Non-protein-bound Iron Levels Research Articles

Early Prediction of Hypoxic-Ischemic Brain Injury by a New Panel of Biomarkers in a Population of Term Newborns.

This research paper is aimed at evaluating the predictive role of a default panel of oxidative stress (OS) biomarkers for the early identification of infants at high risk of HIE and their validation through the correlation with MRI findings. A multicenter prospective observational study was performed between March 2012 and April 2015 in two European tertiary NICUs. Eighty-four term infants at risk for HIE (pH < 7, BE < −13 mmol/L, and 5′ Apgar < 5) were enrolled. Three were excluded for chromosomal abnormalities and one due to lack of blood samples. The final population was divided according to the severity of perinatal hypoxia into 2 groups: mild/moderate HIE and severe HIE. Advanced oxidation protein products (AOPP), non-protein-bound iron (NPBI), and F2-isoprostanes (F2-IsoPs) were measured in blood samples at P1 (4–6 hours), P2 (24–72 hours), and P3 (5 days), in both groups. MRIs were scored for the severity of brain injury, using a modified Barkovich score. The mean GA was 39.8 weeks (SD 1.4) and the mean birth weight 3538 grams (SD 660); 37 were females and 43 males. Significantly lower 5′ Apgar score, pH, and BE and higher Thompson score were found in group II compared to group I at birth. Group II showed significantly higher AOPP and NPBI levels than group I (mean (SD) AOPP: 15.7 (15.5) versus 34.1 (39.2), p = 0.033; NPBI 1.1 (2.5) versus 3.9 (4.4), p = 0.013) soon after birth (P1). No differences were observed in OS biomarker levels between the two groups at P2 and P3. A regression model, including adjustment for hypothermia treatment, gender, and time after birth, showed that AOPP levels and male gender were both risk factors for higher brain damage scores (AOPP: OR 3.6, 95% CI (1.1–12.2) and gender: OR 5.6, 95% CI (1.2–25.7), resp.). Newborns with severe asphyxia showed higher OS than those with mild asphyxia at birth. AOPP are significantly associated with the severity of brain injury assessed by MRI, especially in males.

Placental histological examination and the relationship with oxidative stress in preterm infants

BackgroundPrenatal conditions of enhanced oxidative stress (OS) linked to inflammation or hypoxia have been associated with impaired fetal growth and preterm delivery. Little is known regarding biomarkers of OS in the cord blood of preterm infants and placental histological patterns. ObjectivesTo test the hypothesis that placental lesions indicating chorioamnionitis (CA) or vascular underperfusion (VU) are associated with increased OS in the offspring. Methods120 neonates born below 29+6 weeks of gestational age (GA) were enrolled. Histological characteristics of placentas from their mothers were classified as normal (CTRL group), histological CA (HCA) and vascular underperfusion (VU). Serum concentrations of isoprostanes (IsoPs), non-protein bound iron (NPBI) and advanced oxidative protein products (AOPP), were determined in cord blood. ResultsIsoPs, NPBI and AOPP were significantly increased in HCA group compared to CTRL group. The multivariable regression model, adjusted for GA, maternal age, parity, maternal diabetes, maternal obesity and presence/absence of fetal growth restriction (FGR), showed a significant association between the presence of HCA and increased OS biomarkers levels in cord blood (IsoPs: p = 0.006; NPBI: p = 0.014; AOPP: p = 0.007). Placental VU lesions were significantly associated with higher umbilical IsoPs, NPBI and AOPP levels (IsoPs: p = 0.008; NPBI: p = 0.002; AOPP: p = 0.040). In the cases of placental VU lesions associations were also found between high AOPP levels and low GA (p = 0.002) and the presence of fetal growth restriction (p = 0.014). ConclusionsPlacental lesions indicating inflammation or impaired perfusion are associated with higher cord blood levels of OS biomarkers explaining the fetal susceptibility to oxidative injury and the need of antioxidant protection.

