Oxidation of hemoglobin F is associated with the aging process of neonatal red blood cells.

Previously, we reported that in cord blood there is a population of very dense, surface area-depleted red blood cells (RBC). We hypothesized that oxidative damage might account for the generation of this cell population because Hb F is known to be mildly unstable in vitro. Accordingly, we examined density-separated subpopulations of neonatal red cells searching for evidence of oxidant injury to Hb in vivo. Cord or adult RBC were separated into populations of varying density and an increased amount of membrane-associated globin was found in the densest fraction of cord RBC. Solubilized ghosts from each fraction were analyzed by thiol-disulfide exchange chromatography for the presence of oxidized Hb and spectrophotometrically for the presence of membrane-bound hemichrome. About four times more oxidized Hb was found in unseparated cord RBC than in adult RBC. This difference was most evident in the densest 10-15% RBC subfractions. Membrane-bound hemichrome levels in cord cells were twice those found in adult cells. We found that in cord membrane skeletons there was 2.5 to 9 times as much globin in the dense fraction as compared to the light fraction. Membrane skeletons from dense and light adult RBC differed little from one another. We postulate that membrane (and perhaps membrane skeleton)-associated oxidized Hb is a marker for more generalized oxidative damage, which may create the population of unusually dense cells found in cord blood and ultimately shorten their life span.     

The aging process of human neonatal erythrocytes

The red blood cell population of the human newborn is more heterogeneous than that of adults. To characterize this heterogeneity, red cells were separated on Stractan gradients into populations according to their density and, therefore, generally according to their age. Lipid content, phospholipid asymmetry, osmotic fragility, and deformability were measured. With increasing density, osmotic resistance and deformability decreased to a greater extent in the neonatal than the adult red cell. There was a marked loss of surface area throughout the life span of the neonatal red cell which was not accompanied by a parallel loss of membrane lipid. This apparent discrepancy could be explained at least in part by internalization of membrane lipid as a result of endocytosis, a process shown to be present in even the densest neonatal red cells.     

Cellular dehydration and immunoglobulin binding in senescent neonatal erythrocytes

The life span of neonatal erythrocytes (60-80 days) is shorter than that of adult erythrocytes (120 days). We studied neonatal red blood cells separated on stractan density gradients to further characterize the aging process and to explore the possibility that senescence antigens play a role in the destruction of neonatal erythrocytes. Quantitation of membrane proteins 4.1a and 4.1b served as a marker for cell age and confirmed an enrichment for senescent red cells in the most dense layers of the gradients. Despite the shorter life span of neonatal erythrocytes, cord blood contained a larger percentage of very dense, K+-depleted red cells than did adult blood. ATP levels in dense neonatal and adult cells were decreased to 50-80% of normal values for unseparated red cells. Levels of reduced glutathione did not fall with increasing cell density. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of red cell membrane proteins showed increased membrane-associated globin in senescent neonatal cells, but such gels run without reducing agents did not show oxidative protein cross-linking. Membrane bound immunoglobulins were detected on senescent neonatal and adult red cells by the rosetting antiglobulin test. We conclude that senescence antigens are revealed during the aging process of neonatal erythrocytes, thereby labeling them for antibody-mediated destruction in the reticuloendothelial system.     

Rheological properties of blood and their possible role in the circulation and development of intracranial hemorrhage in preterm infants

Blood viscosity and other rheological properties of blood have important effects on blood circulation. In preterm infants, blood viscosity (at given hematocrit), plasma viscosity and red cell aggregation are decreased compared to term neonates and adults. In addition, the Fahraeus-Lindqvist effect (i.e., decrease in blood viscosity with decreasing vessel diameter) is more pronounced in preterm infants. This suggests that blood flow through arteries, arterioles and veins is facilitated in preterm infants. The large neonatal red cells are less filterable and less able to enter small capillaries than adult red cells. This suggests that the passage of neonatal red cells through narrow capillaries and splenic pores is impaired. The high risk of intracranial hemorrhage in small preterm infants may in part result from poor red cell aggregation, impeded passage of their large red cells through narrow cerebral capillaries and raised blood viscosity due to rapid transfusion of adult blood.     

