Iron delocalization or misregulation of iron metabolism may play a critical role in the pathology of hypoxic ischemic encephalopathy (HIE).
To study iron metabolism and lipid peroxidation in newborn infants and to correlate non-protein-bound iron (NPBI) concentration with the severity of the post-asphyxial injury and subsequent short-term outcomes.
Concentrations of NPBI and malondialdehyde (MDA) in the serum and in the cerebrospinal fluid (CSF) were measured in eight healthy newborn infants and nine newborn infants suffering from moderately severe HIE. Short-term outcomes (death, survival with or without neurological abnormality) were noted at hospital discharge.
Serum and CSF concentrations of both NPBI and MDA were significantly increased in HIE infants when compared to controls. Serum iron was significantly increased and total iron binding capacity was significantly decreased in HIE infants compared to controls. Out of the nine HIE infants, four infants died and two infants survived with abnormal neurological findings at hospital discharge. These six infants with clinical sequels had significantly increased concentrations of NPBI in the serum and in the CSF; and increased concentrations of MDA in the CSF when compared to the other three who survived without short-term abnormalities.
We conclude that hypoxia ischemia alters iron metabolism and lipid peroxidation in newborn infants; and that NPBI and MDA in the CSF are increased in infants with HIE. This study supports a role for iron in oxidative injury to the central nervous system after hypoxic ischemic insults.
Birth asphyxia continues to be the leading cause of neonatal brain injury.1, 2 Hypoxic ischemic encephalopathy (HIE) occurs over two distinctive phases; an initial hypoxic ischemic insult followed by an oxygenation-reperfusion injury. The initial hypoxic insult cannot be reversed, but the brain damage that follows during the reperfusion phase is potentially preventable. Therefore, it becomes crucial to clearly understand physiological and biochemical changes that accompany reperfusion to protect the neonatal brain from further damage. A substantial portion of the injury during reperfusion has been attributed to the excessive formation of reactive oxygen species by the mitochondria, such as superoxide and hydrogen peroxide.3, 4, 5, 6 The increased hydrogen peroxide, and also the acidosis induced by ischemia can release iron from its binding proteins leading to an increased concentration of non-protein-bound iron (NPBI) in plasma.7, 8, 9
Iron delocalization or misregulation of iron metabolism may play a critical role in brain injury.10, 11, 12 As a powerful pro-oxidant, NPBI has the ability to convert hydrogen peroxide into the highly toxic hydroxyl radical.13 In addition, NPBI reacts with oxygen in what is described as ‘the Fenton reaction’ to create potent reactive oxygen species that damage cell membranes, alter intracellular signaling cascade and induce apoptosis.14 Once NPBI crosses the blood–brain barrier, neuronal cell damage can potentially get worse because cerebrospinal fluid (CSF) contents of transferrin is miniscule and, hence, has no capacity to bind NPBI.5
Studies on infants with HIE demonstrated the following: (a) an increased free NPBI in their cord blood; (b) an association between the increased NPBI and the presence of the thiobarbituric reactive oxygen species in the plasma, as an index of lipid peroxidation14 and (c) an association between the increased NPBI in the plasma and the development of adverse clinical outcomes.10 In addition, iron chelating agents are shown to protect the brain when administered after HIE in animal studies.15, 16 Thus, the role of iron in HIE of the newborn is very plausible; but surprisingly such relation remains speculative.14 Currently, there is no confirmation on the iron status in the CSF of infants with HIE; apart from a single study.17 Furthermore, the relation of NPBI and lipid peroxidation within the central nervous system compartment has never been studied.
We, therefore, designed this prospective controlled study on the CSF of infants with HIE following birth asphyxia. Our aim was to examine the status of iron metabolism, lipid peroxidation and their subsequent relation with short-term clinical outcomes. We used malondialdehyde (MDA) concentration in the CSF as an index for lipid peroxidation in brain cells.
