Free Radical Biology & Medicine 40 (2006) 907–915www.elsevier.com/locate/freeradbiomed

Original Contribution

Oxidative stress and autologous immunoglobulin G binding toband 3 dimers in newborn erythrocytes

Viviana Rossi a, Silvia Leoncini a, Cinzia Signorini a, Giuseppe Buonocore b, Patrizia Paffetti b,Donatella Tanganelli b, Lucia Ciccoli a, Mario Comporti a,⁎

a Department of Pathophysiology, Experimental Medicine and Public Health, University of Siena, Via Aldo Moro, 53100 Siena, Italyb Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Siena, Italy

Received 26 July 2005; revised 22 November 2005; accepted 28 November 2005Available online 27 December 2005

Abstract

Since birth-induced oxidative stress (OS) results in the removal of erythrocytes from the blood stream, we studied the binding of autologous IgG toerythrocyte band 3 dimers (the 170-kDa band, which marks the erythrocytes for removal) in preterm and term newborns and in adults. The 170-kDaband 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 haerobic incubation), the appearance of the band occurred much faster with erythrocytes from newborns (particularly preterm) than with those fromadults. When the blots for the 170-kDa band were quantified by scanning densitometry, it was seen that the 0 time values were significantly higher inpreterm compared to term and adult values. After aerobic incubation a progressive increase in the optical density was observed in each group and thedensities 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 F2-isoprostanes (markers of OS) and O2

S− production in the nonincubated (0 time) erythrocytes were much higher in newborn (preterm and term) than inadult erythrocytes. Plasma free F2-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 arefound under conditions in which OS can be detected in erythrocytes or in plasma: namely in newborns or in aged erythrocytes.© 2005 Elsevier Inc. All rights reserved.

Keywords: Oxidative stress; Newborns; Band 3 dimers; Iron release; Membrane esterified F2-isoprostanes; Superoxide anion; Plasma F2-isoprostanes; Non-protein-bound iron

Introduction

Erythrocyte ageing is associated with a decrease in theactivity of several enzymes [1] and with modifications inmembrane proteins [2]. In particular modifications in membrane

Abbreviations: DCI, desferrioxamine chelatable iron; MetHb, methemoglo-bin; IgG, immunoglobulin G; NPBI, non-protein-bound iron; Hepes, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; HPLC, high-performanceliquid chromatography; DFO, desferrioxamine; PBS, phosphate-buffered saline;MCLA, methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-α]pyrazin-3-one; BHT, butylated hydroxytoluene; PGF2α, prostaglandin F2α; GC/NICI-MS/MS, gas chromatography/negative-ion chemical ionization tandem massspectrometry; C18, octadecylsilane; NH2, aminopropyl; NTA, nitrilotriaceticacid; CP22, 3-hydroxy-1propyl-2-methyl-pyridin-4-one.⁎ Corresponding author. Fax: +39 0577 234009.E-mail address: comporti@unisi.it (M. Comporti).

0891-5849/$ - see front matter © 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.freeradbiomed.2005.11.021

band 3 protein, by proteolytic cleavage, clustering or exposureof unusual epitopes, trigger the binding of specific anti-band 3autoantibodies, marking the cell for removal [3–7].

Our previous studies have shown that iron is released fromhemoglobin [8] in a desferrioxamine-chelatable (DCI) formwhen erythrocytes are exposed to oxidative stress, such asincubation with oxidizing agents [9–11], or prolonged aerobicincubation in physiological buffer (a model of rapid in vitroageing) [12]. Iron release is accompanied by methemoglobin(MetHb) formation [10–12] and by oxidative alterations ofmembrane proteins, in particular band 3 protein [12]. Ironrelease seems to be the cause of oxidation of membrane proteinswhich promotes the autologous immunoglobulin G (IgG)binding [12,13]. In fact, cell-permeable iron chelators (such asferrozine, quercetin, and fluor-benzoil-pyridoxal hydrazone)prevent both membrane protein oxidation and autologous IgG

Table 1Clinical characteristics of babies

Preterms Terms

Number 23 37Gestational age (weeks) 34.7 ± 0.47 (28–36) 38.70 ± 0.22 (37–41)Male/Female 12/11 19/18Birth weight (kg) 2.45 ± 0.12 ⁎ 3.04 ± 0.08Vaginal delivery (n) – 16Elective cesarean section (n) 17 20Emergency cesarean section (n) 6 1Apgar-1 min score 6.74 ± 0.46 ⁎⁎ 8.32 ± 0.27Apgar-5 min score 8.78 ± 0.39 ⁎⁎ 9.72 ± 0.10pH 7.31 ± 0.01 7.30 ± 0.01Peack of bilirubin (mg/dl) a 11.3 ± 0.68 11.2 ± 0.74

The data are the means ± SE.a As measured by Bilicheck (from Burke and Burke).⁎ P b 0.001 preterms vs terms.

⁎⁎ P b 0.01 preterms vs terms.