Differences in adhesion receptor expression between immature and older platelets and red blood cells of neonates and adults

PURPOSE: To examine the hypothesis that reticulated platelets and reticulocytes show elevated adhesion receptor expression compared with mature cells in both adult and neonatal cells. METHODS: Flow cytometry was used to examine laminin, fibronectin (VLA-6), and thrombospondin (glycoprotein IV [GPIV]) expression in reticulated red cells, reticulated platelets, and older peripherally circulating mature red cells and mature platelets in seven newborn cord blood samples and blood samples from eight adult volunteers. RESULTS: The difference in the neonatal reticulated platelet percentage of 9.2+/-14.8% was not statistically significant from the adult reticulated platelet percentage of 5.0+/-1.5% in this small population. There was a statistically significant difference between the reticulated cord blood red cell mean of 7.7+/-1.8% and the adult mean of 3.1+/-0.43%. Mean expression of VLA-6 was 96% in adult reticulated platelets, 79% in adult mature platelets, 81% in cord reticulated platelets and 65% in cord mature platelets. Mean expression of GPIV was similar, with corresponding values of 90%, 71%, 78%, and 57%. Reticulated red cells in adults averaged 44% VLA-4 and 46% GPIV; cord reticulocytes were 9% and 15%, respectively. CONCLUSIONS: Reticulated cells newly released from the bone marrow express more adhesive receptors than mature cells in both groups. Cord blood samples showed hypoexpression of both receptor types in red blood cells and platelets.     

Shorter PFA-100® closure times in neonates than in adults: role of red cells, white cells, platelets and von Willebrand factor

In neonates, despite poor platelet function in various in vitro tests, closure times (CTs) in PFA-100^® measurements are shorter than in adults. Neonates have a higher polymeric von Willebrand factor (vWF). They also have a higher haematocrit and higher white blood cell count than adults, which may interfere with the evaluation of platelet and vWF function by means of the PFA-100 in neonates. To assess the role of different blood constituents on neonatal CTs, red blood cell, platelet and white blood cell counts in cord blood were modified. These modifications did not provide any evidence that the difference in number between adult and neonatal blood cells was responsible for shorter neonatal CTs. In further experiments, platelets and/or vWF were inhibited by means of abciximab and anti-vWF antibody, and mixing experiments with neonatal platelet-rich and platelet-poor plasma were performed. The results showed that short cord blood PFA-100 CTs were caused by a constituent of neonatal platelet-poor plasma, probably the neonatal high multimeric vWF. Conclusion: This study demonstrates that CTs in neonates are dependent on the same components, platelets and vWF, as in adults, making it likely that the PFA-100 can be used in neonates in the same way as in adults to investigate platelet and vWF function.     

Increased neonatal platelet deposition on subendothelium under flow conditions: the role of plasma von Willebrand factor

In vitro platelet function of umbilical cord blood and neonatal peripheral vein blood from full-term newborns was compared with that of adults. Citrated whole blood was subjected to shear stress (1300 s(-1)) on subendothelial extracellular matrix (ECM)-coated wells in a cone and plate(let) analyzer. Adhered platelets on the ECM were quantitated by image analyzer. Both umbilical cord and neonatal peripheral blood platelets demonstrated more extensive adhesion than adult platelets, and similar aggregate formation on ECM. The ability of neonatal platelets to form aggregates on ECM was confirmed by scanning electron microscopy. Similar activation of neonatal and adult platelets after subjection to shear stress, in the suspension phase, was established by flow cytometry, which showed an increase in fibrinogen binding and a decrease in glycoprotein Ib expression on platelet membrane. The difference in adhesion rates between neonatal and adult platelets was preserved even when the hematocrit level of the neonatal blood was adjusted to that of adults. Reconstitution of neonatal or adult platelet-rich plasma with autologous or heterologous red packed cells yielded no change in adhesion and aggregation. When von Willebrand factor-covered plates were used to prevent deposition of plasma von Willebrand factor on the surface, no difference in platelet adhesion was seen between neonatal and adult blood. In conventional aggregometry assay, the response to ristocetin of washed platelets of either neonatal or adult source was higher on addition of plasma from neonates than from adults. Our data suggest that the extensive neonatal platelet deposition on ECM is mediated by plasma von Willebrand factor, which is known to be more multimerized and, therefore, more active in neonates than in adults. This mechanism may provide balanced primary hemostasis in neonates despite the platelet hyporeactivity to agonists without application of shear stress.     

The effect of hyperoxic exposure on antioxidant enzyme activities of alveolar type II cells in neonatal and adult rats.