The study was approved by the Institutional Review Board at Mansoura University Children's Hospital in Egypt, and parental consents were obtained before enrollment of subjects. A total of 17 infants born between May 2004 and January 2006 were included in the study. Of them nine newborns were diagnosed with birth asphyxia that met the following criteria: (a) presence of fetal distress such as; abnormal heart rate patterns or meconium-stained amniotic fluid, (b) Apgar score at 5 min that was ⩽6, (c) resuscitation in the delivery room that required the use of positive pressure ventilation for ⩾2 min and (d) profound acidosis with pH<7.1 or base deficit >12 mmol in their cord blood samples. The other eight newborns (control group) were admitted for observation of possible early neonatal sepsis that was later dismissed. All included subjects (n=17) were admitted to the neonatal unit at the Mansoura University Children's Hospital at a postnatal age <24 h, and had their CSF obtained within 72 h. Lumbar punctures were medically indicated in all subjects as part of septic workups to exclude meningitis.
All included infants did not have any of the following: (a) congenital heart disease or major malformations; (b) documented chromosomal abnormalities; (c) grossly bloody CSF or macroscopic hemolysis in CSF supernatant after centrifugation and (d) positive bacterial cultures of blood or CSF.
Neurological examinations were performed for all subjects within 24 h of age and at the time of hospital discharge for those who survived. They were evaluated by the same attending physician who was blinded to their laboratory values.
CSF and blood sampling
Cerebrospinal fluid samples were withdrawn by lumbar puncture within the first 72 h of birth, they were immediately centrifuged to remove cell components and the supernatant was stored at −80 °C until analyzed. Simultaneously, blood samples were collected in heparinized (2 ml) and non-heparinized tubes (2 ml).
The former was used to determine NPBI in plasma and the latter was used for the measurement of serum iron, total iron binding capacity (TIBC) and MDA. Blood was immediately centrifuged and the separated fluid was stored at −80 °C until analysis. Plasma or serum samples that showed pink discoloration (hemolysis) were excluded from the study.
Determination of NPBI and iron indices
Modified Singh's method18 was used for direct quantization of non-transferrin-bound iron in the plasma and CSF by adding excess NTA (nitrilitriacetic acid disodium salt; Sigma Chemical Co., St Louis, MO, USA) to plasma and CSF to form an Fe–NTA complex, then ultrafiltered using a Amicon microfilter with a cutoff value at 30 000. (Micro-30, Amicon Inc. Danvers, MA, USA).19 For the separation of non-transferrin-bound iron and colorimetric quantitation, 100 mM NTA in 1 M HEPES buffer (pH 7.0) was added to serum and allowed to stand for 20 min at room temperature. The solution was then ultrafiltrated using Amicon microfilter with cap and was centrifuged under 3500 × g for 60 min at 4 °C. Then, 1 M TGA was added to the ultrafiltrate for reduction of Fe3+ to Fe2+ and then 0.5 M BPT was added for colorimetry of Fe2+ at a wavelength 537 nm. The ultrafiltrate from normal human serum was used as a blank. Various concentrations of iron standard were obtained from an iron standard and used to plot an iron standard curve to obtain the results.
Serum iron was measured by calorimetric determination without deproteinization in an acid medium by Ferrimat-kit, Bio Merieux, France.20 TIBC was determined in human serum after saturation of the transferrin by an iron solution and absorption of the excess iron on magnesium hydroxyl carbonate, iron determination was then performed by Ferrimat-kit, Bio Merieux, France.21
Measurement of MDA
Malondialdehyde, a lipid peroxidation product, was measured using TBARS (thiobarbituric acid reactive substances) assay. MDA is an end-product of the oxidation and decomposition of polyunsaturated fatty acids containing three or more double bonds. MDA was derived from TBARS to form the MDA–TBARS product, which was measured spectrophotometrically.22
Data were analyzed using SPSS for Windows Statistical Package (SPSS Inc., Chicago, IL, USA). Summary statistics of data were expressed as mean±s.d., median and range. The Kolmogrov–Smirnov test was performed to check normal distribution of data. Non-parametric data were assessed by the χ2 test and the Mann–Whitney U test for continuous variables. Independent samples T-test was used for parametric data. For correlation analysis, Spearman's correlation coefficients were calculated. A P-value <0.05 was considered statistically significant.
