William J. Cashore

Determination of reserve albumin-equivalent for ligand binding, probing two distinct binding functions of the protein

Abstract A method is reported for determination of albumin binding capacity for various ligands in 50-μl sample volumes. A small amount of a radioactively labeled test ligand is added to the undiluted sample and the rate of dialysis of the free ligand into an identical sample without added ligand is measured. The reserve albumin-equivalent concentration is defined as the concentration of a standard albumin preparation which in buffered solution gives the same rate of dialysis and hence the same ratio of free/bound concentrations of the added ligand. It is shown that the reserve albumin-equivalent concentration, thus defined, is identical with the sum of concentrations of carrier species, each multiplied by the first stoichiometric binding constant of the test ligand to the carrier and divided by its first stoichiometric binding constant to the standard albumin. Determinations of this parameter are suitable for studies of the chemical potential and transfer affinities of individual ligands and for determination of interaction among several binding substances. Two test ligands have been used, monoacetyldiaminodiphenyl sulfone and diazepam. The former is bound competitively with bilirubin while diazepam engages another, independent binding function. The method can thus be used for separate determinations of the degree of saturation of two distinct binding functions of albumin. Complex mixtures of several carrier proteins with interacting ligands can be studied.

Clostridia colonization and clostridial toxin in neonatal necrotizing enterocolitis

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.

BILIRUBIN AND JAUNDICE IN THE MICROPREMIE

Although it has been customary to treat neonatal jaundice at lower serum bilirubin levels in low-birth weight infants than in term infants, the threshold bilirubin levels and long-term benefits for early treatment of preterm infants have not been validated. This article summarizes and evaluates existing evidence and strategies for early treatment of bilirubin in micropremies and recommends a conservative but flexible approach to early monitoring and phototherapy for jaundice in extremely low-birth weight infants.

Acceleration of fetal lung maturation by aminophyllin in pregnant rabbits.

Extract: 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 μg/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 μg/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.Speculation: Our data do not permit extrapolation into the clinical application of utilizing xanthine derivatives for the enhancement of fetal lung maturation. However, they provide evidence that there may be several pharmacologic agents that might enhance fetal lung maturation by various mechanisms. These data may also provide impetus for some centers to review the statistical correlation of respiratory distress syndrome in infants born to mothers who have received xanthine derivatives during the latter part of pregnancy for the treatment of such conditions as bronchial asthma. If the latter clinical evidence is a positive one, it may be justified to initiate a clinical trial to evaluate the effect of xanthine derivative treatment of the mother for the purpose of preventing respiratory distress syndrome in the newborn infant.

Behavioral changes correlated with brain-stem auditory evoked responses in term infants with moderate hyperbilirubinemia

The purpose of this study was to test the hypotheses that newborn infants with moderate serum bilirubin concentrations have depressed Brazelton scores and increased brain-stem conduction time and that serum bilirubin levels correlate with Brazelton behavior scores and brain-stem auditory evoked response changes. Fifty term infants who were enrolled into either a low serum bilirubin group (less than 8 mg/dl) or a moderate serum bilirubin group (10 to 20 mg/dl) were tested with the Brazelton Neonatal Behavioral Assessment Scale and a brain-stem auditory evoked response test. Partial correlation analysis controlling for phototherapy revealed that increased bilirubin concentration correlated negatively with the Brazelton orientation and with state range clusters and individual Brazelton test items that involve auditory processing. Increased bilirubin concentration correlated with an increased latency of brain-stem auditory evoked response wave 4, 5. An increased interpeak 1-5 (brain-stem conduction time) correlated with the decreased animate visual and auditory item. We conclude that moderate hyperbilirubinemia in term infants affects both infant behavior, as measured by specific components of the Brazelton test, and brain-stem conduction time, as measured by the brain-stem auditory evoked response test.

Abnormal brain-stem function (brain-stem auditory evoked response) correlates with acoustic cry features in term infants with hyperbilirubinemia.

We hypothesized that changes in brain-stem auditory evoked responses related to bilirubin would be associated with changes in cry because of the anatomic proximity in the brain stem of cranial nerves 8 (auditory) and 9 to 12 (vagal complex, which controls cry). Brain-stem auditory evoked responses and computerized cry analysis were used to study the concurrent effects of moderate hyperbilirubinemia on auditory function and cry. Fifty term infants were divided equally into two groups on the basis of serum bilirubin concentrations: low (less than 8 mg/dl; 136) mumol/L and moderate (10 to 20 mg/dl, 170 to 342 mumol/L). Forty-three infants had successful tracings of brain-stem auditory evoked responses recorded with a Cadwell model 5200A evoked response unit during two successive trials, and a cry recording of each infant was analyzed by computer. The moderate serum bilirubin group had an increase in percent cry phonation (p less than 0.02) and an increase in the variability of the first formant (p less than 0.04) in comparison with the low serum bilirubin group. Serum bilirubin values correlated positively with brain-stem conduction time (r = 0.36, p less than 0.01), percent phonation (r = 0.42, p less than 0.004), and variability of the first formant (r = 0.39, p less than 0.02). Percent phonation, the voiced component produced by increased neural control, correlated with the interpeak of waves latencies I to III (r = 0.32, p less than 0.03) and brain-stem conduction time (wave I to V) (r = 0.35, p less than 0.01). We conclude that hyperbilirubinemia affects adjoining areas of the brain stem that control hearing and cry production.

