Influence of Drugs on Albumin and Bilirubin Interaction

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 (K_D= 5.9 times 10³ M-¹ and 4.5 times 10³ M-¹, respectively) as strongly as Na salicylate (K_D= 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 (K_D= 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.     

Potential of moxalactam and other new antimicrobial agents for bilirubin-albumin displacement in neonates

The effects of several novel antibiotics on in vitro binding of bilirubin to human serum albumin were investigated. At physiologic bilirubin-albumin ratios and pH values, aztreonam, imipenem, azlocillin, enoxacin and ciprofloxacin did not compete with bilirubin at drug concentrations less than 900 micrograms/mL. Cefoperazone caused an apparent increase in unbound bilirubin only at concentrations greater than 35 microM (330 micrograms/mL). Moxalactam, however, caused a linear increase in unbound bilirubin, greater than that seen with sulfisoxazole, over the entire range of antibiotic concentrations. These results may have implications for the use of these newer antimicrobial agents in neonatal infections.     

Bilirubin binding in premature infants from birth to 3 months.

The effect of postnatal age on serum bilirubin binding measurements was studied prospectively in extremely premature infants of 25-28 weeks'' gestation. Serum was obtained from 10 infants at birth, 2-4 days of age, 1 week, 2 weeks, 4 weeks, 8 weeks, and 10-13 weeks. Using peroxidase oxidation, the apparent unbound bilirubin concentration (AUBC) was measured and plotted versus the molar ratio of total bilirubin:albumin (R) using the empiric power curve AUBC = aRb. The apparent unbound bilirubin concentration at bilirubin:albumin ratio 0.6 was used to compare relative binding ability among specimens. This value, as well as the apparent association constants, showed dramatic deterioration after birth, which persisted without improvement until 8 weeks of age. This pattern of recovery correlated in general with the resolution of clinical problems. Binding values equivalent to adult serum were achieved by 10-13 weeks. This study emphasises that diminished bilirubin binding by the sera of premature infants can persist for a prolonged period.     

D-PENICILLAMINE, A NON-BILIRUBIN-DISPLACING DRUG IN NEONATAL JAUNDICE

Abstract. Brodersen, R., Lakatos, L. and Karmazsin, L. (Institute of Medical Biochemistry, University of Aarhus, Denmark, and Pediatric Clinic, University Medical School, Debrecen, Hungary). D-penicillamine, a non-bilirubin-displacing drug in neonatal jaundice. Acta Paediatr Scand, 69:31, 1980.—D-penicillamine, a drug used clinically for the treatment of neonatal hyperbilirubinaemia, was tested for interference with the binding of bilirubin to human serum albumin by three methods: 1) The peroxidase technique, investigating the effect of D-penicillamine on the equilibrium concentration of unbound bilirubin in a solution containing a molar excess of albumin; 2) the MADDS method, measuring the concentration of vacant bilirubin binding site on albumin in a solution of pure albumin, or infant blood serum, with added D-penicillamine; and 3) injection of D-penicillamine into Gunn rats and determination of any decrease of plasma biiirubin which would be caused by displacement of the pigment. Results were negative in all cases. Quantitatively, the doses of D-penicillamine used clinically cannot displace bilirubin from its binding to albumin. The ameliorating effect on hyperbilirubinaemia in the newborn must be due to some other mechanism     

Influence of gestational age on cord serum bilirubin binding studies

The effect of gestational age on bilirubin binding was studied using cord serum from 22 preterm infants and 13 term infants and serum from 17 adults. Using the peroxidase oxidation method, a bilirubin titration curve was obtained by adding bilirubin to serum and measuring the apparent unbound bilirubin concentration. The resultant curve was analyzed using the least-squares fit of the empiric equation Y = aXb. After correction for albumin concentration by plotting the apparent unbound bilirubin concentration against the molar ratio of total bilirubin/albumin, term and preterm infants had identical titration curves, which remained inferior to that of adults. In addition, the apparent primary bilirubin association constant Ka'1 was similar for all infants but was two to three times less than that for adults. We conclude that bilirubin binding by cord serum is equivalent regardless of gestational age. However, adult serum binds bilirubin qualitatively better than does serum from infants of all gestational ages. We suggest that the adverse effect of prematurity on bilirubin binding noted in previous studies may have reflected postnatal complications rather than gestational age as such.     

Severely high serum unbound bilirubin level after abdominal surgery in a newborn

We report a newborn with intestinal malrotation who developed a severely high serum unbound bilirubin level and a low serum albumin level without a marked increase in serum total bilirubin level after abdominal surgery, which required exchange transfusion and albumin supplementation. The serum unbound bilirubin level may be highly relative to the serum total bilirubin level in newborns who have undergone abdominal surgery soon after birth and are hypoalbuminemic after surgery.     

