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This preterm female infant abruptly developed diarrhea and bowel distention at 25 days of age and died within 60 h with fulminating pseudomembranous colitis and proctitis. Toxin-producing C. difficile was recovered from the stool. The pseudomembranous colitis developed 12 days after the last dose of ampicillin was given to the baby. Necrotizing enterocolitis, a condition pathologically distinct from pseudomembranous colitis, was also present.

Modification of the standard negative-pressure respirator, which easily permits application of intermittent negative-pressure ventilation to the infant of less than 1,000 gm, has been described. Previously encountered problems of fixation of the infant, of maintenance of oxygen concentrations, and of inability to obtain chest films of the infant while in the device have been overcome.

• Total bilirubin-binding capacity was measured by Sephadex G-25 gel filtration in 43 clinically well and 45 clinically ill newborn infants between 26 and 41 weeks' gestation. In the well patients, bilirubin-binding capacity, serum albumin concentration, and the molar-binding ratio of bilirubin to albumin were directly related to gestational age. In the sick patients, serum bilirubin-binding capacity and albumin concentration also correlated with gestational age; however, from 32 to 41 weeks' gestation, the mean values for the sick infants were significantly lower than for the well infants. In the sick infants, the molar binding ratio of bilirubin to albumin was also lower than in the well patients and did not correlate with gestational age. The data are in agreement with previous clinical findings indicating an increased risk for low-bilirubin kernicterus among immature infants and suggest that some critically ill term infants may be at risk for kernicterus at serum bilirubin levels below 20 mg/100 ml. ( Am J Dis Child 131:898-901, 1977)

Free bilirubin concentration, bilirubin-binding capacity, and bilirubin-binding affinity were determined by peroxidase oxidation in 66 newborn infants. Twelve healthy term infants whose unconjugated bilirubin concentration was 15.8 +/- 3.7 mg/dl (mean +/- SD) had a binding capacity of 31.9 +/- 3.7 mg/dl (bilirubin: albumin molar ratio = 0.89 +/- 0.07) and Ka = 28 +/- 11 x 10(7)/M. Twelve term infants with clinical complications of asphyxia, acidosis, respiratory distress, or sepsis, and 17 preterm infants with no complications had lower serum albumin concentrations and slightly reduced binding capacity and affinity compared to the healthy term infants. Free bilirubin concentrations were similar in these three groups, averaging 8 to 9 nmol/l in each group. Twenty-five preterm infants with complications had significantly higher free bilirubin (19 +/- 11 nmol/l), lower binding capacity, and lower binding affinity than any of the other three groups (P less than 0.01 for all comparisons). Five of the 25 sick preterm infants had kernicterus at autopsy. These five infants were similar to the other 20 in birth weight, gestational age, serum bilirubin, and serum albumin level, but had significantly higher free bilirubin and significantly lower binding capacity and affinity. The data suggest that serious neonatal illness is associated with a marked reduction in bilirubin-binding capacity and affinity and an increased risk of kernicterus in preterm infants. The mechanism by which neonatal morbidity decreases bilirubin binding is not known.

Twenty-eight drugs, including cephem antibiotics and anti-inflammatory agents currently used or considered potentially useful in neonatal intensive care nurseries in Japan, were examined to determine their influence on albumin and bilirubin interaction by means of a glucose oxidase - peroxidase method, using an automated analyzer (Arrows) for unbound bilirubin (U.B.). The apparent binding constant for drugs to the high-affinity site on albumin (KD) was determined. Of cephem. antibiotics, latamoxef sodium (LMOX) and cefazolin sodium (CEZ) were found to displace bilirubin from albumin (KD= 5.9 times 103 M-1 and 4.5 times 103 M-1, respectively) as strongly as Na salicylate (KD= 6.8 times 103 M-1). Mephenamate and indomethacin, which are used for medical closure of patent ductus arteriosus in premature infants, were also found to be stronger bilirubin displacers (Kn = 1.3 times 105 M-1 and 1.2 times 105 M-1, respectively) than sulfisoxazole (KD= 1.6 times 104 M-1). Maximal displacement factors (MDF's) were also estimated in reference to protein binding (%) and effective serum concentration (M) of each drug in human adults. Of these drugs, mephenamate showed a higher risk of bilirubin displacement (MDF = 3.79) than sulfisoxazole (MDF = 2.58) and LMOX had a higher risk of displacement (MDF = 1.97) than Na salicylate (MDF = 1.85). On the other hand, indomethacin and CEZ showed minimal effects on displacement at therapeutic levels (MDF = 1.03 and 1.00, respectively). At therapeutic serum levels, mephenamate and LMOX may possess the potential for displacing bilirubin from albumin and increasing the risk of bilirubin encephalopathy, in a manner similar to sulfisoxazole.

