Anal. Chem. 1994,66, 1086-1089
Quantitation of Dehydroascorbic Acid by the Kinetic Measurement of a Derivatization Reaction Hans Goldenberg,+ Leopold Jlrovetz,* Paul KraJnlk,sWllhelm Mosgoller,ll Thomas Moslinger, and Esther Schwelnzer'*t*s Departments of Medical Chemistry, Pharmaceutical Chemistry, Radiation and Photochemistry, Histology and Embryology, and Medical Physiology, Universitv of Vienna, Waehringerstrasse 10, A- 1090 Wien, Austria Micromolar concentrationsof dehydroascorbicacid in aqueous solutions react with methanol-containing phosphate or citrate buffers to form a mixture of derivatives with an absorption maximum at 346nm. The compounds, different methyl ketals, were analyzed using fast atom bombardment mass spectrometry. After calibration, kinetic analysis of the formation rates can be used for fast and simple determination of dehydroascorbic acid concentrations. In samples containing both forms of vitamin C, ascorbate and dehydroascorbic acid, autoxidation of ascorbate can be inhibited by addition of transition metal complexing agents; thus, only formation rates accounting for the amounts of dehydroascorbic acid are measured.
measure of the concentration of DHA. Since oxidation of ascorbate to DHA can be prevented, this assay might be used as a simple method for quantifying DHA in the presence of the reduced form of vitamin C.
MATERIALS AND METHODS Immediately before each experiment DHA was prepared using the halogen oxidation method described by Levine's group.4 Deferioxamine (from Ciba Geigy) was a gift from Dr. E. Penner, Dept. of Gastroenterology and Hepatology, General Hospital, Vienna, Austria. Deuterated chemicals for NMR were purchased from Aldrich (Steinheim, Germany). All other reagents were obtained either from Merck (Darmstadt, Germany) or from Sigma (Deisenhofen, Germany) and were of analytical grade. In blood and tissues, vitamin C is found mainly in its reduced state, i.e., ascorbate.' However, knowledge about the fraction Dehydroascorbic acid and ascorbic acid were dissolved 5 present in the oxidized form, i.e., dehydroascorbic acid (DHA) mM in water and then added to the indicated buffer solution is often important. For example, increased levels of DHA to yield the desired concentrations. Since aqueous solutions may be found in some pathological states such as diabetes.2 of oxidized or reduced vitamin are very unstablelo and sensiAdditionally, DHA has also recently gained attention due to tive to light exposure or higher temperatures, they were stored its protective activity on low-density lipoproteins in in vitro at 4 OC in the dark but never for longer than 1 h. systems3 as well as its strongly enhanced accumulation by Spectrophotometric measurements were carried out on a human neutrophils4 after activation of the respiratory burst. Perkin-Elmer Lambda-2 spectrophotometer in a cuvette at Most assays for determination of the DHA concentration are 37 OC. When fetal calf serum (GIBCO) was used in the not ideal, since they lack either sensitivity or specifi~ity.~,~ assays, it was depleted of vitamin C by heat inactivation.' Unintentional interconversion of DHA and ascorbic acid Mass spectrometry measurements were performed either during sample processing or measurement may lead to false on a MS-Engine (Hewlett-Packard Co.) with positive FAB result^.^ However, reliable methods are laborious and require mode (NaC1-glycerol 1:l matrix) or on an Autospec mass the use of expensive equipment.*v9 We show here that DHA, spectrometer (VG Instruments) with positive FAB mode (pwhich has no detectable absorption maximum in aqueous nitrobenzoic acid-glycerol 1:1 matrix). solutions, forms a spectrophotometrically determinable mixture of derivatives when diluted to methanol-containing RESULTS phosphate buffers. Its rate of formation can be taken as a When an aqueous solution of DHA was mixed with a methanol-containing phosphate buffer in a pH range from 6 t Department of Medical Chemistry.
* Department of Pharmaceutical Chemistry.
