PEDIATRICS Vol. 108 No. 4 October 2001, pp. 956-959

Onset of Jaundice in Glucose-6-Phosphate Dehydrogenase-Deficient Neonates

Michael Kaplan, MB ChB^{*, §}, Nurit Algur, MSc, and Cathy Hammerman, MD^{*, §}

From the ^* Department of Neonatology and  Clinical Biochemistry Laboratory, Shaare Zedek Medical Center; and ^§ the Faculty of Medicine of the Hebrew University, Jerusalem, Israel.

	ABSTRACT

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Objective.  We asked whether neonatal jaundice associated with glucose-6-phosphate dehydrogenase (G-6-PD) deficiency commences either in utero or in the immediate postnatal period and whether this perinatal bilirubinemia is the precursor of the subsequent neonatal jaundice and hyperbilirubinemia.

Methods.  Mandatory serum total bilirubin (STB) determinations were performed within 3 hours of birth, to reflect the in utero state (first STB), and on the third day of life (second STB), with additional determinations as clinically necessary, on healthy, term male neonates at high risk for G-6-PD deficiency. G-6-PD Mediterranean mutation was determined by molecular means. G-6-PD-deficient neonates were compared with control participants. The relationship of first STB values to second STB and subsequent hyperbilirubinemia (defined as STB 256 µmol/L [15.0 mg/dL]) was determined.

Results.  Both first and second STB values were significantly higher in the G-6-PD-deficient neonates (n = 52) than in control participants (n = 166; 50 ± 12 µmol/L vs 44 ± 10 µmol/L [2.9 ± 0.7 mg/dL vs 2.6 ± 0.6 mg/dL] and 174 ± 52 µmol/L vs 152 ± 52 µmol/L [10.2 ± 3.1 mg/dL vs 8.9 ± 3.0 mg/dL] for the first and second STB values, respectively). The rate of rise between these 2 points was greater in the G-6-PD-deficient neonates (2.6 ± 0.9 µmol/L/h vs 2.2 ± 0.9 µmol/L/h [0.15 ± 0.05 mg/dL/h vs 0.13 ± 0.05 mg/dL/h). Sixteen (30.8%) of the G-6-PD-deficient neonates developed hyperbilirubinemia compared with 10 (6%) of control participants (relative risk: 5.11; 95% confidence interval: 2.47-10.56). In both G-6-PD-deficient and normal populations, first STB values correlated significantly with both second STB values and with those who subsequently developed hyperbilirubinemia. Significantly more G-6-PD-deficient neonates with a first STB value greater than or equal to the mean developed hyperbilirubinemia compared with those with first STB less than the mean: 13 of 28 neonates versus 3 of 24 (relative risk: 3.7; 95% confidence interval: 1.20-11.51). This difference did not reach statistical significance in the control group.

Conclusions.  Higher first STB values, an increased risk of hyperbilirubinemia in G-6-PD-deficient neonates with first STB value greater than or equal to the mean, and significant correlation between first STB values and second STB values and hyperbilirubinemia suggest that jaundice in G-6-PD-deficient neonates commences in the immediate perinatal period, most likely in utero.  Key words:  bilirubin, jaundice, hyperbilirubinemia, glucose-6-phosphate dehydrogenase deficiency, perinatal, in utero.

An association between umbilical cord blood bilirubin levels, reflecting the in utero state, and serum total bilirubin (STB) values in early neonatal life has been recognized in some neonates for many years.¹ This concept has been used reliably in the prediction of hyperbilirubinemia attributable to Rh isoimmunization.² Although the success in identifying infants who are at risk for subsequent development of hyperbilirubinemia has not been universal,^3,4 in some series of ABO incompatibility^5-7 and also in nonhemolytic conditions,^8-11 use of umbilical cord blood STB determination has shown some success in the prediction of severe jaundice. These observations suggest that neonatal jaundice frequentlybut not consistentlyhas its origins in utero.

Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency is a commonly occurring enzyme defect that is associated with a high incidence of severe neonatal hyperbilirubinemia with the potential of irreversible bilirubin encephalopathy if not treated in time.¹² The pathogenesis of this hyperbilirubinemia is different from that in G-6-PD-normal neonates: decreased bilirubin conjugation, the result of an interaction between G-6-PD deficiency and promoter polymorphism of the gene that controls the bilirubin conjugating enzyme UDP glucuronosyltransferase, is a crucial factor.^13,14 To elucidate further the pathogenesis of the associated bilirubinemia, we therefore asked whether the jaundice associated with the G-6-PD Mediterranean mutation, as in some other conditions, commences in the perinatal period and whether this in utero or very early bilirubinemia is the precursor of subsequent jaundice or hyperbilirubinemia in these neonates.

	METHODS

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Study Protocol

The study was approved by the Institutional Review Board of the Shaare Zedek Medical Center. A cohort of consecutively born healthy boys who were born at 37 weeks' gestation at the Shaare Zedek Medical Center to Sephardic Jewish mothers whose families originated in Asia Minor were studied. This subgroup of the Israeli population has been shown to have an exceptionally high incidence of G-6-PD deficiency.^15,16 Blood was drawn for mandatory STB determinations within the first 3 hours after birth, to reflect the in utero status (first STB), and again at the time of routine metabolic screening on the third day of life (second STB). Simultaneously with 1 of these determinations, blood was collected for DNA extraction.

Routine medical care for these neonates included screening for G-6-PD deficiency on the first day of life, blood group determination, and direct Coombs' testing for infants born to Rh-negative or O blood group mothers. Infants were monitored visually as inpatients by our medical and nursing staff for the development of jaundice with additional STB determinations if warranted clinically. Those with a second STB value 50th percentile for hour of life¹⁷ and therefore at high risk for subsequent hyperbilirubinemia were scheduled to be followed as outpatients, whereas those with lower predischarge STB values, at low risk for hyperbilirubinemia, were evaluated at well-infant clinics or by family pediatricians or ritual circumcisers (mohel) and referred for evaluation if deemed necessary. Finally, any parent who had any doubt as to their infant's jaundice status was able to return for an STB at any time during the first week of life. The patient compliance in our population was excellent, and we are confident that we were aware of virtually all infants with an STB 256 µmol/L (15.0 mg/dL). These neonates were followed by us until stabilization of the STB values. When phototherapy became necessary after discharge, the infants were readmitted to our unit. Phototherapy was commenced in G-6-PD-deficient newborns when STB values exceeded 15.0 mg/dL. Breastfeeding was encouraged, although mothers were warned of the dangers of eating fava beans or taking drugs known to be triggers of hemolysis in G-6-PD-deficient individuals while nursing. Infants with any other condition that was likely to exacerbate hyperbilirubinemia, such as cephalhematoma, direct Coombs'-positive isoimmunization, maternal diabetes, sepsis, or Down's syndrome, were excluded from the study.

Laboratory Methods

DNA was extracted from peripheral blood leukocytes using a high-salt extraction procedure.¹⁸ For G-6-PD genotyping, DNA was shipped to The Scripps Research Institute (La Jolla, CA) for molecular classification. Polymerase chain reaction followed by allele-specific oligonucleotide hybridization was used to determine the presence or absence of nt 563, the nucleotide mutated in G-6-PD Mediterranean.¹⁹ Details of the procedure have been published elsewhere.¹³

STB values were determined by reflectance spectrophotometry using an Ektachem analyzer (Vitros 700c/750XRC Chemistry System; Johnson and Johnson Clinical Diagnostics, Rochester, NY). Blood group determinations and direct Coombs' testing were performed by routine laboratory techniques.

Data Analysis

The G-6-PD genotype was used to classify the infants into G-6-PD-deficient hemizygote (study) and normal hemizygote (control) groups. Hyperbilirubinemia was defined as a serum total bilirubin 256 µmol/L (15.0 mg/dL) in the first week of life, for standardization with our previous studies.^13,17 Results were compared using Student t test, ² analysis, or linear correlation, as appropriate. Significance of these tests was determined as P < .05. Evaluation of the effect of either G-6-PD deficiency or first STB greater than or equal to the mean value on the subsequent development of hyperbilirubinemia was determined by calculating the relative risk and 95% confidence intervals (CI). Significance in these cases was defined as a 95% CI range that did not include the digit 1. Rate of rise of STB was calculated as the difference between the first and second STB values divided by the number of hours between these tests.