908 V. Rossi et al. / Free Radical Biology & Medicine 40 (2006) 907–915

binding, suggesting the possibility that a metal-catalyzedoxidation of membrane protein underlies erythrocyte ageing[12–15]. Furthermore, in two conditions in which an acceler-ated removal of erythrocytes occurs, namely β-thalassemia(major and intermedia) [16] and perinatal period [17], theerythrocyte DCI content and even more the release of iron after24 h of aerobic incubation are greatly increased as compared tonormal adult erythrocytes [18,19], suggesting that β-thalasse-mic and newborn erythrocytes are subjected to an oxidativestress more than normal adult erythrocytes. β-Thalassemic andnewborn erythrocytes show a high content in fetal hemoglobin(HbF) (even higher in preterm infants). HbF, at least undermany experimental conditions, has greater oxygen affinity thanadult hemoglobin and is more subjected to denaturation andoxidation [20–23]. A positive correlation between HbF andDCI content (and release) in thalassemia major and intermediahas also been observed [18], suggesting that the presence ofHbF is a condition favourable to iron release. The erythrocyteDCI content and release after incubation are higher in pretermthan in term newborns [19]. Birth is an oxidative challenge forthe newborn, due to the sharp postnatal transition from therelatively low oxygen intrauterine pressure (pO2 20–25 Torr) tothe significantly higher oxygen extrauterine environment (pO2

100 Torr) [24–26]. Such oxidative challenge is exacerbated bythe low efficiency of natural antioxidant systems in thenewborns, particularly in preterm babies [26,27]. In additionin most newborns [28–30] non-protein-bound iron (NPBI), aform of non-transferrin-bound iron, is detectable in plasma, acondition similar to that occurring in plasma of patients withiron overload such as primary or secondary hemocromatosis[31,32]. Plasma NPBI is higher in preterm than term newbornsand is not detectable in healthy adults [19]. Furthermore, F2-isoprostanes, prostaglandin F2-like compounds formed by freeradical-catalyzed lipid peroxidation of phospholipid-boundarachidonic acid and considered the most reliable marker ofoxidative stress [33–35], are significantly higher in plasma ofnewborns than of healthy adults and are higher in preterm thanin term newborns [36].

Since an accelerated removal of erythrocytes from the bloodstream occurs in preterm and term newborns, we investigatedwhether neonatal erythrocyte exposure to oxidative stressresults in the binding of autologous IgG to band 3 dimers in ahigher percentage of newborns as compared to adults and ofpreterm as compared to term babies. The susceptibility ofneonatal and adult erythrocytes exposed to in vitro ageing tobind autologous IgG was also investigated. The release of iron(DCI) and the formation of esterified F2-isoprostanes inerythrocytes and the occurrence of free F2-isoprostanes and ofNPBI, as markers of oxidative stress in the erythrocytes andplasma, respectively, were also studied.

Materials and methods

Materials

Desferrioxamine (DFO) was supplied by Ciba-Geigy.Centrifugal filter devices (CentriplusR, YM-30) were from

Amicon. The reservoirs for silicic acid column chromatographywere from Varian. The solvents used for HPLC were of HPLCgrade. The methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-α]pyrazin-3-one (MCLA) was from Molecular Probes.The nitrocellulose Hybond-C extra was supplied by AmershamLife Science. The secondary antibody was goat anti-human IgG(Fc-specific) alkaline phosphatase conjugate from SigmaImmunochemicals. 5-Bromo-4-cloro-3-indolyl phosphate/nitroblue tetrazolium liquid substrate system (BCIP/NBT) wasfrom Sigma-Aldrich. Tetradeuterated 8-epi-PGF2α wereobtained from Cayman (Ann Arbor, MI), Sep-Pak VacR C18

(500 mg) and Sep-Pak VacR NH2 (500 mg) cartridges werepurchased from Waters (Milford, MA). Nitrilotriacetic acid(NTA) was from Sigma.

Subjects studied

One hundred thirty subjects were randomly selected from 9am to 2 pm to allow preliminary blood processing on the sameday, from January to October 2004. Thirty-five were excludeddue to the lack of parental consent, 23 were excluded forsuspected sepsis, thalassemias or other abnormal hemoglobins(screened by HPLC) or glucose-6-phosphate dehydrogenasedeficiency (estimated by a Biotech kit), 12 were not enrolleddue to the impossibility to draw cord blood. Sixty newborninfants (37 term and 23 preterm) were examined at birth.Seventy-five percent of preterm newborns received antenatalcorticosteroids. Four milliliters of heparinized blood wascollected from the umbilicain vein immediately after cordclamping. All tests were performed in cord blood immediatelyafter cord clamping in healthy term and in preterm babieswithout any severe illness at birth (for more detailedinformation see Table 1). All postnatal procedures cannotinfluence obviously cord blood data.

Venous blood was also drawn from 50 healthy adult subjects.Informed consent was obtained from the parents of the

newborn infants and from the adults. The study was approvedby the Human Ethics-Deontology Committee of the MedicalFaculty of the University of Siena.

909V. Rossi et al. / Free Radical Biology & Medicine 40 (2006) 907–915

The blood samples of newborn or adult subjects wereimmediately centrifuged at 600 g for 10 min at roomtemperature; the plasma was saved and buffy coat removedby aspiration. The erythrocytes were washed twice withphysiological solution and resuspended in Ringer solution(125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 32 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (Hepes), 5mM glucose, 1 mM CaCl2), pH 7.4 as a 50% (vol/vol)suspension and incubated for 24 and 48 h at 37°C under aerobicconditions, for erythrocyte ageing [12]. The incubations werecarried out in a shaker apparatus in the presence of antibiotics(20 units penicillin and 20 μg streptomycin/ml of buffer).Samples were withdrawn at the indicated times, for thedetermination of DCI, MetHb [37], hemolysis [9], andautologous IgG binding to band 3 dimers [38]. Plasma wasused for free F2-isoprostanes and for NPBI determinations andfor the opsonization step in IgG binding determination. Sampleprocessing was done immediately after each sampling andimmediately after each incubation time, when an incubation wasperformed.

Erythrocyte DFO-chelatable iron (DCI)

DCI was determined as a DFO–iron complex (ferrioxamine)as previously reported [9]. Briefly at 0′ time and at 24 and 48h of the incubation, 25 μM DFO was added to the samples. Theerythrocytes were then lysed by adding water (1 vol) and byfreeze (−70°C)–thawing. The hemolysate was ultrafiltered incentrifugal filter devices (CentriplusR YM-30; Amicon, Milli-pore) and the excess of DFO was removed by silicic acidcolumn chromatography. The DFO–iron complex was deter-mined by HPLC.

