Developmental changes in NO bioavailability in fetal erythrocytes

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Developmental Changes in NO Bioavailability in Fetal Erythrocytes Harry Bard,* Ann M English,† Karine Bellemin,* and Carmen Gagnon* S-nitrosohemoglobin (HbSNO), where hemoglobin (Hb) is nitrosated at Cys␤93, presumably controls delivery of the vasorelaxant nitric oxide (NO) to hypoxic tissues in an oxygen-sensitive manner. Little is known about how Hb regulates NO bioavailability during fetal development. A study was planned to determine the levels of HbSNO and HbFeIINO (NO bound to FeII of heme) in the cord blood of newborn infants of different gestational ages and establish their relationship with the levels of fetal Hb (HbF). Blood samples were collected from umbilical cord obtained from normal newborns between 24 and 41 weeks of gestation. Determinations of HbSNO and HbFeIINO were performed using chemiluminescence. The proportion of HbF was determined by HPLC. There were 11 preterm (24-34 weeks of gestation) and 11 term infants (37-41 weeks of gestation). The levels of HbSNO varied from 0.37 to 1.72 ⴛ 10ⴚ5 mol/mol heme. There was a significant correlation with gestational age (r2 ⴝ 0.46, P ⴝ 0.0005) due to the effect of the decrease in the amount of HbF (r2 ⴝ 0.81, P < 0.0001). The relationship of HbFeIINO was not affected by gestational age or the level of HbF (mean 1.68 ⴞ 1.15 ⴛ 10ⴚ5 mol/mol heme). Under physiological in utero conditions, fetal erythrocytes have lower levels of HbSNO, which increase in the later stage of fetal development. The levels of HbSNO in the fetal red cell are dependent on the level of adult Hb (HbA). The low HbSNO levels at physiological fetal O2 saturations during early development could protect the fetal circulation from an excess release of NO and O2. © 2004 Elsevier Inc. All rights reserved. itric oxide (NO) produced by endothelial cells regulates vascular tone and diffuses into the bloodstream, where it reacts with molecules in the plasma and hemoglobin (Hb) in erythrocytes. NO is eliminated by its reaction with the iron-containing heme groups of oxyhemoglobin producing methemoglobin and nitrate. However, not all of the NO reacts with Hb in the same manner. It also reacts with the heme groups of deoxyhemoglobin to form iron-nitrosyl hemoglobin (HbFeIINO) and with the cysteine at position 93 of the ␤-chain of Hb to form S-nitrosohemoglobin (HbSNO). Stamler and his colleagues1 have demonstrated that HbSNO is an important source of bioactive NO and a crucial component of the cardiorespiratory cycle.


From the *Pediatric Department, University of Montreal, St. Justine’s Hospital; and †Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada. This work was supported by the Canadian Institutes of Health Research Grant MOP-49464. Address reprint requests to Dr. Harry Bard, Research Center, Hospital Sainte-Justine, 3175, Coˆte-Sainte-Catherine, Montreal, Quebec, H3T 1C5, Canada; e-mail: [email protected] © 2004 Elsevier Inc. All rights reserved. 0146-0005/04/2804-0000$30.00/0 doi:10.1053/j.semperi.2004.08.009


They proposed that HbSNO forms at high oxygen tensions, whereas at low oxygen saturations, NO is released and dilates blood vessels. It should be noted that Stamler’s concept is controversial. There are others who claim that basal vascular tone is regulated by on-site NO production as opposed to NO delivery from red blood cell (RBC) HbSNO.2 S-nitrosohemoglobin is capable of transferring NO to low molecular weight thiols, providing a route for delivery of bioactive NO equivalents from the erythrocyte to targets that affect smooth muscle relaxation. Notably, allosteric structural transitions of Hb, triggered by changes in oxygen tension in vivo, contribute to the molecular rearrangement that determine NO addition and release from Hb-Cys␤93. Snitrosation of Hb takes place preferably in the relaxed (R) structure of Hb, which prevails at high oxygen tension (ie, in lung), and release of NO from the resultant HbSNO occurs with the transition to the tense (T) structure at low oxygen tension (ie, in capillaries). The net effect is that when O2 is released from Hb in regions of low pO2, the shift in Hb to the T structure favors NO release. By means of transnitrosation, this