	RESULTS

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A total of 225 infants were enrolled in the study. Seven (6 Coombs' positive, 1 maternal diabetes) were recognized not to meet study criteria after enrollment. Thus, the cohort that met study criteria comprised 218 infants, 52 of whom were hemizygotes for G-6-PD Mediterranean 563T and 166 of whom did not have this mutation. Demographic data for these infants are summarized in Table 1. Despite that significantly fewer G-6-PD-deficient neonates were nursed (Table 1), 16 (30.8%) of the G-6-PD-deficient neonates developed hyperbilirubinemia, compared with 10 (6%) of the control participants (relative risk: 5.11; 95% CI: 2.47-10.56; P < .0001).

Results of the first and second STB tests (mean ± SD) and the age at sampling are summarized in Table 2. Despite the similarity in times of sampling between study and control groups, both first and second STB values and the rate of rise between these 2 determinations were significantly higher in the G-6-PD-deficient neonates than in the control group.

In both G-6-PD-deficient and control groups, first STB values correlated with second STB values (r = 0.6, P < .0001 and r = 0.5, P < .0001 for the G-6-PD-deficient and control infants, respectively), with those who developed serum bilirubin values 256 µmol/L (15.0 mg/dL; r = 0.34, P = .01 and r = 0.21, P = .01, respectively), and with the rate of rise (r = 0.56, P < .0001 and r = 0.39, P < .0001, respectively).

In the G-6-PD-deficient cohort, a significantly greater number of neonates among those with a first STB value greater than or equal to the mean developed hyperbilirubinemia compared with those with a first STB less than the mean: 13 (46%) of 28 neonates versus 3 (12.5%) of 24 neonates (relative risk: 3.7; 95% CI: 1.20-11.51; P = .02). This difference did not reach statistical significance in the control group: 9 (9.6%) of 94 versus 1 (1.4%) of 72, respectively (relative risk: 6.89; 95% CI: 0.89-53.18; P = .06).

	DISCUSSION

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Some studies of umbilical cord blood bilirubin values have shown that both nonhemolytic jaundice^8-11 and hemolysis attributable to Rh isoimmunization² or ABO incompatibility^5-7 commence in utero. In these conditions, the cord blood bilirubin values correlate with subsequent hyperbilirubinemia and have been used to predict its severity. To understand further the pathophysiology of G-6-PD deficiency-associated neonatal jaundice, we studied its onset to determine whether it commences perinatally, ie, either during fetal life or in the immediate postnatal period, as in some hemolytic conditions noted above, or well into the neonatal period, as with other nonhemolytic bilirubinemias. In the current study, already immediately after birth, most likely reflecting the in utero status, the G-6-PD-deficient neonates had significantly higher serum bilirubin values than control participants. Significantly higher STB values were evident again on the third day. The rate of rise of STB and the incidence of hyperbilirubinemia were similarly increased in the G-6-PD-deficient neonates compared with control participants.

Higher umbilical cord blood STB levels^20,21 or increased STB values on the first day of life^22,23 have been shown in previous studies of G-6-PD-deficient neonates, suggesting either in utero or a very early postnatal onset of jaundice. However, as there was no attempt at correlation between this early bilirubinemia and subsequent jaundice in these studies, it cannot be concluded that the former was the forerunner of hyperbilirubinemia. Our demonstration of a significant correlation between very early STB values and later bilirubin values and the significantly higher relative risk of first STB result greater than or equal to the mean in the subsequent development of hyperbilirubinemia now demonstrates that this perinatal bilirubinemia is the precursor of subsequent jaundice and hyperbilirubinemia.