THE ROLE OF OXIDATIVE STRESS IN THE NECROTIZING ENTEROCOLITIS OF PRETERM

Introduction: Oxidative stress (OS) is strongly envolved in the pathogenesis of many preterm newborn diseases; this is due to the low efficiency of natural antioxidant systems unable to counteract the harmful effects of free radicals (FRs). Necrotizing Enterocolitis (NEC) is a multifactorial disease and it is part of the so called free radicals related diseases. Hypoxic-ischaemic events and inflammation, involved in NEC pathogenesis, are responsible of the overproduction of free radicals (FRs), generating OS. Aim: To test the hypotesis that OS marker levels in cord blood may predict the onset of NEC in high risk infants. Materials and methods: 91 preterm newborns of gestational age between 24 and 33 weeks and birth weight between 460 and 2540 grams were consecutively recruited in two italian neonatal intensive care units. Markers of potential oxidative stress risk, Non Protein Bound Iron (NPBI), and free radicals damage, Advanced Oxidation Protein Products (AOPP) and Total Hydroperoxide (TH), were measured in the cord blood. Associations between NEC and OS markers have been checked through inferential analysis (univariate logistic regression). Results: Out of 91 preterm babies, 8 developed NEC. Babies with NEC had a birth weight (BW) and a gestational age (GA) significantly lower than healthy babies (BW=1100,52 &plusmn; 444,30 vs 1320,53 &plusmn; 462,09; GA=29,02 &plusmn; 2,09 vs 30,14 &plusmn; 2,33, respec- tively. p&lt;0.005). Cord blood levels of TH and NPBI were higher in babies with NEC than in babies without, but not significantly. AOPP cord blood levels were significantly higher in babies with NEC than the babies without (AOPP=29,15 &plusmn; 20,02 vs 16,72 &plusmn; 7,34; p&lt;0.05). AOPP demonstrated a significant value for the identification of the risk of NEC (OR=1.13, CI 95%= 1.001-1.282). Conclusions: OS is strongly associated with NEC. The determination of biochemical OS markers in cord blood can be useful in identifying babies at high risk to develop NEC and in devising new strategies to bring consistent benefits to premature babies.

Non-protein-bound iron and 4-hydroxynonenal protein adducts in classic autism

A link between oxidative stress and autism spectrum disorders (ASDs) remains controversial with opposing views on its role in the pathogenesis of the disease. We investigated for the first time the levels of non-protein-bound iron (NPBI), a pro-oxidant factor, and 4-hydroxynonenal protein adducts (4-HNE PAs), as a marker of lipid peroxidation-induced protein damage, in classic autism. Patients with classic autism (n=20, mean age 12.0±6.2years) and healthy controls (n=18, mean age 11.7±6.5years) were examined. Intraerythrocyte and plasma NPBI were measured by high performance liquid chromatography (HPLC), and 4-HNE PAs in erythrocyte membranes and plasma were detected by Western blotting. The antioxidant defences were evaluated as erythrocyte glutathione (GSH) levels using a spectrophotometric assay. Intraerythrocyte and plasma NPBI levels were significantly increased (1.98- and 3.56-folds) in autistic patients, as compared to controls (p=0.0019 and p<0.0001, respectively); likewise, 4-HNE PAs were significantly higher in erythrocyte membranes and in plasma (1.58- and 1.6-folds, respectively) from autistic patients than controls (p=0.0043 and p=0.0001, respectively). Erythrocyte GSH was slightly decreased (−10.34%) in patients compared to controls (p=0.0215). Our findings indicate an impairment of the redox status in classic autism patients, with a consequent imbalance between oxidative stress and antioxidant defences. Increased levels of NPBI could contribute to lipid peroxidation and, consequently, to increased plasma and erythrocyte membranes 4-HNE PAs thus amplifying the oxidative damage, potentially contributing to the autistic phenotype.

