{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T07:51:46Z","timestamp":1789977106434,"version":"4.0.1"},"reference-count":37,"publisher":"Oxford University Press (OUP)","issue":"7","license":[{"start":{"date-parts":[[2006,7,1]],"date-time":"2006-07-01T00:00:00Z","timestamp":1151712000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/journals\/pages\/open_access\/funder_policies\/chorus\/standard_publication_model"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2006,7,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:p>Background: Reduced glutathione (GSH) and its redox forms, glutathione disulfide (GSSG) and glutathionylated proteins (PSSG), are biomarkers of oxidative stress, but methodologic artifacts can interfere with their measurement. We evaluated the importance of correct sample handling during the preanalytical phase for GSH, GSSG, and PSSG measurement.<\/jats:p>\n               <jats:p>Methods: We used human blood for in vitro experiments with oxidants [tert-butylhydroperoxide (t-BOOH), diamide, and menadione]. For in vivo experiments, we used rats in which we cannulated the jugular and femoral veins for both oxidant administration and blood collection. We measured GSH, GSSG, and PSSG with HPLC with or without sample pretreatment with N-ethylmaleimide (NEM) to prevent artifacts. We also measured malondialdehyde (MDA) with HPLC, and protein carbonyls (PCO) with spectrophotometric procedures.<\/jats:p>\n               <jats:p>Results: When methodologic artifacts were prevented by pretreatment with NEM, GSSG results increased up to 3-fold over the basal concentrations, even in the presence of 5 \u03bcmol\/L t-BOOH or diamide and 20 \u03bcmol\/L menadione. PSSG increased by \u223c50% at 20 \u03bcmol\/L t-BOOH or diamide and at 50 \u03bcmol\/L menadione. PCO and MDA remained unchanged. In vivo oxidation treatments elicited immediate and significant increases in GSSG and PSSG over basal values (up to 200-fold), whereas PCO and MDA showed only slight variation 120 or 180 min after treatment.<\/jats:p>\n               <jats:p>Conclusions: With the use of artifact-free measurement methods, GSH, GSSG, and PSSG are potentially powerful and reliable biomarkers of oxidative stress status and can be used to evaluate whether, and to what extent, oxidative stress may be involved in various diseases.<\/jats:p>","DOI":"10.1373\/clinchem.2006.067793","type":"journal-article","created":{"date-parts":[[2006,5,12]],"date-time":"2006-05-12T07:56:19Z","timestamp":1147420579000},"page":"1406-1414","source":"Crossref","is-referenced-by-count":133,"title":["Oxidized Forms of Glutathione in Peripheral Blood as Biomarkers of Oxidative Stress"],"prefix":"10.1093","volume":"52","author":[{"given":"Ranieri","family":"Rossi","sequence":"first","affiliation":[{"name":"Department of Neuroscience, Pharmacology Section, University of Siena, Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isabella","family":"Dalle-Donne","sequence":"first","affiliation":[{"name":"Department of Biology, University of Milan, Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aldo","family":"Milzani","sequence":"first","affiliation":[{"name":"Department of Biology, University of Milan, Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniela","family":"Giustarini","sequence":"first","affiliation":[{"name":"Department of Neuroscience, Pharmacology Section, University of Siena, Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2006,7,1]]},"reference":[{"key":"2020022801245632000_R1","doi-asserted-by":"crossref","unstructured":"Dalle-Donne I, Rossi R, Colombo R, Giustarini D, Milzani A. Biomarkers of oxidative damage in human disease [Review]. Clin Chem2006;52:601-623.","DOI":"10.1373\/clinchem.2005.061408"},{"key":"2020022801245632000_R2","doi-asserted-by":"crossref","unstructured":"Pryor WA eds. Bio-assays for Oxidative Stress Status [BOSS]2001:286 Elsevier Science BV