Evaluation of an acidic deproteinization for the measurement of ascorbate and dehydroascorbate in plasma samples

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	Abstract

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The most popular pretreatment method of plasma samples for the measurement of ascorbate (AsA) and dehydroascorbate (DHA) has been an acidic deproteinization via metaphosphoric acid or trichloroacetic acid. In general, DHA is absent in plasma samples prepared from human blood in a conventional manner. However, when these plasma samples were subjected to acidic deproteinization, DHA was detected in the acidified sample solutions. In the present study, we demonstrate that the oxidation of AsA to DHA in the solutions was promoted by at least two mechanisms, one involving catalysis by ferric ion released from transferrin, and the other involving catalysis by plasma hemoglobin. In the acidified transferrin solution by trichloroacetic acid, an oxidation of AsA to DHA proceeded with standing time, whereas the oxidation was not observed in that by metaphosphoric acid. This oxidation appeared to be catalyzed by ferric ion released from transferrin. In contrast, plasma hemoglobin functioned as a catalyst for AsA oxidation in both metaphosphoric acid and trichloroacetic acid solutions. Therefore, DHA content in the trichloroacetic acid-treated plasma sample was markedly higher than that in the metaphosphoric acid-treated one. These results suggest that DHA detected in acidified plasma samples is an artifact resulting from AsA oxidation.

	Introduction

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Vitamin C is a vital substance for humans, existing as a reduced form, ascorbate (AsA), and an oxidized form, dehydroascorbate (DHA), in vivo. An oxidative stress is thought to be responsible for the AsA loss and the resulting increase in DHA concentration in plasma. On the basis of these results, many investigators have studied the physiological roles and toxicities of DHA (1)(2)(3). However, in our previous study (4), we demonstrated that DHA was quickly degraded to 2,3-diketogulonate in freshly prepared plasma samples (t_1/2 = 1–2 min). Furthermore, we have presented evidence that bicarbonate plays a role as catalyst in the degradation of DHA in plasma (5). Because of the degradation system for DHA in plasma, DHA should not be detectable in plasma samples, which were prepared from whole blood in a conventional manner (4). On the contrary, many reports suggested that the DHA content of human plasma samples was in the range of 20–40% of AsA content (6)(7)(8)(9). Interestingly, in almost all of these reports, an acidic deproteinization was adopted as a pretreatment of plasma samples for the quantitation of AsA and DHA. Commonly used acids include metaphosphoric acid and trichloroacetic acid [reviewed by Washko et al. (10)]. To clarify the contradictory results among previous studies, we thoroughly investigated the oxidation of AsA in acidified plasma samples. Through these investigations, we observed that oxidation of AsA to DHA in acidic solutions proceeded remarkably in the presence of transferrin or hemoglobin. In general, hemoglobin as well as transferrin exists in human plasma (11)(12)(13), and the hemoglobin content in plasma increases with a concomitant hemolysis in plasma preparation. In the present report, we reveal the actions of transferrin and hemoglobin as catalysts for oxidation of AsA to DHA in acidic solutions including metaphosphoric acid, trichloroacetic acid, and perchloric acid.

	Materials and Methods

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materials
Ascorbic acid was purchased from Wako Pure Chemicals. Dehydroascorbic acid was purchased from Aldrich Chemical Co. Human holo-transferrin and human ceruloplasmin were purchased from Sigma Chemical Co. All other chemicals were of reagent grade. Asahipak GS-320 7E (7.6 mm i.d. x 250 mm) and GS-520 7G (7.6 mm i.d. x 500 mm) were purchased from Showa Denko Co. A standard solution of DHA (100 mmol/L) was prepared as follows. Dehydroascorbic acid was dissolved in 0.1 mol/L acetate buffer (pH 4.0), and the solution was standardized by HPLC using a solution of known ascorbic acid concentration as standard. Peak response of DHA in the solution was compared with that of DHA obtained from a standard solution of ascorbic acid after oxidation in 0.1 mol/L acetate buffer containing 10 mmol/L cupric sulfate for 1 h at room temperature.