The clinical characteristics of infants in the two groups were similar, except for decreased Apgar scores at 5 min in the HIE group (Table 1). Serum levels for iron, TIBC, NPBI and MDA were all different, but hemoglobin concentrations were similar in the two groups (Table 2). A significant positive correlation was demonstrated between serum NPBI and each of the following: CSF NPBI (r=0.99, P=0.0001), serum MDA (r=0.98, P=0.0001) and CSF MDA (r=0.98, P=0.0001).
In the CSF, the concentrations of NPBI (μmol l−1) and MDA (μmol l−1) were significantly increased in the HIE group when compared to controls (P=0.003 and P=0.004, respectively) (Figure 1). NPBI was detectable in the CSF of five out of eight CSF samples in the control group and in all CSF samples of the asphyxiated groups (n=9).
Within the HIE group, the concentrations of NPBI in the CSF correlated significantly with serum NPBI (r=0.96, P=0.0001). NPBI concentrations in the CSF and serum, and MDA concentration in the CSF were significantly increased in infants who later died or suffered abnormal neurological examination at hospital discharge when compared to those who survived without neurological sequels (P=0.026, P=0.016 and P=0.038, respectively).
According to Sarnat staging, encephalopathy was moderate in six HIE infants and severe in three HIE infants. Concerning the short-term outcome, all of the severely asphyxiated infants were supported on mechanical ventilation beyond the resuscitation period, one infant had convulsions requiring anticonvulsive therapy and three received dopamine infusion to treat hypotension during the study period. All the three severely asphyxiated infants developed major neurological abnormalities and died during the neonatal period. For the six moderately asphyxiated infants, one died during the neonatal period, two had abnormal neurological examinations and three were neurologically normal at discharge. All the control infants were neurologically normal at discharge. Short-term morbidities correlated positively with serum NPBI (r=0.7, P=0.002), CSF NPBI (r=0.7, P=0.002), serum MDA (r=0.7, P=0.002) and CSF MDA (r=0.7, P=0.002).
This study demonstrated increased concentrations of NPBI and MDA in the CSF of infants with HIE when compared to control infants. Short-term mortality and neurological impairment correlated with increased CSF concentrations of both NPBI and MDA. NPBI was detectable in the CSF of five (63%) control infants; but we did not find any particular difference in the clinical characteristics of the control infants with and without NPBI in the CSF. Our findings in the CSF of HIE infants strengthen the previously suggested pathway for cell injury induced by NPBI. It is conceivable that iron delocalization following hypoxia contributes to the excessive production of NPBI in the CSF, which further increases lipid peroxidation.10, 23 To our knowledge, this is the first report to link NPBI and MDA in the CSF of infants with HIE.
Newborn infants are particularly at an increased risk for oxidative damage through mechanisms that involve iron. As shown in our study, TIBC was low and NPBI was detectable even in the plasma of healthy newborns at a concentration that was generally higher than reported at any other age.10, 24 Plasma concentrations of NPBI were further increased in the HIE group when compared to controls. Such finding is consistent with a number of studies that demonstrate iron delocalization in the setting of hypoxia,25 ischemia reperfusion26, 27 and cardiac arrest.28 The protein-bound iron is considered a safe vehicle for iron transport and storage because it is not capable of inducing a free radical reaction.5 However, the lower pH in the plasma following asphyxia enables transferrin to liberate its iron, thereby inducing free radical production.29 These free radicals are capable of releasing more iron by mobilizing it from ferritin.30 Reperfusion and reoxygenation that follow hypoxia produce large amounts of nitric oxide in neonatal brain tissue, that can release more iron from its binding protein.7 By these mechanisms a cascade of iron release and production of free radicals can be activated, which lead to extensive cell damage.
