MATERIALS AND METHODSutero, fetal bilirubin is cleared by the placenta. At birth the neonatal liver must assume detoxification and elimination of bilirubin. The mechanisms responsible for development of bilirubin conjugation after birth are poorly understood. Evidence for substrate-dependent stimulation of bilirubin conjugation in heterozygous newborn Gunn rats has been reported by Thaler (8) but studies of bilirubin conjugation in human neonates have not been performed due to unavailability of adequate methods. The gestation age at which the capacity for bilirubin conjugation is acquired and the types of conjugates initially formed are unknown. A highly specific and sensitive method for the measurement of unconjugated bilirubin and bilirubin mono-and diester conjugates in biologic fluids has been developed in our laboratory (9, 10). By utilizing this procedure, we investigated the appearance of bilirubin conjugates in serum of human neonates during the period of physiologic jaundice.Collection of serum specimens. Forty-three serial serum samples obtained in the course of routine laboratory evaluation from 13 premature infants (six male, seven female) of 25-36 wk gestational age were compared with 25 samples from 11 fullterm newborns (six male, five female) of 37-41 wk gestational age during the first 3 days oflife. Birth weights for the premature infant group ranged from 590-3000 g while full-term infant's birth weights ranged between 2500-3900 g. Infants with evidence of hemolysis or liver disease were excluded from the study.For comparison, serum samples from 22 healthy adults, seven pregnant women at term and their corresponding infant's cord blood at delivery, 46 cord blood specimens obtained at unselected deliveries, and five cord blood specimens from neonates with intrauterine hyperbilirubinemia due to blood group incompatibility or hypoxia also were examined.All specimens were protected from light in containers wrapped in aluminum foil, and were assayed immediately or after storage in the light-shielded containers at -12°C for less than I wk.Analytical methods. All procedures were performed in dim light. The AM-HPLC method for analysis of serum bilirubins has been previously described (9, 10). About 60 mg of sodium ascorbate, 2-3 mg of disodium ethylene diamine tetraacetate, 4 ml of methanol, and 2 ml of methanol containing the internal standard xanthobilirubic acid methyl ester were mixed with 0.6 ml of serum. The mixture was treated with 6 ml of 2% (w/v) KOH in methanol and immediately vortex-mixed. After reaction for 60-90 s at 20-25°C, 6 ml of chloroform and 12 ml of g1ycine/ HCI buffer (004 M HCI brought to pH 204 with solid glycine) were added sequentially. Organic and aqueous phases were separated by brief centrifugation, the organic phase was transferred to a dry tube, and the extract evaporated to dryness under nitrogen at 30°C. The residue was stored under argon at -12°C and analyzed within I wk. 947 Abbreviation AM-HPLC, alkaline methanolysis-high-performance liquid chromatography ABSTRACf. Bi...
This newly developed and highly specific and sensitive procedure was applied to the determination of unconjugated bilirubin and its ester conjugates in rat serum and human amniotic fluid. Bilirubin conjugates in biological samples are converted to methyl esters by alkaline methanolysis, extracted into chloroform, and the unconjugated bilirubin and esterified pigment derivatives are fractionated by "high-performance" liquid chromatography. The separated pigments are measured spectrophotometrically. Bilirubin and its mono- and di-conjugates are readily quantitated, even in previously undetectable concentrations. Linearity was established from 0.07 to 121.2 mumol/L for unconjugated bilirubin, 0.07 to 34.6 mumol/L for the C-8 monoconjugate, 0.06 to 69.3 mumol/L for the C-12 monoconjugate, and 0.17 to 43.7 mumol/L for the di-conjugate fraction. The detection limit was 0.03 mumol/L for unconjugated bilirubin and for each monoconjugate, and 0.1 mumol/L for the di-conjugated pigment.
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