apparatus
The postcolumn HPLC assembly consisted of a HPLC pump (Hitachi, L-6000), a sample injector (Rheodyne, 7725), a double-plunger pump (Shimamura Instrument Co., PSU-2.5W), a dry reaction bath (Shimamura Instrument Co., DB-5), a fluorescence spectrophotometer (Hitachi, F-1050), and a chromato-integrator (Hitachi, D-2500).

preparation of human plasma samples
Human plasma samples were prepared according to a conventional method as follows. Freshly collected human blood from healthy volunteers (22–38 years of age) was treated with an anticoagulant and then centrifuged at 1000g for 15 min at room temperature. The supernatant was used in the present study. When deproteinization was required, one-half volume of 200 g/L trichloroacetic acid solution, 3 mol/L perchloric acid solution, or 200 g/L metaphosphoric acid solution was added to one volume of plasma, and the mixture was stirred vigorously. After centrifuging at 4000g for 5 min, the supernatant was subjected to the assay.

preparation of human hemoglobin from red blood cells
Human red blood cells were hemolyzed by diluting five times with water, and then the hemolysate was centrifuged at 10 000g for 30 min. The supernatant was applied on the gel-filtration HPLC: column, Asahipak GS-520 7G; eluent, 20 mmol/L Tris-HCl buffer (pH 7.4) containing 0.13 mol/L NaCl (1.0 mL/min); and detection, 540 nm. The fraction of hemoglobin was collected and then used in the present study.

quantitation of ASA AND DHA
AsA and DHA were assayed by a postcolumn HPLC using the following chromatographic conditions (4): column, Asahipak GS-320 7E, and eluent, 0.1 mol/L acetic acid containing 0.5 mmol/L EDTA (1.0 mL/min). The postcolumn reaction conditions using o-phenylenediamine as a fluorometric reagent (14) were as follows: reagent 1, 0.1 mol/L acetate buffer (pH 4.0) containing 20 mmol/L o-phenylenediamine (0.25 mL/min); reagent 2, 0.1 mol/L acetate buffer (pH 4.0) containing 5 mmol/L cupric acetate (0.25 mL/min); reaction temperature, 55 °C; reaction time, 1 min; and detection, fluorescence spectrophotometer (Ex.345 nm and Em.410 nm). Plasma samples were applied directly to HPLC without pretreatment.

	Results

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oxidation of ASA TO DHA IN ACIDIFIED PLASMA SAMPLES
To know the real concentrations of AsA and DHA in plasma, we established a HPLC method that required no pretreatment of sample solutions (4). We applied the method to human plasma samples prepared from heparinized whole blood in a conventional manner. When the plasma samples were directly injected to HPLC, a peak corresponding to DHA was not detected (<0.1 µmol/L). Furthermore, the method was applied to the deproteinized plasma samples by acids including metaphosphoric acid, trichloroacetic acid, and perchloric acid. As shown in Table 1 , a considerable amount of DHA was detected in the acidified plasma samples in compensation for the decrease of AsA content. DHA content in trichloroacetic acid-treated or perchloric acid-treated plasma samples was remarkably higher than that in the metaphosphoric acid-treated one. These results suggest that an acidification of plasma samples promotes an oxidation of AsA to DHA.

oxidation of ASA TO DHA CATALYZED BY FERRIC ION RELEASED FROM TRANSFERRIN IN ACIDIC SOLUTION
In general, ferric ions and cupric ions are known to accelerate AsA oxidation under neutral conditions. Both ferric ion and cupric ion are essential elements in plasma, existing in bound form to transferrin and ceruloplasmin, respectively. In contrast, the binding forces between these metal ions and proteins are comparatively low in acidic solution; thus, acidifying the plasma samples facilitates the release of metal ions. To examine whether ferric ions and cupric ions accelerate AsA oxidation in acidic solutions, AsA was incubated with these ions in acidic solutions including metaphosphoric acid, trichloroacetic acid, and perchloric acid. The time courses of AsA and resulting DHA in the solutions are shown in Fig. 1 . Cupric ions did not act as a catalyst at all, whereas ferric ions accelerated the oxidation of AsA to DHA in trichloroacetic acid and perchloric acid solutions but not in metaphosphoric acid solution.

