Skip to main content
PMC home page
Journal of Clinical Laboratory Analysis logoLink to Journal of Clinical Laboratory Analysis
. 2006 Jul 27;20(4):139–148. doi: 10.1002/jcla.20122

Parameters of oxidative stress status in healthy subjects: their correlations and stability after sample collection

Omidreza Firuzi 1, Přemysl Mladěnka 1, Valeria Riccieri 2, Antonio Spadaro 2, Rita Petrucci 3, Giancarlo Marrosu 3, Luciano Saso 1,✉
PMCID: PMC6807518  PMID: 16874810

Abstract

It has been proposed that sample storage may have some influence on the parameters of oxidative stress status (OSS) in biological fluids. We measured four important OSS parameters in plasma of 23 healthy subjects and repeated the measurements in the same samples kept at –70°C after different time intervals. Hydroperoxides and total antioxidant capacity (TAC) were determined by ferrous ion oxidation in presence of xylenol orange (FOX) and ferric reducing antioxidant power (FRAP) assays, respectively. Sulfhydryls and carbonyls were measured spectrophotometrically. In fresh samples, OSS seemed to increase with age and relatively good correlations were found among different parameters. The meanvalues of hydroperoxides (6.08 µM), TAC (0.334 mM Trolox equivalent), and sulfhydryls (0.562 mM) in fresh samples did not show any significant change after 1, 7, and 30 days of storage. Mean carbonyl concentration determined after 1 day storage (2.0 nmol/mg protein) did not change after 30 days. However, extents of changes in hydroperoxide concentrations varied considerably from one individual to another, even after 1 day. A similar phenomenon was observed in TAC, but after 7 days. We suggest measuring hydroperoxides in fresh samples and TAC maximally after 1 week. Sulfhydryls and carbonyls showed more stability and can be measured at least 1 month after sample collection. J. Clin. Lab. Anal. 20:139–148, 2006. © 2006 Wiley‐Liss, Inc.

Keywords: oxidative stress, storage, hydroperoxide(s), total antioxidant capacity, sulfhydryl(s), carbonyl(s)

