Published online April 21, 2008
PEDIATRICS Vol. 121 No. 5 May 2008, pp. e1348-e1351 (doi:10.1542/10.1542/peds.2007-2215)
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ARTICLE

Erythroid Apoptosis in Idiopathic Neonatal Jaundice

Hasan Özkan, MDa, Hale Ören, MDb, Mansur Tatli, MDa, Halil Ates, MScc, Abdullah Kumral, MDa and Nuray Duman, MDa

a Departments of Pediatric Neonatology
b Pediatric Hematology
c Hematology Laboratory, Dokuz Eylül University Faculty of Medicine, Izmir, Turkey


   ABSTRACT
TOP
 ABSTRACT
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OBJECTIVES. The objectives of this study were to evaluate the contribution of erythroid apoptosis to neonatal idiopathic pathologic jaundice and to determine whether a measurement of the erythroid apoptosis value at birth could predict the development of hyperbilirubinemia during the first 15 days of life.

PATIENTS AND METHODS. Three groups were defined: group 1 (n = 101), healthy newborns whose erythroid apoptosis value and serum total bilirubin levels were detected from birth to day 15; group 2 (n = 24), newborns who were hospitalized for jaundice (serum total bilirubin level: >12.9 mg/dL) without any identifiable pathologic cause; and group 3 (control group, n = 24), healthy newborns whose serum total bilirubin levels were ≤12.9 mg/dL. Erythroid apoptosis value was assessed by flow cytometry using an annexin-V fluorescein isothiocyanate kit.

RESULTS. In group 1, there was no correlation between the erythroid apoptosis value and serum total bilirubin levels obtained at birth and at the fourth and 15th days of life; the erythrocyte apoptosis value obtained at birth was not significantly different between the neonates whose serum total bilirubin levels were >12.9 and ≤12.9 mg/dL and who had prolonged and nonprolonged jaundice during follow-up. The erythroid apoptosis value differed significantly between the newborns in groups 2 and 3. There was no significant correlation between the erythroid apoptosis value and serum total bilirubin levels of the infants in groups 2 and 3.

CONCLUSIONS. The erythroid apoptosis value obtained at birth could not predict the development of hyperbilirubinemia in neonates, but it was increased significantly in jaundiced neonates whose serum total bilirubin levels were >12.9 mg/dL. In these infants, increase in the erythroid apoptosis value may be a result of the toxic effect of bilirubin or of a protective mechanism of neonates to increase heme turnover and bilirubin production to diminish oxidative stress.

Key Words: apoptosis • erythrocyte • jaundice • hyperbilirubinemia • newborn

Abbreviations: STB—serum total bilirubin

Jaundice is one of the most common neonatal clinic conditions occurring in 60% to 70% of term infants and >80% of preterm infants.1–3 The mechanism of this bilirubinemia is multifactorial; increased production of bilirubin, deficiency of hepatic uptake, impaired conjugation of bilirubin, and increased enterohepatic circulation of bilirubin account for most cases of pathologic jaundice in newborn infants during the first week of life.4,5 In newborns, there is an increase in the production of bilirubin mainly because of a shortened red blood cell life span combined with an increased red blood cell mass. Eighty to 90% of bilirubin is derived from the breakdown of hemoglobin from senescent or hemolyzed red blood cells, and the proportion of bilirubin arising from hemoglobin may increase significantly in the circumstance of hemolysis or ineffective erythropoiesis.5,6 Some neonates may develop jaundice although they have no high-risk situations, such as congenital hemolytic anemia or ABO blood group heterospecificity.

Neonatal hyperbilirubinemia condition requires urgent clinical attention because of the increased risk of kernicterus. Because many newborns are discharged early from the hospital after birth, and jaundice may develop in most of them, screening the newborns for their risk of developing significant hyperbilirubinemia is important before hospital discharge.7 The objectives of our study were to evaluate the contribution of erythroid apoptosis to neonatal jaundice in infants with no identifiable pathologic cause of hyperbilirubinemia and to determine if a measurement of erythroid apoptosis value obtained from cord blood of healthy newborns at birth could predict the development of hyperbilirubinemia during the first 15 days of life.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
This study was performed prospectively in the Dokuz Eylül University Hospital Departments of Neonatology and Pediatric Hematology. Study protocol was approved by the Dokuz Eylül University Faculty of Medicine Human Clinical Investigation Committee, and informed consent was obtained from the parents of the infants. Newborns were eligible for the study if they were healthy and their gestational age was ≥37 weeks. Exclusion criteria were newborns having any perinatal complications, infectious disease, congenital or acquired hemolytic anemia, hypoxia, respiratory distress, intrauterine growth retardation, direct hyperbilirubinemia, or congenital anomalies.