Free bilirubin concentrations and bilirubin-binding affinity in term and preterm infants

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.

Ontogeny of bilirubin-binding capacity and the effect of clinical status in premature infants born at less than 1300 grams.

BACKGROUND. Bilirubin is toxic to the brain and enters the brain in unbound form. Serum unconjugated, unbound bilirubin may be a good predictor of bilirubin encephalopathy. Unbound bilirubin levels may depend on the bilirubin-binding capacity of albumin, which has not been described for neonates of <28 weeks’ gestation. OBJECTIVE. The purpose of this work was to determine the ontogeny of bilirubin-binding capacity and the effect of clinical status in very preterm neonates. METHODS. A total of 152 neonates (23–31 weeks’ gestational age; 440–1300 g) were enrolled prospectively. At 5 days of age, total serum bilirubin and unbound bilirubin were measured with the unbound bilirubin-A1 analyzer (Arrows Co, Osaka, Japan) and albumin with the Bromocresol-purple method. Scatchard plots were used to estimate bilirubin-binding affinity and capacity. Clinical status for each infant was rated as high, moderate, or low risk by using a modified Score for Neonatal Acute Physiology model. Low risk was considered clinically stable. RESULTS. Unbound bilirubin has a significant, direct correlation to total bilirubin and is greater in unstable than in stable neonates. For the entire cohort, bilirubin-binding capacity had a direct relationship to gestational age. The bilirubin-binding capacities of infants in the low- and high-risk groups also had a direct relationship to gestational age. Bilirubin-binding capacity was greater in the low-risk group (20.8 ± 4.6 mg/dL; 356 ± 79 μmol/L) than in the moderate- (17.8 ± 3.5 mg/dL; 304 ± 60 μmol/L) or high- (17.3 ± 3.4 mg/dL; 296 ± 58 μmol/L) risk groups. Bilirubin-binding affinity did not differ by clinical risk status or gestational age. CONCLUSIONS. In very preterm, very low birth weight infants, bilirubin-binding capacity is directly proportional to gestational age. Bilirubin-binding capacity is lower and unbound bilirubin higher in unstable than in stable neonates. These data may be useful in guiding the management of hyperbilirubinemia in very low birth weight infants.

RESERVE ALBUMIN AND BILIRUBIN TOXICITY INDEX IN INFANT SERUM

ABSTRACT. Reserve albumin concentration (the concentration of albumin available for binding of unconjugated bilirubin) was determined in 95 sera from 76 subjects by dialysis with 14C‐monoacetyl diamino diphenyl sulfone (MADDS). An index, I of bilirubin toxicity in the plasma was calculated for each subject, based on the bilirubin and reserve albumin concentrations, the affinity of bilirubin for serum albumin, and the pH‐dependent solubility of bilirubin in the plasma. The values of reserve albumin and of I varied significantly with gestational age, clinical condition (whether sick or well), and serum bilirubin level. The value of reserve albumin was decreased and I was increased in association with clinical factors (e. g., hyperbilirubinemia, hypoxia, acidosis, or sepsis) recognized as increasing the risk for bilirubin encephalopathy. The lowest values of reserve albumin and the highest values of I were found in the least mature and sickest infants.

Permeability of the blood brain barrier for 125I-albumin-bound bilirubin in newborn piglets.

ABSTRACT: Permeability of the blood brain barrier (BBB) for bilirubin and 125I-albumin was studied in 2-d- and 2-wk-old piglets. 125I-albumin was given by bolus at the beginning of each study. Hyperbilirubinemia was produced by an initial bolus infusion of bilirubin and sustained at a plasma bilirubin:albumin molar ratio of approximately 1.0 by continuous infusion of bilirubin for 3 h. During the study period, arterial pH and blood gas tensions, serum osmolarity, and mean arterial blood pressure were within the physiologic range for age in both groups. Serum albumin and serum total and unbound bilirubin concentrations were higher in the 2-wk-old piglets. Brain bilirubin concentrations and permeability ( P · S product) of the BBB for bilirubin were higher in the 2-d-old than in the 2-wk-old piglets, but the values of P · S for albumin were not different between the two groups. In 2-d-old piglets, regional brain bilirubin concentrations and permeability of the BBB were higher in subcortical regions (cerebellum and brainstem) than in the cerebral cortex. Regional brain albumin concentrations and BBB permeability to albumin in 2-d-old piglets were higher only in the cerebellum. In all regions, the bilirubin:albumin molar ratio was higher in the brain tissues than in the blood. In 2-wk-old piglets, the brain concentrations and P · S products for bilirubin were lower and the regional differences were less marked than for 2-d-old animals. We conclude that in 2-d-old piglets the blood brain barrier is more permeable to bilirubin than to albumin, that brainstem and cerebellum are more permeable to bilirubin than cortical regions, and that by 2 wk the permeability of the BBB to bilirubin decreases while permeability to albumin remains unchanged.