Nonprotein-Bound Iron and Plasma Protein Oxidative Stress at Birth

0031-3998/05/5806-1295 PEDIATRIC RESEARCH Copyright © 2005 International Pediatric Research Foundation, Inc. Vol. 58, No. 6, 2005 Printed in U.S.A. Nonprotein-Bound Iron and Plasma Protein Oxidative Stress at Birth BARBARA MARZOCCHI, SERAFINA PERRONE, PATRIZIA PAFFETTI, BARBARA MAGI, LUCA BINI, CHIARA TANI, MARIANGELA LONGINI, AND GIUSEPPE BUONOCORE Department of Pediatrics, Obstetrics, and Reproductive Medicine [B.Mar, S.P., P.P., C.T., M.L., G.B.], Department of Molecular Biology (B.Mag, L.B.), University of Siena, 53100 Siena, Italy ABSTRACT We previously reported plasma nonprotein-bound iron (NPBI) as a reliable early indicator of intrauterine oxidative stress (OS) and brain injury. We tested the hypothesis that albumin, an NPBI serum carrier, is the major target of NPBIinduced OS. Twenty-four babies were randomly selected from 384 newborns constituting the final cohort of a prospective study undertaken to evaluate the predictive role of NPBI in cord blood for neurodevelopmental outcome. Twelve were selected in the group with lowest NPBI levels (0 –1.16 ␮M) and good neurodevelopmental outcome and 12 in the group with highest NPBI levels (ⱖ15.2 ␮M) and poor neurodevelopmental outcome. Protein carbonyl groups were identified in cord blood samples by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Western blotting with anti-2,4-dinitrophenyl (DNP) antibodies. Two series of immunoreactive spots, corresponding to serum albumin and ␣-fetoprotein, were found only in the group with highest NPBI levels. We found an association between NPBI and Proteins are an important target for oxidative modification; oxidatively modified forms of proteins accumulate during OS induced by free radical (FR) generation. The modification is a consequence of oxidation of amino acid residues on proteins to form protein CG. CG form during normal aging (1) and in neonates receiving oxygen ventilation (2). Protein CG content is the most widely used marker of oxidative modification of the proteins and the term carbonyl stress has been used to describe excess formation of CG under physiologic and pathologic conditions, such as hypoxia-induced NPBI (3– 6). NPBI indicate a low molecular mass iron form, free of high-affinity binding to transferrin, that seems to occur in plasma, complexed to citrate, lactate, or phosphate or loosely Received February 2, 2005; accepted April 21, 2005. Correspondence: Giuseppe Buonocore, M.D., Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Policlinico “Le Scotte,” V.le Bracci 36, 53100 Siena, Italy: e-mail: Supported by grants from PAR (Piano di Ateneo per la Ricerca) 2004, University of Siena, Siena, Italy. DOI: 10.1203/01.pdr.0000183658.17854.28 carbonylated proteins in babies with highest NPBI levels. Since NPBI may produce hydroxyl radicals through the Fenton reaction, the major target of OS induced by NPBI is its carrier: albumin. Oxidation of albumin can be expected to decrease plasma antioxidant defenses and increase the likelihood of tissue damage due to OS in the newborns. (Pediatr Res 58: 1295–1299, 2005) Abbreviations 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis CG, carbonyl groups DHP, 1,2-dimethyl-3-hydroxy-4(1H)-pyridone DNP, 2,4-dinitrophenyl DNPH, 2,4-dinitrophenyl hydrazine FR, free radicals NPBI, nonprotein-bound iron OS, oxidative stress bound to albumin or other proteins (7). In blood, NPBI causes release of hydroxyl radical (OH·) by superoxide and hydrogen peroxide, possibly via iron-oxygen complexes (8). OH· is an extremely powerful oxidizing species. It attacks all classes of biologic macromolecules depolymerizing polysaccharides, breaking DNA strands, inactivating enzymes, and peroxidating lipids (9 –11). NPBI is released from hemoglobin when erythrocytes are challenged by an oxidative stress (12,13). The newborn is very susceptible to NPBI-induced oxidative stress (14). Recently we reported that NPBI released by erythrocytes in vitro is much higher with hypoxic erythrocytes from newborns compared with that from adults (6). Asphyxia could affect iron metabolism and lead to a significant increase in NPBI and lipid peroxidation in plasma of newborns with hypoxic ischemic encephalopathy, indicating that iron delocalization induced by asphyxia plays a role in the brain injury of asphyxiated infants (15). We demonstrated that plasma NPBI is a reliable early indicator of intrauterine OS and brain injury (5). The aim of present study was to detect protein oxidant stress in the pres- 1295 1296 MARZOCCHI ET AL. ence of NPBI in plasma by identifying carbonylated proteins. There are several methods of measuring carbonylated protein; in all of them, 2,4-dinitrophenyl hydrazine (DNPH) is allowed to react with protein carbonyls to form the corresponding hydrazone, which can be analyzed immunochemically. Oxidation is measured by evaluating overall protein carbonylation but not oxidation of individual plasma proteins. This evaluation is useful for establishing specific pathways of OS in vivo and for determining potential functional consequences of exposure to FR. High-resolution two-dimensional electrophoretic separation of plasma proteins combined with Western blot analysis was recently suggested as an appropriate technique (16). This method made it possible to evaluate the simultaneous presence of all CG in all plasma proteins. We tested the hypothesis that albumin, the carrier of NPBI in plasma, is the major target of NPBI-induced OS. MATERIALS AND METHODS Patients. Twenty-four babies were selected from 384 newborns constituting the final cohort of a prospective study undertaken to evaluate the predictive role of NPBI in cord blood for neurodevelopmental outcome (5). In that study, 400 of the 2902 babies born in the Neonatology Division, Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena (Italy) in the period June 1, 1997 to May 31, 2000 were enrolled according to a random algorithm using the Minitab statistical software package (Mintab Inc., State College, PA). The number lost to follow-up during the study period was 16. The final cohort consisted of 384 newborns with gestational ages from 24 to 42 weeks: 225 ⬎36 weeks, 69 ⬍ 32 weeks, and birth weight ⬍1500 g (range, 490 –5300 g). Neonatal neurodevelopmental examinations were performed at a mean postmenstrual age of 38 weeks according to the criteria of Allen and Capute (17). Follow-up visits were scheduled at age 1, 2, 4, 12, and 24 months. Physical and neurologic examinations were performed by the same neonatal neurologist. Motor development was assessed in the areas of muscle tone, primitive reflexes, automatic reactions, and head and trunk tone. The evaluation included quantitative changes and quality of performance in developmental patterns and milestones. Three to four transfontanellar cranial ultrasound examinations were performed within the first 6 – 8 months of follow-up by one experien (...truncated)