Oxidative Stress in Fetal and Neonatal Brain Development

Free radicals (FRs) are normally generated as by-products of aerobic respiration and various metabolic processes. Oxidative stress (OS) occurs when the production of FRs exceeds the capacity of antioxidant defenses. Nevertheless, a certain amount of OS is required to allow for the normal progression of embryonic and fetal growth. OS has been implicated in the regulation of reproductive processes in both animal and human, such as follicular development, ovulation, fertilization, embryogenesis, placental differentiation and growth. In contrast OS has emerged as a likely promoter of several pregnancy-related disorders, such as spontaneous abortions, embryopathies, preeclampsia, fetal growth restriction, preterm labor and low birth weight. Clinical studies suggest a link between maternal and fetal infection/inflammation and brain damage. The most common view is that intrauterine infection causes a fetal systemic inflammatory response resulting in injury to cerebral white matter. At birth, newborn is exposed to a relatively hyperoxic environment and additional FRs sources such as inflammation, hypoxia, ischemia-reperfusion, glutamate and non protein bound iron (NPBI) release may enhance OS. The susceptibility of the developing brain in extreme low gestational age newborn is due to the intrinsic vascular derangements in the autoregulation of cerebral blood flow and the local metabolic vulnerability, furthermore asphyxia causes an increase in NPBI, that significantly correlates with neurodevelopmental outcome. As matter of fact, a positive correlation exists between the development of IVH and NPBI levels in cord blood suggesting the dangerous potential of NPBI on oligodendrocytes. Brain injury is a leading cause of mortality and disability. The improvement in understanding the pathophysiological mechanisms involved in perinatal brain lesions will allow to identify new neuroprotective strategies.

Cord Blood Cardiac Troponin T and Nonprotein-Bound Iron Levels in Newborns of Mild Pre-Eclamptic Mothers

Background: In hypoxic newborns, cardiac troponin T (cTnT) was shown to be an indicator of cardiac damage and increased levels of nonprotein-bound iron (NPBI), an indicator of increased free radical production and perinatal brain damage. Objective: The aim of this study was to determine cord blood cTnT and NPBI levels in neonates of mild pre-eclamptic mothers. Methods: The study included 50 babies of mild pre-eclamptic mothers and 50 babies of healthy mothers. cTnT and NPBI levels were measured in cord blood. Results: The mean gestational age in the pre-eclamptic and healthy groups were 36.1 ± 3.5 and 38.1 ± 1.9 weeks, mean birth weights were 2,456 ± 945 and 3,059 ± 493 g. Cord blood median cTnT level was significantly higher in the pre-eclamptic group (0.024 vs. 0.015 ng/ml). Serum cTnT in the 95th percentile was 0.047 ng/ml in the healthy group. cTnT levels of preterm babies in the pre-eclamptic group was found to be significantly higher compared to term babies in the control group (0.038 vs. 0.013 ng/ml). It could not be demonstrated whether there is a statistically significant relation between cTnT levels and respiratory distress, gestation, type of delivery, sex and birth weight. The median NPBI level was higher in the control group (3.26 vs. 1.86 µmol/l). Conclusions: Increased levels of cTnT may be a biochemical marker of cardiac involvement in babies of mild pre-eclamptic mothers. In this study, no correlation was found between cTnT levels and NPBI levels.

Effects of Nicotine Infusion on Striatal Glutamate and Cortical Non-Protein-Bound Iron in Hypoxic Newborn Piglets

Background: Perinatal asphyxia triggers a large cascade of mechanisms leading to brain damage. Release of glutamate and increased oxidative stress play substantial roles. Non-protein-bound iron (NPBI), which contributes to the production of free radical species through the Fenton reaction, increases in hypoxic-ischemic brain damage. Results from in vitro and adult animal studies show that nicotine can decrease extracellular levels of NPBI and glutamate. Nicotine’s effects have further been shown to be dose-dependent, with lower doses showing neuroprotective, and higher doses showing neurotoxic effects. Objectives: We wished to assess nicotine’s effect on levels of NPBI and glutamate in an animal model of neonatal hypoxic-ischemic brain damage. Methods: 47 anesthetized newborn piglets were randomized to one of four infusions after hypoxia (nicotine 130 µg/kg/h, 260 µg/kg/h, adrenaline 0.05 µg/kg/min, saline 2.6 ml/kg/h). Glutamate in striatum and NPBI in cortex were analyzed in microdialysate. Results: Striatal glutamate presented a significant rise for all the animals from baseline to the end of hypoxia (p < 0.001). There was a significant difference for nicotine 130 µg/kg/h versus saline (p = 0.002) 2 h after hypoxia. Cortical NPBI presented a significant rise from baseline to the end of hypoxia for all the animals (p < 0.001), and a significant difference between nicotine 130 µg/kg/h versus saline 2 h after hypoxia (p = 0.013). Conclusion: Our findings support the hypothesis that nicotine can decrease extracellular levels of glutamate and NPBI in a neonatal model of hypoxic-ischemic brain damage. This suggests possible neuroprotective effects of a low dose of nicotine in neonates, as it has already been shown in adult models.