In patient classification, 3 groups were defined: group 1, healthy newborns whose erythroid apoptosis value and serum total bilirubin (STB) levels were detected from birth to day 15; group 2 (n = 24), newborns who were hospitalized for jaundice (STB level >12.9 mg/dL) without any identifiable pathologic cause; and group 3 (control group, n = 24), healthy newborns whose STB levels were ≤12.9 mg/dL.

Of the 107 newborns who were enrolled in the first group, 101 completed the study. Six neonates were excluded from the study because of inadequate follow-up. Cord blood samples of these infants were obtained at birth to analyze erythroid apoptosis value, STB level, hematocrit measurement, blood type, direct Coomb's test, and peripheral blood smear. These newborns were called back to the hospital for a control and blood sampling at days 4 and 15; their physical features were noted, and blood samples were obtained to analyze erythroid apoptosis value, STB level, and hematocrit measurement. Some of them were jaundiced, and some had to be hospitalized for hyperbilirubinemia to receive phototherapy. In infants who needed to be hospitalized for jaundice, additional blood samples had to be obtained to analyze whole blood count, peripheral blood smear, direct Coomb's test, and glucose-6-phosphate-dehydrogenase enzyme level. Jaundice was considered to be prolonged if the STB level was >10 mg/dL on the 15th day of life.

Twenty-four infants, who were 3 to 15 days old and admitted to our hospital for jaundice were enrolled in the second group. In these newborns, previously reported well-known risk factors, such as polycythemia, infection, hematoma, hypothyroidism, Rh immunization, ABO blood group heterospecificity, or red cell enzyme and membrane defects were excluded.5 Blood sample for erythroid apoptosis value was obtained when blood was drawn from the infant for other tests necessary for evaluating the cause of jaundice.

Twenty-four newborns were enrolled in the third group (control group). This group was a convenience sample of infants of similar age (3–15 days old) who required admission to our neonatology unit for health control or other reasons, such as phenylketonuria screening and mild jaundice.

In all of infants, 2 mL of blood was drawn into tubes containing EDTA and sent immediately to a laboratory to assess erythroid apoptosis value. Annexin V labeled with fluorescein isothiocyanate-conjugated apoptosis kit (BioSource International, Camarillo, CA) was used for cell staining.8–11 Propidium iodide was used to distinguish apoptotic cells from necrotic cells.9–11 The cells were analyzed by flow cytometry (Cell Quest Analysis Software, Becton Dickinson, San Jose, CA) within an hour of staining. The percentage of early apoptotic erythrocytes (annexin V fluorescein isothiocyanate-conjugated positive and propidium iodide negative) was evaluated and noted for each infant as his or her erythroid apoptosis value. These procedures were performed by the same investigator during the study.

Statistical analyses were performed by using SPSS for Windows Release Program (SPSS Inc, Chicago, IL). All of the values are given as the mean ± SD. Student's t test was used to compare mean values. Mann-Whitney U test was used to compare nonparametric values. Pearson correlation test and Spearman correlation test were used for correlation analysis of parametric and nonparametric values. {chi}2 test was conducted to determine the differences between the 2 groups. P values <.05 were accepted as statistically significant.


   RESULTS
TOP
 ABSTRACT
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The mean gestational age and weight of neonates in group 1 were 39.2 ± 0.7 weeks and 3444 ± 439 g, respectively. Their mean hematocrit value was 47.7%. Mean STB levels of cord blood, fourth, and 15th days were 1.5 ± 0.8, 7.8 ± 4.3, and 4.4 ± 3.1 mg/dL, respectively. Mean cord blood erythroid apoptosis value was 0.45% ± 0.43%. Table 1 gives the erythroid apoptosis values of these neonates according to their gender and characteristics of hyperbilirubinemia. There was no correlation between the erythroid apoptosis value and STB levels obtained from cord blood at birth and from venous blood at the fourth and 15th days of life (r = 0.06, P = .54; r = 0.14, P = .83; and r = –0.31, P = .76, respectively). There was a negative correlation between erythroid apoptosis value and hematocrits obtained at birth and at the fourth day of life (r = –0.23; P = .02). In group 1, the cord blood erythrocyte apoptosis value was not statistically significantly different between the neonates whose STB levels were >12.9 mg/dL and ≤12.9 mg/dL and who had prolonged and nonprolonged jaundice during follow-up (Table 1).


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  		TABLE 1 Erythroid Apoptosis Value of the Jaundiced Neonates in Group 1 According to Gender and to the Characteristics of Hyperbilirubinemia

 
Of the 24 jaundiced infants in group 2, 15 (62.5%) were boys and 9 (37.5%) were girls. In group 3 (control group), 10 (41.7%) were boys and 14 (58.3%) were girls. Gestational week, birth weight, STB levels, hematocrit measurements, age at blood sampling, and erythroid apoptosis value of these infants are given in Table 2. Erythroid apoptosis value and hematocrit measurements differed significantly between the newborns in group 2 and the newborns in group 3 (P = .001 and P = .03, respectively). There was no significant correlation between the erythroid apoptosis value and STB levels of the infants in groups 2 and 3 (r = 0.176, P = .412; and r = 0.276, P = .191, respectively).