of Radiation and Photochemistry. Department of Histology and Embryology. Department of Medical Physiology. (1) Rose, R. C. Biochim. Biophys. Acta 1988, 947, 335-366. (2) Som, S.; Basu, S.; Mukherjee, D.; Deb, S.; Choudhury, P. R.; Mukherjee, S.; Chatterjee, S. N.; Chatterjee, I. B. Metabolism 1981, 30, 572-577. (3) Retsky, K. L.; Freeman, M. W.; Frei, B.J. Biol. Chem. 1993,268,1304-1309. (4) Washko, P. W.; Wang, Y.; Levine, M. J. Biol. Chem. 1993, 268, 1553115535. ( 5 ) Cooke, J. R.; Moxon, R. E. D. In Vitamin C;Counsell, J. N., Hornig, D., Eds.; Applied Science: London, 1981; pp 167-198. (6) Rose, R. C.; Nahrwold, D.Anal. Biochem. 1981, 114, 14C-145. (7) Washko, P. W.; Welch, R. W.; Dhariwal, K. R.; Wang, Y.; Levine, M. AnaLBiochem. (1992) 204, 1-14. (8) Dhariwal, K. R.; Washko, P. W.; Levine, M. Anal. Biochem. 1990,189. 1823. (9) Deutsch, J. C.; Kolhouse, J. F. Anal. Chem. 1993, 65, 321-326. 1 Department
1086 AnalflicalChemistry, Vol. 66,No. 7, April 1. 1994
(10) Lewin, S. VitaminC: Its Molecular Biology andMedicalPotenrial; Academic Press: New York, 1976; pp 63-72. (11) Huelin, F. E. Aust. J. Sci. Res. 1949, 82, 346-354. (12) Sauberlich, H. E.; Green, M.D.; Omaye, S. T. In Ascorbic Acid Chemisfry. Metabolism and Uses;Seib, N. A., Tolbert, B. M., Eds.; American Chemical Society: Washington,DC, 1982; pp 199-221. (13) Roc, J. H.; Kuether, C. A. J. Bid. Chem. 1943, 147, 399407. (14) Omaye, S. T.; Turnbull, J. D.; Sauberlich, H. E. Methods Enzymol. 1979,62, 3-11. (15) Okamura, M. Clin. Chim. Acta 1980, 103, 259-268. (16) Doner, L. W.; Hicks, K. B. Anal. Biochem. 1981, 115, 225-230. (17) Farber, C. M.; Kancngiser,S.; Stahl, R.; Liebes, L.;Silber, R. Anal. Biochem. 1983, 134, 355-360. (18) Washko, P. W.; Hartzell, W. 0.;Levine, M. Anal. Biochem. 1989,181,276282. (19) Levine, M.; Asher, A.; Pollard, H.; Zinder, 0. J. Biol. Chem. 1983, 258, 13111-13115.
0003-2700/94/036& 1086$04.50/0
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Figure 1. (a) Spectra of 70 pM DHA at beginning (A) and 5 (B), 10 (C), 20 (D), 30 (E), and 40 (F) mln after mixing with methanokontaining phosphate buffer (18% methanol, 0.2 M sodium phosphate, pH 7.2). Spectrum A was used as a blank. (b) Same as (a), but with 50 pM ascorbic acid instead of DHA at 0 (A), 60 (B), and 120 (C) min. (c) Increaseof absorbance at 346nm after mixing 60 pMdehydroascorbic acid with 18% methanol, 0.2 M phosphate, pH 7.1.
to 8 for the original alcohol-free stock solution, changes in the absorption spectrum could be observed. After a short lag phase, the absorptionsat 346 and 224 nm started to rise (Figure 1a). The increaseof the absorptionat 224 nm was considerably smaller than that at 346 nm and was not further investigated. Ascorbic acid was diluted in the same way and monitored spectrophotometrically (Figure 1b). The absorption maximum at 265 nm is characteristic for the deprotonized form, the ascorbate anion.10 After a lag phase (which was always longer than with DHA), the same two peaks appeared whereas theconcentration ofascorbatedeclined. Therise in absorption at 346 nm was still observable after complete disappearance of the absorption of ascorbate at 265 nm. The rate of consumption of ascorbate was dependent on the purity of the water used: the purer the water, the slower was the disappearance of ascorbate. This reaction could be accelerated by placing the cuvette containing the sample in the light beam of the photometer (not shown). Ascorbate therefore must
undergo autoxidation to DHA,20 which subsequently reacted with the buffer compounds. Autoxidation is greatly accelerated by traces of transition metals; therefore, the influence of the purity of water can be expected (see also Figure 5 and corresponding text below). DHA was diluted in the methanol-containing phosphate buffer and the increase of absorption was monitored at 346 nm. The above-mentioned lag phase was followed by linear increase of the absorption which then flattened to a plateau (Figure IC). The length of the linear phase was dependent on the concentrations of the methanol, the phosphate, and the DHA. The slope will in the following be referred to as "the formation rate". Mass spectrometrical analyses revealed that the reaction product is not a single substance, but consists of several compounds, predominantly ketals. This mixture is composed of approximately 65% of the molecule shown in Figure 2. Diand trimers which are linked via the side chain appear in minor quantities. Hemiketals were not found at all. In order to confirm the structure of the products we tried to measure NMR and IR spectra. The mixture of products was unstable and the individual compounds could not be separated: All concentration and separation procedures, Le., evaporation, extraction, and chromatographic methods (column chromatography on silica and Al2O3) resulted in irreversible decomposition. For this reason, DHA was derivatized with deuterated reagents (D20, CDsOD, D3P04,NaOD),which should also, simplify the spectra, because all methyl and hydroxyl groups in the