Neonatal animals of several species are more tolerant of hyperoxic exposure than are adults, but the mechanisms of increased neonatal tolerance are unknown, as are the cell types, if any, that contribute to oxygen resistance. We studied the effect of in vivo exposure to 85% oxygen for 72 h on the activities of the antioxidant enzymes, glutathione peroxidase, catalase and superoxide dismutase (SOD), in alveolar type II cells and whole lung from adult and neonatal rats. Baseline antioxidant enzyme activities were generally lower in neonatal type II cells compared with adults. Baseline enzyme activities did not differ in neonatal type II cells and lung homogenates except for lower catalase activity in type II cells. Hyperoxic exposure resulted in 35-38% increases in antioxidant enzyme activities in neonatal whole lung. In neonatal type II cells, SOD activity increased by 170% after hyperoxia, whereas catalase and glutathione peroxidase were not significantly changed. In the adult whole lung, hyperoxic exposure resulted in increases in only glutathione peroxidase activity, whereas in adult type II cells there was a significant decrease in SOD activity after O2 exposure. Therefore, although baseline antioxidant enzyme activities were not higher in neonatal type II cells compared with whole lung, there were differences in the antioxidant enzyme responses of adult and neonatal type II cells to hyperoxia, particularly with respect to SOD. The ability of the neonatal type II cell to respond to hyperoxia with an early increase in SOD activity may contribute to the enhanced oxygen tolerance of the neonate.     

In vivo lability of red cell phosphofructokinase in term infants: the possible molecular basis of the relative phosphofructokinase deficiency in neonatal red cells.

Cord blood erythrocytes from nine term infants were separated by density gradient centrifugation into cohorts of intact cells of progressively increasing density and compared with red cells treated in a similar manner from four healthy adults. Pyruvate kinase (PK), an age-dependent enzyme, progressively decreased in activity from the lightest to the heaviest fractions, in both neonatal and adult red cells, indicating that red cells from newborn infants exhibit the same relationship between red cell age and density that had previously been demonstrated in red cells from adults. The rate of decline of red cell PK activity was essentially the same in neonates and adults, whereas phosphofructokinase (PFK) activity in cord erythrocytes decreased at a significantly faster rate when compared to adults. These data suggest that PFK has an accelerated rate of in vivo decay in neonatal red cells and is an unstable enzyme in the newborn.     

Circulatory changes in newborn lambs with experimental polycythemia: comparison between fetal and adult type blood

Hemodynamic effects of increased hematocrit were compared in two groups of newborn lambs. In the first group (fetal type blood), exchange transfusions were carried out using packed red blood cells obtained from newborn lambs within one to two hours after birth. In the second group (adult type blood), the same procedure was carried out using adult sheep blood. In both groups, hematocrit values ranging between 70% and 80% were reached. The increase in hematocrit caused a decrease in cardiac output due to an increase in peripheral resistance. Pulmonary resistance increased more than systemic resistance. However, the increase in pulmonary resistance was significantly greater in the polycythemic newborn lambs with adult blood. A right-to-left shunt through a patent ductus or a foramen ovale was noted in six of the eight lambs included in this group. On the other hand, none of the seven polycythemic newborn lambs with fetal blood developed signs of right-to-left shunting. It is concluded that during neonatal polycythemia, the level of hematocrit is not the sole factor responsible for the hemodynamic changes observed. Other unknown influences related either to the red cells or the plasma must impinge upon the pulmonary circulation to alter vascular resistance.     

Herpes simplex virus adsorption to and survival within placental lymphoid populations

Neonatal herpes simplex virus (HSV) infections are frequently characterized by widespread dissemination and high morbidity and mortality. To assess the potential role of the lymphocyte in permitting or promoting dissemination of HSV, we studied lymphoid populations of placental and adult blood with respect to HSV survival. Adsorption of HSV to the lymphocyte surface did not differ between placental and adult cells. Placental blood permitted greater survival of HSV than adult lymphocytes. T-cell-enriched lymphocytes appeared to be slightly more permissive than T-cell-depleted lymphocytes, but these differences were not statistically significant and apparently less than adult-placental blood differences. These findings lend support to the hypothesis that intracellular changes common to various neonatal lymphoid populations may permit or promote dissemination of HSV in the neonate.     

