Anal. Chem. 1997, 69, 216-220
Measurement of Ascorbate and Dehydroascorbate Contents in Biological Fluids Ichiro Koshiishi and Toshio Imanari*
Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi, Inage, Chiba-shi, Chiba 263 Japan
Instabilities of ascorbate and dehydroascorbate throughout sample processing are clearly a significant aspect of quantifing of them. Contents of ascorbate in biological fluids decrease with measurable oxidation occurring within minutes to hours. Similarly, dehydroascorbate disappears with chemical or enzymatic degradation within minutes. The half-life of dehydroascorbate in human heparinized plasma was ∼2 min. These results indicated that the amount of dehydroascorbate present in sample solutions is a function of both the oxidation of ascorbate and the degradation of dehydroascorbate during the processing of biological fluids. To quantify ascorbate and dehydroascorbate concentrations in biological fluids including circulating blood plasma and urine, we established a high-performance liquid chromatographic method, which requires no pretreatment of sample solutions. Ascorbate is a water-soluble antioxidant vitamin present in significant amounts in the tissues and body fluids of humans and other mammals. It has been observed that excessive oxidative stress is responsible for the ascorbate loss and the resulting increase in dehydroascorbate concentration in biological fluids. Thus, the ratio of plasma dehydroascorbate to ascorbate concentration may be useful in clinical studies linking oxidative stress to common human illnesses such as cancer,1 diabetes mellitus,2 and hepatic diseases.3,4 Accurate quantification of ascorbate and dehydroascorbate in biological solutions is important to define the role of ascorbate in vivo redox status. So far, several types of quantitative methods have been reported (refer for a review5). The best acceptable method for assaying may be a HPLC with electrochemical detection,6-11 where dehydroascorbate is measured indirectly and apparently negative values for dehydroascorbate content sometimes occur.10 In contrast, to quantify dehydroascorbate directly, a precolumn HPLC with derivatization of dehydroascorbate by
o-phenylenediamine12,13 and 2,4-dinitrophenylhydrazine14,15 and postcolumn HPLC with on-line derivatization by o-phenylenediamine16,17 or its analogue18 have been established. Even though numerous quantitative methods have been applied to biological fluids, definitive values of their dehydroascorbate concentration are not known yet, because of assay reliability. Many authors have shown that addition of reducing agent to plasma increased the ascorbate concentration, owing to reduction of dehydroascorbate to ascorbate,5,8,11 while some authors have demonstrated the absence of dehydroascorbate in the sample.18-20 Both ascorbate and dehydroascorbate are unstable in solution at neutral pH with measurable oxidation and degradation, respectively, occurring within minutes to hours. The point at issue appears to be the transformation of two analytes during sample preparation, sample pretreatment, and assay procedure.21 The present study was undertaken to establish a postcolumn HPLC, where sample pretreatment, such as deproteinization, was not required; stability studies of endogenous ascorbate and dehydroascorbate in biological fluids during sample preparations were also performed. EXPERIMENTAL SECTION Materials. Ascorbic acid was purchased from Wako Pure Chemicals. Dehydroascorbic acid was purchased from Aldrich Chemical Co. All other chemicals were of reagent grade. Asahipak GS-320H (7.6 mm i.d. × 250 mm) was purchased from Asahi Chemical Industry. A standard solution of dehydroascorbate (100 mM) was prepared as follows: dehydroascorbic acid was dissolved in 0.1 M acetate buffer (pH 4.0), and the solution was standardized by HPLC using a solution of known ascorbic acid concentration as the standard. Peak response of dehydroascorbate in the solution was compared with that of dehydroascorbate obtained from a standard solution of ascorbic acid after oxidation in 0.1 M acetate buffer containing 10 mM cupric sulfate for 1 h at room temperature.