Binding of autologous IgG to band 3 dimers

The method of Turrini et al. [38] was used to evaluate thebinding of autologous IgG to oxidatively modified band 3(dimers). The advantage of the isolation of autologous IgGbound to band 3 dimers (as described in the method [38])resides in the fact that, of all the autologous IgG antibodies, justthe small fraction which specifically binds to the band 3 dimersis to be used, and therefore the method is very sensitive andreliable.

Briefly, the nonincubated samples (0′ time) and theincubated (24 and 48 h) ones were centrifuged, washed twicewith Hepes-buffered saline (130 mM NaCl, 10 mM Hepes, 10mM glucose pH 7.4) and suspended at 10% hematocrit with thesame buffer containing 30% (vol/vol) autologous plasma andincubated for 30 min at 37°C, to allow the binding ofautologous IgG (opsonization step). The erythrocytes werethen washed twice in Hepes and the erythrocyte membraneswere prepared according to Dodge et al. [39]. Autologous IgGwere then eluted from erythrocyte membrane and used asprimary antibody. At this end an aliquot of membranes (90 μl)was incubated for 5 min at 4°C with 50 mM glycine (pH 3.0)and then centrifuged at 17,500g in an Eppendorf centrifuge; thesupernatant containing IgG was neutralized with 1 M Tris-HCl,

pH 7.5. Membrane proteins were quantified according to Lowryet al. [40]. Membrane proteins (10 μg) were separated bysodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDS-PAGE: 10% acrylamide) under nonreducing conditionsaccording to Laemmli [41] and then transferred to nitrocelluloseaccording to Towbin et al. [42]. After blocking for 1 h in PBScontaining 1% (wt/vol) bovine serum albumin (BSA), the firstantibody (diluted 1:100) was added to PBS and the nitrocel-lulose was incubated for 1 h at room temperature. Afterwashing, the nitrocellulose was incubated for 30 min at roomtemperature with the second antibody diluted 1:1000 in PBS.The second antibody was anti-human IgG conjugated toalkaline phosphatase. The blot was developed with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium for alkalinephosphatase. Positive control was obtained by preparing asample in which 10% erythrocyte suspension was incubatedwith H2O2 (5 mM final concentration) for 30 min at 4°C.Afterward the cells were washed and incubated with autologousplasma as reported above. Negative controls were the samplesnot exposed to autologous plasma. The membrane proteins wereblotted and directly incubated with the second antibody(antihuman IgG).

Superoxide anion production

The superoxide anion (O2S−) production in erythrocytes was

investigated according to Kondo et al. [43] with minormodifications, using the chemiluminescence probe MCLA (aluciferin derivate), which is highly specific and sensitive tosuperoxide anion. Briefly, the erythrocytes from newborn andadult subjects at 0 time were resuspended in Ringer solution at6%. MCLA (1 mg/ml) was diluted 1:400 with the incubationbuffer and 100 μl was added to the erythrocyte suspension (25μl). Buffer was added to obtain 2 ml final volume.Phenylhydrazine (Phz) was added to reveal O2

S− production,and MCLA-dependent chemiluminescence was measuredbefore and after addition of 100 μl Phz (690 μM finalconcentration) in neonatal and adult erythrocytes. Chemilumi-nescence was measured using a luminometer (LUMACBiocounter M 2010).

Erythrocyte membrane esterified F2-isoprostanesdetermination

The nonincubated samples (0′ time) were washed twice withHepes and were lysed in Dodge buffer [39]. The membraneswere prepared as described for binding of autologous IgG, in thepresence of 90 μM butylated hydroxytoluene (BHT) and storedat −70°C.

A 100 μl aliquot of the sample was resuspended with H2O(0.9 ml) and 1 N KOH (500 μl) was added for the basichydrolysis according to the method used by Nourooz-Zadeh etal. [44] for the tissutal isoprostanes assay. After incubation at45°C for 45 min, the sample was acidified to pH 3 with HCl 1 N(500 μl), spiked with tetradeuterated prostaglandin F2α(PGF2α) (500 pg in 50 μl of ethanol) and extracted with 10ml of ethyl acetate. The upper organic layer, obtained after

Fig. 1. Percentages of preterm and term newborns and of adults showing thebinding of autologous IgG to band 3 dimers in the erythrocytes immediatelyafter the drawing (0′ time) (A) and after in vitro ageing (B). The results reportedderive from determinations carried out in 23 preterms, 37 terms and 50 adults.

910 V. Rossi et al. / Free Radical Biology & Medicine 40 (2006) 907–915

centrifugation at 1000 g for 5 min, was applied onto anaminopropyl (NH2) cartridge preconditioned with 10 ml ofhexane. After derivatization [44] the determination of F2-isoprostanes was accomplished by gas chromatography/nega-tive-ion chemical ionization tandem mass spectrometry (GC/NICI-MS/MS) analysis [45]. Esterified F2-isoprostanes werenormalized for membrane proteins, measured with the Bradfordreagent [46] using 0.2% Triton X-100 to dissolve themembranes. The concentration of hemoglobin in the samemembranes was assayed by measuring the absorbance at 541nm [47].

Plasma free F2-isoprostane determination

Plasma was added with BHT (90 μM) and stored at −70°C.The purification and the quantitation of the plasma free F2-isoprostanes were carried out according to Nourooz-Zadeh et al.[48] and Signorini et al. [45], respectively. In particular,immediately after being thawed, the plasma (1 ml) was spikedwith tetradeuterated PGF2α (500 pg in 50 μl of ethanol) as aninternal standard. The sample was acidified with water (pH 3)and was applied on an octadecylsilane (C18) cartridge. Lipidswere eluted with a solvent mixture, applied on an aminopropyl(NH2) cartridge, and then derivatized [48] and examined by GC/NICI-MS/MS.