spectra would be deuterated. Nevertheless, 'HNMR spectra showed rather complicated patterns of peaks which could not be identified. For 13CNMR, the solutions could not be concentrated sufficiently without the appearance of undesired side reaction products. IR spectra (for the detection of C=O bands) could not be obtained because of the high phosphate concentrations. For constant concentrations of methanol and phosphate, the formation rate was linearly dependent on the concentration of DHA in a range from 3.3 to 231 pM, and the resulting slopes increased with the concentrations of methanol (Figure 3a) or phosphate (Figure 3b). These slopes were plotted against the concentrations of phosphate or methanol, respectively, and a linear relationship was found for the alcohol (Figure 4a), while a saturation curve was obtained for the phosphate (Figure 4b). Further increase in the concentrations of methanol and/or phosphate was limited by the insolubility of phosphate salts in methanol. When deferioxamine and EDTA were present in the methanol-containing buffer, the autoxidation of ascorbate by the transition metals20 was inhibited. Monitoring a mixture of ascorbate and DHA under these conditionsyielded formation rates accounting only for the concentration of DHA, while ascorbate remained stable (Figure 5 ) . DHA was derivatized similarily when phosphate was exchanged with citrate (Figure 6). Various other anions (sulfate, acetate, chloride, hydrogencarbonate)did not catalyze derivatization. To test the applicability of the assay on biological matrices, additional experiments were done with 25% (v/v) heatinactivated fetal calf serum or white wine in both buffers. (20) Halliwell.
B.; Gutteridge, J. M. C. Merhods Enzymol.
1990, 186, 1-85.
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MasslCliargc Flgurr 2. Posltive fast atom bombardment mass spectrum of the compounds formed by mixing of DHA and methanol-phosphate buffers. Inset: NaCi-glyceroi matrix, to be subtracted from the total spectrum. M, methyl diketai. Main peaks with m/z ratios of 31 1 (Na2MH+),267 (MH+), 192 ([MH+] 2CH&CH3), 131 (192 side chaln and partly glycerol matrix), and 93 (glycerol matrix and partly side chain) can be identified.
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Linear correlations between the formation rates and the added amounts of DHA were found (Figure 7).
DISCUSSION The biological activity of vitamin C is based on the interchange of DHA and ascorbate by easy and reversible reduction or oxidation. Unfortunately, this is the main source of error in assays measuring one of the two forms, since they may interconvert unintentionally during sample processing.8 DHA, which in principle is highly unstable and is rapidly degraded in buffer solutions at physiological pH and temperature," has no defined absorption spectrum in the nearUV or visible range.I2 Its light brown color is due to a broad smear of bands throughout the lower wavelength part of the visible spectrum. Classical methods for derivatization into visible products13J4are tedious and sometimes badly reproducible.8 While methods for determination of ascorbate are abundant,8 no simple and sensitive procedure exists for the determination of DHA. Its concentration is most often calculated from that of total vitamin C before and after complete reduction with a strong reductant15 or by similar difference estimations.16J7 This is also true for most HPLC methods,' because the detection of ascorbate is more sensitive and accurate due to the possibility of electrochemical detection'* or due to its high optical density in the UV range.19 Since the concentration of DHA is usually much smaller than that of ascorbate itself,' this creates the inherent problem of 1000
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[Dml ( W Flgwo 3. (a, top) Dependence of absorbance increase at 346 nm at different concentrations of dehydroascorbic acid on different concentrations of methanol in 0.2 M phosphate buffer, pH 7.1: 5 (.),lo (0).15 (A),25 (01,30 p),and (0)35% (v/v) methanol. (b, bottom) Same as Figure 2 but with varying concentrations of phosphate and a flxed concentratlon of 18% (v/v) methanol: 0.025 (0),0.05 (+),O,l (O), 0.2 (A),0.3 (A),0.4 M phosphate. F for linear correlations >0.990 in ail cases.
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Figure 8. Dependenceof absorbance Increase at 346 nm at different concentratlons of dehydroascorbic ackl on different concentrations of cltrate (0.1 (A),0.2 (0),0.3M (0))W i t h 18% (v/v) methanol. 3.O 2.5
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[Dml (W Flgure 1. Formation rates measured with several concentratlons of DHA in the presence of 0 (X), 25 (0),50 ( O ) ,75 (O), and 100 pM (.) ascorbate In 18% (vlv) methand, 0.2 M phosphate, pH 6.6, with 100 pM deferioxamlne and 100 mM EDTA. Data are from three experiments each: error bars were smaller then symbols. Ascorbate concentratlons were monitored by absorbance measurements at 265 nm. The changes of the absorbance always were
