Measurementofoxysterolsand a-tocopherolinplasmaand tissuesamplesasindicesofoxidantstressstatus L.Iuliano, a, * F.Micheletta, a S.Natoli, a S.GinanniCorradini, b M.Iappelli, c W.Elisei, b L.Giovannelli, b F.Violi, d andU.Diczfalusy e a Department of Internal Medicine, University La Sapienza, Rome, Italy b Department of Clinical Medicine, University La Sapienza, Rome, Italy c Department of Surgery, University La Sapienza, Rome, Italy d Department of Experimental Medicine, University La Sapienza, Via del Policlinico 155, 00185 Rome, Italy e Department of Laboratory Sciences and Technology, Karolinska Institut, Huddinge, Sweden Received2August2002 Abstract Oxidantstressseemstoplayaroleinseveralsettingofhumanpathology,suchasatherosclerosis,cancer,andaging.Thestudyof oxidantstressinhumandiseaseshouldbebasedontheevaluationofeithersensitiveandspecificmarkersofenhancedoxidantstress, suchasoxysterols,orantioxidantdefense,bymeasuring a-tocopherol.Wehavedevelopedarapidmethodtomeasuretheoxysterols 7b-hydroxycholesteroland7-ketocholesterolinplasma(50healthysubjects)andtissueasanindexofoxidantstressinvivo,and fromthesamesample a-tocopherolcontent.Themeanplasmaconcentrationof7b-hydroxycholesteroland7-ketocholesterolwas 4:6  1:1and13:4  7:6ng/mL,respectively.Plasma a-tocopherolconcentrationwas5:8  1:0 lmol/molcholesterol.Samplesfrom atherosclerotic plaques contained 20 times more cholesterol, about 45 times higher oxysterols levels, and 600 times more a-to- copherolcomparedtonormalarteries.Nosignificantdifferenceincholesterolandoxysterolcontentwasobservedbetweencirrhotic andnormalliver.However,cirrhoticlivercontainedsignificantlysmallerconcentrationof a-tocopherolcomparedtonormalliver. Inconclusion,wehavedevelopedarapidandreliablemethodfortheassayofcholesteroloxidationproductsand a-tocopherolin plasmaandtissueusefulforestimationofoxidantstress/antioxidantbalance. Ó 2003ElsevierScience(USA).Allrightsreserved. Keywords: Cholesterol;Oxysterols; a-Tocopherol;Oxidantstress Oxidant stress is believed to play a role in several clinical settings, such as atherosclerosis, cancer, and aging. Atherosclerosis is the most important lesion of vessels and is responsible for the majority of deaths in western societies, i.e., cardiovascular mortality. Several studiesconductedinthepasttwodecadeshaverevealed that via the LDL 1 oxidation pathway oxidant stress plays a central role in the context of atherosclerosis [1–3]. Further support of this concept comes from the use of synthetic and natural antioxidants to inhibit atherosclerosis in experimental animal models [4,5]. Theapplicationofthesedataataclinicallevelpromoted theuseofantioxidantsinclinicaltrialswiththesupport of epidemiological data which showed the favorable effect of antioxidants on cardiovascular disease [6]. However, results of these trials were inconclusive and thereasonforthisdiscrepancyisnotyetknown.Clinical trials, mostly based on a-tocopherol, ignored some pharmacological notions and did not study the effects of a-tocopherolsupplementationonreliablemarkersof oxidantstress[7].Thus,itisimportanttocollectfurther basic information at the clinical level about oxidant stress and antioxidants. Several studies, including some from this laboratory, provided evidence that markers of oxidant stress in plasma and/or urine can be modulated by a-tocopherol [8–11]. Among these AnalyticalBiochemistry312(2003)217–223 www.elsevier.com/locate/yabio ANALYTICAL BIOCHEMISTRY * Correspondingauthor.Fax:+39-06-49970103. E-mail address: luigi.iuliano@uniroma1.it (L.Iuliano). 1 Abbreviations used: LDL, low-density lipoprotein; ABAP, 2,2 0 - azo-bis(2-amidinopropane)hydrochloride; ABVN, 2,2 0 -azo-bis(2,4- dimethylvaleronitrile);BHT,2,6-di-tert-butyl-4-methylphenol. 0003-2697/02/$-seefrontmatter Ó 2003ElsevierScience(USA).Allrightsreserved. PII:S0003-2697(02)00467-0