Plasma non-protein-bound iron determination

Plasma non-protein-bound iron measurement was performedessentially according to Singh and co-workers [49]. A largeexcess of nitrilotriacetic acid, which is a low-affinity ligand foriron and complexes the iron nonspecifically bound to plasmaproteins, but does not remove that bound to transferrin orferritin, was added to plasma. The Fe–NTA complex wasultrafiltered as reported [50] and quantified using an HPLCprocedure in which on-column derivatization with a high-affinity iron chelator (3-hydroxy-1propyl-2-methyl-pyridin-4-one, CP22) was used. In this way, iron bound to NTA isconverted to form the colored (CP22)3–Fe complex whichabsorbs in the visible region at 450 nm.

Statistical analysis

Results are reported as means ± SE. Comparisons betweengroups were carried out by Student's t test. Correlationcoefficients were determined by the Pearson test. All testswere two-tailed. The value of P b 0.05 was consideredstatistically significant.

Results

Fig. 1A shows that the 170-kDa band indicating theautologous IgG binding to band 3 dimers is present in 73.9%of preterm newborns, in 21.1% of term newborns, and in 10.0%of healthy adults. Therefore, at birth a large part of theerythrocytes is ready to be eliminated by the phagocytic systemand this reflects an accelerated turnover of red blood cells.

When the susceptibility of the erythrocytes to bindautologous IgG was studied during the in vitro ageing in thethree groups of examined subjects, it was observed (Fig. 1B)that while erythrocytes from the term babies and from adultsaged at a comparable degree, those from preterms babies show aseeming lower degree of ageing, due to the very high percentageof preterms with aged erythrocytes at birth.

Fig. 2 shows typical Western blots of erythrocyte membraneproteins of preterm (A), term (B), and adult (C), at zero time andafter 24 and 48 h of aerobic incubation. Western blotserythrocyte membrane proteins were quantified using scanningdensitometry and the values are reported in Fig. 3A. Zero timevalues were significantly higher in preterm as compared to termand adult values. After aerobic incubation a progressiveincrease in the optical density was observed in each group ofexamined subjects and the densities were higher in preterm thanin the other groups.

The results of iron release during the erythrocyte ageing inthe three groups of subjects are reported in Fig. 3B. As can beseen, the zero time values were higher in preterm than in termand adult erythrocytes. During the aerobic incubation ironrelease progressively increased in each group of subjects, beinghigher in term and preterm than in adult erythrocytes at 24 h. At48 h the release was higher in the preterm group than in theothers. When the values for the binding of autologous IgG toband 3 dimers (scanning densitometry, Fig. 3A) were plottedagainst the values for iron release (Fig. 3B) for each incubationtime, a highly significant correlation was found at 0′ and 48

Fig. 2. Western blots of membrane proteins isolated from preterm (A), term (B), and adult (C) erythrocytes. A typical sample of preterm, term, and adult at 0′ time andduring in vitro ageing (aerobic incubation for 24 and 48 h) is shown. A positive control (H2O2) is also shown.

911V. Rossi et al. / Free Radical Biology & Medicine 40 (2006) 907–915

h (Figs. 3C and D, respectively). Methemoglobin formationroughly paralleled iron release (Fig. 4) and was correlated(r = 0.999; P b 0.02) with the IgG binding values at 0′ time. Noconsistent hemolysis was seen in the 0 time and 24 h samples.Higher hemolysis (15–17%) was seen in the 48 h of pretermerythrocyte samples.

No release of isoprostanes from the erythrocytes into theincubation medium was found during the erythrocytes ageing(not shown). However, the determination of esterified F2-isoprostanes (that is, the isoprostanes still bound to membranephospholipid) in the nonincubated (0′ time) erythrocytesshowed (Fig. 5A) that such isoprostanes were much higher innewborn (preterm and term) than in adult erythrocytes.Similarly, the erythrocyte production of superoxide anion(Fig. 5B) was much greater with newborn (preterm and term)than with adult erythrocytes.

The determinations of free F2-isoprostanes and of NPBI asmarkers of oxidative stress in plasma gave results consistentwith the overall feature: plasma F2-isoprostanes were signifi-cantly higher in preterm than in term and in term than in adults(Fig. 6A); plasma NPBI was higher in preterm than in termnewborns and not detectable in adults (Fig. 6B).

Discussion

The present work shows that in erythrocytes autologous IgGare bound to band 3 dimers in a much greater percentage ofnewborns compared to adults and of preterm compared to terminfants. This suggests that a removal of red blood cells isongoing in the newborns.

Since in previous works [8–13,18,19,51] we have suggestedthat the primary event resulting from oxidative stress of theerythrocyte is the intracellular release of redox-active iron, wedetermined such a release in the various groups of subjectsstudied. A higher content of DCI was seen in newbornerythrocytes compared to adult cells and in preterm as comparedto term erythrocytes. This is consistent with our previous studies

[19]. During the erythrocyte ageing the DCI release increasedmore quickly in newborn than in adult erythrocytes, showing abehavior similar to that of the binding of autologous IgG toband 3 dimers. Recently Ando et al. [52] confirmed that DFO-chelatable iron is higher in senescent than in young erythrocytesas separated by Percoll density gradient centrifugation.Methemoglobin formation, roughly paralleling iron release,was the most direct evidence of erythrocyte oxidative stress.The higher (15–17%) hemolysis seen in the 48 h samples ofpreterm erythrocytes was probably due to the prolongedincubation of cells with a higher fragility [20].