The effect of metabolic and respiratory acidosis on bilirubin and albumin entry into the brain was studied in 24 awake and unanesthetized rats. Hyperbilirubinemia was established by infusion of unconjugated bilirubin at a rate of 30 mg/kg/h for three hours. After two hours, metabolic acidosis was produced in eight rats by infusion of 0.5 N hydrochloric acid at a rate of 0.02 mL/g/h. This reduced the pH level to 7.03 ± 0.01 (mean ± SEM) with a normal value for Pco2. Respiratory acidosis was produced in another group of eight animals who breathed 20% CO2 in a balanced gas mixture for the last hour of the study period. This resulted in a reduction of pH to 7.04 ± 0.01 with Pco2 of 100.4 ± 2.3 mm Hg. A third group of eight rats served as controls and were given equal volumes of saline infusion. No increase in brain bilirubin or brain albumin was found in the group with metabolic acidosis, but in the group with respiratory acidosis both bilirubin and albumin concentrations in the brain increased significantly. No significant differences were found between the groups in serum total or apparent unbound bilirubin, albumin, or osmolality. The results indicate that a brief period of acidosis per se does not increase bilirubin entry into the brain, but hypercarbia does so by opening of the blood-brain barrier.

ON SEPTEMBER 26 AND 27, 1977, a workshop was held at Newport, R.I.; under the sponsorship of Brown University and the National Foundation-March of Dimes, on the methodology, terminology, and clinical applications of serum bilirubin-binding determinations in the newborn infant. Participants included those involved in current research in the areas of bilirubin-albumin interactions and bilirubin encephalopathy (see appended list).

To test the hypothesis that fetal lung maturation can be accelerated by one of the xanthine derivatives, aminophyllin was given to 40 pregnant rabbits beginning on the 20th gestational day for a period of 7-10 days. The fetuses were delivered by cesarean section and fetal lung maturity was assessed by determining the biochemical, functional, and ultrastructural characteristics of aminophyllin-treated vs. control animals. The phospholipid content of the lung tissue homogenate from the aminophyllin-treated group was significantly higher than in the control subjects (saline injected) at 28 days of gestation (421 +/- 9 vs. 368 +/- 12 mug/mg wet wt, mean +/- SEM) and at 29 days of gestation (531 +/- 10 vs. 475 +/- 20). The alveolar wash phospholipid content of the aminophyllin-treated group was higher at 30 days (167 +/- 9 mug/mg dry wt, mean +/- SEM vs. 117 +/- 17). The lung compliance derived from pressure volume curves was also significantly higher in the aminophyllin-treated group when compared with controls at 27 days of gestation (0.023 +/- 0.0005 ml/cm H2O, mean +/- SEM vs 0.010 +/- 0.0002) and at 28 days of gestation (0.048 +/- 0.0003 vs 0.035 +/- 0.0006). There was no significant difference in the number of lamellar bodies in the type II cells between the aminophyllin-treated and the control groups. The data show that aminophyllin has accelerating effects on fetal lung maturation in rabbits when the drug is given to pregnant rabbits during the last 7-10 days of gestation.

Using a Sephadex G-25 separation technique, the total bilirubin-binding capacity was measured in 18 jaundiced infants before and after phototherapy. The initial total bilirubin-binding capacity obtained at 20 to 113 hours of age averaged 17.5 +/- 4.4 mg/dl (mean +/- SD) and was unchanged during phototherapy at 34 to 135 hours of age. In ten infants, the total bilirubin-binding capacity was measured before, during, and 24 hours after the cessation of phototherapy. Here too, phototherapy did not significantly influence the TBBC. These data indicate that in vivo, phototherapy does not alter the serum bilirubin binding capacity of newborn infants.

