title: Non-protein-bound iron detection in small samples of biological fluids and tissues.
abstract: Interest in the pro-oxidative nature of non-protein-bound-iron (NPBI) led to the development of an assay for its detection. The aim was to set up a reliable method of detecting NPBI in small samples of biological fluids and tissue. The method was based on preferential chelation of NPBI by a large excess of the low-affinity ligand nitrilotriacetic acid. To separate NPBI, a two-step filtration procedure was used. All glassware and plasticware were treated to minimize iron contamination. Measurements were performed in plasma, amniotic fluid, bronchoalveolar lavage, and brain tissues. The analytic system detected iron as ferric nitrate standard down to a concentration of 0.01 microM. The 1,2-dimethyl-3-hydroxy-4(1H)-pyridone-Fe(DHP-Fe) complex eluted with a retention time of about 2.6 min. The standard curve for the DHP-Fe complex was linear between 0.01 and 400 microMin water as well as in plasma, bronchoalveolar lavage, brain tissue, and amniotic fluid. The detection limit was 0.01 muM for all biological fluids and brain tissue. The data show that reliable measurements of NPBI are possible in studies on oxidative stress under experimental and clinical conditions. The possibility of investigating NPBI involvement in free-radical injury might be useful in all human diseases in which oxidative stress occur.
authors.affiliation: Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Siena, Italy.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Paffetti
authors.forename: Serafina
authors.initials: S
authors.lastname: Perrone
authors.forename: Mariangela
authors.initials: M
authors.lastname: Longini
authors.forename: Antonio
authors.initials: A
authors.lastname: Ferrari
authors.forename: Donatella
authors.initials: D
authors.lastname: Tanganelli
authors.forename: Barbara
authors.initials: B
authors.lastname: Marzocchi
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Buonocore
mesh_terms: D000328:Adult; D000653:Amniotic Fluid / Q000737:chemistry; D000818:Animals; D001826:Body Fluids / Q000737:chemistry*; D001923:Brain Chemistry; D018893:Bronchoalveolar Lavage; D006801:Humans; D002534:Hypoxia, Brain; D007231:Infant, Newborn; D007501:Iron / Q000032:analysis* / Q000097:blood; D011506:Proteins; D012680:Sensitivity and Specificity; D013552:Swine
chemical_list: D011506:Proteins; D007501:Iron