Non-Protein-Bound Transition Metals and Hydroxyl Radical Generation in Cerebrospinal Fluid of Newborn Infants with Hypoxic Ischemic Encephalopathy

0031-3998/03/5304-0594 PEDIATRIC RESEARCH Copyright © 2003 International Pediatric Research Foundation, Inc. Vol. 53, No. 4, 2003 Printed in U.S.A. Non-Protein-Bound Transition Metals and Hydroxyl Radical Generation in Cerebrospinal Fluid of Newborn Infants with Hypoxic Ischemic Encephalopathy TOHRU OGIHARA, KAZUYA HIRANO, HIROMI OGIHARA, KIRYO MISAKI, MAYO HIROI, TAKAO MORINOBU, HAN-SUK KIM, SATORU OGAWA, RYOICHI BAN, MASASHI HASEGAWA, AND HIROSHI TAMAI Department of Pediatrics, Division of Neonatology, Osaka Medical College, Osaka, Japan ABSTRACT Among various hypothetical mechanisms for the in vivo production of reactive oxygen species, transition metal– catalyzed reactions in cooperation with a biologic reducing agent like ascorbic acid or superoxide may be some of the most important. In the present study, we retrospectively examined the existence of non-protein-bound metal ions, an essentially hazardous pro-oxidant form of various transition metals, and the occurrence of metal-catalyzed reactive oxygen species production in cerebrospinal fluid (CSF) of 10 infants with hypoxic ischemic encephalopathy (HIE) subsequent to perinatal asphyxia and 12 control infants within 72 h of birth. Non-protein-bound iron was detected in eight out of 10 CSF samples from the HIE infants and its level was significantly correlated with Sarnat’s clinical stage, whereas none of the control infants had detectable non-protein-bound iron levels. Non-protein-bound copper was below the detection limit in all CSF samples from both groups. Ascorbic acid was significantly increased in the CSF of HIE infants when compared with that of controls (means, 664.9 versus 449.4 ␮M, p ⫽ 0.008). ortho-Tyrosine and meta-tyrosine, which are highly specific and sensitive markers of protein oxidation induced by hydroxyl radicals, were significantly higher in HIE infants than in controls when evaluated by the ratio relative to their source amino acid, phenylalanine [means, 110.5 versus Advanced perinatal and obstetric management cannot yet prevent brain damage in newborn infants after perinatal asphyxia. Current therapeutic strategies for HIE occurring after perinatal asphyxia are not specific and are only supportive. Numerous studies have suggested that free radicals could have a key role in causing hypoxic ischemic damage to the brain, especially during the reoxygenation/reperfusion phase (1, 2). Received February 22, 2002; accepted October 16, 2002. Correspondence: Tohru Ogihara, M.D., Ph.D., Department of Pediatrics, Division of Neonatology, Osaka Medical College, 2-7, Daigaku-machi, Takatsuki, Osaka 569-8686, Japan; e-mail: DOI: 10.1203/01.PDR.0000054685.87405.59 75.4, p ⫽ 0.018 for ortho-tyrosine/phenylalanine; 104.6 versus 67.7 (nM/␮M ⫻ 102), p ⫽ 0.048 for meta-tyrosine/phenylalanine]. Both ratios were significantly correlated with non-proteinbound iron, but not with ascorbic acid. Our preliminary observations provide direct evidence that hydroxyl radicals are generated in the CNS during asphyxiation. Iron chelation therapy could be worth developing as a neuroprotective strategy for perinatal asphyxia. (Pediatr Res 53: 594–599, 2003) Abbreviations ROS, reactive oxygen species HIE, hypoxic ischemic encephalopathy CSF, cerebrospinal fluid NPBI, non-protein-bound iron NPBC, non-protein-bound copper o-Tyr, ortho-tyrosine m-Tyr, meta-tyrosine Phe, phenylalanine AA, ascorbic acid DHAA, dehydroascorbic acid UA, uric acid GC/MS, gas chromatography-mass spectrometry Among various hypothetical mechanisms for the production of ROS in living organisms, transition metal– catalyzed reactions may be some of the most important (3). When transition metals like iron or copper exist in their non-protein-bound “free” form in vivo, these metals can convert less reactive radicals to more reactive species. Thus, the human body is normally very careful to sequestrate these metal ions to be incorporated into specific metal-binding proteins. However, it is known that iron can be liberated from iron-storage ferritin when environmental pH decreases sufficiently, which may occur during perinatal asphyxia (4). Several studies have detected NPBI in the plasma of newborn infants, who show immature iron metabolism (5–7). In 594 HYDROXYL RADICALS IN BIRTH ASPHYXIA infants with severe perinatal asphyxia, a close association has been reported between an adverse outcome and the plasma concentration of NPBI (8). In addition, CSF essentially has no significant binding capacity for iron or copper because its content of transferrin and ceruloplasmin is very low (9), and it was recently reported that NPBI is elevated in the CSF of preterm infants with posthemorrhagic ventricular dilatation (10). In this context, several animal studies have shown the effectiveness of iron chelation therapy for post hypoxia/ ischemia-reperfusion injury of the brain (11, 12). In the brain, various nonradical compounds, such as catecholamines, ascorbic acid, thiols, and lipid peroxides, can produce reactive radical species in the presence of transition metal ions (9). Among them, AA is known to be strongly concentrated in the brain tissue and CSF by active transport mechanisms (13). In neonates with birth asphyxia, the AA concentration of CSF is known to increase markedly above the normal nonasphyxia level (14). AA, together with UA, is an integral water-soluble antioxidant that seems to serve as the main defense against oxidizing species in the aqueous phase of human body fluids. However, it has also been well confirmed that AA can contribute to the formation of highly reactive hydroxyl radicals in cooperation with redox active metal ions (15). Thus, we hypothesized that ROS formation catalyzed by redox-active transition metals may take place when HIE occurs after perinatal asphyxia. In the present study, we measured the concentrations of NPBI, NPBC, and AA in the CSF of infants with HIE and matched controls, together with the oxidation products of AA and UA (DHAA and allantoin) as markers of ROS generation (16), F2␣-isoprostane as a marker of lipid peroxidation (17), and the levels of o-Tyr and m-Tyr as highly specific and sensitive markers of protein oxidation induced by hydroxyl radicals (18, 19). We also analyzed whether these parameters were correlated with other well-established markers of HIE, including hypoxanthine (a marker of brain ischemia/hypoxia) (20), neuron-specific enolase (a marker of neuronal damage) (21), and excitatory amino acids (22). MATERIALS AND METHODS Patients. Among the infants admitted to the Neonatal Intensive Care Unit of Osaka Medical College Hospital between April 1999 and March 2001, term infants (gestational age ⱖ37 wk) who had abnormal neurologic signs as described by Sarnat and Sarnat (23), such as increased irritability and jitteriness, abnormal tone, abnormal primitive reflexes, altered consciousness, or convulsions, within the first 24 h of life and underwent lumbar puncture within 72 h of birth to rule out me (...truncated)