**3.7.2 Erythrocyte membrane protein band 3**

The human erythrocyte has a lifespan of about 120 days, being removed from the circulation, mainly, by the spleen. Mature erythrocyte has no nuclei and organelles, and presents a very limited biosynthetic capacity, accumulating physical and/or chemical changes throughout its life span. Several modifications occur with cell aging, namely, reduction in cell volume, enzyme activity, antioxidant capacity and deformability.

The erythrocyte membrane protein band 3 is a transmembrane protein, also known as an anion channel, as it mediates the exchange of HCO-3 and Cl ions, which is important for the transport of CO2 from the tissues to the lungs. Band 3 links the cytoskeleton to the membrane lipid bilayer, participating in the maintenance of cell morphology. Band 3 is also involved in the removal of senescent/damaged erythrocytes (Wang, 1994).

The development of oxidative stress may occur when exogenous oxygen metabolites diffuse through the membrane or when oxygen metabolites result from autoxidation of hemoglobin, and the red blood cell (RBC) is unable to detoxify the cell, due to depletion in antioxidant defenses. Accumulation of oxygen metabolites can cause hemoglobin oxidation that has a high affinity for the cytoplasmic domain of band 3 (Fig. 5) (Low et al., 1985). This linkage causes band 3 oligomerization and/or aggregation (Waugh et al., 1987), which is recognized by natural autoantibodies anti-band 3 IgG (Fig. 5). The autoantibodies anti-band 3 have a higher affinity for Band 3 oligomers than for band 3 monomers (Lutz, 1992). The band 3 aggregates will act as a neoantigen on the erythrocyte membrane surface, marking the aged or injured erythrocyte for removal by macrophages of the reticulo-endothlial system. Thus, an increase in membranebound hemoglobin (MBH) (Santos-Silva et al., 1998) and in band 3 aggregation are good markers of erythrocyte senescence and/or damage.

Changes in band 3 profile (% of band 3 monomers, aggregates and proteolytic fragments), besides that associated to erythrocyte aging, were also reports in different inflammatory models, namely, in myocardial infarction (Santos-Silva et al., 1995), ischemic stroke (Santos-Silva et al., 2002) and in high competition physical exercise (Santos-Silva et al., 2001). In

Umbilical Cord Blood Changes in Neonates from a Preeclamptic Pregnancy 285

Fig. 6. Erythrocyte band 3 profile in maternal (M) blood and umbilical cord blood (UCB), in

As was observed in PEc mothers, their newborns had significantly higher MBH and evidences of an erythropoietic stimulation, with an increase in erythrocytes, in nucleated red blood cell (NRBC), reticulocyte count and RPI (Catarino et al., 2009b). This erythropoietic stimulation can occur in response to tissue hypoxia, due to the reduced placental perfusion, with lower oxygen transfer to fetal erythrocytes. Erythropoietic stimulation may also be triggered by tissue hypoxia resulting from increased erythrocyte removal, due to increased RBC injury and/or aging. There were no significant differences in the band 3 profile (Fig. 6) (Catarino et al., 2009b). As already mentioned, the increase of NRBC and reticulocytes, a younger erythroid population, prominent in the fetal circulation, may mask the injury observed in the mature erythrocyte population. The increase in MBH in PEc cases, shows an increase of oxidative stress in erythrocytes, as a result in

oxygen

PE is a hypertensive disorder of pregnancy that affects several organs and systems. Although important quantitative information exists regarding maternal blood modifications in PE, few studies have addressed the influence of this syndrome in newborns from PEc mothers. Moreover, many of the results available in the literature are controversial. The main changes that our group observed in biochemical and hematological umbilical cord blood of newborns from PEc pregnancy when compared with the newborn from normal

The placental dysfunction associated with hypoxia/reoxygenation of the placenta in PE, in the maternal circulation appears to trigger endothelial dysfunction (Fig. 7). To this dysfunction may contribute the hypertriglyceridemia, oxidative stress and the exacerbation of the inflammatory condition. Placental dysfunction, associated with the changes observed in maternal blood, seem to limit the transfer of oxygen and nutrients and, eventually, an abnormal release of products synthesized by the placenta to the fetal circulation; these changes seem to trigger a fetal response in order to adapt to this condition, with the development of endothelial dysfunction, dyslipidemia, and inflammatory response, similar to what occurs in the mother, although less intense. The reduction of oxygen transfer to the fetus associated with oxidative stress, determines oxidative damage in erythrocytes and an

normal pregnancy and preeclampsia (PE) (Adapted from Catarino et al., 2009b)

raised hemoglobin oxidation with subsequent membrane binding.

**4. Concluding remarks** 

pregnancy are described in table 3.

erythropoietic stimulation (Fig. 7).

vitro studies showed that neutrophil activation and elastase, a leukocyte activation product, trigger changes in band 3 profile that are similar to those found in inflammatory conditions and senescent erythrocyte (Santos-Silva et al., 1998). Senescent and/or damaged erythrocyte show an increase in band 3 aggregates and a decrease in band 3 monomers and fragments.