Although umbilical cord blood sampling undoubtedly would have offered a more accurate representation of the in utero status, for logistical reasons and because of difficulties in coordinating the study with the delivery room staff, we chose to obtain samples from the infants within 3 hours of delivery to reflect the in utero situation. Although we cannot exclude categorically a very early postnatal onset of the bilirubinemia, we are confident that this method of sampling reliably reflected the in utero state. Presuming that the hourly rates of rise during the first days of life reflected the rise during the first 3 hours of life as well, the first STB values should have been only marginally elevated over the actual cord blood values and should not have affected the comparisons between the study and control groups to any major degree. The slightly higher but clinically insignificant mean first STB values for the control groups than those reported by others for cord blood samples may represent a combination of the timing of the sample and interlaboratory variation between our laboratory's STB determination and those that have been used by other laboratories in the past.²⁴

Although the current and previous studies showed that umbilical cord or first-day STB determinations²⁵ may have some predictive value for the subsequent development of hyperbilirubinemia, the aim of this study primarily was to shed light on the pathophysiology of G-6-PD deficiency-associated neonatal jaundice. It was not designed as a study of prediction of subsequent hyperbilirubinemia and should not be interpreted as such. Accurate prediction can be accomplished by predischarge STB testing at the time of metabolic screening, as recently described both for normal²⁶ and G-6-PD-deficient¹⁷ newborn populations.

G-6-PD deficiency is estimated to affect hundreds of millions of people not only in areas in which the condition is indigenous but also with a potential for serious complications in North America.²⁷ Awareness of the condition and its dangers and an understanding of the differing pathophysiology of the associated jaundice compared with that of G-6-PD-normal individuals are essential if the potential of bilirubin encephalopathy is to be limited.

	ACKNOWLEDGMENTS

This study was supported at Shaare Zedek Medical Center by grants for neonatal jaundice research from The Golden Charitable Trust, London, United Kingdom, and the Mirsky Research Fund.

We thank Ernest Beutler, MD, for the genotype analysis of G-6-PD Mediterranean mutation and Chana Amsalem for technical assistance.

	FOOTNOTES

Received for publication May 17, 2000; accepted Feb 7, 2001.

Presented in part at the Pediatric Academic Societies-Society for Pediatric Research Annual Meeting, San Francisco, CA, May 1-4, 1999.

Reprint requests to (M.K.) Department of Neonatology, Shaare Zedek Medical Center, Box 3235, Jerusalem 91031, Israel. E-mail: kaplan{at}cc.huji.ac.il

	ABBREVIATIONS

STB, serum total bilirubin; G-6-PD, glucose-6-phosphate dehydrogenase; CI, confidence interval.