Amsterdam. .","DOI":"10.1016\/B978-0-444-50957-4.50029-6"},{"key":"2020022801245632000_R3","doi-asserted-by":"crossref","unstructured":"Meagher EA, FitzGerald GA. Indices of lipid peroxidation in vivo: strengths and limitations [Review]. Free Radic Biol Med2000;28:1745-1750.","DOI":"10.1016\/S0891-5849(00)00232-X"},{"key":"2020022801245632000_R4","doi-asserted-by":"crossref","unstructured":"Stadtman ER, Levine RL. Free radical-mediated oxidation of free amino acids and amino acid residues in proteins [Review]. Amino Acids2003;25:207-218.","DOI":"10.1007\/s00726-003-0011-2"},{"key":"2020022801245632000_R5","unstructured":"Halliwell B, Gutteridge JMC. Antioxidant defences. Halliwell B Gutteridge JMC eds. Free Radicals in Biology and Medicine1999:105-245 Oxford University Press Oxford. ."},{"key":"2020022801245632000_R6","doi-asserted-by":"crossref","unstructured":"Schafer FQ, Buettner GR. Redox environment of the cell as viewed through the redox state of the glutathione disulfide\/glutathione couple [Review]. Free Radic Biol Med2001;30:1191-1212.","DOI":"10.1016\/S0891-5849(01)00480-4"},{"key":"2020022801245632000_R7","doi-asserted-by":"crossref","unstructured":"Pastore A, Federici G, Bertini E, Piemonte F. Analysis of glutathione: implication in redox and detoxification [Review]. Clin Chim Acta2003;333:19-39.","DOI":"10.1016\/S0009-8981(03)00200-6"},{"key":"2020022801245632000_R8","doi-asserted-by":"crossref","unstructured":"Rossi R, Milzani A, Dalle-Donne I, Giustarini D, Lusini L, Colombo R, et al. Blood glutathione disulfide: in vivo factor or in vitro artifact?. Clin Chem2002;48:742-753.","DOI":"10.1093\/clinchem\/48.5.742"},{"key":"2020022801245632000_R9","doi-asserted-by":"crossref","unstructured":"Giustarini D, Dalle-Donne I, Colombo R, Petralia S, Giampaoletti S, Milzani R, et al. Protein glutathionylation in erythrocytes [Technical Brief]. Clin Chem2003;49:327-330.","DOI":"10.1373\/49.2.327"},{"key":"2020022801245632000_R10","doi-asserted-by":"crossref","unstructured":"Giustarini D, Dalle-Donne I, Colombo R, Milzani A, Rossi R. An improved HPLC measurement for GSH and GSSG in human blood. Free Radic Biol Med2003;35:1365-1372.","DOI":"10.1016\/j.freeradbiomed.2003.08.013"},{"key":"2020022801245632000_R11","doi-asserted-by":"crossref","unstructured":"Carru C, Zinellu A, Sotgia S, Marongiu G, Farina MG, Usai MF, et al. Optimization of the principal parameters for the ultrarapid electrophoretic separation of reduced and oxidized glutathione by capillary electrophoresis. J Chromatogr A2003;1017:233-238.","DOI":"10.1016\/j.chroma.2003.08.020"},{"key":"2020022801245632000_R12","doi-asserted-by":"crossref","unstructured":"Steghens JP, Flourie F, Arab K, Collombel C. Fast liquid chromatography-mass spectrometry glutathione measurement in whole blood: micromolar GSSG is a sample preparation artifact. J Chromatogr B Analyt Technol Biomed Life Sci2003;798:343-349.","DOI":"10.1016\/j.jchromb.2003.10.007"},{"key":"2020022801245632000_R13","doi-asserted-by":"crossref","unstructured":"Nemeth I, Orvos H, Boda D. Blood glutathione redox status in gestational hypertension. Free Radic Biol Med2001;30:715-721.","DOI":"10.1016\/S0891-5849(00)00516-5"},{"key":"2020022801245632000_R14","doi-asserted-by":"crossref","unstructured":"Rossi R, Milzani A, Dalle-Donne I, Giannerini F, Giustarini D, Lusini L, et al. Different metabolizing ability of thiol reactants in human and rat blood. J Biol Chem2001;276:7004-7010.","DOI":"10.1074\/jbc.M005156200"},{"key":"2020022801245632000_R15","doi-asserted-by":"crossref","unstructured":"De Maria N, Colantoni A, Fagiuoli S, Liu GJ, Rogers BK, Farinati F, et al. Association between reactive oxygen species and disease activity in chronic hepatitis C. Free Radic Biol Med1996;21:291-295.","DOI":"10.1016\/0891-5849(96)00044-5"},{"key":"2020022801245632000_R16","doi-asserted-by":"crossref","unstructured":"Richard MJ, Guiraud P, Meo J, Favier A. High-performance