Bilirubin Conjugation, Reflected by Conjugated Bilirubin Fractions, in Glucose-6-Phosphate Dehydrogenase-deficient Neonates: A Determining Factor in the Pathogenesis of Hyperbilirubinemia

Background and Objective.Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency is frequently associated with neonatal hyperbilirubinemia, which in severe cases may cause kernicterus and death. Because G-6-PD-deficient individuals frequently undergo acute, trigger-induced hemolytic episodes, increased hemolysis has frequently been implied in the pathogenesis of this neonatal hyperbilirubinemia. However, in Sephardic Jewish G-6-PD-deficient neonates, the rate of hemolysis, reflected by blood carboxyhemoglobin values corrected for inspired carbon monoxide, has been shown to be elevated, not only in those who developed hyperbilirubinemia, but also, to a similar extent, in those who remained only moderately jaundiced. Because at any point, serum total bilirubin values reflect a balance between bilirubin production on the one hand and bilirubin conjugation and elimination on the other, we suspected bilirubin conjugation to be a key factor in the pathogenesis of the hyperbilirubinemia. Physiologically, a fraction of conjugated bilirubin refluxes from the hepatocyte to the serum, and accurate determination of serum conjugated bilirubin fractions can be used to mirror intrahepatocytic bilirubin. Using this principle, we previously demonstrated a decreased diconjugated bilirubin fraction in hyperbilirubinemic G-6-PD-deficient neonates compared with hyperbilirubinemic G-6-PD-normal controls, suggesting diminished bilirubin conjugation. This conjugated bilirubin pattern probably reflects the recently described interaction between G-6-PD deficiency and the variant promoter for the gene encoding the bilirubin conjugating enzyme UDP glucuronosyltransferase, as seen in Gilbert's syndrome. Therefore, we postulated that efficiency of bilirubin conjugation is a crucial factor in the development of hyperbilirubinemia in G-6-PD-deficient neonates. We hypothesized that those G-6-PD-deficient neonates who develop hyperbilirubinemia would have decreased bilirubin conjugation ability, whereas those with a more efficient conjugating system would have a lesser degree of bilirubinemia.Methods.Term, healthy, male, G-6-PD-deficient neonates with no other obvious predisposing cause for hyperbilirubinemia were selected at random when their serum diazo total bilirubin values ranged from 171 to 254 μmol/L (10–14.9 mg/dL). At this point, simultaneous with the diazo bilirubin determination, serum was collected and frozen for high-performance liquid chromatography (HPLC) measurement of serum bilirubin fractions. The infants were followed clinically and with serum diazo bilirubin determinations until they either did not exceed a serum diazo bilirubin value of 254 μmol/L (14.9 mg/dL) (nonhyperbilirubinemic) or until bilirubin values rose above this level (hyperbilirubinemic), by a process of self-selection. A method of alkaline methanolysis, followed by reverse-phase HPLC, was used to measure unconjugated bilirubin and the mono- and diconjugated fractions of serum conjugated bilirubin. Total HPLC bilirubin and total conjugated bilirubin values were calculated from these measured bilirubin fractions. Patients also were classified according to the serum total conjugated bilirubin value as low bilirubin conjugators (serum total conjugated bilirubin less than median) or as high bilirubin conjugators (serum total conjugated bilirubin greater than median). The data were analyzed by comparing serum conjugated bilirubin fractions between the hyperbilirubinemic and nonhyperbilirubinemic groups and the risk of developing hyperbilirubinemia in the low bilirubin conjugators, relative to that of the high bilirubin conjugators.Results.Neonates were sampled at 53 ± 12 and 58 ± 12 hours for the subsequently hyperbilirubinemic and nonhyperbilirubinemic groups, respectively (NS). Initial (ie, at the time of sampling) serum total diazo bilirubin values (mean ± SD) were almost identical for the subsequently hyperbilirubinemic and nonhyperbilirubinemic groups (214 ± 27 μmol/L [12.5 ± 1.6 mg/dL] vs 212 ± 19 μmol/L [12.4 ± 1.1 mg/dL], respectively, NS), as were the HPLC-determined serum total bilirubin values (162 ± 32 μmol/[9.5 ± 1.9 mg/dL] vs 160 ± 16 μmol/L [9.4 ± 0.9 mg/dL, respectively, NS). However, despite similarity in the simultaneously drawn serum diazo bilirubin values, HPLC-determined conjugated bilirubin fractions (mean [range]), measured from the same serum samples, were lower in those infants who ultimately became hyperbilirubinemic than in those who remained nonhyperbilirubinemic: total conjugated bilirubin 0.82 (0–2.07) μmol/L vs 1.24 (0.6–11.0) μmol/L,; monoconjugated bilirubin 0.80 (0–2.07) μmol/L vs 1.24 (0.60–1.23) μmol/L; and diconjugated bilirubin 0.00 (0.00–0.42) μmol/L vs 0.11 (0–1.78) μmol/L. The diconjugated bilirubin fraction was especially affected; 18 (69%) neonates in the subsequently hyperbilirubinemic group had no detectable diconjugate compared with 8 (36%) in the nonhyperbilirubinemic neonates. Conversely, more of those neonates with serum total conjugated bilirubin fraction less than the median value of 1.06 μmol/L (0.06 mg/dL) developed hyperbilirubinemia than those with greater than the median (17/24 [71%] vs 9/24 [37.5%]), respectively (relative risk: 1.89; 95% confidence interval: 1.06–3.36).Conclusions.Although serum total bilirubin levels at the time of sampling were virtually identical, those neonates who subsequently developed hyperbilirubinemia had significantly lower serum conjugated bilirubin fractions than those who remained within the nonhyperbilirubinemic range. The diconjugated bilirubin fraction was especially affected. Those infants with serum total conjugated bilirubin fraction less than the median had a greater risk of developing hyperbilirubinemia than those with greater than the median. These findings reflect inefficient bilirubin conjugation ability in G-6-PD-deficient neonates who develop hyperbilirubinemia. We believe that the current findings are the functional manifestation of the interaction previously reported between G-6-PD deficiency and the variant gene promoter, as seen in Gilbert's syndrome. Diminished bilirubin conjugation ability appears to be a determining factor in the pathogenesis of G-6-PD deficiency associated neonatal hyperbilirubinemia.