Plasma Esterified F2-Isoprostanes and Oxidative Stress in Newborns: Role of Nonprotein-Bound Iron

Nonprotein-bound iron (NPBI) and F2-isoprostanes, reliable markers of oxidative stress, are increased in plasma of newborns and inversely correlated to the gestational age. Because NPBI represents a pro-oxidant stimulus in plasma, we test the hypothesis that the entity of lipid peroxidation is related with NPBI concentrations. Plasma levels of free, esterified, and total F2-isoprostanes were investigated in relation to NPBI levels in 59 newborns and 16 healthy adults. The pro-oxidant role of iron was ascertained in vitro, by measuring all the forms of F2-isoprostanes after incubation with ammonium iron sulfate. Significant positive correlations were found between NPBI and total as well as esterified F2-isoprostanes in plasma of the newborns. The addition of ammonium iron sulfate induced a marked increase in all the forms of F2-isoprostanes after 2 hours of incubation. The higher NPBI concentration, the higher F2-isoprostanes levels. An increase NPBI dose dependent in total F2-isoprostanes formation was observed in dialyzed low density lipoprotein from adult plasma. The results clearly show that once NPBI is generated, whatever its source, it is capable of inducing oxidative stress. NPBI-induced oxidative stress may contribute to the morbidity in preterm infants that are particularly susceptible to free radical damage.

Nonprotein-Bound Iron and Plasma Protein Oxidative Stress at Birth

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 NPBI-induced 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 microM) and good neurodevelopmental outcome and 12 in the group with highest NPBI levels (>or=15.2 microM) 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 alpha-fetoprotein, were found only in the group with highest NPBI levels. We found an association between NPBI and 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.

Non-protein bound iron release during chemotherapy in cancer patients.

Non-protein bound iron (NPBI) is able to catalyse oxidative reactions, causing damage to vital structures. Adverse effects induced by cisplatin seem, in part, to be mediated by free radicals. In the present study, we have measured plasma NPBI, various other iron parameters and antioxidants in 28 cancer patients undergoing cisplatin-based chemotherapy at various time points before and during chemotherapy. No NPBI was present prior to therapy, but within 1-4 days following the first administration of chemotherapy, mean NPBI rose significantly to 10.6+/-6.6 micromol/l (range, 0.6-21.3 micromol/l) in 18 (64.3%) of the 28 patients measured. The rise in NPBI was accompanied by a significant rise in total plasma iron and ferritin and a marked decrease in the latent iron-binding capacity. Concomitantly, plasma vitamins C and E decreased significantly, indicating consumption of antioxidants. Similar observations were also made during the fourth chemotherapy cycle. The increase in NPBI preceded and correlated significantly with chemotherapy toxicity, such as a decrease in leucocyte count and haemoglobin, with a transient rise in various liver enzymes and with known cisplatin-related toxicity, i.e. the loss of renal and hearing function. In conclusion, cisplatin chemotherapy induces oxidative damage which rapidly leads to release of iron from intracellular proteins and the appearance of NPBI. Bone marrow, red blood cells, liver and kidney seem to be a likely source of NPBI. The observed high levels of NPBI may be a major causative determinant in chemotherapy-induced toxicity.

