Liver repopulation with bone marrow derived cells improves the metabolic disorder in the Gunn rat.

BACKGROUND: Reversible ischaemia/reperfusion (I/R) liver injury has been used to induce engraftment and hepatic parenchymal differentiation of exogenous beta2-microglubulin(-)/Thy1(+) bone marrow derived cells. AIM: To test the ability of this method of hepatic parenchymal repopulation, theoretically applicable to clinical practice, to correct the metabolic disorder in a rat model of congenital hyperbilirubinaemia. METHODS AND RESULTS: Analysis by confocal laser microscopy of fluorescence labelled cells and by immunohistochemistry for beta2-microglubulin, 72 hours after intraportal delivery, showed engraftment of infused cells in liver parenchyma of rats with I/R, but not in control animals with non-injured liver. Transplantation of bone marrow derived cells obtained from GFP-transgenic rats into Lewis rats resulted in the presence of up to 20% of GFP positive hepatocytes in I/R liver lobes after one month. The repopulation rate was proportional to the number of transplanted cells. Infusion of GFP negative bone marrow derived cells into GFP positive transgenic rats resulted in the appearance of GFP negative hepatocytes, suggesting that the main mechanism underlying parenchymal repopulation was differentiation rather than cell fusion. Transplantation of wild type bone marrow derived cells into hyperbilirubinaemic Gunn rats with deficient bilirubin conjugation after I/R damage resulted in 30% decrease in serum bilirubin, the appearance of bilirubin conjugates in bile, and the expression of normal UDP-glucuronyltransferase enzyme evaluated by polymerase chain reaction. CONCLUSIONS: I/R injury induced hepatic parenchymal engraftment and differentiation into hepatocyte-like cells of bone marrow derived cells. Transplantation of bone marrow derived cells from non-affected animals resulted in the partial correction of hyperbilirubinaemia in the Gunn rat.

Regenerative medicine: an insight.

Regenerative Medicine is a rapidly evolving field of therapy integrating different scientific and technological areas, including cell biology, biomedical and computer engineering, and clinical medicine, thus creating an interdisciplinary exchange network of skill, ideas, materials and efforts between basic and clinical research. Even if significant achievements have been obtained particularly in Plastic Surgery, Ophthalmology and Orthopedics, the field is still experimental and so far has failed to meet the expectations. The present article reviews the major hurdles that are still hampering the translational "bench to bedside" process and limiting the availability of these innovative therapeutic tools.

Neonatal bilirubin production-conjugation imbalance: effect of glucose-6-phosphate dehydrogenase deficiency and borderline prematurity.

OBJECTIVE: To evaluate relations between production and conjugation of bilirubin in the pathophysiology of jaundice in glucose-6-phosophate dehydrogenase (G6PD) deficient neonates. METHODS: Term and borderline premature (35-37 weeks gestational age), healthy, male, G6PD deficient neonates were studied close to the beginning of the 3rd day. Blood carboxyhaemogobin corrected for inspired CO (COHbc; an index of bilirubin production) and serum total conjugated bilirubin (TCB; a reflection of bilirubin conjugation) were measured in simultaneously drawn blood samples by gas chromatography and reverse phase high performance liquid chromatography respectively. A bilirubin production-conjugation index comprising COHbc/TCB was determined; a high index reflects imbalance between the bilirubin production and conjugation processes. COHbc and TCB individually and the production-conjugation index were studied in relation to serum total bilirubin (STB) concentration. RESULTS: Fifty one G6PD deficient neonates were sampled at 51 (8) hours. COHbc values did not correlate with STB (r=0.22, p=0.15). TCB did correlate inversely with STB (r=-0.42, p=0.004), and there was a positive correlation between the production-conjugation index and STB (r=0.45, p=0.002). The production-conjugation index (median (interquartile range)) was higher in the premature (n=8) than term neonates (2.31 (2.12-3.08) v 1.05 (0.53-1.81), p=0.003). This difference was the result of changes in TCB. CONCLUSIONS: The data show that jaundice in G6PD deficient neonates is the result of an imbalance between production and conjugation of bilirubin with a tendency for inefficient bilirubin conjugation over increased haemolysis in its pathogenesis. Borderline premature infants are at special risk of bilirubin production-conjugation imbalance.

Effect of withdrawal of pravastatin on biliary lipid composition in humans.