Bilirubin inhibits in vitro oxidative phosphorylation and glycolysis. This study investigated the in vivo effect of bilirubin on cerebral oxygen, glucose, and lactate uptake in newborn piglets. Seventeen 2- to 4-day-old piglets were divided into three groups and examined as follows: group 1 = control (C); group 2 = control with sulfisoxazole; and group 3 = experimental, given bilirubin with sulfisoxazole. In the experimental group, bilirubin was infused for 4 h. The cerebral bilirubin content in the bilirubin-infused group was 11.0 +/- 1.4 nmol/g of cerebral cortex (mean +/- SEM), consistent with levels found in infants with kernicterus. However, this level of brain bilirubin had no major, acute effects on cerebral uptake of oxygen, glucose, or lactate despite producing lethargy and ataxia which were consistent with bilirubin intoxication. This suggests that mitochondrial changes may not be involved in vivo in acute bilirubin encephalopathy.

Summary: In 35 neonatal sera, comparisons were made of bilirubin binding capacity determined by Sephadex gel filtration and by peroxidase oxidation techniques. The serum indirect bilirubin concentration was 11.1 ± 4.6 mg/100 ml (190 ± 79 μM/liter); the serum concentration of unbound bilirubin was 50 ± 60 μg/100 ml (0.85 ± 1.03 μM/liter) by gel filtration and 0.77 ± 0.66 μg/100 ml (0.013 ± 0.011 μM/liter) by peroxidase oxidation. Binding capacity averaged 22.9 ± 5.6 mg/100 ml (392 ± 96 μM/liter) by gel filtration and 22.1 ± 6.1 mg/100 ml (378 ± 104 μM/liter) by peroxidase oxidation. The binding capacity, expressed as moles of bilirubin bound per mole of albumin, was 0.80 mol bilirubin/mol albumin by the Sephadex method and 0.77 by the peroxidase method. Individual values for binding capacity were in close agreement (r = 0.961, P < 0.001). At saturation of the first bilirubin binding site, the unbound bilirubin concentration was 190 ± 80 μg/100 ml (3.25 ± 1.37 μM/liter) by gel filtration and 1.87 ± 0.54 μg/100 ml (0.032 ± 0.009 μM/liter) by peroxidase oxidation. There was no correlation between unbound bilirubin concentrations estimated by the two methods. The peroxidase method requires a smaller volume of serum than the Sephadex method and is more sensitive to small changes in free bilirubin concentration. Speculation: Both Sephadex gel filtration and peroxidase oxidation techniques appear capable of predicting the indirect bilirubin concentration at which the first or “tight” bilirubin binding site will be saturated, and above which free bilirubin concentration will be greatly increased. In neonatal sera, the two methods showed close agreement for individual determinations of binding capacity over a wide range of molar ratios. The lower limit for the neurotoxic concentration of unbound or “free” bilirubin is not precisely known and may vary in the face of multiple clinical factors. However, it seems likely that free bilirubin concentrations in excess of those measured at saturation of the first binding site would be potentially neurotoxic, and that bilirubin bound to secondary binding sites would be more readily dissociable.

Auditory brain stem evoked responses (ABR) in newborn infants are altered by elevated serum bilirubin levels and may be used as an indicator of bilirubin toxicity. The hypothesis of our study was that hyperbilirubinemia may affect ABR's because of bilirubin deposition in the brain stem. We examined this hypothesis by studying 21 male adult rats divided into three groups: group A, control (n = 8); group B, low bilirubin (n = 6); and group C, high bilirubin (n = 7). Each experimental group was studied over 150 min. The control group received a buffer solution. The low bilirubin group received a low dose bilirubin bolus of 50 mg/kg followed by continuous infusion of 20 mg/kg/h. The high bilirubin group received a bolus of 100 mg/kg bilirubin followed by continuous infusion of 40 mg/kg/h. All groups received sulfisoxazole (50 mg/kg X 3) during the second hour of the study. Auditory evoked potentials were recorded at 0 and 150 min. At the end of the study, the brains were analyzed for bilirubin content. Bilirubin deposition in the 3 groups was 0.6 +/- 0.28 micrograms/g, 0.93 +/- 0.07 micrograms/g and 3.2 +/- 2.2 micrograms/g for the control, low bilirubin and high bilirubin groups, respectively. Bilirubin deposition in the brain was associated with a significant amplitude reduction of Wave I and III, but had no effect on absolute latencies and interpeak latencies. Very high brain bilirubin concentrations were associated with absence of Waves I and IV. Wave I was also significantly reduced in the low bilirubin group where there was slightly increased bilirubin deposition in the brain. We conclude that ABR changes in the form of wave amplitude reduction were associated with brain stem and cerebellum bilirubin deposition. We speculate that previously reported observations of abnormal ABR changes in hyperbilirubinemic newborn infants are associated with bilirubin deposition.