liquid chromatographic separation of malondialdehyde-thiobarbituric acid adduct in biological materials (plasma and human cells) using a commercially available reagent. J Chromatogr1992;577:9-18.","DOI":"10.1016\/0378-4347(92)80593-F"},{"key":"2020022801245632000_R17","doi-asserted-by":"crossref","unstructured":"Gallemann D, Eyer P. Formation of hydrogen peroxide during precipitation of red cells with perchloric acid: a cautionary note for precise determination of pyruvate, GSH and NAD(P)H. Anal Biochem1990;191:347-353.","DOI":"10.1016\/0003-2697(90)90230-7"},{"key":"2020022801245632000_R18","doi-asserted-by":"crossref","unstructured":"Kosower NS, Kosower EM, Wertheim B. Diamide, a new reagent for the intracellular oxidation of glutathione to the disulfide. Biochem Biophys Res Commun1969;37:593-595.","DOI":"10.1016\/0006-291X(69)90850-X"},{"key":"2020022801245632000_R19","doi-asserted-by":"crossref","unstructured":"Lii CK, Hung CN. Protein thiol modifications of human red blood cells treated with t-butyl hydroperoxide. Biochim Biophys Acta1997;1336:147-156.","DOI":"10.1016\/S0304-4165(97)00020-2"},{"key":"2020022801245632000_R20","doi-asserted-by":"crossref","unstructured":"Di Monte D, Ross D, Bellomo G, Eklow L, Orrenius S. Alterations in intracellular thiol homeostasis during the metabolism of menadione by isolated rat hepatocytes. Arch Biochem Biophys1984;235:334-342.","DOI":"10.1016\/0003-9861(84)90206-6"},{"key":"2020022801245632000_R21","doi-asserted-by":"crossref","unstructured":"Dalle-Donne I, Rossi R, Ceciliani F, Giustarini D, Colombo R, Milzani A. Proteins as sensitive biomarkers of human conditions associated with oxidative\/nitrosative stress. Dalle-Donne I Scaloni A Butterfield DA eds. Redox Proteomics: From Protein Modifications to Cellular Dysfunction and Diseases2006:487-525 John Wiley & Sons Hoboken, NJ. (in press).","DOI":"10.1002\/0471973122.ch16"},{"key":"2020022801245632000_R22","doi-asserted-by":"crossref","unstructured":"Rossi R, Barra D, Bellelli A, Boumis G, Canofeni S, Di Simplicio P, et al. Fast reacting thiols in rat hemoglobin can intercept damaging species in erythrocytes more efficiently than glutathione. J Biol Chem1998;273:19198-19206.","DOI":"10.1074\/jbc.273.30.19198"},{"key":"2020022801245632000_R23","doi-asserted-by":"crossref","unstructured":"Sampathkumar R, Balasubramanyam M, Sudarslal S, Rema M, Mohan V, Balaram P. Increased glutathionylated hemoglobin (HbSSG) in type 2 diabetes subjects with microangiopathy. Clin Biochem2005;38:892-899.","DOI":"10.1016\/j.clinbiochem.2005.06.009"},{"key":"2020022801245632000_R24","doi-asserted-by":"crossref","unstructured":"Maeso N, Garcia-Martinez D, Ruperez FJ, Cifuentes A, Barbas C. Capillary electrophoresis of glutathione to monitor oxidative stress and response to antioxidant treatments in an animal model. J Chromatogr B2005;822:61-69.","DOI":"10.1016\/j.jchromb.2005.05.015"},{"key":"2020022801245632000_R25","doi-asserted-by":"crossref","unstructured":"Herzenberg LA, De Rosa SC, Dubs JG, Roederer M, Anderson MT, Ela SW, et al. Glutathione deficiency is associated with impaired survival in HIV disease. Proc Natl Acad Sci U S A1972;94:1967-1972.","DOI":"10.1073\/pnas.94.5.1967"},{"key":"2020022801245632000_R26","doi-asserted-by":"crossref","unstructured":"Pirmohamed M, Williams D, Tingle MD, Barry M, Khoo SH, O\u2019Mahony C, et al. Intracellular glutathione in the peripheral blood cells of HIV-infected patients: failure to show a deficiency. AIDS1996;10:501-507.","DOI":"10.1097\/00002030-199605000-00008"},{"key":"2020022801245632000_R27","doi-asserted-by":"crossref","unstructured":"Naito C, Niwa T. Analysis of glutathionyl hemoglobin levels in diabetic patients by electrospray ionization liquid chromatography-mass spectrometry: effect of vitamin E administration. J Chromatogr B2000;746:91-94.","DOI":"10.1016\/S0378-4347(00)00121-3"},{"key":"2020022801245632000_R28","doi-asserted-by":"crossref","unstructured":"Gladwin