Measurement of Ascorbic Acid and Dehydroascorbic Acid in Biological Samples

Jens Lykkesfeldt1

1 Royal Veterinary and Agricultural University, Copenhagen, Denmark
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 7.6
DOI:  10.1002/0471140856.tx0706s12
Online Posting Date:  August, 2002
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Abstract

Ascorbic acid and dehydroascorbic acid are commonly used biomarkers of oxidative stress in a variety of experimental models. However, the accurate measurement of these labile compounds remains a challenge both in terms of sample collection and analysis. Determination of dehydroascorbic acid most commonly involves indirect measurement. The concentration is calculated by subtraction of the measured ascorbic acid concentration from that of total ascorbic acid analyzed after reduction of the dehydroascorbic acid present; a method referred to as the subtraction method. Consequently, successful determination of dehydroascorbic acid is dependent upon proper sample handling, quantitative reduction of the compound, and accurate quantification of both ascorbic acid and total ascorbic acid. The unit presents a detailed introduction to ascorbate analysis in biological samples and discusses common problems and pitfalls. The analytical method described is based on reversed‐phase HPLC with coloumetric detection. This method includes co‐analysis of isoascorbic acid and uric acid. Where applicable, uric acid can conveniently be used as an endogenous intrasample standard that significantly improves the accuracy of the subsequent dehydroascorbic acid calculation.

     
 
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Table of Contents

  • Basic Protocol 1: Analysis of Ascorbic Acid, Dehydroascorbic Acid, Isoascorbic Acid, Dehydroisoascorbic Acid, and Uric Acid by HPLC with Coulometric Detection
  • Collection and Storage of Biological Samples Intended for Ascorbic Acid and Dehydroascorbic Acid Analysis
  • Support Protocol 1: Preparation of Plasma Samples
  • Support Protocol 2: Preparation of Isolated‐Cell Samples
  • Support Protocol 3: Preparation of Tissue Samples
  • Support Protocol 4: Passivation of an HPLC System
  • Support Protocol 5: Quantification of Ascorbic Acid and Dehydroascorbic Acid using Subtraction Methods with Endogenous and/or Internal Standards
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Analysis of Ascorbic Acid, Dehydroascorbic Acid, Isoascorbic Acid, Dehydroisoascorbic Acid, and Uric Acid by HPLC with Coulometric Detection

  Materials
  • Mobile phase (see recipe)
  • Plasma (see protocol 2), isolated‐cell (see protocol 3), or tissue homogenate sample (see protocol 4) stabilized with meta‐phosphoric acid (MPA)
  • 1 mM Na 2EDTA prepared from 10 mM Na 2EDTA (372 mg/100 ml)
  • TCEP solution (see recipe)
  • McIlvaine buffer (see recipe)
  • Standard mixture (see recipe)
  • 5% MPA/EDTA solution (see recipe)
  • High‐performance liquid chromatography (HPLC) system (for passivation see protocol 4), including:
    • Isocratic pump (Agilent 1100 series, or equivalent low‐pulsation‐type pump)
    • Vacuum degasser (e.g., Agilent 1100 series), optional
    • Column thermostat (e.g., Agilent 1100 series), 30°C
    • Thermostatted autosampler, 10‐µl injection volume (e.g., Agilent 1100 series), 5°C
  • Uninterruptible power supply (UPS) for HPLC system (e.g., Smart‐UPS 3000VA with extended battery pack, part nos. SU3000INET and SU48BP, respectively; American Power Conversion), optional, but highly recommended
  • Coulometric detector (ESA Coulochem II; ESA) and accessories, including:
    • HP 35900E interface module (Agilent)
    • Model 5020 guard cell (ESA)
    • Model 5010 dual analytical cell (ESA)
  • HPLC column: 7.5‐cm × 4.6‐mm (i.d.) Luna C18(2) 3‐µm particle size (Phenomenex)
  • Guard column: 4.0‐mm × 3.0‐mm (i.d.) SecurityGuard C18 (Phenomenex)
  • Microcentrifuge (e.g., Eppendorf 5417R or equivalent), 4°C
  • Amber HPLC vials with caps (e.g., Agilent), inserts may be required for small volumes

Support Protocol 1: Preparation of Plasma Samples

  Materials
  • Anticoagulated blood sample (e.g., collected in an EDTA‐ or heparin‐containing vacutainer)
  • 10% MPA/EDTA solution (see recipe), 4°C
  • Microcentrifuge (e.g., Eppendorf 5417R or equivalent), 4°C

Support Protocol 2: Preparation of Isolated‐Cell Samples

  Materials
  • Isolated cells in suspension (e.g., 2 × 106 hepatocytes/ml)
  • 10% MPA/EDTA solution (see recipe), 4°C
  • Microcentrifuge (e.g., Eppendorf 5417R or equivalent), 4°C

Support Protocol 3: Preparation of Tissue Samples

  Materials
  • Isolated intact organ
  • PBS ( appendix 2A) or 1.15% (w/v) potassium chloride, 4°C
  • 5% MPA/EDTA solution (see recipe), 4°C
  • 2‐ml conical Potter‐Elvehjem homogenizer, on ice
  • Stirring motor for homogenization (e.g., IKA Eurostar digital, part no. 2482000; IKA‐Werke)
  • Microcentrifuge (e.g., Eppendorf 5417R or equivalent), 4°C

Support Protocol 4: Passivation of an HPLC System

  • 6 N nitric acid
  • Glacial acetic acid
  • Unions appropriate for the high‐performance liquid chromatography (HPLC) system
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Figures