Seminars in Perinatology, Vol 28, No 4 (August), 2004: pp 312-316

RBC Nitric Oxide in the Fetus

system would allow NO bioactivity to be delivered to vascular smooth muscle as S-nitrosoglutathione (GSNO), a form that is protected from heme scavenging. Moreover, it would enable NO to be released preferentially where pO2 is lowest, dilating vessels and directing blood flow to the most ischemic tissues.3-5 Very little is known how fetal Hb (HbF) participates in the regulation of blood flow and the influx of oxygen into the fetal tissues by the transportation of the vasorelaxant NO. In the developing fetus, the placenta plays a role analogous to that of the lungs in the adult.6 Oxygen is transferred across the placenta to HbF (␣2␥2), which has the same ␣-chain as does HbA, but the ␤-chain is replaced by a ␥-chain that differs from the adult ␤-chain at 39 residues.7 Hb-␥ also contains a highly conserved Cys93 residue.7 As in other tissues, NO release plays an important role in determining vascular resistance in the human umbilical–placental circulation where it is a potent vasodilator contributing to maintenance of low basal tone and attenuating the action of vasoconstrictors such as endothelin and thromboxane.8 Nitric oxide may play a significant role in the fetus since evidence suggests that the human placental circulation is maximally dilated under normal circumstances.9 Consistent with a role for NO in fetal circulation, NO synthesis has been shown to occur in the placenta, and endothelial constitutive nitric oxide synthase (ecNOS) mRNA has been localized in syncytiotrophoblast.10 Positive ecNOS immunostaining was also found in stem villous blood vessels and in the endothelium of arteries and veins of the umbilical cord and chorionic plate.11 A distinct property of fetal arterial blood is its low pO2. The pO2 in the blood that carries oxygen from the placenta to the fetus via the umbilical vein is around 40 mm Hg, and fetal venous blood has a pO2 of 20 to 25 mm Hg.12 These pO2 values are much less then the normal 90 mm Hg found in adult arterial blood at sea level. The function of the allosteric transition (from R to T) of Hb during fetal life is unknown. Fetal arterial blood red cells even when well oxygenated may be only 70% saturated12 and 45 to 50% saturated during labor.13,14 The release of large amounts of NO from fetal blood cells in an untimely manner would be life-threatening to the fetus (eg, by causing hypotension). Thus,


there must be differences in the allosteric function of HbFSNO and HbASNO. To further the understanding of the function of HbSNO and HbFeIINO during fetal development, the RBC levels of HbSNO and HbFeIINO and their relationship to HbF and HbA in cord blood of newborn infants of different gestational ages were determined.

Method Blood samples were collected from umbilical cord obtained from normal (appropriate in weight for gestational age) newborns without medical or obstetrical complication delivering between 25 and 41 weeks of gestation. Twentytwo samples were obtained, 11 of which were from preterm (25-34 weeks) and 11 from term (37-41 weeks) infants. Determinations of HbSNO and HbFeIINO were performed essentially as described by Gladwin and coworkers.15 In brief, umbilical vein samples were drawn into prechilled EDTA collection tubes and centrifuged. The RBC pellet was washed three times in 10 mL of PBS-EDTA. Red cells were frozen and thawed, and the hemolysate was diluted 1:2 in 0.5 mmol/L EDTA. The dilute hemolysate was devided into two portions, one was treated with 100-fold molar excess of KCN/K3Fe(CN)6 which removes NO only from HbFeIINO. After 35 minutes of incubation, the treated Hb sample was passed through a Sephadex G-25 column NAP-10 (1.3 ⫻ 2.6-cm) (Amersham Pharmacia Biotech) and eluted with 0.5 mmol/L EDTA to remove nitrite, low molecular weight thiols and KCN/K3Fe(CN)6. The concentration of Hb in the Sephadex G-25 effluent was determined following conversion to cyanomethemoglobin, 540 ⫽ 11 per heme. The two Hb samples (200 ␮L) were injected into reaction mixtures containing 7 mL of glacial acetic acid, 2 mL of distilled water, and 50 mg of KI. A crystal of I2 was added to yield a concentration of 20 mmol/L. Reaction with I3⫺ formed in solution releases NO for chemiluminescence detection using a Sievers NO analyzer model 280i (Ionics, Boulder, CO). Standard curves were obtained using S-nitrosoglutathione (GSNO) (Cayman, Ann Arbor, MI). The background NO signal from wash water eluted the Sephadex G-25 column was subtracted from samples. The sample values were divided by the


Bard et al

Hb concentration and expressed as mol/mol heme. Total nitrosylated Hb concentrations were determined in samples that were not treated with KCN/K3Fe(CN)6. By subtracting HbSNO values from the total, HbFeIINO concentrations were obtained. The proportion of HbF was determined by HPLC with a gradient of 20 to 70% of acetonitrile/0.1%TFA.16 All procedures were approved by the Institutional Review Board of Hoˆpital Ste-Justine. Informed consent was obtained from the mothers of the infants included in the study.

Statistical Analysis Linear regression analysis was used to determined relationships between variables. Values of P ⱕ 0.05 were considered as statistically significant.

Figure 2. The levels of HbFeIINO in relation to gestational age in cord blood of newborn infants (mean 1.68 ⫾ 1.15 ⫻ 10⫺5 mol/mol heme).