Abrupt withdrawal of HMG-CoA reductase inhibitors is associated with increased excretion of cholesterol into bile, but this phenomenon has not been investigated in humans. In order to evaluate whether patients interrupting these hypolipidemic drugs are at increased risk of forming gallstones, pravastatin (40 mg twice a day) or placebo was randomly administered to 16 bile fistula patients for 5 days. Biliary lipid composition was determined in basal conditions and for 5 consecutive days after drug withdrawal. Both biliary cholesterol concentration and saturation increased significantly on the second day after pravastatin withdrawal, but tended to decrease thereafter. Biliary bile acids and phospholipids were not affected. This short-lasting effect on biliary cholesterol excretion was probably the result of a transient increase of hepatic cholesterol synthesis by the up-regulated HMG-CoA reductase in the absence of the inhibitory drug. These results are consistent with the hypothesis that, also in humans, biliary cholesterol excretion could be dependent on the hepatic free cholesterol pool.

Serum and bile bilirubin pigments in the differential diagnosis of Crigler-Najjar disease.

OBJECTIVE: To differentiate between Crigler-Najjar (CN) disease types 1 and 2. DESIGN: The patterns of serum bilirubins, bile pigment composition, and phenobarbital response were studied. PATIENTS: Three infants, affected by high serum unconjugated bilirubin concentrations, previously classified as type 1 CN. METHODS: Serum and bile bilirubin pigment composition, both before and after phenobarbital (PB) treatment, were determined by alkaline methanolysis and high-pressure liquid chromatography. PB was given for at least 3 weeks by oral administration (5 mg/kg bw per day). RESULTS: No diconjugated bilirubin was found either before or after PB treatment in the serum of the three studied infants. In two patients traces of monoconjugated bilirubin were detected before PB therapy, and the ratio of conjugated/total bilirubin (percent) was increased by the PB response. In the third patient, traces of monoconjugated bilirubin appeared only after PB administration. However, the serum unconjugated bilirubin concentration decreased significantly only in the second patient, following the second cycle of PB treatment, leading to the diagnosis of type 2 CN. The analysis of the methyl ester derivatives of bile pigments was also performed on bile samples obtained in two patients by Entero-Test (R) both before and after PB treatment. An absolute increment in monoesterified bilirubin concentration was found after PB administration, although the percent concentration increased in one case and decreased in the other. No diesterified bilirubin was detected in the bile samples. CONCLUSIONS: The present results show that in types 1 and 2 CN disease it is possible to detect traces of monoconjugated but not diconjugated bilirubin both in serum and in bile. Whereas PB treatment is effective in slightly increasing the serum monoconjugated bilirubin concentration even in type 1 CN disease, the diagnosis of type 1 or 2 is based on finding a substantial decrease of serum unconjugated bilirubin following PB administration.

Serum bile acids in patients with liver failure supported with a bioartificial liver.

BACKGROUND: Serum bile acids are increased in liver failure, but the composition of the bile acid pool in this condition has not been studied in detail. This information is of interest because of dihydroxy bile acid toxicity. METHODS: We measured serum bile acids by gas chromatography-mass spectrometry in 13 patients with fulminant liver failure and five patients with acute-on-chronic liver failure. Furthermore, serum bile acids were analysed in the same patients after 6 h of treatment with a bioartificial liver, consisting of a hollow-fibre cartridge with microcarrier-attached porcine hepatocytes and a charcoal column. RESULTS: Pre-bioartificial liver serum bile acids demonstrated a high dihydroxy/trihydroxy ratio and were higher in patients with acute-on-chronic liver failure than in those with fulminant liver failure (452.8 +/- 98.6 vs. 182.1 +/- 39.7 micro mol/L; P < 0.05). Bioartificial liver treatment decreased significantly serum bile acids in patients with fulminant liver failure (-38.8%) and acute-on-chronic liver failure (-35.8%), with a decreased dihydroxy/trihydroxy ratio. In vitro, porcine hepatocytes in the bioreactor cleared most conjugated bile acid species from pooled patient plasma. CONCLUSIONS: Acute liver failure is associated with very high serum levels of toxic bile acids that could contribute to the pathogenesis of the syndrome. Bioartificial liver treatment reduces both serum bile acid concentrations and the hydrophobicity of the bile acid pool.

Effect of tauroursodeoxycholic acid on biliary lipid composition. A dose-response study.