  Abbreviations
DNPH

2,4‐dinitrophenylhydrazine
DTNB

5,5′‐dithiobis(2‐nitrobenzoic acid)
FOX

ferrous ion oxidation in presence of xylenol orange
FRAP

ferric reducing antioxidant power
OSS

oxidative stress status
ROOH

hydroperoxide
SH

sulfhydryl
TAC

total antioxidant capacity

REFERENCES

  • 1. Heinecke JW. Oxidative stress: new approaches to diagnosis and prognosis in atherosclerosis. Am J Cardiol 2003;91:12A–16A. [DOI] [PubMed] [Google Scholar]
  • 2. Pratico D, Zhukareva V, Yao Y, et al. 12/15‐lipoxygenase is increased in Alzheimer's disease: possible involvement in brain oxidative stress. Am J Pathol 2004;164:1655–1662. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Giasson BI, Duda JE, Murray IV, et al. Oxidative damage linked to neurodegeneration by selective alpha‐synuclein nitration in synucleinopathy lesions. Science 2000;290:985–989. [DOI] [PubMed] [Google Scholar]
  • 4. Baskol G, Demir H, Baskol M, et al. Assessment of paraoxonase 1 activity and malondialdehyde levels in patients with rheumatoid arthritis. Clin Biochem 2005;38:951–955. [DOI] [PubMed] [Google Scholar]
  • 5. Brown NS, Bicknell R. Hypoxia and oxidative stress in breast cancer. Oxidative stress: its effects on the growth, metastatic potential and response to therapy of breast cancer. Breast Cancer Res 2001;3:323–327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Kruidenier L, Verspaget HW. Review article: oxidative stress as a pathogenic factor in inflammatory bowel disease—radicals or ridiculous? Aliment Pharmacol Ther 2002;16:1997–2015. [DOI] [PubMed] [Google Scholar]
  • 7. Pryor WA. Measurement of oxidative stress status in humans. Cancer Epidemiol Biomarkers Prev 1993;2:289–292. [PubMed] [Google Scholar]
  • 8. Prior RL, Cao G. In vivo total antioxidant capacity: comparison of different analytical methods. Free Radic Biol Med 1999;27:1173–1181. [DOI] [PubMed] [Google Scholar]
  • 9. Nourooz‐Zadeh J, Tajaddini‐Sarmadi J, Wolff SP. Measurement of plasma hydroperoxide concentrations by the ferrous oxidation‐xylenol orange assay in conjunction with triphenylphosphine. Anal Biochem 1994;220:403–409. [DOI] [PubMed] [Google Scholar]
  • 10. Beier J, Beeh KM, Kornmann O, Buhl R. Stability of glutathione in induced sputum: impact of freezing. Respiration 2003;70:523–527. [DOI] [PubMed] [Google Scholar]
  • 11. Hectors MP, van Tits LJ, de Rijke YB, Demacker PN. Stability studies of ubiquinol in plasma. Ann Clin Biochem 2003;40:100–101. [DOI] [PubMed] [Google Scholar]
  • 12. Morrow JD, Roberts LJ II. Quantification of noncyclooxygenase derived prostanoids as a marker of oxidative stress. Free Radic Biol Med 1991;10:195–200. [DOI] [PubMed] [Google Scholar]
  • 13. Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal Biochem 1996;239:70–76. [DOI] [PubMed] [Google Scholar]
  • 14. Firuzi O, Lacanna A, Petrucci R, Marrosu G, Saso L. Evaluation of the antioxidant activity of flavonoids by “ferric reducing antioxidant power” assay and cyclic voltammetry. Biochim Biophys Acta 2005;1721:174–184. [DOI] [PubMed] [Google Scholar]
  • 15. Hu ML. Measurement of protein thiol groups and glutathione in plasma. Methods Enzymol 1994;233:380–385. [DOI] [PubMed] [Google Scholar]
  • 16. Levine RL, Garland D, Oliver CN, et al. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol 1990;186:464–478. [DOI] [PubMed] [Google Scholar]
  • 17. Firuzi O, Giansanti L, Vento R, et al. Hypochlorite scavenging activity of hydroxycinnamic acids evaluated by a rapid microplate method based on the measurement of chloramines. J Pharm Pharmacol 2003;55:1021–1027. [DOI] [PubMed] [Google Scholar]
  • 18. Nourooz‐Zadeh J. Ferrous ion oxidation in presence of xylenol orange for detection of lipid hydroperoxides in plasma. Methods Enzymol 1999;300:58–62. [DOI] [PubMed] [Google Scholar]
  • 19. Nourooz‐Zadeh J, Smith CC, Betteridge DJ. Measures of oxidative stress in heterozygous familial hypercholesterolaemia. Atherosclerosis 2001;156:435–441. [DOI] [PubMed] [Google Scholar]
  • 20. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein‐dye binding. Anal Biochem 1976;72:248–254. [DOI] [PubMed] [Google Scholar]
  • 21. Macart M, Gerbaut L. An improvement of the Coomassie Blue dye binding method allowing an equal sensitivity to various proteins: application to cerebrospinal fluid. Clin Chim Acta 1982;122:93–101. [DOI] [PubMed] [Google Scholar]
  • 22. Halliwell B, Chirico S. Lipid peroxidation: its mechanism, measurement, and significance. Am J Clin Nutr 1993;57:715S–724S. [DOI] [PubMed] [Google Scholar]
  • 23. Nourooz‐Zadeh J, Rahimi A, Tajaddini‐Sarmadi J, et al. Relationships between plasma measures of oxidative stress and metabolic control in NIDDM. Diabetologia 1997;40:647–653. [DOI] [PubMed] [Google Scholar]