The brain may be especially at risk from free radical-mediated injury because TIBC in the CSF is low. In addition, most of the iron could be in its active ferrous form because of the high concentrations of vitamin C and low concentrations of ceruloplasmin in the CSF.31, 32 Low level of ceruloplasmin leads to decreased ferroxidase activity.33 Ferroxidase catalyzes the oxidation of ferrous iron to ferric iron, and this process is essential for ionic forms of iron to be incorporated into transferrin. The pro-oxidant NPBI has the ability to catalyze the formation of free radicals, (24) particularly at the neuronal compartment, which is rich in polyunsaturated fatty acids.5
We demonstrated a significant link between NPBI and MDA concentrations in the CSF with short-term neurological outcome. Previous studies reported a close association between an increased plasma concentration of NPBI within 8 h after birth and adverse outcomes of neonatal asphyxia.10, 24 Our study adds an important piece of information to the previous reports. It confirms the increased concentrations of NPBI in the CSF that correspond to its plasma concentrations; and it associates this abnormality to an increased level of MDA as a marker for lipid peroxidation in the CSF.
In conclusion, iron metabolism and lipid peroxidation in newborn brain appears to be involved in the pathogenesis of HIE. Furthermore, NPBI concentration in the serum and CSF, and MDA concentration in the serum are associated with the severity of the post-asphyxial injury.
Freeman JM, Nelson KB . Intrapartum asphyxia and cerebral palsy. Pediatrics 1988; 82: 240–249.
Volpe JJ . Neurology of the Newborn. 3rd ed WB Saunders: Philadelphia, 1995; pp 211–369.
Saugstad OD . Oxygen toxicity in the neonatal period. Acta Paediatr 1990; 79: 881–892.
Traystman RJ, Kirch JR, Koehler RC . Oxygen radical mechanisms of brain injury following ischemia and reperfusion. J Appl Physiol 1991; 71: 1185–1195.
Halliwell B . Reactive oxygen species and the central nervous system. J Neurochem 1992; 59: 1609–1623.
McCord J, Roy R, Schaffer S . Oxygen-derived free radicals in post-ischemic tissue injury. N Engl J Med 1985; 312: 159–163.
Reif DWX, Simmons RD . Nitric oxide mediates iron release from ferritin. Arch Biochem 1990; 283: 537–541.
Henry Y, Lepoivre M, Drapier JC, Ducrocq C, Boucher JL, Guissani A . EPR characterization of molecular targets for NO in mammalian cells and organelles. FASEB J 1993; 7: 1124–1134.
Anggard E . Nitric oxide: mediator, murderer, and medicine. Lancet 1994; 343: 1199–1206.
Dorrepaal CA, Berger HM, Benders MJ, van Zoeren-Grobben D, van de Bor M, van Bel F . Non-protein-bound iron in post-asphyxial reperfusion injury of the newborn. Pediatrics 1996; 98: 883–889.
Dorrepaal CA, van Bel F, Moison RM, Shadid M, van de Bor M, Steendijk P et al. Oxidative stress during post-hypoxic-ischemic reperfusion in the newborn lamb: the effect of nitric oxide synthesis inhibition. Pediatric Res 1997; 41: 321–326.
van Bel F, Shadid M, Moison RM, Dorrepaal CA, Fontijn J, Monteiro L et al. Effect of allopurinol on post-asphyxial free radical formation, cerebral hemodynamics, and electrical brain activity. Pediatrics 1998; 101: 185–193.
Yu T, Kui LQ, Ming QZ . Effect of asphyxia on non-protein-bound iron and lipid peroxidation in newborn infants. Dev Med Child Neurol 2003; 45: 24–27.
Georgieff MK . Iron in the brain: its role in development and injury. Neoreviews 2006; 6: 344–352.
Palmer C, Roberts RL, Bero C . Desferoxamine post treatment reduces ischemic brain injury in neonatal rats. Stroke 1994; 25: 1039–1045.
Groenendaal F, Shadid M, Mc Growan JE, Mishra OP, van Bel F . Effects of deferoxamine, a chelator of free iron, on Na+, K+ ATPase activity of cortical brain cell membrane during early reperfusion after hypoxia ischemia in newborn lambs. Pediatr Res 2000; 48: 560–564.