	REFERENCES

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1. Davidson LT, Merrit KK, Weech AA Hyperbilirubinemia in the newborn. Am J Dis Child 1941; 61:958-980
2. Allen FH, Diamond LK. Erythroblastosis Fetalis. Boston, MA: Little, Brown; 1958
3. Haque KN Value of measuring cord blood bilirubin concentration in ABO incompatibility. Br Med J 1978; 2:1604-1605
4. Jacobson M, Bernstein H Limited diagnostic value of routine cord blood bilirubin determinations. Clin Pediatr 1982; 21:610-612
5. Risemberg HM, Mazzi E, MacDonald MG, Peralta M, Heldrich F Correlation of cord bilirubin levels with hyperbilirubinemia in ABO incompatibility. Arch Dis Child 1977; 57:219-222
6. Desjardins L, Blajchman MA, Chintu C, Gent M, Zippursky A The spectrum of ABO hemolytic disease of the newborn infant. J Pediatr 1979; 95:447-449 [CrossRef][Medline]
7. Peevy KJ, Wiseman HJ ABO hemolytic disease of the newborn: evaluation of management and identification of racial and antigenic factors. Pediatrics 1978; 61:475-478 [Abstract]
8. Knudsen A Prediction of the development of neonatal jaundice by increased umbilical cord blood bilirubin. Acta Paediatr Scand 1989; 78:217-221 [Medline]
9. Knudsen A, Lebech M Maternal bilirubin, cord bilirubin, and placenta function at delivery and the development of jaundice in mature newborns. Acta Obstet Gynecol Scand 1989; 68:719-724 [Medline]
10. Rosenfeld JA Umbilical cord bilirubin levels as a predictor of subsequent hyperbilirubinemia. J Fam Pract 1986; 23:556-558 [Medline]
11. Law L-K, Pang C-P, Cheung K-L, Fok T-F Cord blood biochemistry and idiopathic neonatal jaundice. Clin Chem 1996; 42:1716-1717 [Free Full Text]
12. Beutler E G6PD deficiency. Blood 1994; 84:3613-3636 [Free Full Text]
13. Kaplan M, Renbaum P, Levy-Lahad E, Hammerman C, Lahad A, Beutler E Gilbert syndrome and glucose-6-phosphate dehydrogenase deficiency: a dose dependent genetic interaction crucial to neonatal hyperbilirubinemia. Proc Natl Acad Sci U S A 1997; 94:12128-12132 [Abstract/Free Full Text]
14. Kaplan M, Hammerman C Severe neonatal hyperbilirubinemia: a potential complication of glucose-6-phosphate dehydrogenase deficiency. Clin Perinatol 1998; 25:575-590 [Medline]
15. Sheba C, Szeinberg A, Ramot B, Adam A, Ashkenazi I Epidemiologic surveys of deleterious genes in different population groups in Israel. Am J Public Health 1962; 52:1101-1105
16. Kaplan M, Hammerman C, Kvit R, Rudensky B, Abramov A Neonatal screening for glucose-6-phosphate dehydrogenase deficiency: sex distribution. Arch Dis Child 1994; 71:F59-F60
17. Kaplan M, Hammerman C, Feldman R, Brisk R Predischarge bilirubin screening in glucose-6-phosphate dehydrogenase-deficient neonates. Pediatrics 2000; 105:533-537 [Abstract/Free Full Text]
18. Miller SA, Dykes DD, Polesky HF A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16:1215 [Free Full Text]
19. Vives-Corrons JL, Kuhl W, Pujades MA, Beutler E Molecular genetics of G6PD Mediterranean variant and description of a new G6PD mutant, G6PD Andalus^1361A. Am J Hum Genet 1990; 47:575-579 [Medline]
20. Valaes T, Karaklis A, Stravrakakis D, Bavela-Stravrakakis K, Perakis A, Doxiadis SA Incidence and mechanism of neonatal jaundice related to glucose-6-phosphate dehydrogenase deficiency. Pediatr Res 1969; 3:448-458
21. Brown WR, Boon WH Hyperbilirubinemia and kernicterus in glucose-6-phosphate dehydrogenase deficient infants in Singapore. Pediatrics 1968; 41:1055-1062 [Abstract/Free Full Text]
22. Lu T-C, Wei H, Blackwell RQ Increased incidence of severe hyperbilirubinemia among newborn Chinese infants with G-6-PD deficiency. Pediatrics 1966; 37:994-999 [Abstract/Free Full Text]
23. Tan KL Glucose-6-phosphate dehydrogenase status and neonatal jaundice. Arch Dis Child 1981; 56:874-877 [Abstract]
24. Vreman HJ, Verter J, Stevenson DK, Interlaboratory variability of bilirubin measurements. Clin Chem 1996; 42:869-873 [Abstract/Free Full Text]
25. Alpay F, Sarici SU, Tosuncuk D, Serdar MA, Inanc N, Gokcay E. The value of first-day bilirubin measurement in predicting the development of significant hyperbilirubinemia in healthy term newborns. Pediatrics. 2000;106(2). Available at: http://www.pediatrics.org/cgi.content/full/2000/106/e2
26. Bhutani VK, Johnson L, Sivieri EM Predictive ability of a predischarge hour-specific serum bilirubin for subsequent significant hyperbilirubinemia in healthy term and near-term newborns. Pediatrics 1999; 103:6-14 [Abstract/Free Full Text]
27. Kaplan M, Hammerman C Glucose-6-phosphate dehydrogenase-deficient neonates: a potential cause for concern in North America. Pediatrics 2000; 106:1478-1479 [Free Full Text]