The production of superoxide anion was much greater withnewborn erythrocytes compared to adult erythrocytes, confirm-ing our previous studies [51]. The greater O2

S− production innewborn than in adult erythrocytes might be related to the highHbF content of newborn RBC. In fact HbF has greater oxygenaffinity than adult hemoglobin and is likely more subjected tooxidation. Watkins et al. [53] showed that purified HbF cangenerate greater amounts of superoxide, peroxide, and hydroxylradicals than HbA. Kondo et al. [43] have shown that neonatalerythrocytes produce about twice as much O2

S− as adulterythrocytes in response to oxidative stress induced byphenylhydrazine. However, according to others [23] thesereactions occur at a very slow rate.

Interestingly, the oxidative stress induced by the birth is ableto cause phospholipid peroxidation in native (nonincubated)newborn erythrocytes, as shown by the higher level of esterifiedF2-isoprostanes as compared to adult cells. Such isoprostanescould not be detected clearly in the incubation mediumrecovered after the in vitro ageing. However, plasma levels ofF2-isoprostanes are much higher in preterms than in terms andadults, which is consistent with our recent results [36], butwhich does not explain the origin of such lipid peroxidation.The origin of plasma NPBI is also unexplained, even if part ofthe released iron can diffuse out of erythrocytes and can befound in the incubation medium [51]. Probably the largeplacental transfer of iron and the relatively low transferrin level

Fig. 3. Binding of autologous IgG to band 3 dimers (A) and iron (DCI) release (B) in erythrocytes from preterm, term newborns, and adults at 0′ time and duringin vitro ageing. Correlation between autologous IgG binding and iron (DCI) release at 0′ time (C) and at 48 h of aerobic incubation (in vitro ageing) (D). (A) Theresults derive from all the samples (see Materials and methods). Western blots were quantified using scanning densitometry. *Statistically significant at P b 0.05level or below; 0′ time preterms vs 0′ time terms and vs 0′ time adults; 48 h preterms vs 48 h terms and vs 48 h adults; 24 h vs 0′ time in terms and 24 h adults.(B) The results reported derive from determinations carried out in 16 preterms, 29 terms, and 29 adults. (It was not possible to run all the determinations in eachsample, due to the limited amount of blood available). *Statistically significant at P b 0.05 level or below; 0′ time preterms vs 0′ time terms and vs 0′ time adults;48 h preterms vs 48 h adults; 24 h vs 0′ time in preterms, terms, and adults; 24 h preterms vs 24 h adults; 24 h terms vs 24 h adults.

912 V. Rossi et al. / Free Radical Biology & Medicine 40 (2006) 907–915

produce a highly iron-saturated transferrin together with a highincidence of NPBI [54].

A number of studies [3–7,38] have shown that thephagocytic removal of senescent, oxidatively damaged ery-throcytes appears to be mediated by the binding of autologousantibodies with anti-band 3 specificity. Native band 3 innonsenescent erythrocytes is physiologically present as amixture of dimers and tetramers [55,56]. Since nonsenescenterythrocytes do not bind anti-band 3 antibodies and are notrecognized by phagocytes, putative band 3 epitopes are notavailable for bivalent antibody binding in native dimers. It hasbeen shown [38] that band 3 is recognized by specific

autologous antibodies if the quaternary structure is modifiedby oxidative cross-linking of cytoplasmic domains, withensuing reorientation of band 3 within the dimers and thatautologous IgG do not recognize nonoxidatively generated,covalently linked band 3 dimers. It has been concluded [38] thatdisulfide-crosslinked band 3 dimers are the minimal band 3aggregates with enhanced affinity for anti-band 3 antibodies.Band 3 has the capacity to transduce specific intracellularmodifications to the external side of the membrane, making theerythrocyte recognizable by macrophages [38]. The presentstudy shows that dimers of band 3 are formed under conditionsin which iron release and MetHb formation are by far most

Fig. 4. Methemoglobin (MetHb) in term and preterm newborns and adulterythrocytes at 0′ time and during in vitro ageing. The results reported derivefrom the same samples as for iron (DCI) release. *Statistically significant atP b 0.05 level or below; 0′ time preterms vs 0′ time terms and 0′ time adults;24 h preterms and terms vs 24 h adults.

Fig. 6. (A) Plasma F2-IsoPs levels in preterm and term newborns and in adults.(B) Plasma non-protein-bound iron (NPBI) levels in preterm and term newbornsand adults. (A) The results are the means ± SE of determinations carried out in19 preterms, 20 terms, and 10 adults. Results are expressed as pg/ml plasma.

913V. Rossi et al. / Free Radical Biology & Medicine 40 (2006) 907–915

elevated: namely in newborn erythrocytes and in “in vitro” agederythrocytes. On the contrary, in adult erythrocytes, band 3dimers as well as iron release and MetHb formation occur inmuch lower amounts (Figs. 3 and 4) and in a much lowernumber of cases (Fig. 1). These results strongly suggest that theiron released from its physiological complexes (Hb, heme) intothe cytoplasm could have an important part in the oxidativecross-linking of the cytoplasmic domains of band 3. It has alsobeen shown [57–59] that iron can be found associated with thecytoplasmic side of the membrane in which several discrete iron

Fig. 5. (A) Esterified F2-isoprostane levels in erythrocyte membranes fromnewborns and adults. (B) Superoxide anion production, measured as MCLA-dependent chemiluminescence, in newborn and adult erythrocytes at 0′ time. (A)The results are the means ± SE of determinations carried out in 9 newborns and4 adults. Results are expressed as pg/μg membrane protein.*P b 0.05 newbornsvs adults. (B) The results (expressed as relative light units (RLU)) are themeans ± SE of determinations carried out in erythrocytes from 6 newborns and6 adults. *P b 0.01 newborns vs adults.