Fig. 5. Illustration of erythrocyte removal mechanism, mediated by band 3 aggregation.

Belo et al. (2002d) reported changes in band 3 profile, in pregnancy and in puerperium. In the first trimester, pregnant women, when compared with non-pregnant women, showed decreased aggregates of band 3 and increased total fragments. During pregnancy, an increase of total fragments was also observed, suggesting a rise in younger erythrocytes (Belo et al., 2002d).

The exacerbated inflammatory process of pregnancy (which can limit the absorption and mobilization of iron for erythropoiesis), the development of oxidative stress (triggering cumulative oxidative damages in erythrocytes) and the changes in placental perfusion (reduction of feto-maternal oxygen transfer and placental hypoxia), all observed in PE, seems to cause oxidative modifications in erythrocytes and disturbances in the erythropoietic process. In a study by our group, PEc mothers showed an increase in erythrocyte number, reticulocyte number and reticulocyte production index (RPI), reflecting an erythropoietic stimulus, which may be triggered by an accelerated removal of aged/damaged erythrocytes (Catarino et al., 2009b). In fact, in PEc women, an increase in MBH and changes in band 3 profile (with an increase in band 3 aggregates) were observed (Fig. 6) (Catarino et al., 2009b). The increase in bilirubin levels, suggests that these lesions led to the early removal of damaged erythrocytes. In fact, high MBH levels are consistent with increased oxidative stress and inflammation associated with PE. The band 3 profile, observed in PE, seems to reflect the existence of a heterogeneous erythrocyte population, ie, a senescent erythrocyte subpopulation (more band 3 aggregates) and a younger erythrocyte subpopulation (reticulocyte, less band 3 aggregates). This reflects a stimulation of the erythropoietic response that seems to mask the erythrocyte injury.

Fig. 6. Erythrocyte band 3 profile in maternal (M) blood and umbilical cord blood (UCB), in normal pregnancy and preeclampsia (PE) (Adapted from Catarino et al., 2009b)

As was observed in PEc mothers, their newborns had significantly higher MBH and evidences of an erythropoietic stimulation, with an increase in erythrocytes, in nucleated red blood cell (NRBC), reticulocyte count and RPI (Catarino et al., 2009b). This erythropoietic stimulation can occur in response to tissue hypoxia, due to the reduced placental perfusion, with lower oxygen transfer to fetal erythrocytes. Erythropoietic stimulation may also be triggered by tissue hypoxia resulting from increased erythrocyte removal, due to increased RBC injury and/or aging. There were no significant differences in the band 3 profile (Fig. 6) (Catarino et al., 2009b). As already mentioned, the increase of NRBC and reticulocytes, a younger erythroid population, prominent in the fetal circulation, may mask the injury observed in the mature erythrocyte population. The increase in MBH in PEc cases, shows an increase of oxidative stress in erythrocytes, as a result in raised hemoglobin oxidation with subsequent membrane binding.

#### **4. Concluding remarks**

284 From Preconception to Postpartum

vitro studies showed that neutrophil activation and elastase, a leukocyte activation product, trigger changes in band 3 profile that are similar to those found in inflammatory conditions and senescent erythrocyte (Santos-Silva et al., 1998). Senescent and/or damaged erythrocyte show an increase in band 3 aggregates and a decrease

Fig. 5. Illustration of erythrocyte removal mechanism, mediated by band 3 aggregation.

Belo et al. (2002d) reported changes in band 3 profile, in pregnancy and in puerperium. In the first trimester, pregnant women, when compared with non-pregnant women, showed decreased aggregates of band 3 and increased total fragments. During pregnancy, an increase of total fragments was also observed, suggesting a rise in younger

The exacerbated inflammatory process of pregnancy (which can limit the absorption and mobilization of iron for erythropoiesis), the development of oxidative stress (triggering cumulative oxidative damages in erythrocytes) and the changes in placental perfusion (reduction of feto-maternal oxygen transfer and placental hypoxia), all observed in PE, seems to cause oxidative modifications in erythrocytes and disturbances in the erythropoietic process. In a study by our group, PEc mothers showed an increase in erythrocyte number, reticulocyte number and reticulocyte production index (RPI), reflecting an erythropoietic stimulus, which may be triggered by an accelerated removal of aged/damaged erythrocytes (Catarino et al., 2009b). In fact, in PEc women, an increase in MBH and changes in band 3 profile (with an increase in band 3 aggregates) were observed (Fig. 6) (Catarino et al., 2009b). The increase in bilirubin levels, suggests that these lesions led to the early removal of damaged erythrocytes. In fact, high MBH levels are consistent with increased oxidative stress and inflammation associated with PE. The band 3 profile, observed in PE, seems to reflect the existence of a heterogeneous erythrocyte population, ie, a senescent erythrocyte subpopulation (more band 3 aggregates) and a younger erythrocyte subpopulation (reticulocyte, less band 3 aggregates). This reflects a stimulation of the erythropoietic response that seems to mask

in band 3 monomers and fragments.

erythrocytes (Belo et al., 2002d).

the erythrocyte injury.