Neocytopheresis: a new approach for the transfusion of patients with thalassaemia major

At present the treatment of thalassaemia major consists of regular blood transfusions coupled with chelation therapy using deferoxamine. A complementary approach to the problem is the use of blood units enriched with young red cells (neocytes), which reduce the transfusional frequency and thereby diminish the risk of iron overload. Young red cell units were collected from blood from 60 volunteer donors using a cell separator (IBM 2997). Donors' blood was anticoagulated and the young red cell harvesting carried out over 4 h at a constant rotor speed of 500 rpm. Three biological criteria were used to evaluate young red cell quality: the number of reticulocytes, the pyruvate kinase activity and the mean corpuscular volume, all of which show an enrichment of young red cells as compared to standard donor units. The ⁵¹Cr young red cell survival in four normal donors and in two splenectomized patients showed an increased red cell half-life compared to the same study performed with standard blood units. Blood consumption was diminished significantly when the two patients were transfused with young red cell units. It must be emphasized that, despite the high cost of this blood product, the efficiency of this transfusion technique, by reducing blood consumption, represents important progress and a hopeful treatment for chronic anaemia.Abbreviations Hb haemoglobin - MCV mean corpuscular volume - PK pyruvate kinase - TQ transfusion quotient - R reticulocytes - ratio reticulocytes/haemoglobin     

Effect of vitamin K on neonatal erythrocytes.

This study investigated the possible oxidative effect of vitamin K3 (menadione) and Vitamin K1 (Konakion) on neonatal erythrocytes by controlled in vitro exposure. Menadione caused only mild morphological changes and did not decrease ATP levels. However, it oxidized intracellular hemoglobin to methemoglobin in neonatal cells more than in adult cells. Reduced glutathione contents were higher in neonatal cells, but less available for antioxidant protection. Konakion did not increase methemoglobin levels in newborn infants after a prophylactic injection. In vitro exposure to Konakion did not affect reduced glutathione and ATP levels, nor did it oxidize hemoglobin. However, extensive morphological changes were observed, attributed to the effect of its solvent. Therefore, it seems that menadione, which is no longer administered to newborns, causes oxidative stress in neonatal cells whereas Konakion, the current vitamin K1, does not, either in in vitro studies or by therapeutic administration.     

Transient neonatal hemolytic anemia due to the novel gamma globin gene mutation HBG2:C.290T>C,p.Leu97Pro (hemoglobin Wareham)

Unstable gamma globin variants can cause transient neonatal hemolytic anemia. We have identified a novel variant in a newborn who presented with jaundice and anemia requiring phototherapy and red blood cell transfusion. The patient was found to be heterozygous for the mutation HGB2:c.290T>C, p.Leu97Pro, which we have termed hemoglobin (Hb) Wareham. This substitution is expected to generate an unstable hemoglobin with increased oxygen affinity based on the homologous mutation previously described in the beta globin gene, which is termed as Hb Debrousse. The patient fully recovered by 9 months of age as expected with the transition from fetal to adult hemoglobin.     

Increased wave latency in auditory brainstem response to anemia in newborn and adult sheep

The objective of the present study was to determine whether phlebotomy-induced anemia in newborn lambs and adult sheep results in prolonged auditory brainstem response (ABR) wave latencies, and if found, whether developmental differences exist between the two groups. Among newborn lambs, hemoglobin (Hb) levels were reduced from 100-120 to 30-50 g/l over a 3-day period. Over the next 2 days, serial red blood cell transfusions were administered to restore Hb to pre-phlebotomy levels. In adult sheep, comparable levels of phlebotomy-induced anemia were achieved within a 24-hour period, after which Hb levels returned to pre-phlebotomy levels. During the induction of anemia and its reversal, a significant (p < 0.05) increase in ABR latency followed by a return to pre-anemia ABR latencies that was most prominent in the peripheral auditory pathway was observed in both newborn and adult sheep as Hb levels fell below 50 g/l (p < 0.05). There was no difference between adults and newborns in ABR latency relative to the Hb level at which ABR wave latencies increased. We speculate that ABR wave latency prolongation might serve as an indicator of the need for red blood cell transfusion.     

Preeclampsia and Na,K-ATPase activity of red blood cell ghosts from neonatal and maternal blood

We determined Na,K-ATPase activity and cholesterol/phospholipid ratio of maternal and cord red blood cell ghosts from either normotensive or preeclamptic pregnant women. The Na,K-ATPase activity of the red cell ghosts from neonatal blood is significantly lower (25-32%) as compared with the ATPase activity of the maternal red cell ghosts, regardless of the presence or not of preeclampsia. This diminution in Na,K-ATPase activity of the neonatal red blood cell ghosts could be due to an increase in the cholesterol/phospholipid molar ratio of the membrane. The Na,K-ATPase activity of the red blood cell ghosts from pregnant women was unaffected by preeclampsia; however, fetal red blood cell ghosts from infants of preeclamptic mothers showed a significantly lower ATPase activity (20%) than fetal red blood cell ghosts from infants of normotensive mothers. A low Na,K-ATPase activity in the neonatal red blood cells from mothers with preeclampsia could be an indication of an important modification of the physiological role of this enzyme.     