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  		TABLE 2 Gestational Age, Birth Weight, Age at Blood Sampling, Hematocrit, STB, and Erythroid Apoptosis Value of the Neonates in Group 2 and Group 3

 

   DISCUSSION
TOP
 ABSTRACT
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Neonates may develop significant hyperbilirubinemia after hospital discharge and to decrease the risk of kern ikterus, clinical risk factor assessment and/or bilirubin measurement before discharge have been recommended.12 Predischarge bilirubin measurement gives more accurate results and wider risk stratification than a clinical risk factor score.7 To our knowledge, this is the first time that the contribution of erythroid apoptosis to neonatal hyperbilirubinemia has been evaluated in jaundiced infants with no identifiable cause. In our study, we showed that erythroid apoptosis value obtained from cord blood of neonates at birth can not predict the development of hyperbilirubinemia during the first 2 weeks of life, but we demonstrated that erythroid apoptosis value differed significantly in jaundiced neonates whose STB levels were >12.9 mg/dL. It may be because of toxic effects of bilirubin to erythrocytes, because bilirubin promotes an accelerated aging of human erythrocytes by inducing loss of membrane lipids.4,13–15 However, we cannot explain the increase in erythroid apoptosis value only with bilirubin toxicity, because we couldn't find a correlation between the STB levels and erythroid apoptosis value, and the hematocrit levels of the neonates in group 2 were slightly higher than the neonates in group 3. In the absence of evident hemolysis features, the relationship between bilirubin production and increased erythrocyte apoptosis may not be the major reason.

Bilirubin is generally regarded as a toxic compound when accumulated at abnormal concentrations.16 Recently, some authors have suggested that unconjugated bilirubin is physiologically useful and can act as an antioxidant.17–21 During the oxidant stress, oxidants such as nitric oxide play an important role in the pathogenesis of human diseases, especially in the neonatal period.22–24 Neonates have limited antioxidant protective capacity against the circulating free radicals, and bilirubin is a potent antioxidant cytoprotectant.17–20,25 Increased oxidative stress in neonates may trigger hyperbilirubinemia, and high serum bilirubin level may protect the cells from oxidative damage.19,25,26 On the other hand, bilirubin is the final product of heme catabolism, and heme oxygenase seems to be an important molecule with roles in oxidative stress, inflammation, immunity, and cellular regulation and signaling.27 Heme oxygenase 1 promoter region contains an antioxidant response element, and induction of heme oxygenase was reported as a general response to oxidant stress.28,29 Carbon monoxide produced through the heme oxygenase reaction leads directly to an increase in carbon monoxide bound to circulating hemoglobin and creating carboxy hemoglobin. It is a sensitive index of heme and bilirubin production.30,31 This mechanism may also affect the rate of erythrocyte apoptosis and increase bilirubin production in neonates to protect them against oxidative stress.

This study demonstrated that the erythroid apoptosis value obtained from cord blood of healthy neonates at birth cannot predict the development of hyperbilirubinemia during the first 2 weeks of life, but it supported that the erythroid apoptosis value differed significantly in jaundiced neonates whose STB levels were >12.9 mg/dL. In these infants, the increase in erythroid apoptosis value may be because of a toxic effect of bilirubin or may be because of a protective mechanism of neonates to increase heme turnover and bilirubin production to diminish oxidative stress.


   FOOTNOTES
 
Accepted Nov 8, 2007.

Address correspondence to Hale Ören, MD, Dokuz Eylül University Faculty of Medicine, Department of Pediatric Hematology, 35340 Balcova, Izmir, Turkey. E-mail: hale.oren{at}deu.edu.tr

The authors have indicated they have no financial relationships relevant to this article to disclose.


What's Known on This Subject

Erythroid apoptosis is a cause of hyperbilirubinemia, and hyperbilirubinemia may cause erythroid apoptosis. Also, a protective mechanism of neonates may increase erythroid apoptosis to increase heme turnover and bilirubin production to diminish oxidative stress.

 

What This Study Adds

This is the first time that the contribution of erythroid apoptosis to neonatal hyperbilirubinemia has been evaluated in neonates and in jaundiced infants who have no identifiable cause of hyperbilirubinemia.

 


   REFERENCES
TOP
 ABSTRACT
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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PEDIATRICS (ISSN 1098-4275). ©2008 by the American Academy of Pediatrics

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