Videos

Literature Cited

Literature Cited
   Ali, M.S. and Phillippo, E.T. 1996. Simultaneous determination of ascorbic, dehydroascorbic, isoascorbic, and dehydroisoascorbic acids in meat‐based food products by liquid chromatography with postcolumn fluorescence detection: A method extension. J. AOAC Int. 79:803‐808.
   Berry, M.N., Edwards, A.M., and Barritt, G.J. 1991. Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 21: Isolated Hepatocytes: Preparation, Properties and Applications. Elsevier/North‐Holland, Amsterdam.
   Bode, A.M., Cunningham, L., and Rose, R.C. 1990. Spontaneous decay of oxidized ascorbic acid (dehydro‐L‐ascorbic acid) evaluated by high‐pressure liquid chromatography. Clin. Chem. 36:1807‐1809.
   Deutsch, J.C. and Kolhouse, J.F. 1993. Ascorbate and dehydroascorbate measurements in aqueous solutions and plasma determined by gas chromatography‐mass spectrometry. Anal. Chem. 65:321‐326.
   Frei, B., England, L., and Ames, B.N. 1989. Ascorbate is an outstanding antioxidant in human blood plasma. Proc. Natl. Acad. Sci. U.S.A. 86:6377‐6381.
   Hagen, T.M., Ingersoll, R.T., Wehr, C.M., Lykkesfeldt, J., Vinarsky, V., Bartholomew, J.C., Song, M.H., and Ames, B.N. 1998. Acetyl‐L‐carnitine fed to old rats partially restores mitochondrial function and ambulatory activity. Proc. Natl. Acad. Sci. U.S.A. 95:9562‐9566.
   Hagen, T.M., Ingersoll, R.T., Lykkesfeldt, J., Liu, J., Wehr, C.M., Vinarsky, V., Bartholomew, J.C., and Ames, B.N. 1999. (R)‐alpha‐lipoic acid‐supplemented old rats have improved mitochondrial function, decreased oxidative damage, and increased metabolic rate. FASEB J. 13:411‐418.
   Iheanacho, E.N., Hunt, N.H., Stocker, R. 1995. 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.
   Kall, M.A. and Andersen, C. 1999. Improved method for simultaneous determination of ascorbic acid and dehydroascorbic acid, isoascorbic acid and dehydroisoascorbic acid in food and biological samples. J. Chromatogr. B Biomed. Sci. Appl. 730:101‐111.
   Lykkesfeldt, J. 2000. Determination of ascorbic acid and dehydroascorbic acid in biological samples by high‐performance liquid chromatography using subtraction methods: Reliable reduction with Tris[2‐carboxyethyl]phosphine hydrochloride. Anal. Biochem. 282:89‐93.
   Lykkesfeldt, J. and Ames, B.N. 1999. Ascorbic acid recycling in rat hepatocytes as measurement of antioxidant capacity: Decline with age. Methods Enzymol. 299:83‐88.
   Lykkesfeldt, J., Loft, S., and Poulsen, H.E. 1995. Determination of ascorbic acid and dehydroascorbic acid in plasma by high‐performance liquid chromatography with coulometric detection—are they reliable biomarkers of oxidative stress? Anal. Biochem. 229:329‐335.
   Lykkesfeldt, J., Prieme, H., Loft, S., and Poulsen, H.E. 1996. Effect of smoking cessation on plasma ascorbic acid concentration. Br. Med. J. 313:91.
   Lykkesfeldt, J., Loft, S., Nielsen, J.B., and Poulsen, H.E. 1997. Ascorbic acid and dehydroascorbic acid as biomarkers of oxidative stress caused by smoking. Am. J. Clin. Nutr. 65:959‐963.
   Lykkesfeldt, J., Hagen, T.M., Vinarsky, V., and Ames, B.N. 1998. Age‐associated decline in ascorbic acid concentration, recycling, and biosynthesis in rat hepatocytes: Reversal with (R)‐alpha‐lipoic acid supplementation. FASEB J. 12:1183‐1189.
   Lykkesfeldt, J., Christen, S., Wallock, L.M., Chang, H.H., Jacob, R.A., and Ames, B.N. 2000. Ascorbate is depleted by smoking and repleted by moderate supplementation: A study in male smokers and nonsmokers with matched dietary antioxidant intakes. Am. J. Clin. Nutr. 71:530‐536.
   Poulsen, H.E., Loft, S., Prieme, H., Vistisen, K., Lykkesfeldt, J., Nyyssonen, K., and Salonen, J.T. 1998. Oxidative DNA damage in vivo: Relationship to age, plasma antioxidants, drug metabolism, glutathione‐S‐transferase activity and urinary creatinine excretion. Free Radic. Res. 29:565‐571.
   Rumsey, S.C. and Levine, M. 2000. Vitamin C. In Chemical Analysis, Vol.154: Modern Analytical Methodologies in Fat‐ and Water‐Soluble Vitamins (W.O. Song, G.R. Beecher, and R.R. Eitenmiller, eds.) pp. 411‐445. John Wiley & Sons, New York.
   Sauberlich, H.E., Wood, S.M., Tamura, T., Freeberg, L.E. 1991. Influence of dietary intakes of erythorbic acid on plasma vitamin C analyses. Am. J. Clin. Nutr. 54:1319S‐1322S.
Key References
   Lykkesfeldt, 2000. See above.
  The method on which the is based. Includes stability data and reduction kinetics for TCEP.
   Rumsey and Levine, 2000. See above.
  Critical and comprehensive review of vitamin C analysis.
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