*P b 0.001 preterms vs terms or adults; **P b 0.01 terms vs adults. (B) Theresults are the means ± SE of determinations carried out in 20 preterms,30 terms, and 28 adults. Results are expressed as nmol/ml plasma. *P b 0.05preterms vs terms. NPBI was not detectable in adult plasma.

compartments (denatured hemoglobin, free heme, moleculariron, etc.) can be demonstrated. In particular, molecular iron[59] would be able to cycle between ferric and ferrous states andthereby participate in several redox reactions with consequentoxidative damage to membrane structures.

In conclusion iron release, MetHb formation, oxidation ofmembrane proteins, and binding of autologous IgG to band 3dimers appears to be strictly related as can be seen whenerythrocytes are exposed to an oxidative stress such as thatoccurring at birth. The formation of band 3 dimers is consideredone of the main pathways of the erythrocyte ageing[3,7,38,60,61]. The accelerated removal of erythrocytes fromthe blood stream during the perinatal period seems to beassociated to various events consequent to oxidative stress.Since the latter is an ongoing process during the life spanwhenever erythrocytes are exposed to oxygen in the bloodcompartment, the continuous formation of aged erythrocytescan be easily guessed. The oxidative challenge at birth due tothe sharp increase in neonatal blood oxygen concentration mayaccelerate the erythrocyte ageing process.

Acknowledgments

The competent advice of Dr. Turrini Francesco (Depart-ment of Genetics, Biology, and Biochemistry, University of

914 V. Rossi et al. / Free Radical Biology & Medicine 40 (2006) 907–915

Torino Medical School) is gratefully acknowledged. ThisResearch was supported by a grant from the Italian Ministryfor Education, University, and Research (F.I.R.B. 2002 prot.RBAU01H4T8_002). We thank the Siena Hospital Adminis-tration for the mass spectrometer purchase.

References

[1] Imanishi, H.; Nakai, T.; Abe, T.; Takino, T. Glutathione-linked enzymeactivities in red cell aging. Clin. Chim. Acta 159:73–76; 1986.

[2] Piccinini, G.; Minetti, G.; Balduini, C.; Brovelli, A. state of glutathione andmembrane proteins in human red cells of different age.Mech. Ageing Dev.78:15–26; 1995.

[3] Lutz, H. U.; Bussolino, F.; Flepp, R.; Stammler, P.; Kazatchkine, M. D.;Arese, P. Naturally occurring anti-band-3 antibodies and complementtogether mediate phagocytosis of oxidatively stressed human erythrocytes.Proc. Natl. Acad. Sci. USA 84:7368–7372; 1987.

[4] Kay, M. M. B.; Goodman, S. R.; Sorensen, K.; Whitfield, C. F.; Wong, P.;Zaki, L.; Rudloff, V. Senescent cell antigen is immunologically related toband 3. Proc. Natl. Acad. Sci. USA 80:1631–1635; 1983.

[5] Kay, M. M. B. Localization of senescent cell antigen on band 3. Proc. Natl.Acad. Sci. USA 81:5753–5757; 1984.

[6] Kay, M. M. B.; Marchalonis, J. J.; Schluter, S. F.; Bosman, G. Humanerythrocyte aging: cellular and molecular biology. Transfus. Med. Rev.5:173–195; 1991.

[7] Hornig, R.; Lutz, H. U. Band 3 protein clustering on human erythrocytespromotes binding of naturally occurring anti-band 3 and anti-spectrinantibodies. Exp. Gerontol. 35:1025–1044; 2000.

[8] Ferrali, M.; Ciccoli, L.; Signorini, C.; Comporti, M. Iron release anderythrocyte damage in allyl alcohol intoxication in mice. Biochem.Pharmacol. 40:1485–1490; 1990.

[9] Ferrali, M.; Ciccoli, L.; Comporti, M. Allyl alcohol-induced hemolysis andits relation to iron release and lipid peroxidation. Biochem. Pharmacol.38:1819–1825; 1989.

[10] Ferrali, M.; Signorini, C.; Ciccoli, L.; Comporti, M. Iron release andmembrane damage in erythrocytes exposed to oxidizing agents, phenylhy-drazine, divicine and isouramil. Biochem. J. 285:295–301; 1992.

[11] Ciccoli, L.; Ferrali, M.; Rossi, V.; Signorini, C.; Alessandrini, C.;Comporti, M. Hemolytic drugs aniline and dapsone induce iron releasein erythrocytes and increase the free iron pool in spleen and liver. Toxicol.Lett. 110:57–66; 1999.

[12] Signorini, C.; Ferrali, M.; Ciccoli, L.; Sugherini, L.; Magnani, A.;Comporti, M. Iron release, membrane protein oxidation and erythrocyteageing. FEBS Lett. 362:165–170; 1995.

[13] Comporti, M.; Signorini, C.; Buonocore, G.; Ciccoli, L. Iron release,oxidative stress and erythrocyte ageing. Free Radic. Biol. Med.32:568–576; 2002.

[14] Ferrali, M.; Signorini, C.; Caciotti, B.; Sugherini, L.; Ciccoli, L.; Giachetti,D.; Comporti, M. Protection against oxidative damage of erythrocytemembrane by the flavonoid quercetin and its relation to iron chelatingactivity. FEBS Lett. 416:123–129; 1997.

[15] Ferrali, M.; Signorini, C.; Ciccoli, L.; Bambagioni, S.; Rossi, V.; Pompella,A.; Comporti, M. Protection of erythrocytes against oxidative damage andautologous immunoglobulin G (IgG) binding by iron chelator fluor-benzoil-pyridoxal hydrazone. Biochem. Pharmacol. 59:1365–1373; 2000.