produce clinical copper deficiency (except for a lower hematocrit at six months) in premature infants fed much lower copper intakes. This study suggests the age at which infants of different gestations can be expected to have an increase in serum copper and ceruloplasmin concentrations. Infants past that age (after 3 months in most very premature infants) who have very low serum copper concentrations may be copper deficient, especially if clinically compatible, and may benefit from additional copper supplies. Prior to that time, serum copper and ceruloplasmin values are probably inadequate indicators of total body copper status, and the benefit to risk ratio of copper supplementation is not established. In the great majority of infants, serum copper concentrations will rise spontaneously as either the liver, gastrointestinal tract, or both, mature.

Many drugs given to mothers and their newborns are bound to albumin. After 25 years of experience in the United States, sulfisoxazole still remains as the only drug clearly associated with an epidemic of kernicterus in newborn infants; however, several other drugs that are often used and most of the newer drugs being developed for possible use in the perinatal period have not been adequately tested for their effects on bilirubin binding, distribution, and metabolism. As the routine use of multiple drugs increases in high-risk newborns, it is appropriate to review the methods available for testing bilirubin-drug interactions. In this presentation we shall review several methods potentially useful for studying bilirubin displacement from albumin by drugs, using examples from our own laboratory.

Serial determinations of bilirubin-binding capacity were performed in 61 newborn infants during the first 10 days of life. 27 infants were classified as term (gestational age greater than or equal to 36 weeks) and 34 as preterm (gestational age less than or equal to 33 weeks); 34 were classified as 'sick' and 27 as 'well'. Bilirubin-binding capacity was measured by Sephadex gel filtration. In relation to postnatal age, total bilirubin-binding capacity (TBBC) remained stable in well term and preterm infants, decreased slightly in sick preterm infants, and decreased significantly in sick term infants. TBBC, serum albumin, and molr binding ratio (B/A) were significantly higher in well than in sick infants in both term and preterm groups; there were no significant differences between sick term and sick preterm infants. Clinical recovery in 16 infants was associated with a significant rise in TBBC and in B/A. The data suggest that in healthy infants, the serum bilirubin-binding capacity remains relatively unchanged during the first 10 days of life. Clinically ill infants show wide patient-to-patient variability in TBBC. Because of the tendency of TBBC to decrease with postnatal age in sick infants, repeated determinations of TBBC may be indicated for the management of sick jaudiced newborns.

We are indeed interested to learn that Higgs et al.1 have achieved early growth rates comparable to those in our study with the use of intravenous fat emulsion in small premature infants. Further information on the metabolic complications and autopsy findings encountered in their study would provide important additional evidence concerning the effectiveness and potential hazards of using intravenous fat emulsions in sick newborns. There was a difference between the two studies in the method of infusing the fat emulsion.

1350 THE EFFECT OF BILIRUBIN (BR) ON CEREBRAL CORTEX (CE) O 2 AND GLUCOSE METABOLISM IN PIGLETS (P)

Urinary concentrating and diluting defects have been described in hyperbilirubinemic Gunn rats. Renal medullary Na and urea concentrations are decreased in these animals (JCI 55:319-329, 1975) suggesting a possible association between solute reabsorption and the urinary defects. While Na transport may be adversely influenced by bilirubin (BR) (Ped Res 16:319A, 1982), the effects of BR on transepithelial fluxes of urea and water have not been studied. Therefore, urea and water flux measurements were made in responsive epithelia (bladders) isolated from the Dominican toad (Bufo marinus). Tissues were acutely exposed to amphibian HCO3- Ringers, pH 8.1 containing BR (0.1 mM) and 0.05% bovine serum albumin (BSA) or BSA alone. Urea permeability (K trans) was calculated from the mucosal to serosal flux of 14C labeled urea in short circuited bladders. Water movement was gravimetrically determined by the Bentley bag technique. Tissues were stimulated with vasopressin (VP) 40 mU/ml for urea and 20 mU/ml for water studies. The hydroosmotic response to VP was likewise unaffected by BR in the serosal bath; control 46.5 ± 2.7 mg/min vs. exp. 45.5 ± 3.3 mg/min (n=9). Although VP induced active Na transport is inhibited by BR, BR appears to have no effect on VP induced cell membrane permeability changes that allow for passive transepithelial movement of urea and water.