Non-Protein-Bound Iron Is Elevated in Cerebrospinal Fluid from Preterm Infants with Posthemorrhagic Ventricular Dilatation

Posthemorrhagic ventricular dilatation (PHVD) is closely associated with white matter injury and neurologic disability in the preterm infant. An important factor in periventricular white matter damage may be the specific vulnerability of iron-rich immature oligodendroglia to reactive oxygen species toxicity. Non-protein-bound iron (NPBI) is a potent catalyst in the generation of hydroxyl radicals (Fenton reaction). Our objective was to determine whether NPBI is increased in cerebrospinal fluid (CSF) from preterm infants with PHVD compared with preterm control infants. Samples of CSF were obtained from 20 infants with PHVD and 10 control subjects. The level of NPBI was determined by a new spectrophotometric method using bathophenanthroline as a chelator. To evaluate the effect of hemolysis, CSF and blood were mixed in different proportions, spun, frozen and thawed, and then analyzed for NPBI. NPBI was found in 75% (15 of 20) of infants with PHVD and in 0% (0 of 10) of control infants (p = 0.0002). Hemolysis induced in vitro did not result in any significant levels of NPBI. Within the group with PHVD, NPBI concentrations in CSF did not correlate with disability, parenchymal brain lesions, or the need for shunt surgery. NPBI was increased in CSF from preterm infants with PHVD, and the increase could not be explained by hemolysis alone. Free iron may help to explain the association between intraventricular hemorrhage and white matter damage.

The capacity of different infusion fluids to lower the prooxidant activity of plasma iron: an important factor in resuscitation?

Prooxidant activity of non-protein-bound iron (NPBI) is an important contributor to reactive oxygen species-induced injury after the resuscitation of critically ill patients. Plasma NPBI occurs in critically ill adults, children, and newborn babies, who often require resuscitation. The ability of the resuscitation fluids to bind iron and lower the patients' NPBI levels in vitro has not previously been studied. In an in vitro model, highly iron-saturated cord blood plasma from 10 preterm and 10 term babies was mixed with FFP, pasteurized plasma protein solution, and 0.9-percent saline. Plasma from 10 healthy adult volunteers was used as a control. Before and after the mixing with any resuscitation fluid, NPBI levels and ceruloplasmin iron-oxidizing and transferrin iron-binding antioxidant capacities were measured. After the in vitro mixing with FFP, the incidence and concentration of NPBI were markedly decreased and the iron-binding antioxidant capacity was increased in the plasma of the preterm and term babies. Being mixed with pasteurized plasma protein solution and 0.9-percent saline did not influence the iron-binding antioxidant capacity of newborn babies' plasma. In the control plasma, results were not changed after the mixing with any resuscitation fluid. In every group, the iron-oxidizing antioxidant capacity was not changed after the mixing with any fluid. Iron-induced oxidative tissue damage may be influenced by resuscitation fluids. In the ongoing debate over the choice of crystalloid or colloid resuscitation fluids, the influence of each fluid on the patient's antioxidant capacity warrants more attention.

Effect of deferoxamine and allopurinol on non-protein-bound iron concentrations in plasma and cortical brain tissue of newborn lambs following hypoxia-ischemia

Reduction of non-protein-bound iron (NPBI) using iron chelators may attenuate hypoxia-ischemia-induced reperfusion injury of the brain. This study investigated whether administration of low-dose deferoxamine and allopurinol, both having NPBI-chelating properties, reduced hypoxia-ischemia-induced NPBI formation in plasma effluent from the brain and in cerebral cortical tissue. Twenty-one newborn lambs underwent severe hypoxia-ischemia. Upon reperfusion and reoxygenation the lambs received either a placebo ( n=7), or deferoxamine 2.5 mg/kg ( n=7) or allopurinol 20 mg/kg ( n=7). The post-hypoxic-ischemic NPBI levels in plasma were significantly lower after deferoxamine but not after allopurinol as compared to placebo-treated lambs. Cortical NPBI levels in both deferoxamine and allopurinol-treated lambs were significantly lower than NPBI levels in placebo-treated lambs. We conclude that deferoxamine effectively lowers NPBI in plasma effluent from the brain, and that both, deferoxamine and allopurinol, lower NPBI in cortical brain tissue.