PE is a hypertensive disorder of pregnancy that affects several organs and systems. Although important quantitative information exists regarding maternal blood modifications in PE, few studies have addressed the influence of this syndrome in newborns from PEc mothers. Moreover, many of the results available in the literature are controversial. The main changes that our group observed in biochemical and hematological umbilical cord blood of newborns from PEc pregnancy when compared with the newborn from normal pregnancy are described in table 3. already in

The placental dysfunction associated with hypoxia/reoxygenation of the placenta in PE, in the maternal circulation appears to trigger endothelial dysfunction (Fig. 7). To this dysfunction may contribute the hypertriglyceridemia, oxidative stress and the exacerbation of the inflammatory condition. Placental dysfunction, associated with the changes observed in maternal blood, seem to limit the transfer of oxygen and nutrients and, eventually, an abnormal release of products synthesized by the placenta to the fetal circulation; these changes seem to trigger a fetal response in order to adapt to this condition, with the development of endothelial dysfunction, dyslipidemia, and inflammatory response, similar to what occurs in the mother, although less intense. The reduction of oxygen transfer to the fetus associated with oxidative stress, determines oxidative damage in erythrocytes and an erythropoietic stimulation (Fig. 7).

Umbilical Cord Blood Changes in Neonates from a Preeclamptic Pregnancy 287

develop, at long term, cardiovascular diseases. Changes in the inflammatory response and endothelial dysfunction observed in umbilical cord blood of newborns from mothers with

It would be especially interesting to study these and other parameters associated with cardiovascular risk in children born from mothers who developed PE during pregnancy at

The authors are grateful to the nursery group of Obstetrics Service of Hospital S. João, in particular to nurse Célia Ribeiro for her generous help in the maternal and cord blood collection. This work was supported by FCT and FSE for PhD grant (SFRH/BD/7056/2001).

Aydin T., Varol F. & Sayin N. (2006). Third trimester maternal plasma total fibronectin levels

Baksu B., Davas I., Baksu A., Akyol A. & Gulbaba G. (2005a). Plasma nitric oxide,

Baksu B., Baksu A., Davas I., Akyol A. & Gulbaba G. (2005b) Lipoprotein(a) levels in

Bar J., Harell D., Bardin R., Pardo J., Chen R., Hod M. & Sullivan M. (2002). The elevated

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*Gynaecol Res*, Vol. 31, No. 3, pp. 277-282, ISSN 1341-8076

**5. Acknowledgements** 

ISSN 0020-7292

6, 588S–595S, ISSN 0731-5724

Vol. 21, No. 1, pp. 1-6, ISSN 0951-3590

Vol. 69, No. 3, pp. 145-51, ISSN 0902-4441

3848

4460

**6. References** 


PlGF, placental growth factor; sVEGFR-1, soluble vascular endothelial growth factor receptor type 1; VEGF, vascular endothelial growth factor; tPA, tissue plasminogen activator; PAI-1, plasminogen activator inhibitor; TG, triglycerides; HDLc, HDL cholesterol; LDLc, LDL cholesterol; Apo, apolipoprotein; CRP, c-reactive protein; sVCAM, soluble vascular cell adhesion molecule; MCV, mean cell volume; MCH, mean cell hemoglobin; NRBC, nucleated red blood cell; RPI, reticulocyte production index; MBH, membrane-bound hemoglobin ↑↑↑ - P<0.001; ↑↑ - P<0.01; ↑ - P<0.05

Table 3. Main changes observed in biochemical and hematological umbilical cord blood of newborns from PEc pregnancy when compared with the newborn in normal pregnancy.

Fig. 7. Schematic of some modifications observed in maternal and cord blood. IUGR, intrauterine growth restriction; SGA, small for gestational age.

In summary, most of the changes observed in the maternal circulation in PE women are also present in the cord blood of their newborns, although these changes are less pronounced.

PE shares several similarities with atherosclerosis, such as modifications in the lipid profile, amplification of the inflammatory process and increased oxidative stress. These changes may contribute to disturbe cell activation with subsequent endothelial dysfunction. Some studies have suggested that pregnant women who developed PE have a predisposition to develop, at long term, cardiovascular diseases. Changes in the inflammatory response and endothelial dysfunction observed in umbilical cord blood of newborns from mothers with PE, may also have an increased risk to develop cardiovascular diseases in the future.

It would be especially interesting to study these and other parameters associated with cardiovascular risk in children born from mothers who developed PE during pregnancy at different stages of its growth and also to study in women with a history of PE.