Bilirubin (BR) can inhibit cyclic AMP-dependent protein kinase activity. To investigate the mechanism of BR neurotoxicity, we studied the effect of BR on production of dopamine, a neuro-transmitter whose synthesis is stimulated by cyclic AMP (c-AMP). We used a synaptosome (SYN) preparation to study dopamine synthesis rate (DSR); in vitro. SYNs, the biochemically functional units of synapses, were isolated by differential centrifugation from the corpus striatum of adult male Sprague-Dawley rats. DSR was quantitated by 14CO2 produced after addition of the labeled precursor, L-(l- 14C) tyrosine. The effect of BR was assessed at 4 different BR concentrations under 2 experimental conditions: basal DSR and 2mM dibutyryl c-AMP (db c-AMP) stimulated DSR. Results as noted in figures, were expressed as % of basal DSR and compared against matched controls. We conclude that while BR has no effect on basal DSR, it does dampen the db c-AMP stimulated synthesis of dopamine in vitro. The data provide evidence for an inhibitory effect of BR on protein kinase-mediated neuro-transmitter synthesis activation.

Free fatty acids (FFA) are strong bilirubin displacers from Albumin (A) at FFA:A ratio >3:1. A model of bilirubin displacement using FFA:A ratio to predict free bilirubin was tested in 21 sera from 14 newborns (8 sick, 6 well). Concentration of FFA was 0.64 ± 0.40 mEq/1, A 0.41 ± 0.09 mEq/1, and FFA:A was 1.6 ± 1.0 (Mean ± S.D.); FFA:A was

Top of pageAbstract The risk of bilirubin-related brain injury may be increased by perinatal or neonatal infection. To determine whether Gram-negative bacteremia acutely increases transfer of bilirubin and albumin from blood to brain, 6 adult rats were injected with 106 E. coli to produce positive blood cultures, and then infused while awake with bilirubin (BR) and 125I albumin (ALB) via indwelling catheters for 180 minutes. 3 of 6 rats with positive blood cultures had lethargy or temperature instability during the experiment. At 180 mins, the animals were sacrificed for brain BR and ALB concentrations. Compared to 5 controls, there were no differences in serum or brain BR and ALB in the bacteremic rats, as shown in the table: In the adult rat, bacteremia does not cause acute changes in permeability of the blood-brain barrier to bilirubin or albumin. If sepsis does increase the risk of bilirubin injury to the central nervous system, systemic metabolic or circulatory changes due to sepsis or acute effects of endotoxin, rather than acute bacterial effects on the blood brain barrier, may be more likely reasons for this association.

The study was designed to investigate the effects of bilirubin on synaptosomes from rat brain. Ten percent suspensions of synaptosomes derived from hippocampus and striatum from male Sprague-Dawley rats were prepared in 0.25 M sucrose. A Krebs-Ringer buffer, pH 7.3, gassed with 95% O2, 5% CO2, was used throughout. 75μl of synaptosome suspension ana 625μl of a buffered bilirubin/ albumin solution (molar ratio 8:1) were incubated for 5 min. at 37°C, after which 40μl of 3H-dopamine and 14C-5HT were added and incubation continued for another 10 min. The following bilirubin concentrations (μM) were studied: 1 - 10 - 20 - 40 - 80 - 160 - 320 - 640. The reaction was stopped and the synaptosomes harvested by filtering through a glass microfibre filter premoistened with ice cold saline. Activities were counted in Insta-gel II in a Packard Tri-carb 460 CD scintillation counter. A significant difference from control values (p

Hypercarbia (HC) induced increases in regional brain blood flow (RBBF) may augment regional brain bilirubin deposition (RBBD) in piglets (P). 10 2-4 day old P were infused with bilirubin (BR) to raise & maintain a serum BR of approximately 8 mg/dl. HC (pCO2=76mmHg) was induced with 15% CO2. 14 control (C) P had similar BR levels without HC. Serum free bilirubin was similar in C & HC P. RBBF (by microspheres) & RBBD were as follows: (M±SEM) *p

