Non-protein-bound iron detection in small samples of biological fluids and tissues.

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.

Oxidative stress and autologous immunoglobulin G binding to band 3 dimers in newborn erythrocytes.

Since birth-induced oxidative stress (OS) results in the removal of erythrocytes from the blood stream, we studied the binding of autologous IgG to erythrocyte band 3 dimers (the 170-kDa band, which marks the erythrocytes for removal) in preterm and term newborns and in adults. The 170-kDa band was present in as much as 74% of preterm, in 21% of term newborns, and in 10% of adults. During erythrocyte ageing "in vitro" (0, 24, and 48 h aerobic incubation), the appearance of the band occurred much faster with erythrocytes from newborns (particularly preterm) than with those from adults. When the blots for the 170-kDa band were quantified by scanning densitometry, it was seen that the 0 time values were significantly higher in preterm compared to term and adult values. After aerobic incubation a progressive increase in the optical density was observed in each group and the densities were higher in preterm than in the other groups. The course of iron release during the various incubations was analogous to that of the 170-kDa band blots, and significant correlations were found at 0 and 48 h. Methemoglobin formation roughly paralleled iron release. Esterified F(2)-isoprostanes (markers of OS) and O(2)(-) production in the nonincubated (0 time) erythrocytes were much higher in newborn (preterm and term) than in adult erythrocytes. Plasma free F(2)-isoprostanes were significantly higher in preterms than in terms and in terms than in adults. Plasma non-protein-bound iron (NPBI) was higher in preterm than in term newborns and not detectable in adults. In conclusion dimers of band 3 with autologous IgG are found under conditions in which OS can be detected in erythrocytes or in plasma: namely in newborns or in aged erythrocytes.

Glutathione recycling and antioxidant enzyme activities in erythrocytes of term and preterm newborns at birth.

We previously demonstrated a high susceptibility of neonatal red blood cells (RBC) to oxidative stress at birth. The aim of this study was to compare the RBC antioxidant capacity and redox cycle enzyme activities as well as glutathione (GSH) recycling in full-term and preterm infants at birth and in normal adults. GSH and GSH disulfide (GSSG) concentrations, GSH/GSSG ratio, and the activities of glucose-6-phosphate dehydrogenase (G-6-PDH), GSH peroxidase, GSH reductase (GR), catalase (CAT), superoxide dismutase (SOD), and hexokinase (HK) were measured in RBC of 25 healthy adults and 56 newborns (23 term, 33 preterm) at birth. The GSH recycling was measured in adult and newborn RBC exposed to oxidative stress (1 mM tert-butylhydroperoxide). The RBC of term and preterm babies showed higher GSH, GSSG, G-6-PDH, GR, and HK levels/activities and lower GSH/GSSG ratios and higher GSH-recycling rates than those of adults. In preterm babies significant correlations were found between G-6-PDH and CAT, GSH, GSH/GSSG ratio, and GSSG (r = -0.67, r = 0.71, r = -0.66, p < 0.01; r = 0.71, p < 0.05, respectively). In term newborns, statistically significant correlations were observed between G-6-PDH and CAT, SOD, and GSH (r = -0.65, r = -0.65, r = -0.69, p < 0.01, respectively). The results indicate the central role of the G-6-PDH activity in antioxidant defenses. We speculate that preterm babies have prompter involvement of antioxidant defenses than term babies.

Activin a plasma levels at birth: an index of fetal hypoxia in preterm newborn.

Activin-A is a growth factor involved in cell growth and differentiation, neuronal survival, early embryonic development and erythropoiesis. Hypoxemia is a specific trigger for increasing activin-A in fetal lamb circulation. We tested the hypothesis that fetal hypoxia induces activin-A secretion in preterm newborn infants. Fifty newborn infants with gestational ages ranging from 26 to 36 wk were enrolled in a prospective study performed at the Pediatrics, Obstetrics and Reproductive Medicine Department, University of Siena, Italy. Heparinized blood samples were obtained from the umbilical vein after cord clamping, immediately after delivery. Activin A, hypoxanthine (Hx), xanthine (Xa) plasma levels and absolute nucleated red blood cell (NRBC) count were measured. Activin-A levels (p < 0.0001) and NRBC (p < 0.0001) were significantly higher in hypoxic than in non hypoxic preterm newborns. Cord activin A levels were significantly related with Hx (taua=0.64, taub=0.64, p < 0.0001) and Xa (taua=0.56, taub=0.57, p < 0.0001) levels, NRBC ((taua=-0.45, taub=-0.46, p < 0.0001) count; pH (taua=-0.47, taub=-0.48, p < 0.0001) and base deficit (taua=-0.36, taub=0.-0.36, p = 0.0002). Preterm newborns with signs of perinatal hypoxia at birth have increased activin-A levels, suggesting that activin-A may reflect indirectly intrauterine hypoxia.