Levels of HbSNO from 0.37 to 1.72 ⫻ 10⫺5 mol/mol heme in relation to gestational age are shown in Fig 1. There was a significant increase in the levels of HbSNO as a function of gestational age (P ⫽ 0.0005). In contrast, HbFeIINO was not affected by gestational age (mean of 1.68 ⫾ 1.15 ⫻ 10⫺5 mol/mol heme) (Fig 2). Fig 3 illustrates the distribution of the levels of HbF in relation to gestational age. There was minimal change in HbF level up to 34 weeks of

gestation, when the switchover from HbF to HbA takes effect.17 There is a significant inverse correlation between the levels of HbF and HbSNO (P ⬍ 0.0001; Fig 4A). To distinguish between the effects of % HbF versus gestational age on HbSNO levels, the study population was subdivided into two gestational age groups. One group contained preterm infants (ⱕ34 weeks of gestation) and the other group the term infants (37-41 weeks of gestation). The HbF levels varied between 91 and 95%, and 75 and 90% in preterm and term infants, respectively. There was no correlation between HbF and HbSNO in the preterm infants (Fig 4B, HbSNO ranged from 0.37 to 0.76 ⫻ 10⫺5 mol/mol heme with a mean of

Figure 1. The levels of HbSNO as mol/mol heme in cord blood of newborn infants in relation to gestational age. r2 ⫽ 0.4602, P ⫽ 0.0005.

Figure 3. The percentage of HbF of total hemoglobin in relation to gestational age in cord blood from newborn infants.


RBC Nitric Oxide in the Fetus


Discussion This study demonstrates the changes in HbSNO as well as HbFeIINO during human fetal development. The levels of HbSNO increase only when the switchover to HbA begins and the levels of HbA raise. This occurs in the later part of fetal development. One of the only studies that has compared the functional properties of blood with erythrocytes with predominantly HbF (newborn cord blood) with blood with erythrocytes containing HbA (adult blood) was performed by Calatayud and coworkers.18 They showed that in vitro relaxation of rat aortic rings by endogenous endothelium derived NO were more inhibited by fetal erythrocytes than adult erythrocytes. These results suggested that fetal erythrocytes have a higher NO scavenging effect than adult erythrocytes. The finding of Calatayud and coworkers18 can be considered in accordance with this report, where the lower level of NO found in fetal erythrocytes could allow for a greater amount of NO scavenging than adult erythrocytes. This study provides information that will contribute to our understanding of the role of NO on oxygen delivery in the fetus. The results can serve as a baseline for comparing NO physiology under normal and pathological prenatal conditions as well as during the postnatal adaptation of the fetal circulation.


Figure 4. (A) The relationship of HbSNO to the percent fetal hemoglobin (HbF) in the cord blood of all infants included in the study (r2 ⫽ 0.81, P ⬍ 0.0001). (B) The relationship of HbSNO to the percent HbF in the newborn preterm infants. There was no correlation between HbF and HbSNO in the preterm infants. The mean value was 0.56 ⫾ 0.13 ⫻ 10⫺5 mol/mol heme. (C) The relationship of HbSNO to the percent of HbF in the term newborn infants (37-41 weeks of gestation) (r2 ⫽ 0.886, P ⬍ 0.0001).

0.56 ⫾ 0.13 ⫻ 10⫺5 mol/mol heme), but a very significant correlation was found for the term infants (HbSNO increased from 0.53 to 1.72 ⫻ 10⫺5 mol/mol heme) (P ⬍ 0.0001; Fig 4C).

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born babies of various populations. Hemoglobin 15:349379, 1991 Myatt L, Brewer AS, Langdon G, et al: Attenuation of the vasoconstrictor effects of thromboxane and endothelin by nitric oxide in the human fetal-placental circulation. Am J Obstet Gynecol 166:224-230, 1992 Mak KK, Gude NM, Walters WA, et al: Effects of vasoactive autacoids on the human umbilical-fetal placental vasculature. Br J Obstet Gynaecol 91:99-106, 1984 Conrad KP, Vill M, McGuire PG, et al: Expression of nitric oxide synthase by syncytiotrophoblast in human placental villi. FASEB J 7:1269-1276, 1993 Myatt L, Brockman DE, Eis AL, et al: Immunohistochemical localization of nitric oxide synthase in the human placenta. Placenta 14:487-495, 1993 Nicolaides KH, Economides DL, Soothill PW: Blood gases, pH, and lactate in appropriate-and small-for-gestationalage fetuses. Am J Obstet Gynecol 161:996-1001, 1989 Goffinet F, Langer B, Carbonne B, et al: Multicenter study on the clinical values of fetal pulse oximetry. I.






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