This study was designed to explore the mechanisms of previously shown enhanced bilirubin brain deposition in urea induced HO. 28 2-4 day old P were infused with bilirubin(B)to raise & maintain serum B(SB) to approximately 8mg/dl. 14 P served as isoosmotic controls(C),7 P received urea infusions to raise serum osmolality to 330mOsm//L(HO330)& 7 P to 375mOsm/L(HO375). During 60 min of HO, SB was similar among the 3 groups(7-9mg/dl). Serum unbound B(UB) was also similar between HO330&C. In contrast, UB was 2.7-fold higher in HO375 vs C(p

Carotid artery injection of Evans Blue or albumin-bound bilirubin (ALB+BR) for 60 sec. in adult rats produced no cortical staining with an intact blood-brain barrier(BBB) and non-uniform “tufted” perivascular staining after barrier opening to ALB by 1.8 Molar Urea × 30 sec. Unbound BR given for 10-60 sec. to 25 rats without barrier opening, followed by carotid perfusion with saline, produced uniform staining, and the uptake of BR measured by chloroform extraction was dose-dependent (r=0.641, p

This study was designed to assess the mechanism of bilirubin entry into the brain in rats when subjected to bilirubin displacement by sulfisoxazole. Hyperbilirubinemia (approximately 170μM/l or 10 mg/dl)) was established by infusion of unconjugated bilirubin at a rate of 30 mg/kg/hr for three hours. After 2 hours of bilirubin infusion, displacement of bilirubin was produced by a bolus infusion of sulfisoxazole at a dose of 50 mg/kg, resulting in a significant but transient increase in the serum concentrations of unbound bilirubin from 1.84±0.29 μg/dl at 120 mins to 3.10±0.38 μg/dl at 130 mins. (Mean ±S.E.M., p

213 of 508 consecutive neonatal ICU admissions (Jan-June, 1983) had gastric aspirate cultures for prolonged rupture of membranes (>18 hrs), maternal fever, or respiratory distress at birth. To evaluate the use of GA cultures as a screen for neonatal sepsis or pneumonia, we compared GA culture results with clinical outcome, autopsy findings, radiologic diagnosis of pneumonia, and results of cultures from other sites. 18 of the 213 infants had confirmed sepsis or pneumonia, with positive and negative GA cultures distributed as shown in the table: 7 of 18 GA cultures (39%) in newborns with serious infections, including 6 of 8 (75%) in newborns with bacteremia, were positive for the specific pathogen found at other sites. 13 of 195 GA cultures (7%) gave "false positive" results in newborns with no subsequent evidence of neonatal infection. 182 of 195 infants (93%) evaluated for infection because of perinatal risk factors, but with no subsequent supporting evidence for perinatal infection, had negative GA cultures. GA is easily obtained for culture and microscopic examination at birth. Bacteriologic examination of GA has a low rate of false positive results and may permit early recognition of bacterial pathogens in some newborns with serious perinatal infections.

1266 TRANCUTANEOUS pO 2 (TCpO 2 ) DURING INTRAVENOUS FAT INFUSIONS IN LOW BIRTH WEIGHT INFANTS

Hypothermia, hypoglycemia, hypoxemia, and acidosis may increase the risk of kernicterus at low serum bilirubin levels. When pH is lowered in vitro, no change in free bilirubin (UB) or in bilirubin binding affinity (Ka) can be detected (Nelson et al. Pediat. Res. 8:963, 1974). In 11 newborns with respiratory distress and acidosis in whom body temperature, oxygenation, and blood glucose were normal, we measured UB and Ka by peroxidase oxidation at a bilirubin:albumin molar ratio (B/A) of 0.8 before and after correction of acidosis with NaHCO3 and/or assisted ventilation. Serum albumin (3.3 vs. 3.1 gm%) and indirect bilirubin levels (2.3 vs. 2.8 mg%) were similar during acidosis and recovery. Results are as follows: Ka of normal infants matched for gestational age is 2.0±0.2 × 108 M−1 in our laboratory. The data indicate a significant decrease in Ka associated with neonatal acidosis; correction of acidosis results in improvement of Ka and reduction in free bilirubin.