Effects of albumin infusion therapy on total and unbound bilirubin values in term infants with intensive phototherapy

BACKGROUND: The purpose of the present study was to evaluate the effect of intravenous albumin administration on the serum total and unbound bilirubin values in term non-hemolytic hyperbilirubinemic neonates during intensive phototherapy. METHODS: Fifty-eight infants (gestational age 39.4 +/- 1.4 weeks; birth weight 3,245 +/- 435 g) were given phototherapy with similar light energy. Twenty infants (control group) received only phototherapy, while 38 others (albumin-treated group) were also given human albumin at 1 g/kg bodyweight, i.v., during the first 2 h of phototherapy. RESULTS: When comparing changes in total and unbound bilirubin values 0, 2, 6 and 24 h after entering the study between the albumin-treated group and the control group, there was a significant reduction in the serum unbound bilirubin values at the end of albumin treatment and at 6 and 24 h. However, there was no significant reduction in total serum bilirubin values during the study period. In the albumin-treated group, the mean serum unbound bilirubin reduction from the baseline level at the end of albumin treatment and at 6 and 24 h was 0.40 +/- 0.19, 0.41 +/- 0.20 and 0.43 +/- 0.20 microg/dL, respectively. CONCLUSIONS: The results suggest that albumin priming may be effective for an immediate reduction in serum unbound bilirubin values, the fraction that is potentially neurotoxic.     

STUDIES ON PHOTOTHERAPY IN NEWBORN INFANTS

ABSTRACT: Windorfer, A., Faxelius, G. and Boréus, L. O. (Departments of Clinical Pharmacology and Paediatrics, Karolinska Hospital, Stockholm, Sweden). Studies on phototherapy in newborn infants: Influence on protein binding of bilirubin and salicylate and on activity of acetylsalicylic acid esterase. Acta Paediatr Scand 64:293, 1975.– Phototherapy of newborn infants with hyperbilirubinemia was shown to result in an increase in hematocrit values and in the activity of the erythrocyte enzyme acetylsalicylic acid esterase. The elevation of the enzyme activity also could be produced in light-treated rabbits and in vitro after illumination of blood from adult volunteers. The binding of bilirubin to serum albumin and of salicylate to plasma proteins did not alter, nor did the concentrations of albumin or total proteins in plasma. It is concluded that light does not increase the unbound fraction of bilirubin in blood.     

Bilirubin-albumin binding affinity and serum albumin concentration during intensive phototherapy (blue double light) in jaundiced newborn infants

Thirty newborn infants with normal birth weights and uncomplicated hyperbilirubinaemia were studied. Twenty three of these were treated continuously for 24h with intensive phototherapy (blue double light), and seven untreated infants served as controls. During the treatment the serum concentrations of total bilirubin and unbound bilirubin in diluted serum measured by the peroxidase method were markedly reduced. The binding affinity of bilirubin to its high affinity site on serum albumin was not affected. During the treatment a slight decrease of the serum albumin concentration occurred, and the possible causes of this observation are discussed.     

Deposition of bilirubin acid in the central nervous system--a hypothesis for the development of kernicterus

On the basis of the concentration of unconjugated bilirubin and available albumin for the binding of bilirubin it is possible to calculate the level of unbound bilirubin in a serum sample. The solubility of bilirubin can further be calculated when the pH is known. In cases of threatened kernicterus the free bilirubin concentration in serum samples from newborn infants surpasses the solubility by a factor close to one hundred. It is hypothesized that deposition of bilirubin in tissues takes place as an ongoing event, the deposited pigment being eliminated by bilirubin oxidase in healthy infants. Kernicterus results when the rate of deposition becomes overwhelming as a result of high bilirubin concentration, low albumin reserve, low pH, after administration of a displacing drug, or if the bilirubin oxidase system has been compromised by preceding birth asphyxia or other forms of central nervous system injury.     

Deposition of Bilirubin Acid in the Central Nervous System–A Hypothesis for the Development of Kernicterus

ABSTRACT. On the basis of the concentration of unconjugated bilirubin and available albumin for the binding of bilirubin it is possible to calculate the level of unbound bilirubin in a serum sample. The solubility of bilirubin can further be calculated when the pH is known. In cases of threatened kernicterus the free bilirubin concentration in serum samples from newborn infants surpasses the solubility by a factor close to one hundred. It is hypothesized that deposition of bilirubin in tissues takes place as an ongoing event, the deposited pigment being eliminated by bilirubin oxidase in healthy infants. Kernicterus results when the rate of deposition becomes overwhelming as a result of high bilirubin concentration, low albumin reserve, low pH, after administration of a displacing drug, or if the bilirubin oxidase system has been compromised by preceding birth asphyxia or other forms of central nervous system injury.     

