BILIRUBIN CONJUGATION, REFLECTED BY CONJUGATED BILIRUBIN FRACTIONS, IN GLUCOSE-6-PHOSPHATE DEHYDROGENASE-DEFICIENT NEONATES - A DETERMININGFACTOR IN THE PATHOGENESIS OF HYPERBILIRUBINEMIA

Background and Objective. Glucose-6-phosphate dehydrogenase (G-6-PD) d eficiency is frequently associated with neonatal hyperbilirubinemia, w hich in severe cases may cause kernicterus and death. Because G-6-PD-d eficient individuals frequently undergo acute, trigger-induced hemolyt ic episodes, increased hemolysis has frequently been implied in the pa thogenesis 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 hyperb ilirubinemia, but also, to a similar extent, in those who remained onl y moderately jaundiced: Because at any point, serum total bilirubin va lues reflect a balance between bilirubin production on the one hand an d bilirubin conjugation and elimination on the other, we suspected bil irubin conjugation to be a key factor in the pathogenesis of the hyper bilirubinemia. Physiologically, a fraction of conjugated bilirubin ref luxes from the hepatocyte to the serum, and accurate determination of serum conjugated bilirubin fractions can be used to mirror intrahepato cytic bilirubin. Using this principle, we previously demonstrated a de creased diconjugated bilirubin fraction in hyperbilirubinemic G-6-PD-d eficient neonates compared with hyperbilirubinemic G-6-PD-normal contr ols, suggesting diminished bilirubin conjugation. This conjugated bili rubin pattern probably reflects the recently described interaction bet ween 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 bil irubin conjugation is a crucial factor in the development of hyperbili rubinemia in G-6-PD-deficient neonates. We hypothesized that those G-6 -PD-deficient neonates who develop hyperbilirubinemia would have decre ased bilirubin conjugation ability, whereas those with a more efficien t conjugating system would have a lesser degree of bilirubinemia. Meth ods. Term, healthy, male, G-6-PD-deficient neonates with no other obvi ous predisposing cause for hyperbilirubinemia were selected at random when their serum diazo total bilirubin values ranged from 171 to 254 m u mol/L (10 -14.9 mg/dL). At this point, simultaneous with the diazo b ilirubin determination, serum was collected and frozen for high-perfor mance liquid chromatography (HPLC) measurement of serum bilirubin frac tions. The infants were followed clinically and with serum diazo bilir ubin determinations until they either did not exceed a serum diazo bil irubin value of 254 mu mol/L (14.9 mg/dL) (nonhyperbilirubinemic) or u ntil bilirubin values rose above this level (hyperbilirubinemic), by a process of self-selection. A method of alkaline methanolysis, followe d by reverse-phase HPLC, was used to measure unconjugated bilirubin an d the mono- and diconjugated fractions of serum conjugated bilirubin. Total HPLC bilirubin and total conjugated bilirubin values were calcul ated from these measured bilirubin fractions. Patients also were class ified according to the serum total conjugated bilirubin value as low b ilirubin conjugators (serum total conjugated bilirubin less than media n) or as high bilirubin conjugators (serum total conjugated bilirubin greater than median). The data were analyzed by comparing serum conjug ated bilirubin fractions between the hyperbilirubinemic and nonhyperbi lirubinemic groups and the risk of developing hyperbilirubinemia in th e low bilirubin conjugators, relative to that of the high bilirubin co njugators. Results. Neonates were sampled at 53 +/- 12 and 58 +/- 12 h ours for the subsequently hyperbilirubinemic and nonhyperbilirubinemic groups, respectively (NS). Initial (ie, at the time of sampling) seru m total diazo bilirubin values (mean +/- SD) were almost identical for the subsequently hyperbilirubinemic and nonhyperbilirubinemic groups (214 +/- 27 mu mol/L [12.5 +/- 1.6 mg/dL] vs 212 +/- 19 mu mol/L [12.4 +/- 1.1 mg/dL], respectively, NS), as were the HPLC-determined serum total bilirubin values (162 +/- 32 mu mol/[9.5 +/- 1.9 mg/dL] vs 160 /- 16 mu mol/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]), measure d from the same serum samples, were lower in those infants who ultimat ely became hyperbilirubinemic than in those who remained nonhyperbilir ubinemic: total conjugated bilirubin 0.82 (0-2.07) mu mol/L vs 1.24 (0 .6-11.0) mu mol/L,; monoconjugated bilirubin 0.80 (0-2.07) mu mol/L vs 1.24 (0.60-1.23) mu mol/L; and diconjugated bilirubin 0.00 (0.00-0.42 ) pmol/L vs 0.11 (0-1.78) mu mol/L. The diconjugated bilirubin fractio n was especially affected; 18 (69%) neonates in the subsequently hyper bilirubinemic group had no detectable diconjugate compared with 8 (36% ) in the nonhyperbilirubinemic neonates. Conversely, more of those neo nates with serum total conjugated bilirubin fraction less than the med ian value of 1.06 pmol/L (0.06 mg/dL) developed hyperbilirubinemia tha n those with greater than the median (17/24 [71%] vs 9/24 [37.5%]), re spectively (relative risk: 1.89; 95% confidence interval: 1.06-3.36). Conclusions. Although serum total bilirubin levels at the time of samp ling were virtually identical, those neonates who subsequently develop ed hyperbilirubinemia had significantly lower serum conjugated bilirub in fractions than those who remained within the nonhyperbilirubinemic range. The diconjugated bilirubin fraction was especially affected. Th ose infants with serum total conjugated bilirubin fraction less than t he median had a greater risk of developing hyperbilirubinemia than tho se with greater than the median. These findings reflect inefficient bi lirubin conjugation ability in G-6-PD-deficient neonates who develop h yperbilirubinemia. We believe that the current findings are the functi onal manifestation of the interaction previously reported between G-6- PD deficiency and the variant gene promoter, as seen in Gilbert's synd rome. Diminished bilirubin conjugation ability appears to be a determi ning factor in the pathogenesis of G-6-PD deficiency associated neonat al hyperbilirubinemia.