Tauroursodeoxycholic acid has been proposed for the treatment of hepatobiliary disease, but data on the enrichment of biliary tauroursodeoxycholic acid pool and on changes of biliary lipids after administration of the compound are scarce. We studied the composition of biliary lipids in a series of 33 patients with radiolucent stones, before and after treatment with tauroursodeoxycholic acid, 3.5 - 16.6 mg/kg/day for 4 - 6 weeks. Duodenal bile was collected with the Entero-Test after gallbladder contraction. Tauroursodeoxycholic acid administration produced the following dose-dependent effects: a linear decrease of cholesterol saturation (r = 0.59, p < 0.001); a non-linear increase of the percent of ursodeoxycholic acid in bile (r = 0.59, p < 0.001); a non-linear increase of the fraction of ursodeoxycholate conjugated with taurine. At the dose of 11 mg/kg per day, cholesterol saturation was 80%, ursodeoxycholic acid represented about 45% of biliary bile acids, and about half of UDCA was conjugated with taurine. Biliary bile acids were repeatedly measured in 6 patients during long-term treatment with 9.7 - 12.1 mg/kg. The fraction of tauroursodeoxycholic acid decreased progressively from 67.6% +/- 10.5 to 29.1% +/- 5. Tauroursodeoxycholic acid is as effective as ursodeoxycholic acid on a molar basis in reducing biliary cholesterol saturation and in enriching bile with ursodeoxycholate. Moreover, tauroursodeoxycholic acid administration is associated with higher concentrations of tauroconjugates in the bile than those previously reported by feeding the free bile acid.

Is there a place for medical treatment in children with gallstones?

BACKGROUND: Medical treatment of gallstones with ursodeoxycholic acid (UDCA) or chenodeoxycholic acid (CDCA) has not been evaluated in children. AIM: The purpose of this study was to assess the effectiveness of UDCA in the treatment of gallstones in children. METHODS: UDCA was used to treat 15 patients, (7 boys and 8 girls; mean age, 7.8 years; range, 3 months to 15 years) for 1 year. All had radiolucent stones with a maximum diameter of 10 mm and a normally contractile gallbladder. RESULTS: The stones disappeared completely in two children but returned later. All symptomatic patients became symptom free. CONCLUSION: UDCA is ineffective in the treatment of gallstones in children except in terms of relieving symptoms while on treatment.

Comparison of pig, human and rat hepatocytes as a source of liver specific metabolic functions in culture systems--implications for use in bioartificial liver devices.

The limited availability of human hepatocytes results in the use of animal cells in most bioartificial liver support devices. In the present work, clinically relevant liver specific metabolic activities were compared in rat, pig and human hepatocytes cultured on liver-derived biomatrix to optimize the expression of differentiated functions. Pig hepatocytes showed higher rates of diazepam metabolism (2.549+/-0.821 microg/h/million cells vs. 0.474+/-0.079 microg/h/million cells rats, p<0.005, and vs. 0.704+/-0.171 microg/h/million cells in man, p<0.005) and of bilirubin conjugation (21.60116+/-8.433237 micromoles/l/24 h vs. 6.786809+/-2.983758 in man, p<0.001 and vs. 9.956538+/-1.781016 micromoles/l/24 h in rats, p<0.005). Urea synthesis was similar in pig and in human hepatocytes (150+/-46.3 vs. 144.8+/-21.46 nmoles/h/million cells) and it was lower in rats (84.38+/-35.2; p<0.001 vs. man, p<0.02 vs. pig). High liver specific metabolic activities in cultured pig hepatocytes further support their use as a substitue for human cells in bioartificial liver devices.

Routine determination of biliary lipid composition with the entero-test.

A simple method for the assay of biliary lipids was used for routine determination of biliary cholesterol saturation index (CSI) in 17 healthy subjects, 40 untreated patients with radiolucent gallstones and 21 gallstone patients treated with ursodiol. The method is based on collection of bile with the Entero-Test, a device for easy sampling of gastrointestinal contents. The procedure was easy to perform and well accepted by the patients. Both CSI and the cholesterol content of bile were higher in untreated gallstone patients than in controls, and were significantly lower in treated than in untreated patients. Normal CSI was found in 35% of untreated gallstone patients, while 24% of healthy subjects had supersaturated bile. Supersaturated bile was still present in 14% of ursodeoxycholic acid treated patients, suggesting inefficacy of therapy. These results demonstrate the possibility of performing easy, routine determination of biliary lipid composition, which can yield useful information for the management of gallstone patients.

Ultrasonic evaluation of gallbladder emptying with ceruletide: comparison to oral cholecystography with fatty meal.

