DHA dehydroascorbate PBS phosphate-buffered saline