EFFECT OF EXCHANGE TRANSFUSION ON SERUM RESERVE ALBUMIN FOR BINDING OF BILIRUBIN AND INDEX OF SERUM BILIRUBIN TOXICITY

ABSTRACT. Ebbesen F. (Department of Neonatology, Rigshospitalet, Copenhagen, Denmark). Effect of exchange transfusion on serum reserve albumin for binding of bilirubin and index of serum bilirubin toxicity. Acta Paediatr Scand, 70:643,.–Seventeen newborn infants, who received their first exchange transfusion due to hyperbilirubinaemia and/or rhesus haemolytic disease, were studied. The exchange transfusions were performed with fresh, citrated blood. During the exchange transfusion a marked increase in the serum reserve albumin concentration for binding of bilirubin measured by the [^,4C]-MADDS method was observed, followed by a smaller decrease after the transfusion. Plasma pH increased both during and after the exchange transfusion. During the exchange transfusion a drastic fall in index of serum bilirubin toxicity was observed, followed by a smaller increase after the transfusion. Citrate was not found to interfere in the binding of bilirubin to albumin. The results are in agreement with the clinical finding that an exchange transfusion performed with fresh, citrated blood effectively reduces the risk of bilirubin encephalopathy. The ratio in serum of binding albumin, i.e. bilirubin plus reserve albumin, to total albumin failed to be increased by the exchange transfusion, and a decrease occurred after the transfusion. These findings indicate the presence in infant serum of non-binding albumin. Donor albumin with intact binding potential is partly transformed into the non-binding variety in the course of one hour after the transfusion. In the most severely rhesus sensitized infant a drastic decline of the serum albumin binding capacity was seen during the first day of life.     

Unbound bilirubin associated with kernicterus: a historical approach

OBJECTIVE: To determine the unbound bilirubin concentration (UBC) associated with kernicterus with the use of clinical data from clusters of kernicterus after sulfisoxazole and benzyl alcohol administration. DESIGN: Sulfisoxazole at 12 mg/dL and benzoate at 10 mmol/L are associated with kernicterus at total bilirubins near 12 and 10 mg/dL, respectively. The concurrent UBC was estimated by first measuring the drug-induced increases in UBC in plasma and artificial sera (peroxidase-diazo method). The increases were then applied to baseline UBC, determined by linear regression analysis of binding data (peroxidase method) from 86 newborns, at total bilirubins of 12 mg/dL for sulfisoxazole and 10 mg/dL for benzoate. Sensitivity and specificity were determined with existing data. RESULTS: Sulfisoxazole and benzoate increased UBC in artificial sera 2.1-fold and 4.1-fold, respectively, and in plasma (sulfisoxazole) 2.4-fold. Benzoate would increase baseline UBC from 0.29 to 1.19 microg/dL and sulfisoxazole from 0.36 to 0.86 microg/dL. The sensitivity and specificity of a UBC of 0.86 microg/dL for predicting kernicterus are 79% and 92% and for 1.19 microg/dL, 50% and 98%, respectively. CONCLUSION: Historic data predict that the unbound bilirubin above which kernicterus becomes likely lies between 0.86 and 1.19 microg/dL, in good agreement with existing information.     

Effect of ibuprofen on bilirubin-albumin binding

Ibuprofen is used for closing the ductus arteriosus in premature newborn infants. Ibuprofen interferes with bilirubin-albumin binding and increases the unbound bilirubin in pooled newborn plasma to levels similar to those produced by sulfisoxazole, a drug that causes kernicterus in premature newborn infants.     

Bilirubin binding in the plasma of newborns: critical evaluation of a fluorescence quenching method and comparison to the peroxidase method

Bilirubin binding affinities and capacities and apparent unbound ("free") bilirubin levels were determined in serum samples from 47 high-risk newborns, in 22 samples of cord serum, and in serum samples from 15 Greek children with marked hyperbilirubinemia, by both fluorescence quenching and peroxidase methods. The free fatty acid:albumin molar ratio was also determined for serum samples from high-risk newborns. In vitro and in vivo measurements suggest that free fatty acids are rarely present at levels that produce significant displacement of bilirubin, which is in agreement with previous studies. The two bilirubin binding assays showed only fair correlation with sizable discrepancies for many specimens. Technical difficulties inherent in the fluorescence quenching method and possible sources of error are discussed. Our observations suggest that routine application of these two assays as the primary criterion for therapeutic intervention (e.g., exchange transfusion) is premature.     