[16] Vigi, V.; Volpato, F.; Gaburro, F.; Conconi, F.; Bargellesi, A.; Pontremoli,S. The correlation between red-cell survival and excess of alpha-globinsynthesis in beta-thalassemia. Br. J. Haematol. 16:25–30; 1969.

[17] Pearson, H. A. Life-span of the fetal red blood cell. J. Pediatr. 70:166–171;1967.

[18] Ciccoli, L.; Signorini, C.; Scarano, C.; Rossi, V.; Bambagioni, S.; Ferrali,M.; Comporti, M. Iron release in erythrocytes from patients with beta-thalassemia. Free Radic. Res. 30:407–413; 1999.

[19] Ciccoli, L.; Rossi, V.; Leoncini, S.; Signorini, C.; Paffetti, P.; Bracci, R.;Buonocore, G.; Comporti, M. Iron release in erythrocytes and plasma non-

protein-bound iron in hypoxic and non hypoxic newborns. Free Radic.Res. 37:51–58; 2003.

[20] Lubin, B. H.; Van de Berg, J. J. M.; Lewis, R. A.; Scott, M. D.; Kuypers,F. A. Unique properties of the neonatal red cell. In: Xanthou, M.; Bracci,R.; Prindull, G., eds. Neonatal haematology and immunology II. ExcerptaMedica, Amsterdam, pp. 79–89; 1993.

[21] Martin, H.; Huisman, T. H. J. Formation of ferrihaemoglobin of isolatedhuman haemoglobin types by sodium nitrate. Nature 200:898–899; 1963.

[22] Robson, N.; Brittain, T. Heme stability in the human embryonichemoglobins. J. Inorg. Biochem. 64:137–147; 1996.

[23] Macdonald, V. W.; Charache, S. Differences in the reaction sequencesassociated with drug-induced oxidation of hemoglobins E, S, A, and F.J. Lab. Clin. Med. 102:762–772; 1983.

[24] Gutteridge, J. M. C.; Westermarck, T.; Halliwell, B. Oxigen radicaldamage in biological system. In: Johnson, J. E., ed. Free radicals agingand degenerative diseases. A.R. Liss, New York, pp. 99–139; 1986.

[25] Saugstad, O. D. Oxygen toxicity in the neonatal period. Acta Paediatr.Scand. 79:881–892; 1990.

[26] Saugstad, O. D. Mechanisms of tissue injury by oxygen radicals:implications for neonatal disease. Acta Paediatr. 85:1–4; 1996.

[27] Bracci, R.; Buonocore, G. The antioxidant status of erythrocytes in pretermand term infants. Semin. Neonatol. 3:191–197; 1998.

[28] Evans, P. J.; Evans, R.; Kovar, I. Z.; Holton, A. F.; Halliwell, B.Bleomycin-detectable iron in the plasma of premature and full-termneonates. FEBS Lett. 303:210–212; 1992.

[29] Berger, H. M.; Mumby, S.; Gutteridge, J. M. C. Ferrous ions detected iniron-overloaded cord blood plasma from preterm and term babies:implications for oxidative stress. Free Radic. Res. 22:555–559; 1995.

[30] Dorrepaal, C. A.; Berger, H. M.; Benders, M. J. N.; Van Zoeren-Grobben,D.; Van de Bor, M.; Van Bel, F. Nonprotein-bound iron in postasphyxialreperfusion injury of the newborn. Pediatrics 98:883–889; 1996.

[31] Loreal, O.; Gosriwatana, I.; Guyader, D.; Porter, J.; Brissot, P.; Hider, R. C.Determination of non-transferrin-bound iron in genetic hemochromatosisusing a new HPLC-based method. J. Hepatol. 32:727–733; 2000.

[32] al-Refaie, F. N.; Wickends, D. G.; Wonke, B.; Kontoghiorghes, G. J.;Hoffbrand, A. W. Serum non-transferrin-bound iron in beta-thalassemiamajor patients treated with desferrioxamine and L1. Br. J. Haematol.82:431–436; 1992.

[33] Morrow, J. D. Roberts, L.J., II. The isoprostanes: current knowledge anddirections for future research. Biochem. Pharmacol. 51:1–9; 1996.

[34] Delanty, N.; Reilly, M.; Pratico, D.; FitzGerald, D. J.; Lawson, J. A.;FitzGerald, G. A. 8-Epi PGF2 alpha: specific analysis of an isoeicosanoidas an index of oxidant stress in vivo. Br. J. Clin. Pharmacol. 42:15–19;1996.

[35] Montuschi, P.; Barnes, P. J. Roberts, L.J., II. Isoprostanes: markers andmediators of oxidative stress. FASEB J. 18:1791–1800; 2004.

[36] Comporti, M.; Signorini, C.; Leoncini, S.; Buonocore, G.; Rossi, V.;Ciccoli, L. Plasma F2-isoprostanes are elevated in newborns and inverselycorrelated to gestational age. Free Radic. Biol. Med. 37:724–732; 2004.

[37] Evelyn, K. A.; Malloy, H. T. Microdetermination of oxyhemoglobin,methemoglobin and sulfhemoglobin in a single sample of blood. J. Biol.Chem. 126:655–662; 1938.

[38] Turrini, F.; Mannu, F.; Cappadoro, M.; Ulliers, D.; Giribaldi, G.; Arese, P.Binding of naturally occurring antibodies to oxidatively and nonoxida-tively modified erythrocyte band 3. Biochim. Biophys. Acta 1190:297–303; 1994.

[39] Dodge, T. G.; Mitchell, R. G.; Hanahan, D. J. The preparation andchemical characteristics of hemoglobin-free ghosts of human erythrocytes.Arch. Biochem. Biophys. 100:119–130; 1963.