Assessment of gallbladder function is required prior to nonsurgical treatment of gallstones. In order to develop a practical and reproducible method of evaluation, gallbladder emptying was studied by ultrasound (US) in 55 gallstone patients after intramuscular administration of ceruletide (0.3 micrograms/kg). In 27 of these subjects, the US procedure was compared to oral cholecystography (OCG) with fatty meal. Maximal percent gallbladder contraction was reached 30 min after ceruletide in all patients. Maximal percent contractions were 47.5 +/- 27.7 during US with ceruletide and 33.9 +/- 16.3 during OCG with fatty meal (p = 0.03). A significant linear relationship was found between the results obtained with the two different procedures (r = 0.57; p = 0.002). Serial US determinations of gallbladder emptying were performed in 16 patients. Individual variation was below 20% in 11 subjects, and in five subjects it ranged between 20 and 40%. Minor, self-limiting side effects were observed in 13 patients. US determination of gallbladder emptying after ceruletide appears to be a practical and reliable method to assess gallbladder function.

Ultrasonic evaluation of gallbladder emptying with ceruletide.

In order to develop a practical and reproducible method to assess gallbladder function, gallbladder emptying was studied by ultrasound in 27 gallstone patients after i.m. administration of ceruletide (0.3 micrograms/kg), and the procedure was compared to oral cholecystography with fatty meal. Maximal percent gallbladder contraction was reached in all patients 30 min after ceruletide. Maximal percent contractions were 47.5 +/- 27.7 during ultrasound with ceruletide and 33.9 +/- 16.3 during oral cholecystography with fatty meal (p = 0.03). A significant linear relationship was found between the results obtained with the two different procedures (r = 0.57; p = 0.002). Serial ultrasound determinations of gallbladder emptying were performed in 16 patients. Day-to-day variation was below 20% in 11 subjects, and it ranged between 20 and 40% in 5 subjects. Minor side effects were observed in 9 patients. Ultrasound determination of gallbladder emptying after ceruletide appears to be a practical and reliable method to assess gallbladder function.

Unconjugated and conjugated bilirubin pigments during perinatal development. III. Studies on serum of breast-fed and formula-fed neonates.

The process of conjugation and secretion of bilirubin was studied in a group of healthy, full-term, exclusively breast-fed newborns and a control group of exclusively formula-fed infants by means of a reverse-phase high-performance liquid chromatographic analysis of bilirubins present in serum. The serum concentrations of unconjugated bilirubin, esterified bilirubin, and the proportion of diesterified bilirubin (as percent of esterified bilirubin) were not significantly different in breast- and formula-fed infants on the 3rd and 5th days of life. These data suggest that both bilirubin production and conjugation are not different in breast-fed and in formula-fed newborns.

Bilirubin inhibits bile acid induced apoptosis in rat hepatocytes.

BACKGROUND AND AIMS: Hydrophobic bile acids contribute to hepatocellular injury in cholestasis and rapidly induce apoptosis in vitro; however, unlike Fas agonists, cholestasis does not cause extensive hepatocyte apoptosis. As antioxidants provide protection against bile acid induced liver injury, our premise was that bilirubin, a free radical scavenger with increased plasma levels in the presence of liver disease, could protect hepatocytes against bile acid induced apoptosis. METHODS: Freshly isolated rat hepatocytes were incubated for four hours with 100 micromol/l glycochenodeoxycholate (GCDC) alone or with increasing concentrations of unconjugated (UCB) or conjugated (CB) bilirubin. RESULTS: Both UCB and CB inhibited GCDC induced apoptosis in a dose dependent fashion and suppressed the generation of reactive oxygen species by hepatocytes. CONCLUSIONS: The antiapoptotic effect of bilirubin associated with its antioxidant properties indicates that hyperbilirubinaemia may have a protective role in liver disease.

A simple method for the determination of lipid composition of human bile.

The Entero-Test, a device for easy sampling of gastrointestinal contents, including bile, has been used for determination of biliary lipid composition. The device consists of a weighted gelatin capsule containing 140 cm of a highly absorbent nylon line. The capsule is swallowed while one end of the string is taped to the face. After 3.5 h, when the line has reached the duodenum, gallbladder contraction is stimulated by intramuscular administration of ceruletide. The line is pulled out, and the last 15 cm are eluted four times in methanol. Total bile acids (by 3 alpha-hydroxysteroid-dehydrogenase assay), individual bile acids (by high performance liquid chromatography), phospholipids (by assay of lipid-soluble phosphorus), and cholesterol (by gas-liquid chromatography) are determined in the eluate. Tests in vitro demonstrated no preferential binding and a good recovery of biliary lipids from the thread. Similar values of biliary cholesterol saturation were obtained by means of duodenal intubation and of the Entero-Test in a series of 12 subjects (r = 0.952). In 5 subjects, individual bile acids were also measured and were found to be similar with both techniques (r = 0.948). When the test was repeated over 3 days in a series of 7 subjects, biliary cholesterol saturation was found to be remarkably reproducible (CV = 7.6%). Thus, the Entero-Test is a convenient technique for the determination of biliary lipid composition, which can be particularly useful in longitudinal studies.