  • 24. Nourooz‐Zadeh J, Tajaddini‐Sarmadi J, McCarthy S, Betteridge DJ, Wolff SP. Elevated levels of authentic plasma hydroperoxides in NIDDM. Diabetes 1995;44:1054–1058. [DOI] [PubMed] [Google Scholar]
  • 25. Nuttall SL, Heaton S, Piper MK, Martin U, Gordon C. Cardiovascular risk in systemic lupus erythematosus—evidence of increased oxidative stress and dyslipidaemia. Rheumatology (Oxford) 2003;42:758–762. [DOI] [PubMed] [Google Scholar]
  • 26. Sodergren E, Nourooz‐Zadeh J, Berglund L, Vessby B. Re‐evaluation of the ferrous oxidation in xylenol orange assay for the measurement of plasma lipid hydroperoxides. J Biochem Biophys Methods 1998;37:137–146. [DOI] [PubMed] [Google Scholar]
  • 27. Holley AE, Slater TF. Measurement of lipid hydroperoxides in normal human blood plasma using HPLC‐chemiluminescence linked to a diode array detector for measuring conjugated dienes. Free Radic Res Commun 1991;15:51–63. [DOI] [PubMed] [Google Scholar]
  • 28. Frei B, Stocker R, Ames BN. Antioxidant defenses and lipid peroxidation in human blood plasma. Proc Natl Acad Sci USA 1998;85:9748–9752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Bub A, Watzl B, Abrahamse L, et al. Moderate intervention with carotenoid‐rich vegetable products reduces lipid peroxidation in men. J Nutr 2000;130:2200–2206. [DOI] [PubMed] [Google Scholar]
  • 30. Cao G, Russell RM, Lischner N, Prior RL. Serum antioxidant capacity is increased by consumption of strawberries, spinach, red wine or vitamin C in elderly women. J Nutr 1998;128:2383–2390. [DOI] [PubMed] [Google Scholar]
  • 31. Castenmiller JJ, Lauridsen ST, Dragsted LO, van het Hof KH, Linssen JP, West CE. beta‐carotene does not change markers of enzymatic and nonenzymatic antioxidant activity in human blood. J Nutr 1999;129:2162–2169. [DOI] [PubMed] [Google Scholar]
  • 32. Erdogan C, Unlucerci Y, Turkmen A, Kuru A, Cetin O, Bekpinar S. The evaluation of oxidative stress in patients with chronic renal failure. Clin Chim Acta 2002;322:157–161. [DOI] [PubMed] [Google Scholar]
  • 33. Wijnberger LD, Krediet TG, Visser GH, van Bel F, Egberts J. Early neonatal antioxidant capacity after preexisting impaired placental function. Early Hum Dev 2003;71:111–116. [DOI] [PubMed] [Google Scholar]
  • 34. Himmelfarb J, McMonagle E, McMenamin E. Plasma protein thiol oxidation and carbonyl formation in chronic renal failure. Kidney Int 2000;58:2571–2578. [DOI] [PubMed] [Google Scholar]
  • 35. Jaswal S, Mehta HC, Sood AK, Kaur J. Antioxidant status in rheumatoid arthritis and role of antioxidant therapy. Clin Chim Acta 2003;338:123–129. [DOI] [PubMed] [Google Scholar]
  • 36. Situnayake RD, Thurnham DI, Kootathep S, et al. Chain breaking antioxidant status in rheumatoid arthritis: clinical and laboratory correlates. Ann Rheum Dis 1991;50:81–86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. McGrath LT, Mallon P, Dowey L, et al. Oxidative stress during acute respiratory exacerbations in cystic fibrosis. Thorax 1999;54:518–523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Roberts JC, Francetic DJ. The importance of sample preparation and storage in glutathione analysis. Anal Biochem 1993;211:183–187. [DOI] [PubMed] [Google Scholar]
  • 39. Telci A, Cakatay U, Salman S, Satman I, Sivas A. Oxidative protein damage in early stage Type 1 diabetic patients. Diabetes Res Clin Pract 2000;50:213–223. [DOI] [PubMed] [Google Scholar]
  • 40. Chevion M, Berenshtein E, Stadtman ER. Human studies related to protein oxidation: protein carbonyl content as a marker of damage. Free Radic Res 2000;33:S99–S108. [PubMed] [Google Scholar]
  • 41. Picklo MJ, Montine TJ, Amarnath V, Neely MD. Carbonyl toxicology and Alzheimer's disease. Toxicol Appl Pharmacol 2002;184:187–197. [DOI] [PubMed] [Google Scholar]
  • 42. Mantle D, Falkous G, Walker D. Quantification of protease activities in synovial fluid from rheumatoid and osteoarthritis cases: comparison with antioxidant and free radical damage markers. Clin Chim Acta 1999;284:45–58. [DOI] [PubMed] [Google Scholar]
  • 43. Winterbourn CC, Chan T, Buss IH, Inder TE, Mogridge N, Darlow BA. Protein carbonyls and lipid peroxidation products as oxidation markers in preterm infant plasma: associations with chronic lung disease and retinopathy and effects of selenium supplementation. Pediatr Res 2000;48:84–90. [DOI] [PubMed] [Google Scholar]
  • 44. Garibaldi S, Aragno I, Odetti P, Marinari UM. Relationships between protein carbonyls, retinol and tocopherols level in human plasma. Biochem Mol Biol Int 1994;34:729–736. [PubMed] [Google Scholar]
  • 45. Mutlu‐Turkoglu U, Ilhan E, Oztezcan S, Kuru A, Aykac‐Toker G, Uysal M. Age‐related increases in plasma malondialdehyde and protein carbonyl levels and lymphocyte DNA damage in elderly subjects. Clin Biochem 2003;36:397–400. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Laboratory Analysis are provided here courtesy of Wiley

RESOURCES