MT, Shelhamer JH, Ognibene FP, Pease-Fye ME, Nichols JS, Link B, et al. Nitric oxide donor properties of hydroxyurea in patients with sickle cell disease. Br J Haematol2002;116:436-444.","DOI":"10.1046\/j.1365-2141.2002.03274.x"},{"key":"2020022801245632000_R29","doi-asserted-by":"crossref","unstructured":"Spooren AAMG, Evelo CTA. A study on the interaction between hydroxylamine analogues and oxyhemoglobin in intact erythrocytes. Blood Cells Mol Dis1997;23:323-336.","DOI":"10.1006\/bcmd.1997.0150"},{"key":"2020022801245632000_R30","doi-asserted-by":"crossref","unstructured":"Yeh CC, Hou MF, Tsai SM, Lin SK, Hsiao JK, Huang JC, et al. Superoxide anion radical, lipid peroxides and antioxidant status in the blood of patients with breast cancer. Clin Chim Acta2005;361:104-111.","DOI":"10.1016\/j.cccn.2005.05.002"},{"key":"2020022801245632000_R31","doi-asserted-by":"crossref","unstructured":"Turi S, Friedman A, Bereczki C, Papp F, Kovacs J, Karg E, et al. Oxidative stress in juvenile essential hypertension. J Hypertens2003;21:1-8.","DOI":"10.1097\/00004872-200301000-00024"},{"key":"2020022801245632000_R32","doi-asserted-by":"crossref","unstructured":"Blankenberg S, Rupprecht HJ, Bickel C, Torzewski M, Hafner G, Tiret L, et al. Glutathione peroxidase 1 activity and cardiovascular events in patients with coronary artery disease. N Engl J Med2003;349:1605-1613.","DOI":"10.1056\/NEJMoa030535"},{"key":"2020022801245632000_R33","doi-asserted-by":"crossref","unstructured":"Schnabel R, Lackner KJ, Rupprecht HJ, Espinola-Klein C, Torzewski M, Lubos E, et al. Glutathione peroxidase-1 and homocysteine for cardiovascular risk prediction: results from the AtheroGene study. J Am Coll Cardiol2005;45:1631-1637.","DOI":"10.1016\/j.jacc.2005.02.053"},{"key":"2020022801245632000_R34","doi-asserted-by":"crossref","unstructured":"Ischiropoulos H, Beckman JS. Oxidative stress and nitration in neurodegeneration: cause, effect, or association? [Review]. J Clin Invest2003;111:163-169.","DOI":"10.1172\/JCI200317638"},{"key":"2020022801245632000_R35","doi-asserted-by":"crossref","unstructured":"Stocker R, Keaney JF, Jr. Role of oxidative modifications in atherosclerosis [Review]. Physiol Rev2004;84:1381-1478.","DOI":"10.1152\/physrev.00047.2003"},{"key":"2020022801245632000_R36","doi-asserted-by":"crossref","unstructured":"Lonn E, Bosch J, Yusuf S, Sheridan P, Pogue J, Arnold JM, et al. Effects of long-term vitamin E supplementation on cardiovascular events and cancer: a randomized controlled trial. JAMA2005;293:1338-1347.","DOI":"10.1001\/jama.293.11.1338"},{"key":"2020022801245632000_R37","doi-asserted-by":"crossref","unstructured":"Vivekananthan DP, Penn MS, Sapp SK, Hsu A, Topol EJ. Use of antioxidant vitamins for the prevention of cardiovascular disease: meta-analysis of randomized trials. Lancet2003;361:2017-2023.","DOI":"10.1016\/S0140-6736(03)13637-9"}],"container-title":["Clinical Chemistry"],"original-title":[],"language":"en","link":[{"URL":"http:\/\/academic.oup.com\/clinchem\/article-pdf\/52\/7\/1406\/32685970\/clinchem1406.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"http:\/\/academic.oup.com\/clinchem\/article-pdf\/52\/7\/1406\/32685970\/clinchem1406.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2020,2,28]],"date-time":"2020-02-28T06:27:00Z","timestamp":1582871220000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/clinchem\/article\/52\/7\/1406\/5626882"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2006,7,1]]},"references-count":37,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2006,7,1]]},"published-print":{"date-parts":[[2006,7,1]]}},"URL":"https:\/\/doi.org\/10.1373\/clinchem.2006.067793","relation":{},"ISSN":["0009-9147","1530-8561"],"issn-type":[{"value":"0009-9147","type":"print"},{"value":"1530-8561","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2006,7,1]]},"published":{"date-parts":[[2006,7,1]]}}}