[40] Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Proteinmeasurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275;1951.

[41] Laemmli, U. K. Cleavage of structural proteins during the assembly of thehead of the bacteriophage T4. Nature 227:680–685; 1970.

[42] Towbin, H.; Staehlin, R.; Fordon, J. Electrophoretic transfer of proteinsfrom polyacrylamide gels to nitrocellulose sheets: procedure and someapplications. Proc. Natl. Acad. Sci. USA 76:4350–4354; 1979.

[43] Kondo, M.; Itoh, S.; Kusaka, T.; Imai, T.; Isobe, K.; Onishi, S. The ability

915V. Rossi et al. / Free Radical Biology & Medicine 40 (2006) 907–915

of neonatal and maternal erythrocytes to produce reactive oxygen speciesin response to oxidative stress. Early Hum. Dev. 66:81–88; 2002.

[44] Nourooz-Zadeh, J.; Liu, E. H.; Yhlen, B.; Anggard, E. E.; Halliwell, B. F4-isoprostanes as specific marker of docosahexaenoic acid peroxidation inAlzheimer's disease. J. Neurochem. 72:734–740; 1999.

[45] Signorini, C.; Comporti, M.; Giorgi, G. Ion trap tandem massspectrometric determination of F2-isoprostanes. J. Mass Spectrom.38:1067–1074; 2003.

[46] Bradford, M. A rapid and sensitive method for the quantition of microgramquantities of protein utilizing the principle of dye-binding. Anal. Biochem.72:248–254; 1976.

[47] Kannan, R.; Labotka, R.; Low, P. S. Isolation and characterization of thehemichrome-stabilized membrane protein aggregates from sickle erythro-cytes. Major site of autologous antibody binding. J. Biol. Chem.263:13766–13773; 1988.

[48] Nourooz-Zadeh, J.; Gopaul, N. K.; Barrow, S.; Mallet, A. I.; Anggard,E. E. Analysis of F2-isoprostanes as indicators of non-enzymatic lipidperoxidation in vivo by gas chromatography-mass spectrometry:development of a solid-phase extraction procedure. J. Chromatogr. B.Biomed. Appl. 667:199–208; 1995.

[49] Singh, S.; Hider, R. C.; Porter, J. B. Separation and identification ofdesferrioxamine and its iron chelating metabolites by high-performanceliquid chromatography and fast atom bombardment mass spectrometry:choice of complexing agent and application to biological fluids. Anal.Biochem. 187:212–219; 1990.

[50] Kime, R.; Gibson, A.; Yong, W.; Hider, R.; Powers, H. Chromatographicmethod for the determination of non-transferrin-bound iron suitable for useon the plasma and bronchoalveolar lavage fluid of preterm babies. Clin.Sci. (Lond.) 91:633–638; 1996.

[51] Ciccoli, L.; Rossi, V.; Leoncini, S.; Signorini, C.; Blanco-Garcia, J.;Aldinucci, C.; Buonocore, G.; Comporti, M. Iron release, superoxideproduction and binding of autologous IgG to band 3 dimers in newbornand adult erythrocytes exposed to hypoxia and hypoxia-reoxygenation.Biochim. Biophys. Acta 1672:203–213; 2004.

[52] Ando, K.; Ogawa, K.; Misaki, S.; Kikugawa, K. Increased release of freeFe ions in human erythrocytes during aging in the circulation. Free Radic.Res. 36:1079–1084; 2002.

[53] Watkins, J. A.; Claster, S.; Caughey, W. S. Enhanced production of oxyradicals and peroxide by hemoglobins S and F. Fed. Proc. 45:1640;1986.

[54] Berger, H. M.; Moison, R. M.; Van Zoeren-Grobben, D.; Conneman, N.;Geerdink, J. Pro-oxidant effects of iron in the newborn period. In:Ziegler, E. E.; Lucas, A.; Moro, G. E., eds. Nutrition of the very low-birthweight infant. LippincottWilliamsWilkins,Philadelphia,pp.121–137;1999.

[55] Schubert, D.; Boss, K.; Dorst, H. J.; Flossdorf, J.; Pappert, G. Thenature of the stable noncovalent dimers of band 3 protein fromerythrocyte membranes in solutions of Triton X-100. FEBS Lett.163:81–84; 1983.

[56] Cuppoletti, J.; Goldinger, J.; Kang, B.; Jo, I.; Berenski, C.; Jung, C. Y.Anion carrier in the human erythrocyte exists as a dimer. J. Biol. Chem.260:15714–15717; 1985.

[57] Repka, T.; Shalev, O.; Reddy, R.; Yuan, J.; Abrahmov, A.; Rachmilewitz,E. A.; Low, P. S.; Hebbel, R. P. Nonrandom association of free iron withmembranes of sickle and β-thalassemic erythrocytes. Blood 82:3204–3210; 1993.

[58] Shalev, O.; Hebbel, R. P. Catalysis of soluble hemoglobin oxidation by freeiron on sickle red cell membranes. Blood 87:3948–3952; 1996.

[59] Browne, P.; Shalev, O.; Hebbel, R. B. The molecular pathobiology of cellmembrane iron: the sickle red cell as a model. Free Radic. Biol. Med.24:1040–1048; 1998.

[60] Lutz, H. U. Elimination of old erythrocytes from the circulation: exposureof a cell-age specific antigen on aging erythrocytes. Schweiz. Med.Wochenschr. 111:1507–1517; 1981.

[61] Turrini, F.; Arese, P.; Yuan, J.; Low, P. S. Clustering of integral membraneproteins of the human erythrocyte membrane stimulates autologous IgGbinding, complement deposition, and phagocytosis. J. Biol. Chem.266:23611–23617; 1991.