Ogihara T, Hirano K, Ogihara H, Misaki K, Hiroi M, Morinobu T et al. Non-protein-bound transition metals and hydroxyl radical generation in cerebrospinal fluid of newborn infants with hypoxic ischemic encephalopathy. Pediatr Res 2003; 53: 594–599.
Singh S, Hider RC, Porter JB . A simple method for calorimetric quantitation of non-transferrin-bound iron in serum. Biochem Trans 1989; 17: 697–698.
Zhang D, Okada S, Kawabata T, Yasuda T . An improved simple colorimetric method for quantitation of non-transferrin-bound iron in the serum. Biochem Mol Biol Int 1995; 35: 635–641.
Carter P . Spectrophotometric determination of serum iron at the submicrogram level with a new reagent (ferrozine). Anal Biochem 1971; 40: 450–458.
Ramsay WNM . The determination of the total-iron-binding capacity of serum. Clin Chem Acta 1957; 2: 221.
Ozaras R, Tahan V, Turkmen S, Talay E, Besirli K, Aydin S et al. Changes in malondialdehyde levels in bronchoalveolar fluid and serum by the treatment of asthma with inhaled steroid and β2 agonist. Respirology 2000; 5: 289–292.
Bromont C, Marie C, Bralet J . Increased lipid peroxidation in vulnerable brain regions after transient forebrain ischemia in rats. Stroke 1989; 20: 918–924.
Buonocore G, Perrone S, Longini M, Paffetti P, Vezzosi P, Gatti MG et al. Non protein bound iron as early predictive marker of neonatal brain damage. Brain 2003; 126: 1224–1230.
Buonocore G, Zani S, Sargentini I, Gioia D, Signorini C, Bracci R . Hypoxia-induced free iron release in the red cells of newborn infants. Acta Paediatr 1998; 87: 77–81.
Krause GS, Nayini NR, White BC, Hoenher TJ, Garritano AM, O'Neil BJ et al. Natural course of iron delocalization and lipid peroxidation during the first eight hours following a 15-min cardiac arrest in dogs. Ann Emerg Med 1987; 16: 1200–1205.
Chiao JJ, Kirschner RE, Fantini GA . Iron delocalization occurs during ischemia and persists on reoxygenation of skeletal muscle. J Lab Clin Med 1994; 124: 432–438.
Komara JS, Nayini NR, Bialick HA, Indrieri RJ, Evans AT, Garritano AM et al. Brain iron delocalization and lipid peroxidation following cardiac arrest. Ann Emerg Med 1986; 15: 384–389.
Siesjo BK . Acidosis and ischemic brain damage. Neurochem Pathol 1988; 9: 31–88.
Biemond P, Swaak AJ, Beindorff CM, Koster JF . Superoxide-dependent and independent mechanisms of iron mobilization from ferritin by xanthine oxidase. Biochem J 1986; 239: 169–173.
Gutteridqe JM . Ferrous ions detected in cerebrospinal fluid by using bleomycin and DNA damage. Clin Sci (Lond) 1992; 82: 315–320.
Gutteridqe JM . Iron and oxygen radicals in brain. Ann Neurol 1992; 32: S16–S21.
Hirano K, Morinobu T, Kim HS, Hiroi M, Ban R, Ogawa S et al. Blood transfusion increases radical promoting non-transferrin bound iron in preterm infants. Arch Dis Child Fetal Neonatal Ed 2001; 84: F188–F193.
About this article
Cite this article
Shouman, B., Mesbah, A. & Aly, H. Iron metabolism and lipid peroxidation products in infants with hypoxic ischemic encephalopathy. J Perinatol 28, 487–491 (2008) doi:10.1038/jp.2008.22
- non-protein-bound iron (NPBI)
Free radicals and neonatal encephalopathy: mechanisms of injury, biomarkers, and antioxidant treatment perspectives
Pediatric Research (2019)
Frontiers in Neuroscience (2019)
Journal of Neurochemistry (2019)
Free Radical Biology and Medicine (2019)
Frontiers in Physiology (2019)