Red cell oxygen affinity, hemoglobin type, 2,3-diphosphoglycerate, and pH as a function of fetal development.

Studies were carried out on fresh cord blood obtained at delivery from nonstressed normal fetuses ranging from 24 to 42 weeks of gestation, to determine the relationship of 2,3-diphosphoglycerate (DPG), the intracellular red cell and extracellular pH, and the proportions of adult and fetal hemoglobin in regulating the position of fetal red cell oxygen affinity in utero. There was a significant positive correlation between P50 and gestational age (r = .62, P less than .001), the linear regression increased from 17.8 to 22.5 mm Hg. There was also a significant positive correlation between P50 and the percentage of adult type hemoglobin (HbA) (r = .67, P less than .001). In contrast gestational age had no effect of 2,3-DPG levels, the mean and SD was 14.86 +/- 2.04 mol/gm of Hb or delta pH between plasma and red cell, the mean was 0.187 +/- SD 0.032. However, there was a significant negative correlation between the intraerythrocyte hydrogen ion concentration and DPG level (r = .5, P less than .025). It is concluded therefore that the decrease in fetal oxygen affinity as gestation progresses is related mainly to the increase in the amount of HbA and the levels of DPG or delta pH between plasma and red cells are not a function of gestational age.     

PLASMA AND RED BLOOD CELL FOLATE IN BREASTFED INFANTS

Abstract. We have studied growth, red blood cell status and folate concentrations in plasma and red cells in a group of 35 breastfed infants during the first year of life. Folic acid supplementation was not given to the mothers during pregnancy or lactation, and none of them developed megaloblastic anaemia. The growth and red blood cell status of the infants were both normal. At birth, and throughout the period of observation, the folate concentrations in plasma and red cells were significantly higher than in the adult reference material. A positive correlation between plasma and red cell folate was demonstrated. During the latter part of pregnancy and lactation the foetuses and infants seem to be protected against folate deficiency. We regard the folate status of normal breastfed infants as optimal. The optimal supply of the vitamin in artificial nutrition should be the amount of folate necessary to maintain plasma and red cell folate concentrations similar to those found in breastfed infants.     

Bupivacaine alters red blood cell properties: a possible explanation for neonatal jaundice associated with maternal anesthesia

Cord blood was incubated with lidocaine, mepivacaine, bupivacaine, or buffer and red blood cell filterability was determined. Only bupivacaine at either 1 or 2 micrograms/ml prolonged filterability by an average of 58 to 65% over red cells treated with buffer alone. Tritiated bupivacaine was bound to a greater extent to red cell ghosts from cord blood (24.6 +/- 5.8%) than to adult red cell ghosts (14.6 +/- 2.6%). Finally, we determined red cell survival in 13-day-old rats injected with bupivacaine or buffer. At 2 h after injection, buffer-treated animals had a red cell survival of 96.9 +/- 3.3%, whereas 2-h survival was reduced to 82.6 +/- 8.7% for the animals injected with bupivacaine. Our results suggest that the neonatal jaundice associated with maternal anesthesia, especially bupivacaine, may be related to the observations that these agents cross the placenta, bind to the red cell membrane and reduce its filterability, resulting in shortened red cell survival.     

Membrane lipid fluidity and filterability of red blood cells from adults and newborns

Membrane lipid fluidity was reexamined in red blood cells and ghosts from adults and newborns. Fluorescence anisotropies of the hydrophobic probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and perylene were significantly and substantially greater in fresh intact red cells from newborns than from adults; however, no significant difference was detected with the polar fluorophores, 12-(9-anthroyl) stearic acid and retinol. These results suggest that probes in the hydrophobic core of the membrane have less motional freedom in red cells from newborns than from adults, whereas probe motional freedom in the polar lipid headgroup regions of the membranes is similar for both cell types. DPH fluorescence anisotropy increased upon making white ghosts or upon storage of blood. Temperature studies indicated that DPH fluorescence anisotropy in fresh intact neonatal red cells is increased by an amount corresponding to that produced by cooling adult red cells by 22 degrees C. Elevated intracellular calcium decreased red cell filterability without affecting DPH fluorescence anisotropy of ghost membranes. This result suggests that the effect of calcium in reducing filterability is independent of alterations in membrane lipid motional freedom. It is unlikely that the decreased lipid motional freedom of red cells from newborns contributes significantly to their decreased filterability.