Unconjugated and conjugated bilirubin pigments during perinatal development. V. Effect of phototherapy on serum conjugated bilirubin in hyperbilirubinemic neonates.

OBJECTIVE: To determine the effect of phototherapy on serum conjugated bilirubin fractions, an index of bilirubin conjugation, in hyperbilirubinemic neonates. METHOD: Serum was sampled from 21 jaundiced (serum diazo total bilirubin > or = 274 micromol/l), term, otherwise healthy neonates prior to starting phototherapy, and 24 h after the commencement of treatment. Alkaline methanolysis followed by reverse-phase, high-performance liquid chromatography, specific for determination of unconjugated bilirubin and the monoconjugated and diconjugated fractions of total conjugated bilirubin in serum, was used for the analysis. Prephototherapy values were compared to those at 24 h. RESULTS: Serum total bilirubin and total conjugated bilirubin values decreased during the study period, from 220 (211-239) to 177 (157-213) micromol/l (median (25-75% range)) p = 0.001, for the former, and from 1.4 (0.87-1.57) to 0.93 (0.69-1.84) micromol/l, p = 0.005, for the latter. These parameters decreased by a similar percentage (-16.6+/-14.8% and - 14.0+/-23.4%, respectively; p > 0.05). Both monoconjugated and diconjugated bilirubin, calculated as a percentage of total conjugated bilirubin, remained constant over the study period (88.2 (72.2-96.4)% before phototherapy and 92.5 (87.3-96.8)% after 24 h, p > 0.05, for monoconjugated bilirubin, and 11.8 (3.6-27.8)% and 7.5 (3.2-12.7)%, respectively, p > 0.05, for diconjugated bilirubin). CONCLUSIONS: Serum total conjugated bilirubin values decreased in parallel to serum total bilirubin levels during phototherapy, maintaining a constant relationship between these two parameters. The ratios of monoconjugated and diconjugated bilirubin to total conjugated bilirubin remained constant. These findings imply that phototherapy does not alter bilirubin conjugation in hyperbilirubinemic neonates.

Effect of tauroursodeoxycholic acid (TUDCA) on biliary lipid composition.

TUDCA has been proposed in the treatment of hepatobiliary disease, but data on the enrichment of the biliary TUDCA pool and changes of biliary lipids after administration of the compound are scarce. We studied the composition of biliary lipids in a series of 33 patients with radiolucent stones, before and after treatment with TUDCA, 3.5 to 16.6 mg/kg/die for 4-6 weeks. Duodenal bile was collected with the Entero-Test after gallbladder contraction. Cholesterol saturation was 147% + 67 before treatment. TUDCA administration produced the following dose-dependent effects: a) a linear decrease of cholesterol saturation (r = 0.59; p); b) a non-linear increase of the percent of ursodeoxycholate in bile (r = 0.59; p); c) a non-linear increase of the fraction of ursodeoxycholate conjugated with taurine. At the dose of 11 mg/kg per day, cholesterol saturation was 80%, ursodeoxycholic acid represented about 45% of biliary bile acids, and about half of urso was conjugated with taurine. These results can be used as guidelines to assess the required daily dosage of TUDCA.

Conjugated bilirubin in neonates with glucose-6-phosphate dehydrogenase deficiency.

We used a system capable of measuring conjugated bilirubin and its monoconjugated and diconjugated fractions in serum to assess bilirubin conjugation in 29 glucose-6-phosphate dehydrogenase (G6PD)-deficient, term, male newborn infants and 35 control subjects; all had serum bilirubin levels > or = 256 mumol/L (15 mg/dI). The median value for diconjugated bilirubin was lower in the G6PD-deficient neonates than in control subjects (0.06 (range 0.00 to 1.84) vs 0.21 (range 0.00 to 1.02) mumol/L, p = 0.006). Diglucuronide was undetectable in 11 (38.9%) of the G6PD-deficient infants versus 3 (8.6%) of the control subjects (p = 0.015). These findings imply a partial defect of bilirubin conjugation not previously demonstrated in G6PD-deficient newborn infants.

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 micromol/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 micromol/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 +/