(1) Weitzman, S. A.; Gordon, L. I. Blood 1990, 76, 655-663. (2) Chatterjee, I. B.; Banerjee, A. Anal. Biochem. 1979, 98, 368-374. (3) Maellaro, E.; Casini, A. F.; Del Bello, B.; Comporti, M. Biochem. Pharmacol. 1990, 39, 1513-1521. (4) Martensson, J.; Meister, A. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 46564660. (5) Washko, P. W.; Welch, R. W.; Dhariwal, K. R.; Wang, Y.; Levine, M. Anal. Biochem. 1992, 204, 1-14. (6) Farber, C. M.; Kanengiser, S.; Stahl, R.; Liebes, L.; Silber, R. Anal. Biochem. 1983, 134, 355-360. (7) Behrens, W. A.; Madere, R. Anal. Biochem. 1987, 165, 102-107. (8) Margolis, S. A.; Davis, T. P. Clin. Chem. 1988, 34, 2217-2223. (9) Lee, W.; Davis, K. A.; Rettmer, R. L.; Labbe, R. F. Am. J. Clin. Nutr. 1988, 48, 286-290. (10) Washko, P.; Rotrosen, D.; Levine, M. J. Biol. Chem. 1989, 264, 1899619002. (11) Barja, G.; Hernanz, A. Methods Enzymol. 1994, 234, 331-337.
(12) Omaye, S. T.; Turnbull, J. D.; Sauberlich, H. E. Methods Enzymol. 1979, 62, 3-11. (13) Speek, A. J.; Schrijver, J.; Schreurs, W. H. P. J. Chromatogr. 1983, 305, 52-60. (14) Roe, J. H.; Mills, M. B.; Oesterling, M. J.; Damron, C. M. J. Biol. Chem. 1948, 174, 201-208. (15) Garcia-Castineiras, S.; Bonnet, V. D.; Figueroa, R.; Miranda, M. J. Liq. Chromatogr. 1981, 4, 1619-1640. (16) Capellmann, M.; Bolt, H. M. Fresenius J. Anal. Chem. 1992, 342, 462-466. (17) Vanderslice, J. T.; Higgs, D. J. J. Micronutr. Anal. 1990, 7, 67-70. (18) Lopez-Anaya, A.; Mayersohn, M. Clin. Chem. 1987, 33, 1874-1878. (19) Okamura, M. Clin. Chim. Acta 1980, 103, 259-268. (20) Levine, M.; Dhariwal, K. R.; Washko, P. W.; DeB Butler, J.; Welch, R. W.; Wang, Y.; Bergsten, P. Am. J. Clin. Nutr. 1991, 54, 1157S-1162S. (21) Bode, A. M.; Cunningham, L.; Rose, R. C. Clin. Chem. 1990, 36, 18071809.
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© 1997 American Chemical Society
Figure 1. Oxidation of ascorbate and cleavage of the lactone ring of dehydroascorbate in buffered solutions. One volume of the solution containing 1 mM ascorbate (A) or 1 mM dehydroascorbate (B) was mixed with 19 volumes of buffered solution: 0.1 M Tris-HCl buffer (pH 7.3) and 0.1 M acetate buffer containing 10 mM EDTA (pH 4.0). After incubating at 37 °C, the mixture was submitted to HPLC.
