Abstract
Human serum contains several antioxidants. The total antioxidant capacity (AOC) is the sum of all the antioxidant activities present in serum. The aim of our study was to investigate whether the AOC of bilirubin (BR), α‐tocopherol (TOH), ascorbic acid (AA), and uric acid (UA) could be measured with good precision and recovery in the presence of human serum albumin (HSA). We measured the AOC of each antioxidant using a Cobas Mira S instrument (Roche Diagnostic Systems, Montclair, NJ) by measuring the inhibitory effect of a given compound on the oxidation of the radical cations of 2,2′‐azino‐di‐(3‐ethylbenzthiazoline 6‐sulphonate) (ABTS) incubated with metmyoglobin and H2O2. The assay had a linear AOC response range of 27–2,000 μmol/L. The within‐ and between‐day coefficients of variation (CVs) did not exceed 3.4% and 4.2%, respectively. The AOC of albumin in serum is much greater than that of BR, TOH, AA, or UA owing to the substantially greater concentration of HSA in serum. An aliquot of a solution of AA, UA, BR, or TOH was added to HSA or distilled water, and the AOC was determined. The AOC of BR, TOH, AA, and UA increased in a linear way with increasing concentrations. However, we found that the magnitude of increase in the AOC of a mixture of HSA and any of these antioxidants was lower than the sum of the AOC of HSA and any one of the following: AA, UA, BR, or TOH (all expressed in μmol/L). J. Clin. Lab. Anal. 18:45–49, 2004. © 2004 Wiley‐Liss, Inc.
Keywords: antioxidants, free radicals, total antioxidant capacity, Trolox, AAPH
REFERENCES
- 1. Wayner DDM, Burton GW, Ingold KU, Barclay LRC, Locke SJ. The relative contributions of vitamin E, urate, ascorbate and proteins to the total peroxyl radical‐trapping antioxidant activity of human blood plasma. Biochim Biophys Acta 1987;924:408–419. [DOI] [PubMed] [Google Scholar]
- 2. Lindeman JHN, van Zoeren‐Grobben D, Schrijver J, Speek AJ, Poorthuis BJHM, Berger HM. The total free radical trapping ability of cord blood plasma in preterm and term babies. Pediatr Res 1989;26:20–24. [DOI] [PubMed] [Google Scholar]
- 3. Ihara H, Hashizume N, Hemmi H, Yoshida M. Antioxidant ability of bilirubin, vitamin E, vitamin C, and albumin in the peroxyl‐radical‐induced hemolysis of human erythrocytes. J Anal Biol Sci 2000;23:425–430. [Google Scholar]
- 4. Miller NJ, Rice‐Evans C, Davies MJ, Gopinathan V, Milner A. A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clin Sci 1993;84:407–412. [DOI] [PubMed] [Google Scholar]
- 5. Aoki Y, Ihara H, Nakamura H, Aoki T, Yoshida M. Delta bilirubin: its isolation by precipitation with ammonium sulfate and properties. Jpn J Clin Chem 1992;21:1–5. [Google Scholar]
- 6. Wayner DDM, Burton GW, Ingold KU. The antioxidant efficiency of vitamin C is concentration‐dependent. Biochim Biophys Acta 1986;884:119–123. [DOI] [PubMed] [Google Scholar]
- 7. Koster JF, Biemond P, Swaak AJG. Intracellular and extracellular sulfydryl levels in rheumatoid arthritis. Ann Rheum Dis 1986;45:44–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Muramoto Y, Matsushita M, Irino T. Reduction of reaction differences between human mercaptalbumin and human nonmercaptalbumin measured by the bromcresol purple method. Clin Chim Acta 1999;289:69–78. [DOI] [PubMed] [Google Scholar]
- 9. Niki E, Saito M, Yoshikawa Y, Yamamoto Y, Kamiya Y. Oxidation of lipids. XII. Inhibition of oxidation of soybean phosphatidylcholine and methyl linoleate in aqueous dispersions by uric acid. Bull Chem Soc Jpn 1986;59:471–477. [Google Scholar]
- 10. Sato K, Niki E, Shimasaki H. Free radical‐mediated chain oxidation of low density lipoprotein and its synergistic inhibition by vitamin E and vitamin C. Arch Biochem Biophys 1990;279:402–405. [DOI] [PubMed] [Google Scholar]
- 11. Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN. Bilirubin is an antioxidant of possible physiological importance. Science 1987;235:1043–1046. [DOI] [PubMed] [Google Scholar]
- 12. Stocker R, Glazer AN, Ames BN. Antioxidant activity of albumin‐bound bilirubin. Proc Natl Acad Sci USA 1987;84:5918–5922. [DOI] [PMC free article] [PubMed] [Google Scholar]