View larger version (27K): [in this window] [in a new window]   Figure 1. Oxidation of AsA to DHA in acidic solution in the presence of ferric ion or cupric ion. One volume of 100 µmol/L ascorbic acid solution containing 25 µmol/L cupric chloride or 25 µmol/L ferric chloride was mixed with one-half volume of 200 g/L metaphosphoric acid, 200 g/L trichloroacetic acid, or 3 mol/L perchloric acid, and then the mixture solution was incubated at 28 °C. AsA and DHA were determined by HPLC.

Furthermore, one volume of 5 g/L transferrin (human holo-transferrin, Sigma) solution containing 100 µmol/L AsA was mixed with one-half volume of 200 g/L metaphosphoric acid, 200 g/L trichloroacetic acid, or 3 mol/L perchloric acid. After centrifuging at 10 000g for 30 s, the supernatant stood at 28 °C. The time courses of AsA and resulting DHA in the supernatant are shown in Fig. 2 . Similarly, an oxidation of AsA to DHA was not observed in metaphosphoric acid-treated transferrin solution, whereas AsA oxidation remarkably proceeded in trichloroacetic acid-treated and perchloric acid-treated transferrin solutions. The oxidation of AsA to DHA in an acidified transferrin solution by trichloroacetic acid was affected by temperature as shown in Fig. 3 . However, a complete depression of AsA oxidation was not achieved by lowering temperature up to 4 °C. These results indicate that, if the biological samples containing transferrin undergo an acidic deproteinization by trichloroacetic acid or perchloric acid, a considerable amount of AsA was oxidized to DHA.

View larger version (26K): [in this window] [in a new window]   Figure 2. Oxidation of AsA to DHA catalyzed by ferric ion released from transferrin in the acidic solution. One volume of transferrin solution (5 g/L) containing 100 µmol/L AsA was mixed with one-half volume of 200 g/L metaphosphoric acid, 200 g/L trichloroacetic acid, or 3 mol/L perchloric acid, and then the mixture solution was incubated at 28 °C. AsA and DHA were determined by HPLC.

View larger version (14K): [in this window] [in a new window]   Figure 3. Effect of temperature on the oxidation of AsA to DHA catalyzed by ferric ion released from transferrin in trichloroacetic acid solution. One volume of transferrin solution (5 g/L) containing 100 µmol/L AsA was mixed with one-half volume of 200 g/L trichloroacetic acid, and then the mixture solution was incubated. AsA and DHA were determined by HPLC. The reaction rate constant (k) was plotted as a function of the reciprocal of the temperature (Arrhenius plot).

oxidation of ASA TO DHA PROMOTED BY HEMOGLOBIN IN ACIDIC SOLUTION
It has been reported that 21–189 µg/mL hemoglobin is presented in healthy human plasma samples (11). Moreover, the hemoglobin content in plasma remarkably increases with a concomitant hemolysis in plasma preparation from heparinized whole blood. Human hemoglobin does not have an AsA oxidase-like activity under neutral conditions. To examine whether hemoglobin accelerates the AsA oxidation in acidic solution, one volume of 200 mg/L hemoglobin solution (corresponding to the hemolysate of 0.18 mL/L red blood cells) containing 20 µmol/L AsA was mixed with one-half volume of 200 g/L metaphosphoric acid solution, and then the mixture solution was kept at 28 °C. The time course of the ratio of DHA concentration to AsA concentration in the solution is shown in Fig. 4 . Furthermore, similar results were obtained when a hemoglobin solution containing AsA was mixed with 200 g/L trichloroacetic acid solution or 3 mol/L perchloric acid. The effect of the hemoglobin content on the oxidation of AsA to DHA in acidic deproteinization is shown in Fig. 5 . The hemoglobin content is correlated with the degree of AsA oxidation. These findings indicate that hemoglobin is capable of catalyzing the oxidation of AsA to DHA in an acidic solution.