Apparatus. The postcolumn HPLC assemble 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 chromatointegrator (Hitachi, D-2500). Quantitation of Ascorbate and Dehydroascorbate. Ascorbate and dehydroascorbate were assayed by a postcolumn HPLC using the following chromatographic conditions: column, Asahipak GS-320H; eluent, 0.1 M acetic acid containing 0.5 mM EDTA (1.0 mL/min). The postcolumn reaction conditions were as follows; reagent 1, 0.1 M acetate buffer (pH 4.0) containing 20 mM o-phenylenediamine (0.25 mL/min); reagent 2, 0.1 M acetate buffer (pH 4.0) containing 5 mM cupric acetate (0.25 mL/min); reaction temperature, 55 °C; reaction time, 1 min; detection, fluorescence spectrophotometer (excitation 345 nm; emission, 410 nm). Plasma and urine samples were applied directly to HPLC without pretreatment. Processing of Biological Fluids Including Plasma and Urine. Fresh human blood samples were collected as follows; a finger was stuck with a sterile needle, and ∼50 µL of blood was collected in a heparin-coated capillary, which had been cooled in a freezer. After centrifuging the blood at 4000g for 1 min at 0 °C, the supernatant was subjected to HPLC. It should be noted that the time period from the collection of blood to the sample injection was kept within 10 min. For the assay of ascorbate and dehydroascorbate in human urine, the sample was transferred immediately to a tube, which was cooled in freezer. If a precipitate was formed in urine, it was centrifuged at 4000g for 1 min at 0 °C. The supernatant was subjected to HPLC. Throughout sample processing, sample solutions was kept below 0 °C. If conventional sample processing was carried out, blood was treated with an anticoagulant and then centrifuged at 1000g for 15 min at room temperature. When deproteinization was required, 50 µL of 20% trichloroacetic acid solution, 3 M perchloric acid solution, or 20% metaphosphoric acid solution was added to 100 µL of plasma, and the mixture was stirred vigorously. After centrifugation at 4000g for 5 min, the supernatant was subjected to HPLC.
Figure 2. Schematic diagram of HPLC for the quantitation of ascorbate and dehydroascorbate. The chromatographic conditions were as follows; column, Asahipak GS-320H (Asahi Chemical Industry Co.); eluent, 0.1 M acetic acid containing 0.5 mM EDTA (1.0 mL/ min). The postcolumn reaction conditions were as follows: reagent 1, 0.1 M acetate buffer (pH 4.0) containing 20 mM o-phenylenediamine (0.25 mL/min); reagent 2, 0.1 M acetate buffer (pH 4.0) containing 5 mM cupric acetate (0.25 mL/min); reaction temperature, 55 °C; reaction time, 1 min; sample volume, 5-50 µL; detector, fluorescence spectrophotometer (excitation, 345 nm; emission, 410 nm).
Figure 3. Chromatograms of standard ascorbate and dehydroascorbate. The chromatographic conditions were as described in Figure 2: (A) 10 µM dehydroascorbate (sample volume, 50 µL); (B) 10 µM ascorbate (50 µL); (C) mixture of 10 µM ascorbate and 10 µM dehydroascorbate (50 µL).
RESULTS HPLC for the Simultaneous Quantitation of Ascorbate and Dehydroascorbate. In order to quantify ascorbate and dehydroascorbate accurately by HPLC, oxidation of ascorbate and degradation of dehydroascorbate are unavoidable during the separation process. In general, ascorbate is unstable in a solution with significant oxidation occurring within minutes, due to molecular oxygen and traces of contaminating metals. When ascorbate was dissolved in Tris-HCl buffer (pH 7.3) and the solution was kept at 37 °C, disappearance of ascorbate was observed within 1 h (Figure 1A). This oxidative reaction was not observed for ascorbate in acidic solution containing EDTA (Figure Analytical Chemistry, Vol. 69, No. 2, January 15, 1997
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Figure 5. Oxidation of ascorbate and cleavage of the lactone ring of dehydroascorbate in human heparinized plasma. The time courses of endogenous ascorbate level (A) and spiked dehydroascorbate level (B) in human plasma were monitored. For dehydroascorbate, 1 volume of 1 mM dehydroascorbate solution was mixed with 99 volumes of the heparinized human plasma. Each plasma sample was kept at the temperature indicated.
Figure 4. Effects of the protein on the quantitation of ascorbate and dehydroascorbate: (A, C) One volume of 1 mM dehydroascorbate and ascorbate standard solutions were mixed with 99 volumes of phosphate-buffered saline (PBS) containing BSA as indicated; (B, D) A 100-fold concentration of dehydroascorbate and ascorbate standard solution was mixed with PBS containing 7% BSA in a volume ratio of 1:99, respectively. Each solution was kept in an ice-water bath throughout sample preparation.