Comparative analysis between albumin-binding capacity and the concentration of unbound bilirubin in hyperbilirubinemic sera of newborn infants

Albumin-binding capacity and the concentration of unbound bilirubin after separation by gel filtration were determined in the sera of 20 newborn infants aged 1 to 7 days. Total serum bilirubin level ranged between 12.0 and 23.8 mg/100 ml. The results are as follows:

1. The albumin-binding capacity ranged between 48 and 100% without any relation to the level of total serum bilirubin.
2. The mean concentration of unbound bilirubin was 0.11 mg/100 ml. No significant correlations were found between unbound bilirubin levels and the albumin-binding capacity or the concentration of total bilirubin.

     

Factors affecting the binding of bilirubin to serum albumins: validation and application of the peroxidase method

The unbound "free" bilirubin concentration (Bf), not the total bilirubin concentration, is the critical determinant of cellular uptake and toxicity of bilirubin. We compared Bf measured by a modified peroxidase method with published data obtained with ultrafiltration and examined conditions that affect the affinity (KF) of human (HSA) and bovine (BSA) serum albumin for bilirubin. The peroxidase and ultrafiltration methods yielded similar KF values that decreased with increasing HSA concentration and the presence of 50 mM chloride. When related to ionic strength, inhibition of BSA-bilirubin binding by chloride, bromide, and sulfate were similar, whereas phosphate buffer had a smaller effect. KF was lower at 37 degrees C than at 25 degrees C for HSA but not for BSA. KF for BSA was similar at pH 7.4 and 8.0. BSA and FCS had similar binding properties. The close agreement of Bf and KF values determined by the peroxidase method with published results obtained by ultrafiltration validates both methods and supports the use of the peroxidase method as a practical technique for measuring Bf under steady state conditions in minimally diluted serum or culture medium.     

The effect of multiple exchange transfusions on bilirubin binding

Three bilirubin binding tests (hydroxybenzene-azobenzoic acid dye binding method, the estimation of unbound bilirubin by horseradish peroxidase assay and the saturation of albumin by the salicylate saturation index) were performed on pre-exchange samples of blood and repeated 24 hours after the procedure. No significant improvement in bilirubin binding was found even in infants receiving as many as four exchange transfusions. Based on these bilirubin binding tests, we find no evidence that the criteria for subsequent exchange transfusions should be different from the first exchange transfusion.     

The Effect of Multiple Exchange Transfusions on Bilirubin Binding

ABSTRACT. Three bilirubin binding tests (hydroxybenzene-azobenzoic acid dye binding method, the estimation of unbound bilirubin by horseradish peroxidase assay and the saturation of albumin by the salicylate saturation index) were performed on pre-exchange samples of blood and repeated 24 hours after the procedure. No significant improvement in bilirubin binding was found even in infants receiving as many as four exchange transfusions. Based on these bilirubin binding tests, we find no evidence that the criteria for subsequent exchange transfusions should be different from the first exchange transfusion.     

Bilirubin-albumin-binding function of 2 human albumin preparations (placental and plasma). Comparison of their efficacy in the icteric premature infant

Two albumin preparations obtained by Cohn fractionation of either plasma of blood donors (plasmatic albumin) or human placental blood (placental albumin) were studied in vitro and in vivo regarding their bilirubin-binding function. Analysis of this function during the industrial processing of the two preparations indicated that alcoholic fractionation and, to a lesser extent, stabilizers, were responsible for the decrease of (a) the association constants between albumin and bilirubin, (b) bilirubin-binding capacity of albumin. Unexpectedly, improvement of bilirubin-binding parameters was observed after the final heating stage. Stabilizers were reversibly bound as suggested by a further improvement of binding function seen after a brief contact of the preparations with red blood cells. The changes were similar for the two preparations. Fifty-one sick premature hyperbilirubinemic neonates were randomly infused either with placental or plasmatic albumin (1.5 g/kg). Albuminemia, bilirubinemia, erythrocytic bilirubin, unbound bilirubin (peroxidase method) were evaluated before and 3 hours after infusion. Improvement of bilirubin-binding parameters was frequently observed but without clear-cut relation with change in bilirubin/albumin molar ratio. No difference was noted between the two albumin preparations. In spite of a decrease of their association constants with bilirubin, the two albumins retained a high binding potency for bilirubin in vivo.