View larger version (24K): [in this window] [in a new window]   Figure 4. Oxidation of AsA to DHA catalyzed by hemoglobin in acidic solution. One volume of 200 mg/L hemoglobin solution containing 20 µmol/L AsA was mixed with one-half volume of 200 g/L metaphosphoric acid, 200 g/L trichloroacetic acid, or 3 mol/L perchloric acid, and then the mixture solution was kept at 28 °C. The solution was directly applied on HPLC.

View larger version (16K): [in this window] [in a new window]   Figure 5. Effect of hemoglobin content on the AsA oxidation in acidic solution. One volume of hemoglobin solutions (the concentration is indicated on figure) containing 20 µmol/L AsA were mixed with one-half volume of 200 g/L metaphosphoric acid solution, and then the solutions were kept at 28 °C for 10 min. After centrifuging at 10,000g for 30 s, the supernatants were applied on HPLC.

To examine whether free ferrous ions affect the AsA oxidation in an acidic solution, one volume of 25 µmol/L FeCl₂ solution containing 100 µmol/L AsA was mixed with one-half volume of 200 g/L metaphosphoric acid solution, and then the solution mixture was kept at 28 °C. The time courses of AsA and resulting DHA in the solution are shown in Fig. 6 . Free ferrous ions did not act as a catalyst in an acidic solution. These findings indicate that hemoglobin acts as an AsA oxidase in an acidic solution.

View larger version (16K): [in this window] [in a new window]   Figure 6. Oxidation of AsA to DHA in acidic solution in the presence of ferrous ion. One volume of 100 µmol/L ascorbic acid solution containing 25 µmol/L ferrous chloride was mixed with one-half volume of 200 g/L metaphosphoric acid, and then the mixture solution was incubated at 28 °C. AsA () and DHA () were determined by HPLC.

	Discussion

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Thus far, an acidic deproteinization using metaphosphoric acid or trichloroacetic acid has been recommended as a pretreatment of plasma samples for the quantitation of AsA and DHA (10). The acidified plasma samples can be stored at -70 °C for a long period with no marked oxidation of AsA (15)(16). The principal advantages of an acidic deproteinization: precipitation of proteins; prevention of AsA oxidation and DHA degradation; and removal of interfering substances (10). Indeed, both AsA and DHA are stable in an acidic solution. However, in the present study, we provide evidence that AsA is unstable in acidified plasma samples. The oxidation of AsA in an acidified plasma sample solution proceeds through at least two different ways: (a) catalysis by ferric ion released from plasma transferrin; and (b) catalysis by plasma hemoglobin. In the trichloroacetic acid-treated plasma, the oxidation of AsA to DHA is accelerated by both transferrin and hemoglobin. In contrast, AsA in the metaphosphoric acid-treated plasma is subject to oxidation catalyzed by plasma hemoglobin but not by transferrin. Therefore, the DHA concentration in the trichloroacetic acid-treated plasma was remarkably higher than that in the metaphosphoric acid-treated plasma.

In the 1930s, Lemberg et al. (17) noted that the coupled oxidation between oxyhemoglobin and AsA (especially at higher concentrations) proceeds by the direct reaction of AsA and oxyhemoglobin. Our findings, coupled with this fact, suggest that a reaction between oxyhemoglobin and AsA is more progressive in acidic solutions.

The evidence presented in this study suggests that an acidic deproteinization method is not applicable to quantitation of AsA and DHA in biological fluids that contain transferrin and/or hemoglobin. To determine AsA and DHA in biological fluids, a HPLC method, which required no pretreatment of sample solution, is most reliable (4).

	Acknowledgments

 
This work was supported by a Grant-in-Aid for Scientific Research (C; no. 08672470) from the Ministry of Education, Science, Sports and Culture, Japan.

	Footnotes

 
Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi, Inage, Chiba-shi, Chiba 263, Japan.

	References

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