1A). In contrast, dehydroascorbate is relatively more unstable at neutral pH, producing 2,3-diketogulonate by a cleavage of lactone ring with a half-life of ∼20 min at 37 °C. To prevent the degradation of dehydroascorbate, we examined the influence of pH on this process. As shown in Figure 1B, it was observed that dehydroascorbate was comparatively stable in weak acidic solution (pH 3.0-4.5) at 37 °C for 20 min. Thus, these findings indicate that the separation of ascorbate and dehydroascorbate on column can be achieved using a weak acidic solution containing EDTA as an eluent without any risk of their transformation. Of the steps required for the sample processing, sample pretreatment is the step at which ascorbate oxidation and dehydroascorbate degradation are most susceptible. To avoid this step, we undertook to establish a HPLC in which biological fluids can be injected directly without sample pretreatment. Along these lines, we examined the poly(ethylene glycol) copolymer as a packing material in the separation column. This copolymer has characteristics of low adsorption of proteins and is suitable for separating low molecular weight organic molecules, so that direct injection of biological fluids is possible. Asahipak GS-320H is a column packed with poly(ethylene glycol) copolymer. Using this column for HPLC, separation of ascorbate and dehydroascorbate was achieved. 218 Analytical Chemistry, Vol. 69, No. 2, January 15, 1997
Figure 6. Chromatograms of ascorbate and dehydroascorbate in human plasma (B) and human urine (C). The sample was processed exactly as described in the Experimental Section. A standard solution (A) containing 50 µM ascorbate and 50 µM dehydroascorbate (sample volume, 5 µL).
The fluorometric reaction of dehydroascorbate with o-phenylenediamine is considered the most convenient method for the postcolumn reaction, since this reaction progresses under mild conditions of pH, reaction temperature, and reaction time. To prevent interferences from proteins in the sample during the postcolumn reaction, we used acetate buffer (pH 4.0) as solvent with a reaction temperature of 55 °C and reaction time of 1 min. For ascorbate, inclusion of cupric ion in reaction solution enabled oxidation to dehydroascorbate, which was detected fluorometrically in the same manner. The flow diagram of the postcolumn HPLC detection is shown in Figure 2, and the chromatograms of standard ascorbate and dehydroascorbate in Figure 3. It should be noted that this system has the capacity to prevent both the
Figure 7. Oxidation of ascorbate to dehydroascorbate in acidified plasma samples: (A) The standard solution containing 50 µM ascorbate and 50 µM dehydroascorbate (sample volume, 5 µL). (B) Human plasma (5 µL). (C) Human plasma mixed with an equal volume of 0.2 M acetate buffer (pH 5.0) and the mixture kept for 10 min at room temperature (10 µL). Two volumes of human plasma mixed with 1 volume of 20% metaphosphoric acid (D), 20% trichloroacetic acid (E), or 3 M perchloric acid (F). After centrifugation the supernatant was subjected to HPLC (7.5 µL).
oxidation of ascorbate and the degradation of dehydroascorbate during the chromatographic run. We examined whether the presence of protein in the sample influences the chromatographic pattern by interacting with ascorbate and dehydroascorbate. Figure 4 shows that the presence of up to 10% bovine serum albumin (BSA) does not affect the correlations between peak responses and injected ascorbate and dehydroascorbate concentrations. In both cases, the calibration curves were found to be linear between 0.2 and 200 µM. Processing of Biological Fluids. Both the oxidation of ascorbate and the degradation of dehydroascorbate progress during treatment of blood with anticoagulants and centrifugation of whole blood to recover plasma. Figure 5A shows the effect of temperature on ascorbate oxidation, suggesting that the oxidation is prevented by performing this process at 0 °C. Similar results were found with dehydroascorbate as well. As shown in Figure 5B, when human plasma samples spiked with dehydroascorbate (final concentration, 10 µM) were incubated at 37 °C, the dehydroascorbate content decreased in an exponential fashion with a half-life of 2 min in human plasma. At present, the mechanism by which dehydroascorbate is degraded in plasma is unclear. However, it is clear that processing plasma at 0 °C and injecting the sample within 10 min after collection of blood prevent the oxidation of ascorbate and the degradation of dehydroascorbate. To examine whether dehydroascorbate is present in biological fluids, we applied the present method to assay human blood and urine. Chromatograms are shown in Figure 6. The ratio of the content of dehydroascorbate to that of ascorbate in human plasma was 7.6 ( 2.8%, and that in human urine 6.4 ( 5.0% (Table 1). In contrast, when human blood underwent the conventional process-
Table 1. Concentrations of Ascorbate and Dehydroascorbate in Human Plasma and Urinea sample
dehydroascorbate (µM)
ascorbate (µM)
dehydroascorbate/ ascorbate (%)
human plasma human urine
4.4 ( 1.7 8.2 ( 3.7
58.1 ( 14.0 181 ( 87
7.6 ( 2.8 6.4 ( 5.0
a Human plasma and urine were collected from seven volunteers (22-37 years old). Each value represents mean (SE.
ing at room temperature, dehydroascorbate was not detected at all (data not shown). DISCUSSION The present study was undertaken to examine the presence of dehydroascorbate in human biological fluids. So far, many investigators have described the quantitation of dehydroascorbate in biological fluids including blood plasma. Most of them, but not all, have shown that the dehydroascorbate content of human plasma was in the range of 20-30% of ascorbate content,8,11,22-26 whereas others have demonstrated the absence of it in human blood plasma.18-20 The evidence presented in this work suggests that this controversy is a result of the sample processing. As demonstrated in this study, dehydroascorbate was not detected (22) Behrens, W. A.; Madere, R. Anal. Biochem. 1987, 165, 102-107. (23) Deutsch, J. C.; Kolhouse, J. F. Anal. Chem. 1993, 65, 321-326. (24) Moeslinger, T.; Brunner, M.; Spieckermann. Anal. Biochem. 1994, 221, 290-296. (25) Nyyssonen, K.; Pikkarainen, S.; Parviainen, M. T.; Heinonen, K.; Mononen, I. J. Liq. Chromatogr. 1988, 11, 1717-1728. (26) Nagy, E.; Degrell, I. J. Chromatogr. Biomed. Appl. 1989, 89, 276-281.
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in plasma samples that were prepared from blood of normal subjects in the conventional manner (see the Experimental Section). Clearly this is caused by the degradation of dehydroascorbate during sample processing. On the other hand, an acidic deproteinization using metaphosphoric acid, trichloroacetic acid, or perchloric acid has been the generally accepted excellent sample pretreatment for ascorbate and dehydroascorbate quantitation.8,27 However, the oxidation of ascorbate by the plasma oxidants is more progressive under acidic conditions, as opposed to neutral conditions. As shown in Figure 7, it seems that a significant quantity of ascorbate (metaphosphoric acid, 12-20% of endogenous plasma ascorbate; trichloroacetic acid, 23-27%; perchloric acid, 24-32%) was therefore irreversibly oxidized when (27) Lunec, J.; Blake, D. R. Free Radical Res. Commun. 1985, 1, 31-39.
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plasma samples were subjected to the acidic deproteinization. These findings indicate that the acidic deproteinization is not suitable for the quantitation of dehydroascorbate in biological fluids. In conclusion, the present method appears to be a satisfactory method for determining the ascorbate and dehydroascorbate content of biological fluids including blood plasma and urine, since both the oxidation of ascorbate and the degradation of dehydroascorbate were prevented throughout sample processing. Received for review July 31, 1996. Accepted November 1, 1996.X AC960704K X
Abstract published in Advance ACS Abstracts, December 15, 1996.
